ENVIRONMENTAL RESEARCH LABORATORY
COL
U, 8. aWHtOIIMIflTAl. PROTECTION ASEftCY
OFFICE OF RESEARCH & DEVELOfSilKNT
BtvmOMMEirrAi RESEARCH LABORATORY
SAQINE ISLAND
GULF BREF.ZE, FLORIDA 32501
-------
ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE
LU
O
T
COLLECTED REPRINTS
1973-1974
U. S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH & DEVELOPMENT
ENVIRONMENTAL RESEARCH LABORATORY
SABINE ISLAND
GULF BREEZE, FLORIDA 32561
-------
TABLE OF CONTENTS
Contribution
No.
126. GERALD E. WALSH and THOMAS E. GROW
Composition of Thalassia testudinum and Ruppia maritima.
131. W. P. SCHOOR
In vivo binding of p,p'-DDE to human serum proteins.
143. GERALD E. WALSH, SISTER REGINA BARRET, GARY H. COOK and
TERRENCE A. HOLLISTER.
Effects of herbicides on seedlings of the red mangrove
Rhizophora mangle L.
147. DAVID J. HANSEN, STEVEN C. SCHIMMEL, AND JAMES M.
KELTNER, JR.
Avoidance of pesticides by Grass Shrimp (Palaemonetes
pugio).
154. GERALD E. WALSH
Mangroves: A Review.
155. PHILIP A. BUTLER
Organochlorine residues in estuarine molluscs, 1965-1972.
156. P. W. BORTHWICK, T. W. DUKE, A. J. WILSON, JR., J. I. LOWE,
J. M. PATRICK, JR., and J. C. OBERHEU
Accumulation and movement of Mirex in selected estuaries
of South Carolina, 1969-71.
157. A. L. JENSEN
The relationship between dynamic pool and surplus produc-
tion models for yield from a fishery.
158. NELSON R. COOLEY, JAMES M. KELTNER, JR., AND JERROLD
FORESTER R
The polychlorinated biphenyls, Aroclor 1248 and 1260:
Effect on and accumulation by Tetrahymena pyriformis.
159. TERRENCE A. HOLLISTER and GERALD E. WALSH
Differential responses of marine phytoplankton to
herbicides,'oxygen inhibition.
163. W. P. SCHOOR
Some aspects of myosin adenosine triphosphatase of pink
shrimp (Penaeus duorarum).
164. DAVID J. HANSEN
Aroclor^ 1254: Effect on composition of developing
estuarine animal communities in the laboratory.
-------
Contribution
No.
165. A. W. BOURQUIN
Estuarine microbes and organochlorine pesticides
(a brief review).
167. MARLINE. TAGATZ
A larval tarpon, Megalops atlanticus, from Pensacola,
Florida.
168. P. W. BORTHWICK, G. H. COOK, and J. M. PATRICK, JR.
Mirex residues in selected estuaries of South Carolina
- June 1972.
169. DAVID L. COPPAGE and EDWARD MATTHEWS
Short-term effects of organophosphate pesticides on
cholinesterase of estuarine fishes and pink shrimp.
170. D. R. NIMMO, J. FORESTER, P. T. HEITMULLER, and G. H. COOK
Accumulations of Aroclor^ 1254 in grass shrimp
(Palaemonetes pugio) in laboratory and field exposures.
172. D. J. HANSEN, P. R. PARRISH, and J. FORESTER
AroclorR 1016: Toxicity to and uptake by estuarine
animals
174. PATRICK R PARRISH
AroclorR 1254, DDT, and ODD, and Dieldrin: Accumulation
and loss by American oyster, (Crassostrea virginica)
exposed continuously for 56 weeks.
175. S. C. SCHIMMEL, D. J HANSEN, and J. FORESTER
Effects of AroclorR 1254 on laboratory-reared embryos
and fry sheepshead minnows (Cyprinodon variegatus) .
176. PHILIP A. BUTLER
Trends in pesticide residues in shellfish.
177. D. J. HANSEN, S. C. SCHIMMEL, and JERROLD FORESTER
AroclorR 1254 in eggs of sheepshead minnows: Effect
on fertilization success and survival of embryos and
fry.
178. P. R. PARRISH, JOHN A. COUCH, J. FORESTER, J. M. PATRICK
JR., and G. H. COOK ' '
Dieldrin: Effects on several estuarine organisms.
179. M. E. TAGATZ, P. W. BORTHWICK, G. H. COOK, and D. L
COPPAGE
Studies of ground applications of malathion on salt-marsh
environments in northwest Florida.
-------
Contribution
No.
180- THOMAS W. DUKE
Criteria for determining importance and effects of
pesticides on the marine environment: A brief over-
view.
181. D. J. HANSEN, S. C. SCHIMMEL, and E. MATTHEWS
Avoidance of Aroclor 1254 by shrimp and fishes.
183. A. L. JENSEN
Leslie matrix models for fisheries studies.
184. A. L. JENSEN
Predator-prey and competition models with state
variables: biomass, number of individuals, and average
individual weight.
190. WILHELM P. SCHOOR ,,
Accumulation of Mirex- C in the adult blue crab
(Callinectes sapidus).
191. PHILIP A. BUTLER
Biological problems in estuarine monitoring.
192. NELSON R. COOLEY
Occurrence of snook on the north shore of the Gulf of
Mexico.
193. GERALD E. WALSH, TERRENCE A. HOLLISTER, and JERROLD
FORESTER
Translocation of four organochlorine compounds by red
mangrove, (Rhizophora mangle L.) seedlings.
195. THOMAS W. DUKE, and DAVID P. DUMAS
Implications of pesticide residues in the coastal
environment.
196. PATRICK R. PARRISH, DAVID J. HANSEN, JOHN A. COUCH,
JAMES M. PATRICK, JR., and GARY H. COOK
Effects of the polychlorinated biphenyl, Aroclor^ 1016,
on estuarine animals.
198. D. R. NIMMO, and L. H. BAHNER
Some physiological consequences of polychlorinated
biphenyl and salinity-stress in penaeid shrimp.
200. L. H. BAHNER
A salinity controller for flowing-water bioassays.
-------
Contribution
No.
201. AL W. BOURQUIN, and GARY H. COOK
Degradation of malathion by estuarine microbes.
202. NELSON R. COOLEY
Effects of pesticides on protozoa.
203. A. W. BOURQUIN and GARY H. COOK
Impact of microbial seed cultures on the aquatic
environment.
204. ALFRED J. WILSON, JR., and JERROLD FORESTER
Methods and problems in analysis of pesticides in the
estuarine environment.
207. STEVEN C. SCHIMMEL, DAVID J. HANSEN, and JERROLD FORESTER
Effects of Aroclor 1254 on laboratory-reared embryos
and fry, Cyprinodon variegatus (Pisces: Cyprinodontldae)
208. W. PETER SCHOOR
Theoretical model and solubility characteristics of
Aroclor^ 1254 in water.- Problems associated with low-
solubility compounds in aquatic toxicity tests.
209. L. KEIFER, H. JANNASCH, K. NEALSON, and A. BOURQUIN
Observations of luminescent bacteria, in continuous
culture.
210. AL BOURQUIN, L. KEIFER, and S. CASSIDY
Microbial response to malathion treatments in salt
marsh microcosms.
211. JOHN A. COUCH
Pathological effects of Urosporidium (Haplosporida)
infection in microphallid metacercariae.
213. JOHN A. COUCH
Free and occluded virus, similar to Baculovirus, in
hepatopancreas of pink shrimp
215. JOHN A. COUCH
An enzootic nuclear polyhedrosis virus of pink shrimp:
Ultrastructure, prevalence, and enhancement.
216. JOHN A. COUCH, and DELWAYNE R. NIMMO
Ultrastructural studies by shrimp exposed to the
pollutant chemical, polychlorinated biphenyl (Aroclor
1254).
219. J. A. COUCH, and D. R. NIMMO
Detection of interaction between natural pathogens and
pollutant chemicals in aquatic animals.
-------
Contribution
No.
220. JOHN A. COUCH
Ultrastruetural and protargol studies of Lagenophrys
callinectes (Ciliophora: Peritrichida).
238. P. W. BORTHWICK
A clinical centrifuge tube for small blood samples.
242. JOHN A. COUCH, and DELWAYNE R. NIMMO
(Abstract). Cytopathology, ultrastrueture, and virus_
infection in pink shrimp exposed to the PCB, Aroclor
1254.
245. PHILIP A. BUTLER
Estuaries.
247. JAMES W. GILLETTE, JAMES HILL, IV., ALFRED W. JARVINEN,
and W. PETER SCHOOR
A conceptual model for the movement of pesticides through
the environment.
250. J. COUCH, G. GARDNER,J. HARSHBARGER, M. TRIPP, and P. YEVICH
Histological and physiological evaluations in some marine
fauna.
251. JELLE ATEMA, CHARLES C. COUTANT, PATRICIA De COURSEY,
DAVID HANSEN, JAMES S. KITTREDGE, JOHN J. MAGNUSON, DON
MILLER and MARK J. SCHNEIDER
Behavioral measures of environmental stress.
-------
EXTRAMURAL RESEARCH PUBLICATIONS
EPA-660.3-75-015 Effects of Mirex and Methoxychlor on striped
mullet, Mug11 cephalus L. May, 1975.
Lee, Jong H., Colin E. Nash and Joseph R.
Sylvester.
(Oceanic Foundation, Makapuu Point, Waimanalo,
Hawaii 96795) .
Grant No. R 802348, Program Element No.
1EA077, ROAP/Task No. 10 AKC/040. David J.
Hansen, Project Officer.
EPA-660/3-75-024 The effect of Mirex and Carbofuran on
estuarine microorganisms. June, 1975. Brown,
Lewis R., Earl G. Alley and David W. Cook
Performing organization: Mississippi State
University, Mississippi State, Miss. 39762
Contract No. 68-03-0288, Program Element
No. 1-EA077. ROAP/Task No. 10 AKC/33.
Al W. Bourquin, Project Officer.
-------
AUTHOR INDEX
Contribution No.
Atema, Jelle 251
Bahner, Lowell H 198
200
Borthwick, Patrick W 156
168
179
238
Bourquin, Al W 165
201
203
209
210
Butler, Philip A. . . 155
176
191
245
Cooley, Nelson R - 158
192
202
Cook, Gary H 143
168
170
178
179
196
201
Coppage, David L 169
179
Couch, John A 178
196
211
213
215
216
219
220
242
250
-------
Contribution No.
Duke, Thomas W [[[ 156
180
195
Dumas , David P [[[ 195
Forester , Jerrold [[[ 158
170
172
175
178
193
204
207
Gillette, James W [[[ 247
Hansen, David J [[[ 147
164
172
175
177
181
196
207
Heitmuller, P. T [[[ 170
Hollister , Terrence A
159
193
Jensen, A. L [[[ ^57
183
184
Keltner, James M [[[ ^-,
158
Kief er, Linda [[[ 209
Lowe , Jack I .............................................. ,
-------
Contribution No.
Panish, Patrick R 172
174
178
196
Patrick, James M 156
168
178
196
Schimmel, Steven C 147
175
177
181
207
Schoor, W. Peter 131
163
190
208
247
Tagatz, Marlin E 167
179
Walsh, Gerald E 126
143
154
159
193
Wilson, Alfred J., Jr 156
204
-------
CONTRIBUTION NO. 126
-------
Composition of Thalassia testudinum and Ruppia maritima
GERALD E. WALSH AND THOMAS E. GROW
LITTLE is known at present about the nutritive value of aquatic
plants, especially in relation to annual variations in their chemical
constituents. Turtle grass (Thalassia testudinum) and widgeon
grass (Ruppia maritima) are common in the inshore waters of
Florida (Phillips, 1960). They are important constituents of estu-
arine nursery grounds for marine animals and many forms of plant
and animal life are associated with them (Hudson et al., 1970).
The seagrasses are eaten by fishes, turtles, and other aquatic ani-
mals (Randall, 1965), and birds (OIney, 1968). Detritus derived
from seagrasses is eaten by small marine animals (Menzies and
Rowe, 1969; Fenchel, 1970. Also, T. testudinum and its epiphytes
are important in biogeochemical cycles in estuarine areas (Parker,
1966).
Both T. testudinum and R. maritima have been used successfully
in preliminary experiments as fertilizers for tomatoes (van Breed-
veld, 1966) and as feed supplements for Sheep (Bauersfeld et aL,
1969).
Because of the importance of T. testudinum and R. maritima to
estuarine ecosystems, we investigated seasonal distributions of pro-
tein, carbohydrate, trace elements, and energy content of their
leaves and rhizomes. Also, the potential nutritive value of the sea-
grasses was evaluated.
METHODS
Thalassia testudinum and R. maritima were collected between 6
June 1969 and 27 May 1970 from a mixed bed at the western edge
of Sabine Island in Santa Rosa Sound near Gulf Breeze, Florida.
They were taken in the morning to avoid possible diel variation in
the factors measured. Abundance of R. maritima was greatly re-
duced in February 1970 and enough plant material could not be
collected for all tests.
After collection, plants were taken immediately to the laboratory,
where the epiphyton was removed. The leaves were separated from
the rhizomes and all were washed quickly in a stream of distilled
-------
98 QUARTERLY JOURNAL OF THE FLORIDA ACADEMY OF SCIENCES
water. Leaves and roots were dried to constant weight in an oven
at 100 C and ground in a Wiley mill to pass the 40-mesh sieve. The
pulverized material was stored in vacua over anhydrous calcium
carbonate until tested.
Ash content was determined by combustion in a muffle furnace
at 55 C for five hours.
Total protein was measured by the method of Strickland and
Parsons (1965) using acetonylacetone (2,5-hexanedione) reagent
and the procedure was standardized against the Kjeldahl-Nessler
method. We report protein content as percentage of dry weight and
of ash-free dry weight.
Total carbohydrate was measured by a variation of the anthrone
method for particulate carbohydrate (Strickland and Parsons, 1965).
Fifty mg of each sample were suspended in 50 ml of 0.2 N H2SO4 in
a 125-ml Erlenmeyer flash. The sample was hydrolysed at 100 C for
90 min. with mixing every 15 min. The hydrolysate was passed
through a glass-fiber filter of 0.45/* porosity and 0.2 ml of the filtrate
was pipetted into a test tube. To this was added 10.8 ml of anthrone
reagent (0.?, g anthrone, 8.0 ml 95 per cent alcohol, 30.0 ml distilled
water, and 100 ml concentrated H2SO4). The solution was heated
at 100 C for five min. and brought quickly to room temperature in
an ice-water bath. After 15 min. the extinction was measured
against distilled water at 6200 A in a one-cm glass cell in a Beckman
DU spectrophotometer. Glucose was used in preparation of stan-
dard carbohydrate solutions. The data are expressed as percentage
carbohydrate in dry weight and in ash-free dry weight.
Concentrations of sodium, potassium, magnesium, iron, man-
ganese, and zinc in leaves and rhizomes were measured by atomic
absorption spectroscopy, using a modification of the method of
David (1958). Approximately 0.01 g of dried plant material was
placed in a 30 ml Kjeldahl digestion flask with two ml of a 1:7
sulphuric acid-perchloric acid mixture and 10 to 12 ml of nitric
acid. Digestion was continued until organic matter was completely
destroyed. Four glass beads were added to each flash to prevent
bumping.
After digestion, the flash was cooled to room temperature and
three ml of distilled water added. After gentle shaking, the contents
were transferred to a 25-ml volumetric flask. This washing pro-
cedure was repeated twice with five ml of distilled water and the
-------
WALSH AND CROW: Composition of Sea Grasses 99
hydrolysate taken to 25 ml with distilled water. The hydrolysate
was analyzed on a Beckman Model 1301 atomic absorption unit
equipped with a Beckman DB-G spectrophotometer. Concentra-
tions of the elements are reported as parts per thousand (ppt) of
dry weight.
Caloric contents were determined on a Parr Series 1200 adi-
abatic calorimeter. Fuse wire and acid corrections were made for
each determination and results are expressed as kilocalories per
gram of ash-free dry weight.
RESULTS AND DISCUSSION
Ash. Annual mean values and ranges of values for ash, protein,
carbohydrate, and energy are given in Table 1. Annual variation in
ash content was not found and analysis of variance indicated that
all mean values were significantly different at the 0.05 leveL The
TABLE 1
Annual means for ash, protein, carbohydrate, and energy contents of Thalassia
testudinum and Ruppia maritima between June 1969 and May 1970.
Component Annual Range
mean
Ash, % dry weight
T. testudinum leaves 24.5 20.6-26.9
T. testudinum rhizomes 23.8 21.4-25.4
R. maritima leaves 18.8 15.8-23.8
R. maritima rhizomes 22.4 18.6-24.8
Protein, % ash-free dry weight
T. testudinum leaves 25.7 13.6-37.1
T. testudinum rhizomes 11.0 7.7-14.7
R. maritima leaves 23.2 13.5-32.6
R. maritima rhizomes 20.0 14.1-26.9
Carbohydrate, % ash-free dry weight
T. testudinum leaves 23.6 18.3-35.8
T. testudinum rhizomes 72.1 54.5-80.3
R. maritima leaves 27.0 24.3-34.3
R. maritima rhizomes 63.6 52.0-73.3
Energy, Kcal/g ash-free dry weight
T. testudinum leaves 4.66 4.47-4.79
T. testudinum rhizomes 4.88 4.76-5.16
R. maritima leaves 4.44 4.28-4.69
R. maritima rhizomes 4.25 4.09-4.38
-------
100 QUARTERLY JOURNAL OF THE FLORIDA ACADEMY OF SCIENCES
values obtained for ash contents were similar to those for most other
aquatic plants (Westlake, 1965) and for leaves of T. testudinum
(Burkholderetal., 1959).
Protein. There was considerable annual variation in the amount
of protein in ash-free dry weight of leaves. The highest value found
for T. testudinum leaves was 2.7 times that of the lowest, while that
for R. maritima leaves was 2.4 times greater. Annual variation in
35
30
25
20
15
10
I
-------
WALSH AND GROW: Composition of Sea Grasses 101
protein in underground parts was less. In both T. testudinum and
R. maritima, the highest concentration in the rhizomes was 1.9 times
that of the lowest.
During the annual cycle, the protein content of the leaves of T.
testudinum was always greater than that of the rhizomes (Figure 1),
the annual mean concentration in leaves being over two times
larger (Table 1). Concentration of protein in the leaves of T. tes-
tudinum increased in the late winter and spring between 9 January
and 16 April. Concentrations decreased rapidly thereafter, and
were lowest in summer on 2 July 1969. In the rhizomes, however,
concentrations of protein increased only slightly between 26 Febru-
ary and 16 April 1970, and fell less precipitously than did those of
the leaves.
The annual mean concentration of protein in the leaves of R.
maritima was slightly greater than that in the rhizomes, but concen-
tration was greater in the rhizomes in the summer months of May,
June, and July. Concentrations in the leaves reached a peak on 8
April 1970, and fell rapidly thereafter. Lowest concentration was
found in the summer on 4 August 1969. Concentration of protein in
the rhizomes of R. maritima rose slowly in the nine-month period
between 4 August and 14 May, with lowest concentrations occuring
in summer in June and early August.
The above findings are related to the functional aspects of leaves
and rhizomes. Leaves generally have a greater amount of protein
than rhizomes because they are largely concerned with biosynthesis
and, consequently, contain large amounts of enzymes and many
membranes. However, rhizomes are storage organs and contain
relatively large amounts of carbohydrate, as will be shown later.
Leaf protein is greatest in spring when biosynthesis is rapid, where-
as concentrations of carbohydrate in rhizomes are greatest in fall
and winter.
Bauersfeld et al. (1969) suggested that T. testudinum may be of
value as a feed additive for domestic animals. They reported that
the leaves of T. testudinum, after a single washing with distilled
water, contained between 9.0 and 14.1 per cent protein on a dry
weight basis, whereas the rhizomes contained 15 per cent. Burk-
holder et al. (1959) reported that the dried leaves of T. testudinum
contained 13.1 per cent protein. Neither study, however, reported
the dates on which samples were taken.
-------
102 QUARTERLY JOURNAL OF THE FLORIDA ACADEMY OF SCIENCES
On a percentage dry weight basis, the protein contents of our
samples were: T. testudinum leaves, 10.3-29.7; rhizomes, 5.8-12.2;
R. maritime leaves, 10.9-28.5; rhizomes, 10.4-18.1. These values are,
in general, higher than those for many other plants. Among the
80
70
60
50
40
30
£ 20
kj
kl
£ 10
x
V> 0
»*
uT SO
k-
70
£ 60
<
I-
O
50
40
30
26
10
0
Fig.
Ruppia
•RUPPIA LEAVES
. RUPPIA RHIZOMES
.-. /
•THALASSIA LEAVES
•THALASSIA RHIZOMES
, --
A S
1969
N 0
MONTH
M
1970
2. Annual variation of carbohydrate in Thalassia testudintim and
marttima.
-------
WALSH AND CHOW: Composition of Sea Grasses 103
aquatic plants, Myrtophyllum sp. contained approximately 7.8 per
cent protein in dry weight (Oelshlegel, 1969) and Spartina dlter-
niflora 8.9 per cent (Hall et al., 1970). Boyd (1970), in a study of
the protein content of 11 freshwater species, reported a range of
from 4.0 per cent (Typha latifolia) to 21.6 per cent (Nuphar ad-
vena). Yee (1970) reported 17.5 per cent protein in Hydritta sp.
and 30.5 per cent in Pistia stratoides. Among foodstuffs, 114 lines
of corn contained 9.8-16.3 per cent protein (Davis et al., 1970), 49
varieties of grain sorghum contained 8.6-16.5 per cent (Virupaksha
and Sastry, 1968) and wheat grain between 8.3-12.4 per cent (Chro-
minski, 1967).
Though high in protein, it is doubtful that these seagrasses could
be used directly as food by humans. The unanimous consensus of
a taste panel at the Gulf Breeze laboratory was that dried leaves
and rhizomes are gritty, and have a strong, unpleasant odor and
flavor.
Carbohydrate. In contrast to protein contents, carbohydrate
contents of rhizomes were greater than those of leaves (Table 1)
because rhizomes are storage organs for starch. Fig. 2 shows that
the carbohydrate contents of rhizomes, as percentage ash-free dry
weight, began to rise in July due to production and storage in sum-
mer, and attained peak concentrations in October and November.
Decrease in spring was probably due to utilization of stored carbo-
hydrate for biosynthesis and respiration.
The carbohydrate contents of the seagrasses tested were similar
to those of other plants. As percentage dry weight, T. testudinum
leaves contained between 12.5 and 25.5 per cent carbohydrate,
whereas the rhizomes contained between 41.5 and 62.9 per cent.
Leaves of R. maritima contained between 20.0 and 27.2 per cent
and rhizomes between 35.8 and 55.1 per cent carbohydrate. Re-
ported values for other plants, as percentage dry weight, are: al-
falfa, 13-25 (Raguse and Smith, 1965, 1966; Grotelueschen and
Smith, 1967); red clover, 14-21 (Raguse and Smith, 1966), and tim-
othy, 48 (Grotelueschen and Smith, 1967). Most of the values for
seagrasses were within these ranges.
Energy. The energy contents (Table 1) of all samples were
very similar to those reported for most other plants (Cummings,
1967) and no annual trends were observed.
Elements. Annual variations in concentrations of sodium, potas-
-------
104 QUARTERLY JOURNAL OF THE FLORIDA ACADEMY OF SCIENCES
50
40
30
20
10
* 12
UJ
0.
6 "
^. 10
0.100
0.075
0.090
0.025
Mg
I
Mn
32.5
24.5
16.5
8.5
0
S.OO
3.T5
2.50
1.25
0
0.050
0.029
Fe
Zn
• LEAVES
• RHIZOMES
JJASONDJ FMAM
MONTH
JJASONDJFMAM
MONTH
Fig. 3. Annual variations of some elements in Thalassia testudinum,
slum, magnesium, iron, manganese, and zinc are shown in Figures
3 and 4. The variations appear to be associated with age and func-
tional aspects of the materials analyzed.
In several aquatic macrophytes, concentrations of some elements
decline with age. For example, concentrations of nitrogen, phos-
phorus, sulfur, calcium, and potassium decline with age in Typha
latifolia and in the bulrush Scripus americanus (Boyd, 1970). Con-
centrations of zinc, manganese and iron are lowest in mature
Spartina akerniflora (Williams and Murdock, 1969), and the
authors suggested that the decrease may be due to dilution of
actively growing tissues by structural material which contained
little of the elements measured.
Table 2 gives concentrations of the elements found in the leaves
-------
WALSH AND GROW: Composition of Sea Grasses
105
so
4O
30
20
10
I o
tfl
O 13
»~
-------
106 QUARTERLY JOURNAL OF THE FLORIDA ACADEMY OF SCIENCES
TABLE 2
Concentrations, in parts per thousands, of
roots of selected plants.
Plant
Carrot
leaves
roots
Soybean
leaves
roots
Sunflower
leaves
roots
Sweet potato
leaves
roots
Tomato
leaves
roots
K
13.3
16.8-59.2
8.0
14.4-15.6
16.2-19.0
13.6-38.0
16.1-23.7
6.8-17.4
5.2-37.6
8.0-34.1
Mg
2.8
1.2-2.4
7.9
10.7-31.8
11.0
1.3-12.7
4.5-5.4
0.6-2.1
6.2-15.5
4.6
some elements in the leaves and
Fe
0.36-0.77
0.04-0.49
0.34
_
_
0.03
_
0.01-0.14
0.28-0.54
-
Mn
0.02-0.20
0.01-0.09
0.03-0.19
0.02-0.15
0.07-1.27
_
_
0.01-0.03
0.05-4.93
-
Zn
0.03
0.01
0.10
_
-
0.02
_
0.01
0.03
-
From Altaian, P. L. and D. S. Dittmer (eds.), Biology Data Book, 1964.
In summary, in relation to other aquatic plants and" food crops,
T. testudinum and H. maritima contain significant amounts of pro-
tein, carbohydrate, energy, and minerals. The nutritive value of T.
testudinum has been established (Bauersfeld et al., 1969) and that
of H. maritima is implied from the work reported here. Annual
variation in chemical composition, however, implies that the nutri-
tional value of seagrasses varies throughout the year.
LITERATURE CITED
ALTMAN, P. L., AND D. S. DrrrMEH (eds.). 1964. Biology data book. Federa-
tion of American Societies for Experimental Biology, Washington, D. C.
xix + 633 pp.
BAUEHSFELD, P., R. R. KIFER, N. W. DURANT, AND J. E. SYKES. 1969. Nutrient
content of turtle grass (Thalassia testudinum). Proc. Intl. Seaweed
Symp., vol. 6, pp. 637-645.
BOYD, C. E. 1970. Arnino acid, protein, and caloric content of vascular aquatic
macrophytes. Ecology, vol. 51, pp. 902-906.
BURKHOLDEH, P. R., L. M. BURKHOLDEH, AND J. A. RrvEHO. 1959. Some chem-
ical constituents of the turtle grass Thalassia testudinum. Bull. Torrey
Bot. Club, vol. 86, pp. 88-93.
CHROMINSKI, A. 1967. Effect of (2-chloroethyl) trimethylammonium chloride
on protein content, protein yield, and some qualitative indexes of winter
wheat grain. Jour. Agr. Food Chem., vol. 15, pp. 109-112.
-------
WALSH AND GROW: Composition of Sea Grasses 107
CUMMTNGS, K. W. 1967. Calorific equivalents for studies in ecological ener-
getics. Pymanting Laboratory of Ecology, University of Pittsburgh. 2nd.
ed., 52 pp.
DAVID, D. J. 1958. Determination of zinc and other elements in plants by
atomic absorption spectroscopy. Analyst., vol. 8, pp. 655-661.
DAVIS, L. W., W. P. WILLIAMS, JR., AND L. CROOK. 1970. Interrelationships
of the protein and amino acid contents of inbred lines of corn. Jour.
Agr. Food Chem., vol. 18, pp. 357-360.
FENCHEL, T. 1970. Studies on the decomposition of organic detritus derived
from the turtle grass Thalassia testudinum. Limnol. Oceanogr., vol. 15,
pp. 14-20.
GHOTELUESCHEN, R. D., AND D. SMITH. 1967. Determination and identification
of nonstructural carbohydrates removed from grass and legume tissue
by various sulfuric acid concentrations, takadiastase, and water. Jour.
Agr. Food Chem., vol. 15, pp. 1048-1051.
HALL, K. J., W. C. WEIMER, AND G. F. LEE. 1970. Amino acids in an estua-
rine environment. Limnol. Oceanogr., vol. 15, pp. 162-164.
HUDSON, J. H., D. M. ALLEN, AND T. J. COSTELLO. 1970. The flora and
fauna of a basin in Central Florida Bay. U. S. Fish Wildl. Ser. Spec.
Sci. Rep. Fish. 604, pp. 1-14.
MENZIES, R. J., AND G. T. ROWE. 1969. The distribution and significance of
detrital turtle grass, Thalassia testudinum, on the deep-sea floor off
North Carolina. Int. Rev. Ges. Hydrobiol., vol. 54, pp. 217-222.
OELSHLEGEL, F. J., JR. 1969. Potential for protein concentrates from alfalfa
and waste green plant material. Jour. Agr. Food Chem., vol. 17, pp.
665-668.
OLNEY, P. J. S. 1968. The food and feeding habits of the pochard, Aythya
ferina. Biol. Conserv., vol. 1, pp. 71-76.
PARKER, P. L. 1966. Movement of radioisotopes in a marine bay: cobalt-60,
iron-59, manganese-54, zinc-65, sodium-22. Publ. Inst. Mar. Sci., Univ.
Texas, vol. 11, pp. 102-107.
PHILLIPS, R. C. 1960. Observations on the ecology and distribution of the
Florida seagrasses. Florida State Bd. Conser., Prof. Papers Ser., No. 2,
St. Petersburg, iv + 72 pp.
RAGUSE, C. A., AND D. SMITH. 1965. Carbohydrate content in alfalfa herb-
age as influenced by methods of drying. Jour. Agr. Food Chem., vol.
13, pp. 306-309.
. 1966. Some nonstructural carbohydrates in forage legume herbage.
Jour. Agr. Food Chem., vol. 14, pp. 423-426.
RANDALL, J. E. 1965. Grazing effect on seagrasses by herbivorous reef fishes in
the West Indies. Ecology, vol 46, pp. 255-260.
STRICKLAND, J. D. H., AND T. R. PARSONS. 1965. A manual of sea water
analysis, 2nd ed. Fish. Res. Bd. Canada. Bull. No. 125. Ottawa, viii +
203 pp.
VAN BREEDVELD, J. F. 1966. Preliminary study of seagrass as a potential source
of fertilizer. Florida State Bd. Conser., Mar. Lab. Spec. Sci. Rep. 9,
pp. 1-20.
-------
108 QUARTERLY JOURNAL OF THE FLORIDA ACADEMY OF SCIENCES
VmupAKSHA, T. K., AND L. V. S. SASTRY. 1968. Studies on the protein content
and amino acid composition of some varieties of grain sorghum. Jour.
Agr. Food Chem., vol. 16, pp. 199-203.
WESTLAKE, D. F. 1965. Some basic data for investigations of the productivity
of aquatic macrophytes. In: C. R. Goldman (ed.), Primary Productivity
in Aquatic Environments. Mem. 1st. Ital. Idrobiol., 18 Suppl., Univer-
sity of California Press, Berkeley, pp. 229-248.
WILLIAMS, R.. B., AND M. B. MURDOCK. 1969. The potential importance of
Spartina altemifloTa in conveying zinc, manganese, and iron into es-
tuarine food chains. In D. J. Nelson and F. C. Evans (eds.), Sym-
posium on Radioecology, 2nd Nat. Symp., Ann Arbor, Michigan, pp.
431-439.
YEE THONG TAN. 1970. Composition and nutritive value of some grasses,
plants, and aquatic weeds tested as diets. Jour. Fish. Biol., vol. 2,
pp. 253-257.
Gulf Breeze Environmental Research Laboratory, Sabine Island,
Gulf Breeze, Florida 32561. An associate laboratory of the National
Environmental Research Center, Corvallis, Oregon. Contribution
No. 126.
Quart. Jour. Florida Acad. Sci 35(2) 1972(1973)
-------
CONTRIBUTION NO. 131
-------
In vivo Binding of p,p'-DDE to Human
Serum Proteing
by W. P. ScHOOR1
Department of Pharmacology, Louisiana State University
Medical Center, New Orleans, La. 70112
Although it is convenient to estimate chlorinated hydrocarbon
pesticide levels in man by determining the amount present in
serum, few investigators have questioned possible interactions of
these compounds with serum proteins and the resulting consequences.
DALE et al. (1965) suggested that the binding of pesticides to
serum protein was the cause of incomplete pesticide recovery from
human serum by hexane extraction, and GUNTHER et al. (1954) pro-
posed molecules were held by proteins with consequent inhibition
of the normal function of these proteins. Binding of dieldrin
and telodrin to serum proteins has been demonstrated by MOSS and
HATHWAY (1964), but the concentrations of these pesticides were
considerably greater than those normally encountered in man and
pH ranges during the separation deviated too far from the physio-
logical norm to allow much speculation on the results. HATANAKE
et al. (1967) attempted to recover pesticides from protein frac-
tions after Sephadex G-50 treatment, but met with inconsistent
results. Careful review of these reports emphasizes that direct
evidence of serum protein binding under physiological conditions
should be obtained.
METHODS
Serum was prepared from the author's blood by allowing it to
clot for 2 hr at 0°C, then removing the liquid portion. Two-mi
samples were kept at -15°C for not more than 10 days.
Ten g of Sephadex G-200 was soaked for 3 days in 1.0 liter
of 0.9% NaCl solution at pH 7.35. This material was placed in a
2.5 cm x 46 cm glass column and allowed to settle under flow.
Two ml of serum were diluted to 10 ml with 0.9% NaCl (pH 7.35),
placed on the column, and eluted with the same solution. Ten-mi
fractions were collected at a flow rate of 2-3 ml/min at constant
Present address: Environmental Protection Agency, Gulf Breeze
Laboratory, Sabine Island, Gulf Breeze, Florida 32561.
70
Bulletin of Environmental Contamination & Toxicoloitv
VoL 9, No. 2, O 1973 by Springer-Verlag New Yo* '
-------
pressure. Protein concentrations were determined with a Zeiss
Model PMQ-II spectrophotometer at 280 mu.
The pooled protein fractions, usually about 150 ml, were ex-
tracted with 200-ml portions of hexane. When a 2.0-ml serum sam-
ple was diluted to 150 ml and extracted in this way, more p,p'-DDE
was recovered and reproducibility was greater than when the serum
was extracted directly by the method of DALE et al. (1965, 1970).
Since further experimentation showed that a reduction in the vol-
ume of the diluted serum from 150 ml to 50 ml caused no significant
reduction in p,p'-DDE recovery, the following procedure was adop-
ted for the routine serum extractions:
Two ml of serum, diluted to 50 ml with 0.9% NaCl solution
(pH 7.35), and 75 ml hexane were shaken vigorously in a 250-ml
separatory flask for 10 sec and the flask inverted and vented.
The shaking and venting process was continued for 1 min, after
which the phases were allowed to separate. The process was re-
peated three times. Any interphase emulsion was broken by centri-
fugation. The extract was reduced to an appropriate volume by
evaporation under a gentle stream of air at 35°C. The p,p'-DDE
was detected by means of a Microtek MBT-220 gas chromatograph,
using an OV-17/QF-1 column for quantitation and a SE-30/QF-1
column for confirmation at 195"C and a tritium-type electron-
capture detector at 205t'C. Further confirmation was achieved
with thinlayer chromatography on silic acid with heptane as sol-
vent.
RESULTS
Only p,p'-DDE was quantitated because it was present in large
enough amounts to be confirmed by thin-layer chromatography. The
p,p'-DDT present was in such low amounts that reproducibility be-
tween chromatograms was very poor. No polychlorinated biphenyl
derivatives (PCB's) were present. Table 1 shows that compared
with the method of DALE et al. (1965, 1970) the extraction method
gives almost a twofold increase in p,p'-DDE recovered as well as
less variability (F-test; p<0.01). Protein recovery after
Sephadex G-200 chromatographic treatment was 95%, and the protein
fractions contained approximately 80% of the original amount of
p,p'-DDE. When p,p'»-DDE in hexane was placed on the column and
the hexane was allowed to evaporate, elution under the conditions
described for serum chromatography did not yield any p,p'-DDE in
the effluent in the range of the protein fractions. Preliminary
data indicate that very little p,p'-DDE is found in the gamma
globulin fraction. Precipitation of serum proteins with ammonium
sulfate and subsequent hexane extraction of the supernatant and
the precipitate yielded no p,p'-DDE in the former and only trace
amounts in the latter. Serum samples kept at -15 C for more
than 10 days showed a decrease in recoverable p,p'-DDE (e.g.,
20% less was recovered from a sample stored for 21 days). Both
71
-------
observations indicate that the degree of binding of p,p'-DDE to
the proteins changes as the conformation of the protein changes.
DISCUSSION
The importance of the binding of drugs to serum proteins and
the concomitant effects on their pharmacological activity cannot
be overemphasized (SELLERS and KOCH-WESER 1969, MEYER and GUTTMAN
1968, CONN and LUCHI 1961, DOLLERY et al. 1961, MACREGOR 1965,
BRODIE 1965, CUCINELL et al. 1965). BRODIE (1965) states,
"Actually, almost all drugs are reversibly bound to. proteins in
plasma or tissue. The bound drug, often a high percentage of
the total, acts as a reservoir, preventing wild fluctuations
between ineffective and toxic levels of the biologically active
unbound fraction." It is believed that the same type of mechanism
can explain the behavior of p,p'-DDE and, very likely, all
chlorinated hydrocarbons, including the PCB's, in the blood.
Although microsomal enzyme induction is usually cited as
cause for the reduced serum levels of pesticides, the following
interpretation should be considered: (1) Serum concentrations
of chlorinated hydrocarbon pesticides normally encountered in
human beings reflect "bound" levels that are relatively inert.
(2) Any compound that can interfere with the binding of the
pesticide may free it for adsorption at a site of toxic action,
metabolic breakdown, or storage, depending on the distribution
constant. (Large amounts of inertly bound pesticides upon libera-
tion could in this fashion become available for binding at the
site of toxic action). (3) The enzyme system responsible for the
breakdown of the pesticides is always present, but cannot function
because the substrate is tightly bound to the serum proteins.
Pesticide metabolism is strictly governed by the difference in
the distribution constants between the two sites.
This hypothesis would explain why aldrin enhanced the reten-
tion of p,p'-DDT and p,p'-DDE in the blood of dogs (DEICHMANN
et al. 1969), the reduced paroxon binding capacity in rat
plasma on oral pretreatment with tr.i-o-tolyl phosphate (LAUWERYS
and MURPHY 1969), and the lowering of serum-bound iodine by
o.p-DDD in humans (MARSHALL and TOMPKINS 1968). It would also
explain the low levels of chlorinated hydrocarbons found in
persons treated with anticonvulsant drugs by DAVIES et al.
(1969) and SCHOOR (1970).
72
-------
TABLE 1
Comparison of recovery of p,,p'-DDE in human serum by different
analytical methods.
Method of DALE Present method
et al. (1965)
Combined protein fraction after
G-200 treatment
Serum
ppm
0.019
0.022
0.024
0.012
0.012
0.017
0.019
0.015
0.019
0.019
Serum
ppm
0.036
0.037
0.037
0.034
0.030
0.034
0.035
0.033
0.034
0.034
0.034
0.036
Serum
ppro
0.025
0.028
0.026
0.029
a
Two ml of serum were extracted in each analysis
73
-------
REFERENCES
BRODIE, B.B.: Proc. Royal Soc. Med., 58, 946 (1965).
CONN, H.L., and R.J. LUCHI: J. Clin. Invest., 40. 509 (1961).
CUCINELL, S.A., A.H. CONNEY, M. SANSUR, and J.J. BURNS: Clin.
Pharmacol. Therap, j>, 420 (1965).
DALE, W.E., A. CURLEY, and C. CUETO: Life Sciences, j>, 47 (1965).
DALE, W.E., J.W. MILES, and T.B. GAINES: J.A.O.A.C., 53, 1287
(1970).
DAVIES, J.E., W.F. EDMUNDSON, C.H. CARTER, and A. BARQUET: The
Lancet, July 5, 7 (1969).
DEICHMANN, W.B., M. KEPLINGER, I. DRESSLER, and F. SALA: Toxi-
col. Appl. Pharmacol., .14, 205 (1969).
DOLLERY, C.T., D. EMSLIE-SMITH, and D.F. MUGGLETON: Brit. J.
Pharmacol., 1_7, 488 (1961).
GUNTHER, F.A., R.C. BLINN, G.E. CARMAN, and R.L. METCALF: Arch.
Biochem. Biophys., _50, 504 (1954).
HATANAKA, A., B.D. HILTON, and R.D. O'BRIEN: J. Agr. Food Chem.,
L5, 854 (1967).
LAUWERYS, R.R., and S.D. MURPHY: Toxicol. Appl. Pharmacol., 14,
348 (1969).
MACREGOR, A.G.: Proc. Royal Soc. Med. 58, 943 (1965).
MARSHALL, J.S., and L.S. TOMPKINS: J. Clin. Endoc. and Metab.,
28, 386 (1968).
MEYER, M.C., and D.E. GUTTMAN: J. Pharm. Sci., 57, 895 (1968).
MOSS, J.A., and D.E. HATHWAY: Biochem. J., 9l_, 384 (1964).
SCHOOR, W.P.: The Lancet, Sept, 5, 520 (1970).
SELLERS, E.M., and KOCH-WESER: The New England J. Med., 281.
1141 (1969).
74
-------
CONTRIBUTION NO. 143
-------
Reprinted from BioScience
Research Reports
Effects of Herbicides on Seedlings of the Red
Mangrove, Rhizophora Mangle L.
"Mangrove" is a general term applied
to a community of shrubs or trees that
grow below the high-tide mark along
tropical shores. The term is also used
with reference to individual plant
species which occur within that com-
munity (Davis 1940, Macnae 1968). The
mangrove community is highly produc-
tive (Golley et al. 1962) and supports a
wide variety of animals which depend
upon plant detritus as a source of food
(Heald 1971, Odum 1971). In Florida,
many commercially important animals
such as pink shrimp (Penaeus duo-
rarum), blue crabs (Callinectes sapidus),
striped mullet (Mugil cephalus), and
spotted seatrout (Cynoscion nebulosus)
use the mangrove for food and as
nursery grounds (Idyll 1965, Idyll et al.
1968, Tabb 1966).
Susceptibility of mangrove to herbi-
cides was first investigated by Truman
(1961). who treated the grey mangrove,
Avicenhia marina, with the auxin-type
herbicides 2,4-dichlorophenoxyacetic
acid (2;4-D) and 2,4,5-trichloropheno-
xyacetic acid (2,4,5-T). A concentration
of only 1 percent in diesel oil distillate
killed all trees when applied to the bark,
and Truman concluded that the grey
mangrove is very susceptible. The con-
cept of high susceptibility of mangrove
to herbicides was extended by Tschirley
(1969)'and Oriaris and Pfeiffer (1970).
They stated that forests in Vietnam
dominated by Rhizophora conjugata,
Bruguiera parviflora, and B. cylindrica
were destroyed after application of
either a combination of 13.8 kg/ha of
the n-butyl ester of 2,4-D and 14.8
kg/ha of the n-butyl ester of 2,4,5-T or
6.72 kg/ha of the triisopropanolamine
salt of 2,4-D in combination with 0.61
kg/ha of the triisopropanolamine salt of
4-amino-3,5,6-trichloropicolinic acid
(picloram). Westing (1971) found that
treated mangrove areas in Vietnam re-
mained uncolonized by plants 6 years
after treatment and suggested that
plants of the intertidal zone are highly
sensitive to hormone-type herbicides.
Re colonization of mangrove in
denuded areas must depend upon estab-
lishment and growth of seedlings. If
herbicide residues remain in the soil,
development of seedlings could be in-
hibited. The purpose of our research
was to describe effects of a commercial
formulation of 2,4-D and picloram on
seedlings of the red mangrove, Rhizo-
phora mangle L.
The genus Rhizophora is circum-
tropical in its distribution. Rhizophora
mangle is common along the shores of
the Gulf of Mexico, Caribbean Sea, West
Africa (Chapman 1970), and Hawaii
(Walsh 1967). The tree is vivaparous
and, in southern Florida, produces seed-
lings throughout the year. The seedling
consists of an elongated hypocotyl up
to 30 cm long, with a short plumule
approximately 0.5 cm long. The
plumule is composed of the first leaves
covered by the cotyledonary stipules.
Leaf development does not occur until
roots become established in the soil.
METHODS
Seedlings 18.2 to 26.5 cm long were
picked from trees near Coral Gables,
Florida, and planted in estuarine mud in
plastic boxes in the laboratory. Salinity
of the water which covered the sedi-
ment was 30 parts per thousand, and pH
of the sediment ranged from 6.4 to 6.7.
Room temperature was maintained at
27°C, and light was from gro-lux
flourescent tubes beside the boxes. The
lighting regime was alternate 12-hour
periods of light and darkness. The herbi-
cide formulation used was Tordon®1
101, which is a combination of the
triisopropanolamine salt of 2,4-D
Publication No. 143 from the Gulf Breeze
Laboratory, Environmental Protection
Agency, Gulf Breeze, Florida 32561,
Associate Laboratory of the National Environ-
mental Research Center, Corvallis, Oreg.
®Registered trademark, Dow Chemical
Company, Midland, Michigan. Reference to
trade names in this publication does not
constitute endorsement by the Environmental
Protection Agency.
(39.6%) and the triisopropanolamine
salt of picloram (14.3%). This formula-
tion is similar to that of Agent White,
which was used in Vietnam. The formu-
lation was added to the surface of the
water so that the amount of each
herbicide within the seedlings after up-
take by the roots could be measured.
Groups of seedlings were treated 3 days
after planting when no leaves were
extended. Later, other groups were
treated after one pair or two pairs of
leaves were extended. Seedlings were
treated at rates of 1.12, 11.2, and 112.0
kg/ha (1, 10, and 100 Ib/acre) of the
commercial formulation. These rates
were equivalent to active ingredient
concentrations of 0.44, 4.40 and 44.0
kg/ha 2,4-D and 0.16, 1.60, and 16.0
1
"UWTMATtD VII ««/*» »J t
Fig. 1. Effects of Tordon 101 on seedlings of
R. mangle treated when no lerret were
present A. 30 days tttet treatment; B. 40
days after treatment; C 80 days afna treat-
ment
June 1973
361
-------
kg/ha picloram. Each test was per-
formed three times and 45 seedlings
were treated at each rate. Forty-five
seedlings were maintained as controls
for each experiment.
For quantitation of residues, herbi-
cides were extracted from plant material
with an acidified mixture of petroleum
ether and ethyl ether (1:1). The acid
salts were removed from the mixture
with a basic aqueous wash. The aqueous
solution was acidified and the free acids
extracted with chloroform. After
evaporation to dryness, the acids were
esterified with diazomethane and the
methyl ester quantitated by electron-
capture gas chromatography. The limits
of quantification were 0.01 parts per
million (ppm) for picloram as picolinic
acid and 0.02 ppm for 2,4-D. Residues
are expressed as the averages for each
treatment.
Sections of at least six leaves and
roots from each experiment were fixed
in formalin-aceto-alcohol, dehydrated in
dioxane, embedded in paraffin, and cut
to a thickness of 10 microns on a rotary
microtome. They were affixed to slides
with Haupt's adhesive and stained with
safranin and fast green (Sass 1958) for
histopathological studies.
RESULTS
Results are summarized in Table 1.
In the first experiments, seedlings were
treated while the first pair of leaves
were enclosed in the cotyledonary
stipules. In Fig. 1A, which shows seed-
lings 30 days after treatment, the leaves
can be seen emerging from the hypoco-
tyls of untreated seedlings and from one
of those treated with 1.12 kg/ha. Seed-
lings treated with 11.2 kg/ha showed
normal root development but were high-
ly chlorotic on the upper one-half of the
hypocotyl. Those treated with 112.0
kg/ha exhibited wide areas of chlorosis
on their upper halves and were dead
after 30 days. Some of these seedlings
had longitudinal splits in the epidermis
and cortex, and callosities protruded
from the surface of the hypocotyl.
After 40 days (Fig. IB) growth of
untreated seedlings had progressed
normally, with further growth of roots
and extension of the first pair of leaves.
Seedlings treated with 1.12 kg/ha were
alive, bul root development was not as
extensive as in the untreated group. The
plumules of most were slightly ex-
panded as before normal leaf extension.
All seedlings treated with 11.2 kg/ha
were dead. As with seedlings treated
with 112.0 kg/ha, these seedlings were
chlorotic, and the plumules of some had
fallen from the hypocotyl. After 80
days, untreated seedlings had three pairs
of leaves and appeared healthy. Those
treated with 1.12 kg/ha were still alive,
and some had as many as two pairs of
leaves (Fig. 1C). However, stems and
leaves were smaller than those of the
untreated groups and root development
was poor.
Residues of herbicides in the
hypocotyls are given in Table 2. Tissue
concentrations were related to applica-
tion rates, but residues, although
present, were too low to quantitate in
seedlings exposed to 1.12 kg/ha. Clear-
ly, such low concentrations of herbi-
cides in the hypocotyls caused impaired
development of seedlings which were
treated before emergence of leaves.
When seedlings with one pair of
leaves were treated with Tordon 101,
those which received 112.0 kg/ha
became chlorotic and died within 15
days after treatment. Within a few days
TABLE 2. Concentrations of 2,4-D and
picolinic acid, in parts per million (± 20%), of
wet tissue in the hypocotyls of R. mangle
seedlings treated with Tordon 101 before
emergence of leaves. Residues were detected
in every analysis of seedlings treated with
1.12 kg/ha but were below the level of
quantification.
Days after treatment
TABLE 1. Summary of responses of Rhizophora mangle to Tordon 101
Pairs of leaves
when treated
Rate of application, kg/ha
1.12
11.2
112.0
Positive phototrophic Positive phototrophic
response response
No other effects Chlorosis of leaves
noted and hypocotyl
Histological abnor-
malities of leaves
and roots
Death approximately
30 days after treat-
ment
Chlorosis of leaves and
hypocotyl
Histological abnormali-
ties of leaves
Defoliation
Death 15 days after
treatment
30 40
Treatment Picolinic Picolinic
kg/ha 2,4-D acid 2,4-D acid
11.2
112.0
0.81 0.14
4.10 0.58
2.26 <0.01
'mi
Retarded development Retarded root Same as with 11.2 kg/ha
No deaths development treatment, except
Chlorosis of hypocotyl death 30 days after
Death 40 days after treatment
treatment
Same as when 1 pair Same as when 2 pairs Same as when 1 pair of F"fr 2. Effects of Tordon 101 on seedlings of
of leaves present of leaves present, oeaves present, except R- fnangle treated when one pair of leaves was
except death with- death within 10 days present A. 10 days after treatment; B. 30
in 40 days days after treatment; C. 60 days after treat-
ment
362
BioScience Vol. 23 No. 6
-------
TABLE 3. Concentrations of 2,4-D and picotinic acid, in parts per mfllion (± 20%), of wet
tissue in organs of R. mangle seedlings treated with Toidon 101 when a single pair of leaves was
present Residues were detected in every analysis of seedlings treated with 1.12 kg/ha but were
below the level of quantification.
Treatment
kg/ha
11.2
112.0
Day
10
30
10
Roots
Picolinic
2,4-D Acid
<0.02
<0.02
1.04
<0.01
<0.01
0.23
Hypocotyl
Picolinic
2,4-D Acid
0.23
0.10
1.55
0.04
0.04
0.41
Stem
Picolinic
2,4-D Acid
0.17
0.27
0.91
0.04
0.08
0.19
1st leaves
Picolinic
2,4-D Acid
<0.02
0.05
0.46
<0.01
0.05
0.10
after treatment, the hypocotyls of seed-
lings treated with 1.12 and 11.2 kg/ha
became markedly bent toward the light
source (Fig. 2A), a reaction that would
be expected after treatment with
hormone-type herbicides as concentra-
tion of auxin generally is greater on the
dark sides of stems. Greater growth or
elongation of cells on the dark side
would cause bending of the stem toward
the light. Thirty days after treatment,
the seedlings had returned to the up-
right position. Table 3 shows that the
amount of 2,4-D in the hypocotyls of
seedlings treated with 11.2 kg/ha
decreased after 30 days. However, this
group was moribund at that time, with
chlorosis of the leaves and hypocotyls
and with callosities along most of the
length of the hypocotyl. After 60 days,
seedlings treated with 1.12 kg/ha ap-
peared normal.
Concentrations of herbicides in
various plant parts are given in Table 3.
Residues were present in seedlings
treated with 1.12 kg/ha but were too
low to quantitate. Response to such low
tissue concentrations of the auxin-type
herbicides was indicated by bending of
the hypocotyl toward the light. At
other application concentrations,
highest tissue residues were usually in
the hypocotyl, which may explain why
effects were greatest in that organ.
When seedlings with two pairs of
leaves were treated at the two higher
concentrations, chlorosis appeared on
the hypocotyl at 10 to 20 days after
treatment (Fig. 3A). Soon thereafter the
leaves became dry and brittle, turned
brown, and curled inward (Fig. 3B). The
signs were those of desiccation and
defoliation (Bovey et al. 1969). Seed-
lings treated with 112.0 kg/ha were
dead within 10 days after treatment.
Those exposed to 11.2 kg/ha were dead
within 40 days. Bending of the
hypocotyl toward the light occurred in
all seedlings treated with 1.12 and 11.2
kg/ha, but this condition lasted less than
30 days. Residues were again usually
greatest in the hypocotyls (Table 4), but
were considerably greater in the leaves
than they were in the previous tests.
Studies were made of histological
abnormalities of leaves and roots as-
sociated with herbicidal treatment. The
leaves of R. mangle have a well-
developed cuticle on both surfaces. Im-
mediately below the upper epidermis is
a single or double layer of cells which
contain tannin. These are underlain in
turn by several layers of hypodermal
cells, palisade parenchyma, spongy
parenchyma, another layer of tannin
cells, and the lower epidermis. Figure
4A shows the normal histology of the
leaf near the midrib. Figure 4B shows
early effects of treatment with 11.2
kg/ha Tordon 101 in the same area. Cell
wall continuity has begun to break
down in the hypodermis, palisade paren-
chyma, and spongy parenchyma. The
normal leaf structure in the region
centrally located between the midrib
and the margin is shown in Fig. 4C. This
is the final stage in leaf degeneration
before it falls from the seedling. A total
loss of structural integrity is shown.
Cortical cells of the root were also
affected by treatment with 11.2 kg/ha
Tordon 101. Histology of the normal
root is shown in Fig. 4E and of the
treated root in Fig. 4F. The figures
demonstrate destruction of the cortex
after treatment with herbicide.
DISCUSSION
Amounts of herbicides required to
kill mangrove appear to be smaller than
those required to kill other species of
tropical trees. In our experiments, a
combination of 4.4 kg/ha 2,4-D and 1.6
kg/ha picloram killed all seedlings.
Bovey et al. (1969) treated a mixed
upland tropical forest in Puerto Rico
with 6.72 kg/ha 2,4-D and 1.68 kg/ha
picloram and obtained 90 percent
defoliation after one month, but
reforestation began after that time.
Truman (1961) reported that complete
defoliation of the grey mangrove in
Australia was caused by application of 1
percent 2,4-D to the bark. The same
TABLE 4. Concentrations of 2,4-D and picoBnic acid, in parts per nuUkm (± 20%), of wet
tissue in organs of R. mangle seedlings treated with Tordon 101 when two pairs of leaves were
present Residues were detected in every analysis of seedlings treated with 1.12 kg/ha but were
below die level of quantification.
Treatment
kg/In Diy
Roota
PlM
1A-D Add
Hypocotyl
ncotinfc
Add
Stem
Pitt>lmk
Add
lit lena
PicoBnic
2,4-D Add
fed lava
HcaUnk
Add
Fig. 3. Effects of Tordon 101 on seedlings of H.l 30 <0.02 <0.01 0.10 0.03 0.02 0.01 <0.02 <0.01 0.13 0.06
R. mangle treated when two pain of leaves 40 <0.02 <0.01 0.23 0.10 0.23 0.10 0.29 0.10 0.35 0.10
were present A. 20 days after treatment; B. u2.o 10 1.23 0.39 1.68 0.49 1.02 0.43 0.63 0.24 0.87 0.41
40 days after treatment . . __
June 1973
363
-------
Fig. 4. Effects of Toidon 101 on microscopic anatomy of R. mangle. A. Section through
untreated leaf near midrib (t=tannin cells, h=hypodermis, p=palisade parenchyma, s=spongy
parenchyma); B. Section through leaf near midrib in seedling treated with 11.2 kg/ha; C.
Section through center of normal leaf; D. Section through center of leaf of seedling treated
with 11.2 kg/ha; E. Section through cortex of normal root; F. Section through cortex of root
of seedling treated with 11.2 kg/ha. Magnification of all sections: 100X.
application rate caused only 9 percent
defoliation of Eucalyptus maculata in
the highlands.
The reasons for this apparent great
sensitivity of mangrove to herbicides are
not clear. Westing (1971) suggested that
susceptibility is related to physiological
attributes that permit growth in the
tropical tidal environment. Scholander
et al. (1966) showed that Rhizophora
regulates ion uptake by a salt-exclusion
mechanism in the roots. Our research
demonstrated destruction of roots by
herbicidal treatment, and it is possible
that, in addition to direct effects of
herbicides, death of seedlings was
caused by disruption of their osmore-
gulatory ability. Further, physical con-
ditions in the tidal environment could
cause greater herbicidal uptake and
activity than in upland regions. The
mangrove environment is very fertile
(Macnae 1968), and it is well known
that high fertility coupled with
abundant water increases the suscep-
tibility of plants to herbicides (Ham-
merton 1967).
Our experiments incidate that rela-
tively low concentrations of auxin-type
herbicides inhibit mangrove develop-
ment. Reclamation of the mangrove
forest may be difficult if low residues
from previous sprayings persist in soil.
REFERENCES
Bovey, R. W., C. C. Dowler, and J. D.
Diaz-Colon. 1969. Response of tropical
vegetation to herbicides. Weed Sci., 17:
28S-29Q.
Chapman, V. J. 1970. Mangrove phyto-
sociology. Trap. Ecol., 1: 1-19.
Davis, J. H., Jr. 1940. The geologic role of
mangroves in Florida. Carnegie Inst. Wash.,
Dept Mar. Biol., Pap. from the Tortugas
Lab., 32: 303^12.
Golley, F. B., H. T. Odum, and R. F. Wilson.
1962. The structure and metabolism of a
Puerto Rican red mangrove forest in May.
Ecology, 43: 9-19.
Hammerton, J. L. 1967. Environmental
factors and susceptibility to herbicides.
Weeds, IS: 330-336.
Heald, E. J. 1971. The production of organic
detritus in a south Florida estuary. Univ.
Miami, Sea Grant Tech. Bull., No. 6, viii,
110pp.
Idyll, C. P. 1965. Shrimp need fresh water
too.Natl. Parks Mag., 10: 14-15.
Idyll, C. P., D. C. Tabb, and B. Yokel. 1968.
The value of estuaries to shrimp. Proc.
Marsh Estuary Management Symp., La/
State Univ., July 1967, pp. 83-90.
Macnae, W. 1968. A general account of the
fauna and flora of mangrove swamps and
forests in the Indo-West Pacific region. Adv.
Mar. Biol., 6: 73-270.
Odum, W. 1971. Pathways of energy flow in a
south Florida estuary. Univ. Miami, Sea
Grant Tech. Bull., No. 7, xi, 162 pp.
Orians, G. H. and E. W. Pfeiffer. 1970.
Ecological effects of the war in Vietnam.
Science. 168: 544-554.
Sass, J. E. 1958. Botanical Microtechnique.
3rd edition, Iowa State Univ. Press, Ames,
228 pp.
Scholander, P. F., E. D. Bradstreet, H. T.
Hammel, and E. A. Hemmingsen. 1966. Sap
concentrations in halophytes and some
other plants.PlantPhysiol., 41: 529-532.
Tabb, D. C. 1966. The estuary as a habitat for
spotted seatrout, Cynoscion nebulosus.
Spec. Publ. Am. Fish. Soc., 3: 59-67.
Truman, R. 1961. The eradication of man-
groves. Aust. J. Sci., 24: 198-199.
Tschirley, F. H. 1969. Defoliation in Viet-
nam. Science, 163: 779-786.
Walsh, G. E. 1967. An ecological study of a
Hawaiian mangrove swamp. Pages 420-431
in G. H. Lauff, ed. Estuaries, AAAS Publ.
No. 83.
Westing, A. H. 1971. Ecological effects of
military defoliation on the forests of South
Vietnam. BioScience, 21: 893-898.
GERALD E. WALSH
REGINA BARRETT
GARY H. COOK
TERRENCE A. HOLLISTEr\
Environmental Protection Agency
Gulf Breeze Laboratory
Sabine Island
Gulf Breeze, Fla. 32561
Associate Laboratory of the
National Environmental Research
Center, Corvallis, Oreg.
364
BioScience Vol. 23 No. 6
-------
CONTRIBUTION NO. 147
-------
Avoidance of Pesticides by Grass Shrimp
(Palaemonetes pugio)1
by DAVID J. HANSEN, STEVEN C. SCHIMMEL, and JAMES M. KELTNER, JR.
Environmental Protection Agency
Gulf Breeze Laboratory, Sabine Island, Gulf Breeze, Flo. 32561
Associate Laboratory of the National Environmental Research Center
Corvallis, Ore.
Some fishes can avoid certain pesticides in water. Green
sunfish, Lepomis cyanellus. was repelled by chlordane but not
lindane (SUMMEKFELT and LEWIS 1967); sheegshead minnows, Cyprinodon
variegatus. avoided DDT, endrin, Dursban Qx , and 2,4-D (BEE) but did
not avoid malathion or Sevin OP (HANSEN 1969); and mosquitofish,
Gambusia affinis, avoided DDT, Dursban, 2,4-D, malathion and Sevin
but not endrin (HANSEN et al. 1973).
Crustaceans are usually more sensitive to pesticides, par-
ticularly insecticides, than are fishes, but little is known
about their ability to avoid pesticide pollution. The purpose of
this study was to evaluate the capacity of the euryhaline grass
shrimp, Palaemonetes pugio, to avoid DDT, endrin, Dursban,
malathion, Sevin and 2,4-D. This shrimp was selected because of
its importance in the food web (WOOD 1967) and its abundance in
local waters.
Experimental Procedure
Grass shrimp, 10-40 mm rostrum-telson length, were seined
from brackish-water ponds on Sabine Island. They were acclimated
for at least 5 days in the laboratory at 20% salinity and 20 C
before they were used in experiments.
The avoidance response was tested in a black plastic apparatus
designed to allow the shrimp to move from a holding area either
into a section which contained water with pesticide or into one
which contained water without pesticide (HANSEN et al. 1973). A gate
was lowered at the junction between the two sections and the
holding area to trap shrimp for counting. When a test was in
progress the apparatus was covered with black acrylic plastic to
exclude light. Filtered sea water diluted with aerated tap water
to 20% salinity and maintained at 20 C entered the upper end of
each of the two sections at a rate of 400 ml/minute and flowed to
1
Gulf Breeze Contribution No. 147
Registered trademark: Dow Chemical Co., (Dursban) and Union
Carbide Corp. (Sevin). Reference to trade names in this publi-
cation does not imply endorsement of the products by the
Environmental Protection Agency.
129
Bulletin of Environmental Contamination & Toxicology,
Vol. 9, No. 3, © 1973 by Springer-Verlag New York Inc.
-------
the drain in the holding area. Pesticides dissolved in acetone
were metered through stopcocks at 0.5 ml/minute into the water
entering one of the two sections. The same amount of acetone
without pesticide was metered into the water entering the other
section. The two upper "Y's" served no specific function in
these tests.
Pesticides selected for avoidance testing included five
insecticides and one herbicide (Table 1). Concentrations of these
chemicals were selected so that one was higher and two or three
were lower than the concentration that flowing water bioassays
indicated would kill 50 percent of the shrimp in 24 hours (LC50).
Concentrations were not checked by chemical analysis.
TABLE 1
Description of chemicals tested and 24-hour LCSO's for grass
shrimp, Palemonetes pugio.
Pesticide
DDT
Endrin
Dursban
Ma lathi on
Sevin (Carbaryl)
2,4-D (butoxyethanol
ester)
Type
Organochlorine
Organochlorine
Organophosphate
Organophosphate
Carbamate
Herbicide
Personal communication, Jack I. Lowe
Agency, Sabine Island, Gulf Breeze,
Percentage
active
ingredient
99
97
99
95
98
70 (acid
equivalent)
, Environmental
Florida 32561,
24 -hour
LC50 (ppm)
0.0007
0.0015
0.0032
0.032
0.038
No effect at
1 0 ppm^
Protection
May 8, 1972.
Static bioassay.
The avoidance response was tested in two phases:
1. The ability of grass shrimp to choose between water that
contained a pesticide and water free of pesticide was tested.
Response to each concentration of pesticide was tested at least
four times; twice with the pesticide entering one section of the
apparatus and twice with the pesticide entering the opposite
section. For each of the four replications, 50 shrimp were
placed in the holding area with the gate lowered. After 30
minutes, the gate was raised to give the shrimp access to both
sections. One hour later, the gate was closed and the number of
shrimp in each section was recorded. This procedure was repeated
when additional data were required to verify the conclusions.
2. The capacity of shrimp to discriminate between concen-
trations of pesticide avoided in the first phase was tested.
130
-------
Experimental procedure was the same as in the first series of
tests except that the shrimp were given a choice between two
concentrations of the same pesticide.
The ability of grass shrimp to avoid pesticides in both
phases was evaluated statistically by the chi-square test on the
assumption that if there was no response to the pesticides, the
shrimp that left the holding area would enter each section with
equal frequency. Preliminary tests without pesticides indicated
that this assumption was correct. Lack of any preference for the
right or left section in avoidance tests (49 vs. 51%) further
corroborated this assumption. Avoidance or preference was con-
sidered significant if the probability that observed distribu-
tions would occur by chance was 0.05 or less. Shrimp remaining
in the circular area after a test was completed were not included
in the statistical analysis because stationary shrimp may not
have been exposed to the two choices and moving shrimp may have
been in transit between areas.
Avoidance
Grass shrimp avoided 1.0 and 10.0 ppm of the butoxyethanol
ester of 2,4-D by seeking water free of this herbicide but did
not avoid any of the five insecticides (Table 2). The avoidance
response of two fishes, sheepshead minnows and mosquitofish, to
these same pesticides was tested identically and 2,4-D was the
most readily avoided (HANSEN 1969, HANSEN et al. 1973). Fish in
TVA reservoirs were apparently repelled by application of 2,4-D
at 40-100 pounds per acre (SMITH and ISOM 1967). Concentrations
of 2,4-D in reservoir water one hour after application reached
0.16 ppm; slightly less than amounts avoided by shrimp in our
tests. Although statistical analysis indicated that grass shrimp
preferred 0.0001 ppm of DDT, this was probably not valid because
preference was observed in only one of three replications, and
shrimp did not respond to greater or lesser concentrations.
Grass shrimp given a choice between two concentrations of
2,4-D selected the lower concentration (Table 3). Up to 78 per-
cent of the shrimp that left the holding area avoided the higher
of the two concentrations. Only 2,4-D was tested in this manner
because it was the only pesticide that shrimp avoided by seeking
water free of toxicant.
131
-------
TABLE 2
Capacity of grass shrimp to seek water free of pesticide"
2
N.S. s not significant.
- P(3.84 = 0.05; 6.63 » 0.01; 10. oj : 0.001.
Pesticide
DDT
Endrin
Dursban
Malathion
Sevin
2,4-D
Concentration
(ppm)
0.01
0.001
0.0001
0.00001
0.01
0.001
0.0001
0.001
0.0001
0.00001
1.0
0.1
0.01
0.1
0.01
0.001
0.0001
10.1
1.0
0.1
Number of
tests
4
4
12
4
4
4
4
4
4
8
8
4
4
4
4
8
4
4
4
4
Number of
In pesticide
60
46
218
78
60
79
76
63
76
133
137
62
71
66
62
129
57
44
51
57
Shrimp*
In water
80
55
165
84
77
64
66
70
88
105
117
61
77
61
51
102
65
91
76
59
Percentage
in water
57.1
54.4
43.1
48.1
56.2
44.8
46.5
52.6
53.6
44.2
46.1
49.6
52.0
48.0
45.1
44.2
53.3
67.4
59.8
50.9
2
~y. value
N.S.
N.S.
7.334
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
16.363
4.921
N.S.
*Does not include shrimp in holding area at end of test.
-------
TABLE 3
Response of grass shrimp exposed to two-different concentrations
of the butoxyethanol ester of 2,4-D. -v = P(3.84 = 0.05; 6.63 =
0.01; 10.83 = 0.001.
Concentrations
High
10.0
10.0
1.0
(ppra)
Low
1.0
0.1
0.1
Number of
In high
cone.
43
24
43
shrimp*
In low
cone.
90
85
67
Percentage
in low
Concentration
67.7
78.0
60.9
-v 2
^
value
16.61
34.14
5.24
*Does not include shrimp in holding area at end of test.
Our study indicates that grass shrimp are less able to avoid
and are more readily affected by pesticides than were the fishes
used in earlier experiments (HANSEN 1969, HANSEN et al. 1973).
Similarly, the European brown shrimp (Cragon cragon) did not avoid
DDT (0.1 ppm), azinphos-methyl (1 ppm), atrazine (10 ppm) and
aminotriazole (1,000 ppm) and were more sensitive to these com-
pounds than were fishes (PORTMAN In press). These data suggest
that shrimp may be extremely vulnerable to pesticide pollution
because they are (1) extremely sensitive to pesticides and (2)
generally are unlikely to avoid water polluted by pesticides.
Consequently it is important that pesticides destined for use in
and near estuaries be tested to determine their toxicity to
shrimp and the capacity of shrimp to avoid them.
Literature Cited
HANSEN, D.J.: Trans. Am. Fish. Soc. 9£, 426 (1969).
HANSEN, D.J., E. MATTHEWS, S.L. NALL and D.P. DUMAS: Bull. Envir-
on. Contam. Toxicol. J3, (In press) (1973).
PORTMAN, J.E.: Proc. FAO Tech. Conf. on Marine Pollution and
its Effects on Living Resources and Fishing, December 1970,
Rome (In press).
SMITH, G.E. and B.C. ISOM: Pestic. Monit. J. JL, 16 (1967).
SUMMERFELT, R.C. and W.N. LEWIS: J. Water Pollut. Contr. Fed.
39, 2030 (1967).
WOOD, C.E.: Contri. Mar. Sci. 12., 54 (1967).
133
-------
CONTRIBUTION NO. 154
-------
Reprinted from
ECOLOGY OF HALOPHYTES
© 1974
ACADEMIC PRESS, INC
MANGROVES: A REVIEW1
Gerald £. Walsh
Environmental Protection Agency
Gulf Breeze Laboratory
Sabine Island
Gulf Breeze, Florida 32561
Associate Laboratory of the National Environmental Research Center,
Corvallis
"The beaches on that coast I had come to visit are treacherous and sandy
and the tides are always shifting things about among the mangrove roots...A
world like that is not really natural...Parts of it are neither land nor sea and so
everything is moving from one element to another, wearing uneasily the queer
transitional bodies that life adopts in such places. Fish, some of them, come
out and breathe air and sit about watching you. Plants take to eating insects,
mammals go back to the water and grow elongate like fish, crabs climb trees.
Nothing stays put where it began because everything is constantly climbing
in, or climbing out, of its unstable environment."
INTRODUCTION
The quotation above from Loren Eisley's eloquent book, "The Night
Country," portrays in poetic terms the fascination of the tropical mangrove
forest for those who have studied and researched that "not really natural"
1 Publication No. 154 from the Gulf Breeze Laboratory, Environmental Protection
Agency, Gulf Breeze, Florida 32561 Associate Laboratory of the National
Environmental Research Center, Corvallis.
51
-------
GERALD E.WALSH
world. In the mangrove ecosystem, where tides and coastal currents bring
unremitting variation to the forest, plants, and animals adapt continuously to
changing chemical, physical, and biological characteristics of their
environment. Many species use the environment dominated by mangrove
trees for food and shelter during part or all of their life cycles. There is
constant movement of living and non-living matter into and out of the
mangrove swamp, and the effects of such movement may be felt miles away
(Heald 1971, Odum 1971). Of course, not all tropical coasts are lined with
mangrove forests; often a mangrove stand is small, or only an occasional tree
dots the shoreline.
The factors which determine development of coastal forests, the ecological
roles of mangroves in estuaries, and their utilization by man have been
studied at length. The references at the end of this review give over 1,200
published accounts on mangroves. I am certain to have missed many
publications in my search, but the number gives testimony to the importance
of mangroves in estuaries. For an historical sketch of published works on
mangrove, see Bowman (1917), who traced the mangrove literature back to
325 B.C. and the chronicle of Nearchus, commander of the fleet of Alexander
the Great. Additional information is given in the reports of Walter and Steiner
(1936) on East African mangroves, Davis (1940b) on the ecology and
geologic roles of mangroves in Florida, and Macnae (1968) on the flora and
fauna of mangrove swamps in the Indo-West-Pacific region. See also the
excellent discussion of ecology of the Rhizophoraceae by van Steenis in Ding
Hou(1958).
Davis (1940b) described "mangrove" as a general term applied to plants
which live in muddy, loose, wet soils in tropical tide waters. According to
Macnae (1968), mangroves are trees or shrubs that grow between the high
water mark of spring tides and a level close to but above mean sea level. They
are circumtropical on sheltered shores and often grow along the banks of
rivers as far inland as the tide penetrates. Chapman (1939, 1940, 1944a)
described silt, sand, peat, and coral reefs as mangrove habitats. On the reef,
seedlings develop in holes and crevices in the porous coral rock, but the trees
are usually stunted and the area occupied by the stand is not large. The reef
may be a habitat only in those areas where tidal height is not great, because
total inundation for extended periods of time can be fatal to seedlings
(Rosevear 1947). Another mangrove habitat, the sand beach, described by
Chapman (1940) supports Rhizophora mangle L. Later, van Steenis (1962)
stated that R. stylosa Griffith is often found in sand in the Indo-Pacific
region. Hathaway (1953) and Moul (1957) reported stands of R. mucronata
Lamk., Sonneratia caseolaris (L.) Engler, and Bruguiera conjugata
(gymnorhiza?) Lamk is sand on several atolls in the Pacific Ocean. I saw R.
mangle growing in sand in Hawaii.
Boughey (1957) described mangroves which grew in two types of lagoons
on the west coast of Africa. In open lagoons, some of the mud around the
margins was exposed daily at low tide. Rhizophora racemosa G.F.W. Meyer
52
-------
ECOLOGY OF HALOPHYTES
and R. harrisonii Leechman grew on the exposed mud. Rhizophora species
grew only in open lagoons which were flooded daily. In closed lagoons,
Avicennia nitida Jacq. was the dominant form in association with Conocarpus
erectus L., Laguncularia racemose Gaertn., and Dodonea viscosa L.
Burtt , Davy (1938) classified tropical woody vegetation types according to
"mature" or "apparently stable" communities. Two of his classes apply to
mangrove vegetation and are given here because much of the nomenclature is
in common usage today.
1. Tropical Mangrove Woodland
Name Suggested for Adoption: Mangrove woodland.
Synonyms: Mangrove, Mangrove swamp, Tidal forest.
Brief Definition: Woodland formation below high-tide mark; sometimes
forest-like. Nearest in form to dry evergreen forest. A subformation of the
littoral swamp forest.
Habitat: Soil flooded with water either permanently or at high tide; water
usually more or less brackish; on estuarine mud.
2. Tropical Littoral Woodland
Name Suggested for Adoption: Littoral woodland.
Synonyms: Strand vegetation, Beach forest, Dune forest.
Brief Definition: Woodland formations in situations mentioned below;
somewhat resembling semi-evergreen forest; open herbaceous vegetation.
General Description: The most characteristic species of this formation in
India and Burma is the evergreen but very light-foliaged Casuarina, which
often forms an almost pure fringe on sandy beaches and dunes along the sea
face. Scattered smaller evergreen trees occur, with fewer deciduous trees, and
these, in the absence of Casuarine, form the dominant canopy. On the east
coast of Tropical Africa are such species as Heritiera littoralis Dryand,
Barringtonia racemosa L., Terminalia catappa L., Phoenix reclivata, and
Diospyros vaughaniae; species of Pandanus and Coco* nucifera L. are
characteristic of this formation, which naturally includes several species
whose seeds or fruits are current-borne. Ipomea pescaprae commonly occurs
as a surface creeper on exposed sand dunes. Xerophytic herbs such as
Sansevieria, Opuntia, Kalanchoe, and Euphorbia are common.
Habitat: Sandy and gravelly seashores; not subject to immersion, but under
constant maritime influence. All around the coast wherever a fair width of
sandy beach occurs, including sandy bars on the sea face of river deltas.
In this discussion, I shall follow Macnae (1968) and use the word
"mangrove" with reference to individual kinds of trees, and the word
"mangal" with reference to the swamp forest community.
It has been estimated that between 60% and 75% of the tropical coastline
is lined with mangrove trees (McGill 1958) though some stands are more
extensive than others. There seem to be five basic requirements for extensive
mangal development. They are:
1. Tropical temperatures. Well developed mangals are found only along
coastal areas where the average temperatures of the coldest month is higher
53
-------
GERALD E. WALSH
than 20°C and the seasonal temperature range does not exceed 5°C (West
1956, van Steenis 1962).
2. Fine-grained alluvium. Mangrove stands are best developed along deltaic
coasts or in estuaries where soft mud comprised of fine silt and clay and rich
in organic matter, is available for growth of seedlings. Quartzitic and granitic
alluvia are generally poor substrata, whereas volcanic soils are highly
productive of mangroves (Schuster 1952, West 1956, Haden-Guest et al.
1956, Macnae and Kalk 1962, Macnae 1968).
3. Shores free of strong wave and tidal action. Mangroves develop best
along protected shores of estuaries because strong wave and tidal actions
uproot seedlings and carry away soft mud (Young 1930, Cockayne 1958).
4. Salt water. Salt water per se is not a physical requirement of mangroves
(Bowman 1917, Warming and Vahl 1925, Rosevear 1947, Egler 1948, Daiber
1960). Mangroves are faculative halophytes that occupy tidal areas where
fresh-water plants, which are intolerant to salt, cannot live (West 1956).
5. Large tidal range. A wide, horizontal tidal range has been cited as
requisite for extensive growth of mangrove (Foxworthy 1910, West 1956)
and Chapman and Trevarthen (1953) stated that a universal scheme for
comparison of different shores can be based only on the tides as a universal
controlling factor. Although the tide per se is probably of little importance in
determining the extent of mangal development, on a shore of gentle gradient
and large tidal range, a wide belt of alluvium will be formed, and with it, a
wide belt of mangrove. Deep tidal penetration would also cause saline water
to be distributed far inland. Davis (1940) described the action of wind in
driving salt water inland in Florida.
These five factors can determine the occurrence of mangroves, the species
present, and the area occupied by a mangal. Once established, mangals
throughout the tropics have many ecological similarities. In the following
pages I attempt to summarize from accounts available to me, what is known
about mangroves and mangals.
GEOGRAPHICAL DISTRIBUTION
Geographical distribution of mangroves is similar in many ways to that of
sea grasses (Den Hartog 1957) and marine angiosperms in general (Good
1953). The main difference is that some mangrove species occur on both sides
of the Atlantic Ocean and on the Atlantic and Pacific coasts of the Americas.
Fig. 1 shows the general geographic distribution of mangroves. Among
individual genera and species, distribution is undoubtedly influenced by
whether or not the plant is viviparous, and the ability of the seedling to
survive in sea water for an extended period of time. Dispersal of resting
seedlings by drift in the open ocean and by alongshore surface currents
permits wide geographic range, and temperature and geomorphological
characteristics determine distribution along individual coasts.
54
-------
ECOLOGY OF HALOPHYTES
Geographical distribution is restricted, in general, to the tropics, but
Oyama (1950) reported a small stand of mangrove on the southern tip of
Kyushu Island at 35°N latitude. Later, van Steenis (1962a) identified the
species there as Kandelia kandel (L.) Druce. Vu Van Cuong (1964) reported
the Ryukyu Islands (about 27°N latitude) to be the northern limit of R.
mucronata, B. gymnorhiza, Avicennia marina (Forsk.) Vierth., Xylocarpus
moluccensis (Lamk.) Roem., X. granatum Koenig, Lumnitzera littorea (Jack)
Voigt, and Lumnitzera racemosa Willd. In the southern hemisphere van
Steenis (1962a) reported the southernmost stand to be on North Island of
New Zealand at "less than 40° south." Chapman and Ronaldson (1958)
reported that dwarfed A. marina grew in abundance in Auckland Harbor at
37°S latitude.
Mangroves are present on the Pacific coast of South America only to about
4°S latitude due to lack of sedimentation below that point. It was once
thought that atmospheric drought caused absence of mangroves from that
and other areas. Van Steenis (1962a) pointed out, however, that drought is
not a factor in distribution as mangroves grow in the Arabian Gulf, the delta
of the Indus River, southern Timor, and western Australia, where large silt
deposits are found on arid coasts. The major differences between mangals on
arid coasts and those on humid coasts is the paucify of the epiflora in the
former.
Every mangal is composed of two classes of plants: (a) genera and higher
taxa which are found only in the mangrove habitat and (b) species that
belong to genera of inland plants but which are adapted for life in the swamp
forest. World distribution of genera that occur in mangrove swamps is given in
Table 1. For a detailed listing of many forms in class "b" above, see Vu van
Cuong (1964). The fern Acrostichium aureum appears to be a circumtropical
associate of mangroves since it has been reported in mangles of Ceylon
(Abeywickrama 1964), India (Biswas 1927), Africa (Bews 1916, Boughey
1957), and the West Indies (Borgesen 1909).
Geographically, mangrove vegetation may be divided into two groups: that
of the Indo-Pacific region and that of western Africa and the Americas. The
Indo-Pacific region is comprised of East Africa, the Red Sea, India, Southeast
Asia, southern Japan, the Philippines, Australia, New Zealand, and the
southeastern Pacific archipelago as far east as Samoa. The West
Africa-Americas region includes the Atlantic coasts of Africa and the
Americas, the Pacific coast of tropical America, and the Galapagos Islands.
Mangroves are not native to Hawaii, but R. mangle, B. sexangula (Lour.)
Poir., S. caseolaris, and Conocarpus erectus have been introduced.
Distributions of several species found only in mangrove swamps are shown
in Fig. 2-8. These figures, with Table i, show that (a) the greatest number of
genera and species occur along the shores on the Indian and western Pacific
oceans, (b) there are no species common to East and West Africa, and (c) the
species of the Americas and West Africa are related taxonomically. Species
found on both the eastern shores of the Americas and the western shore of
55
-------
Table 1. Distribution of plant genera that occur only in mangrove swamps
(Chapman 1970).
Families and Genera
Rhizophoraceae
Rhizophora
Bruguiera
Ceriops
Kandelia
Avicenniaceae
Avicennia
Myrsinaceae
Aegiceras
Meliaceae
Xylocarpus
Combretaceae
Laguncular ia
Conocarpus
Lumnitzera
Bombacaceae
Camptostemon
Plumbaginaceae
Aegiatilis
Total
species
7
6
2
1
11
2
?10
1
1
2
2
2
Indian Ocean
W. Pacific
5
6
2
1
6
2
?8
0
0
2
2
2
Pacific
America
2
0
0
0
3
0
?
1
1
0
0
0
Atlantic
America
3
0
0
0
2
0
2
1
1
0
0
0
West
Africa
3
0
0
0
1
0
1
1
1
0
0
0
Con't on next page
56
-------
Table 1 con't
Palmae
Nypa
Myrtaceae
Osbornia
Sonneratiaceae
Sonneratia
Rubiaceae
Scyphiphora
55 44
57
-------
9Q 6O 3O Q 3O 6O 9O HO ISO 1«O
90 60 30 O
6O 9O HO 15O 1BO
Figure 1. World distribution of mangroves (after Chapman 1970). ••••less than
five species present; - • - • — • five to twenty species present;
_____ more than 20 species present.
90 60 30
3O 6O 9O I7O 15O ISO
30 60 90 120 150 ISO
Figure 2. World distribution of Rhizophora species (after Ding Hou 1960, Vu Van
Cuong 1964, and Chapman 1970). R, mangle L.; _,AA R
racemosa F f.Vf. Meyer; • • • R. harrisonii Le^hm • g^
mucronata Lamk.; O O O R. apiculata Blume; + + + R. stylosa Grif-
fith; • • R. lamarckii Montr.
58
-------
9O
6O
130
HO I«O
ISO ItO
Figure 3. World distribution of Avicennia species (after Vu Van Couong 1964 and
Chapman 1970). + + + A. nitida (germinans?) Jacq.; AAA A. schauerana
Stapft; C
-------
150 180
30
60
120
150
180
Figure 5. Distribution of Sonneratia species (after Vu Van Cuong 1964 and Chap-
man 1970). S. alba J. Smith; • • • • S. caseolaris (L.) Engler
* + * S. ovata Backer; A A A S. griffithii Kurz.; - • ^ • - &_
apetala Buch.-Ham. -
30
60
90
120
150
3O
150
180
Figure 6. Distribution of.
Cenoi
(Pen.) C. B. Rob.; C.
decandra (Griffith) Ding Hou; ••••KandeBa Kandel (L.) Druce (after~
Vu Van Cuong 1964).
60
-------
»<> «0 30 O 30 60 90 i?O
Figure 7. World distribution of Xylocarpus species (after Vu Van Cuong 1964).
X.granatum Koenig; X.moluccensis(Lamk.) Roem.;XX — X.
gangeticus Park; AAA X. minor Ridley; ®® X. parvifolius Ridley;
••• X. australasicum Ridley; ^A X. guianensis; • • • * X. benadirensis
Moll.
9O 6O 3O
3O 6O 9O 12O ISO ISO
90 6O 30
3O 6O 9O I JO ISO ISO
Figure 8. Distribution of ^onocarpus ereotus L.; + + + Laguncularis racemosa
Gaertn.: • • • • Lumnitzera racemosa Willd.; and Lumnitzera littorea
(Jack.) Voigt.
61
-------
GERALD E. WALSH
Africa are R. mangle, R. racemosa, R. harrisonii, Laguncularia racemosa, and
C. erectus. Pelliciera rhizophorae Planchon and Triana, a member of the tea
family (Theaceae), is found only on the Pacific coast of tropical America. It
occurs in small communities on exposed areas such as the seaward tips of
point bars in estuaries or in spots having hard, clay soils (West 1956). Fuchs
(1970) reported pure stands of P. rhizophorae on firm, sandy ground of low
salinity. Pelliciera was associated with Rhizophora on low, muddy ground but
in this habitat, trees of both genera were small.
There is confusion concerning taxonomy and distribution of Avicennia
species on the shores of the eastern and western Atlantic Ocean. It was
commonly held that a single species, A. nitida, occurred on both sides of the
Atlantic Ocean. Moldenke (1960), however, recognized A. nitida in the
Americas and A. africana Moldenke in West Africa. Vu van Cuong (1964)
discarded the species nitida and recognized A. germinans in the Americas and
A. africana in West Africa. Both Chapman (1970) and Vu van Cuong (1964)
recognized four species in the Americas and only one, A. africana, in West
Africa, and toxonomists at this time seem to agree that the American and
West African species are closely related. Chapman (1970) speculated that the
reason for confusion is that speciation is now occurring within the genus on
both sides of the Atlantic.
There is also confusion in the common names of some mangroves. Those of
the genus Rhizophora are called "red" mangrove in both the Americas and
Africa. Avicennia, called the "black" or "honey" mangrove in the Americas,
is known as the "white" mangrove in West Africa. Laguncularia is called the
"white" mangrove in America.
From taxonomic and distributional considerations, Ding Hou (1960) and
van Steenis (1962) concluded that Rhizophora, Avicennia, Xylocarpus,
Lumnitzera, and Laguncularia arose in the Indo-Malaysian region and spread
westward to East Africa and (except Laguncularia) eastward to the Pacific
coast of the Americas. The genera reached the Caribbean Sea sometime
between the Upper Cretaceous Period and the Lower Miocene Epoch, when
the Isthmus of Panama was an open seaway. After establishment on eastern
American shores, the trees reached West Africa when seedlings were carried
across the ocean by surface currents.
To explain why the mangrove floras of East and West Africa are separate,
van Steenis (1962a) postulated that the climate of South Africa during the
Upper Cretaceous Period was not tropical and that mangroves could not have
been distributed from east to west around the Cape of Good Hope.
As an interesting sidelight to the problem of distribution, Ding Hou (1960)
pointed out that broad areas of the Pacific Ocean, to which favorable currents
flow, do not contain mangrove. He attributed this to lack of suitable coasts
for successful implanting of seedlings. He also described R. mangle, a native
of the Americas, as present in New Caldonia, Fiji, and Tonga (see Fig. 2).
Chapman (1970) speculated that early man carried seedlings from Pacific
62
-------
ECOLOGY OF HALOPHYTES
America to those islands for growing trees to serve as a source of tannin.
ECOLOGY
Mangrove swamp forests are complex ecosystems that occur along
intertidal accretive shores in the tropics. Dominated by estuarine trees, they
draw many of their physical, chemical, and biological characteristics from the
sea, inflowing fresh water, and upland forests. Mangrove swamps serve as
ecotones between land and sea, and elements from each are stratified both
horizontally and vertically between the forest canopy and subsurface soil.
The canopy is inhabited by floristic and faunistic elements from the
tropical rain forest, including epiphytes, insects, reptiles, birds and mammals.
Phytotelmata, filled with rain water, support a variety of algae, protozoa, and
immature insects. Below the canopy, portions of tree stems are immersed, in
relation to the tidal cycle, for various periods throughout the day. An
extreme example is Inhaca Island in southeastern Mozambique where stems
are often immersed for 8 to 12 hours per day at depths up to 2 m (Mogg
1963). The surface soil of swamps is alternately inundated and drained. It
supports animals such as crabs, amphibians, reptiles, air-breathing fishes, and
mammals, whose distributions are governed by degree of tidal penetration
and by the nature of the substratum. At the landward edge of the swamp,
typically fresh-water forms such as frogs, monitors, and crocodiles may be
found (Macnae 1968), and I have observed the toadBufo marinus in salinities
up to 10 ppt (parts per thousand) in a mangal in Hawaii.
At the seaward edge, the mud surface is often a truly marine mid-littoral
soft-bottom environment (Rutzler 1969) and supports crabs, shrimp,
shellfish, etc. Numerous permanent and semi-permanent pools contain
insects, shellfish, amphibians, and fish. Throughout the mangal is a network
of rivulets, creeks, channels, and often rivers which change in depth with tidal
eb"b and flow. These contain numerous sessile forms such as algae, fungi,
tunicates, sponges, and shellfish which live on mangrove prop- and
aerial-roots. Mobile forms such as worms, crabs, shrimp, and fish migrate
within the waterways in relation to the tidal cycle and nature of the
substratum.
Jennings and Bird (1967) gave six environmental factors which affect
geomorphological characteristics in estuaries and, therefore, the flora and
fauna. The characteristics were: (1) aridity, (2) wave energy, (3) tidal
conditions, (4) sedimentation, (5) mineralogy, and (6) neotectonic effects.
All have been cited as factors in mangrove establishment. Troll and
Dragendorff (1931) stated that water, salt, and oxygen contents of the soil
are also important. On a short-term basis, tropical storms are very disruptive
to mangals (West 1956, Alexander 1967) and are the greatest single sources of
repeated set backs to the vegetation (Exell 1954). On the other hand, storms
may carry propagules further inland than would normal tides (Egler 1952),
and Mullan (1933) stated that seeds of mangroves are dispersed widely during
the monsoon in Malaya.
63
-------
GERALD E. WALSH
Tides and type of substratum are probably the most important factors that
govern the nature of intertidal communities (Chapman and Trevarthen 1953).
In the case of mangroves, salinity of the' surface and soil waters are also very
important (Davis 1940), as are temperature, rainfall, rate of evaporation,
topography, and geomorphology.
Surface Water
One of the distinctive features of mangrove vegetation is the ability to live
in salt water as faculative halophytes. In such situations as reefs, lagoons, and
the Florida Everglades, the surface water environment of mangroves is fairly
stable in terms of physical and chemical composition. In mangals, salt and
nutrient concentrations of surface water, whether in waterways or covering
the swamp floor at high tide, is regulated by (1) inflow of fresh water from
upland areas, (2) inflow and outflow of seawater with each tidal cycle, (3)
precipitation, and (4) humidity.
Chemical and physical data on the surface waters of mangrove swamps
have been reported from the Great Barrier Reef (Orr and Moorehouse 1933);
Inhaca Island, Mozambique (Macnae and Kalk 1962); Cananeira, Brazil
(Teixeria and Kutner 1963, Teixeria et al. 1965, Okuda et al. 1965); Tabasco,
•Mexico (Thorn 1967); Hawaii (Walsh 1967); and Trinidad (Bacon 1968,
1971). Davis (1966) reported salinities up to 43 ppt and temperatures to
39.5°C in a mangrove salt-water pool in Jamaica. Examples of extreme
conditions in a single swamp were given by Walsh (1967) who analyzed the
water at six stations located between the landward and seaward edges' of a
swamp in Hawaii. At the landward edge, tidal effect was minimal or
non-existant between August 1961 and November 1962, and the water was
always fresh. Oxygen content of the water was that of a dystrophic body,
averaging 0.67 ml/L throughout the sampling period. None of the factors
measured were subject to large diel, monthly, or annual changes. Proceeding
from the landward to the seaward stations in the swamp, diel changes became
greater. At the seaward edge, water chemistry at high tide was similar to that
of open bay water with great variations in relation to the tidal cycle. At low
tide the water was fresh, whereas at high tide salinity was always greater than
25 ppt. In spite of such great differences in physical and chemical properties
of the surface water, R. mangle grew in a dense stand between the landward
and seaward edges of the swamp.
Bacon (1968) gave similar data for a mangrove swamp in Trinidad. In
addition, he reported diel variations in concentrations of dissolved nitrate,
phosphate, silicate, and suspended solids. Increased concentrations of nitrate'
and silicate occurred at low tide. Bacon suggested that either the inflowing
fresh water was richer in nutrients than tidal water or that nutrients were
released from the mud at low water. Walsh (1967) found a similar
phenomenon for both nitrate and phosphate, and ascribed this to greater
solubility of the substances in fresh water than salt water. He also
demonstrated the affinities of the various types of swamp substrata for
64
-------
ECOLOGY OF HALOPHYTES
nitrate and phosphate. In addition, nitrate and phosphate contents of swamp
waters at low tide were greater than those of inflowing water. All of this
indicates a dynamic system in which nitrate and phosphate are released from
or taken up by sediments covered by surface water.
Watson (1928), DeHaan (1931), Walter and Steiner (1936), Macnae and
Kalk (1962), and Macnae (1966) proposed schemes for classification of
zonation of vegetation within mangals based upon tidal inundation and
salinity. The details of these proposals were reviewed by Macnae (1968). In
each scheme, vegetation of southeastern Asia was related to salinity of the
water and Macnae (1968), using the data of Watson (1928), showed the
general preference of Avicennia intermedia for coastal seawater and of A. alba
for less saline water around the mouths of rivers. Rhizophora mucronata lived
in water of greater salinity than R. apiculata. Bruguiera sexangula occurred in
water of greater salinity than B. cylindrica, B. parviflora, and 5. gymnorhiza.
The three species of Xylocarpus lived in the less saline areas of the swamp.
Davis (1940) related salinity of surface water to distribution of trees in a
swamp in southern Florida. American swamps are simpler floristically than
those of Malaya and Davis demonstrated the relationship between tidal
penetration and salinity to horizontal zonation. In Florida, R. mangle and
Luguncularia racemosa are mixohaline, but the former is the pioneer species
on seacoasts. Rhizophora mangle was found by Davis to grow in salinities that
ranged from fresh water in the Everglades to 34.9 ppt along the seashore.
Laguncularia grew in "nearly fresh water" to water of 45.8 ppt, and was
usually found in association with the other mangrove species. Davis stated
"no particular habitat is definitely most suitable for Laguncularia." This
concept was extended by Thorn (1967), who observed that Laguncularia
racemosa in Tabasco formed communities with other mangroves and had less
stringent habitat requirements than they.
Davis (1940) reported A. nitida growing in the field in salinities between
36.8 ppt and 38.6 ppt although it can grow in fresh water in the laboratory.
This species seems to be adapted for survival in swamp areas with great
salinity fluctuations. The community is not flooded deeply by tidal water and
salt is concentrated by evaporation during dry periods. During periods of rain,
the surface water is diluted greatly so that the Avicennia zone has a greater
range of salinity than any other. Conversely, Conocarpus erectus grew only
where salinity was low and the ground'covered only occasionally by tidal
water. Many Conocarpus localities had no surface water; where there was
surface water, salinity averaged less than 2 ppt. An important factor for
survival of C. erectus seemed to be high salinity of the soil water. This will be
discussed in the next section.
Sedimentation and Soil
According to the nature of the substratum, mangroves may be classified as
reef, sand, mud, and peat types (Chapman 1940, Rutzler 1969). Also, some
are found occasionally among boulders, having roots within cracks or other
niches, and use tidal water as the source of nutrients. The typical sediments
65
-------
GERALD E. WALSH
of swamps are composed of peaty, soft, sandy or clayey mud. They are
similar to the sediments of salt marshes, which occupy the same
sedimentological position at higher latitudes. Mineralogy of mangrove
sediments is concerned mainly with clastic detritus from rivers and calcareous
debris formed either biologically by shelled organisms or by inorganic
precipitation. Algae and bacteria can also function in precipitation and pyrite
is often abundant in swamps, usually embedded within or attached to plant
remains. Along some shores, where tidal and alongshore currents control the
character of the sediments, siliceous and quartzitic sand may predominate.
Mangroves advance seaward only where sedimentary processes prepare
shallow water areas for growth of seedlings Mangals often are associated with
lateral accretion of sediment along tropical shores, and location, size, and
shape of swamps are influenced strongly by the pattern of coastal
sedimentation. Hagen (1890) and van Steenis (1941) stated that natural
coastal accretion by mud-silting is the major factor responsible for
development of large mangals. Although the trees do not aid appreciably in
lateral extension of shores, they do aid in accumulation of sediment with
subsequent build-up of soils (Curtiss 1888, Vaughan 1909, Watson 1928,
Holdridge 1940, Egler 1952, West 1956, Boughey 1957, Vann 1959, Stoddart
1962, Scholl 1963, Thorn 1967, Macnae 1968). During high tide, brackish,
sediment-laden water overflows the numerous creeks and channels of the
swamp. Alluvium is deposited on the swamp floor and, with autochthonous
organic and inorganic detritus, aids in land elevation. Freise (1938) stated
that the black color of mangrove mud in Brazil was due to the presence of
iron sulphide. The black mud was often covered by a 1-5 cm deep layer of
grey-brown mud which was either deposited during tidal inundation or
affected chemically by oxygen in tidal waters.
Accretion of sediment along alluvial coasts is regulated mainly by
physiographic-geomorphic processes such as (a) the rate at which sediment is
brought into an area by rivers, and tides, (b) the angle of slope of the shore,
(c) sedimentary distributional patterns, (d) subsidence or emergence of the
coast, (e) other factors associated with changes in sea level, and (f) tidal-river
channel development. As in other estuaries, the coarser sediments of
mangrove swamps are generally in the channels, the finer sediments along the
shores of the channels (Walsh 1967). Also, when a river reaches the estuary,
the heavier particulate elements have been sorted out above the mangal, so
that the predominent sediments within the swamp are of fine-grained alluvium.
Near the mouth of the estuary, coarser sediments may again be found. These
originate from tidal and alongshore currents which have enough energy for
deposition of small sand particles and calcareous detritus. The contribution of
inorganic detritus from seawater is usually small, however, because strong
currents do not allow seedling development. River-borne sediment is the
greatest source of allochthonous material in most waamps and appears to be
especially important in the Indo-Malayan region (Watson 1928, Schuster
1952, Macnae 1968).
66
-------
ECOLOGY OF HALOPHYTES
Sedimentation of autochthonous matter is an important factor for
mangroves which are not influenced greatly by fresh-water inflow. Davis
(1940) stated that the authochthonous mangals of Florida develop over three
primary soil types; namely, (1) siliceous sands, (2) calcareous sands, and (3)
calcareous mud marls. Mature R. mangle trees are thus sometimes found on
nearly bare rock with only small pockets for rooting, but more often grow on
deep peat soils. Although the general physical and morphological features of
soils vary greatly between mangals, the halotropic peats so often found with
mangroves are composed mainly of calcium compounds from shells,
biologically precipitated calcite and aragonite, and organic matter of floral
and faunal origin.
Extensive autochthonous mangrove swamps have developed along the
western side of Andros Island in the Bahamas where the rate of carbonate
mud precipitation is great. Burkholder and Burkholder (1958) described the
autochthonous sediments of Bahia Fosforescente in Puerto Rico. The
sediments contained large amounts of mangrove roots, stems, and leaves, and
the authors stressed the important influence of mangrove detritus on the
chemistry and biology of the bay. At the present time, autochthonous peat
swamps are developing along the southwestern coast of Florida because of the
paucity of sediments from rivers and streams.
In southeast Asia, where large numbers of rivers drain uplands of volcanic
origin, large allochthonous swamps form in deltas, estuaries, lagoons, and
along sheltered open coasts. These allochthonous swamps are the most highly
developed mangals in the world (Watson 1928, Macnae 1968).
Mixed authochonous-allochthonous swamps occur along the Pacific coast
of Colombia where there is low to intermediate supply of river-borne
sediment (West 1956).
Several systems have been proposed for the classification of mangrove
swamp soils. Aubert (1954) and Dubois (1954) classified mangrove soils in
relation to hydromorphic characteristics and salinity. Bonfils and Faure
(1961) related halomorphic soil types to the degree of salt-and fresh-water
flooding and to the relative concentrations of chlorides and sulphates.
D'Hoore (1963) typed mangrove swamp soils as "juvenile soils on marine
alluvium" and called them "weakly developed soils." This general
classification was accepted by Giglioli and Thornton (1965), who suggested
further subdivision for agricultural purposes according to soil texture, water
regime, degree of gleying and/or mottling, amount of oxidizable sulfur, and
relative amounts of chlorides and sulphates.
Grant (1938), Davis (1940b), Chapman (1940), Thorn (1967), Walsh
(1967), and Giglioli and King (1965) discussed the evolution of mangrove
swamps in relation to silting and plant succession. Davis (1940b) listed three
main factors which promote soil accretion: (l)mplar, (2) physicochemical,
and (3) biotic.
Molar factors are mainly tides, littoral currents, and winds. The first stages
of accretion consist of marine and estuarial sedimentation, resulting in
67
-------
GERALD E. WALSH
formation of shoals, bars, and flats in the shallow water. At the same time,
deposition of sediments and physico-chemical precipitation of dissolved
substances occurs when fresh and salt water mix (Jackson 1958). This adds
carbonates, phosphates, nitrates, and other substances to the developing soil.
Hesse (1961b) reported that R. racemosa swamps in Sierra Leone were
comprised of fibrous mud, whereas A. germinans swamp soils were
non-fibrous. Also, Rhizophora swamps had higher pH values, C/N ratios, and
contents of oxidizable sulfur, nitrogen, phosphorus, and carbon. Giglioli and
Thornton (1965) described the early phases of swamp evolution in the
Gambia, West Africa, where R. racemosa pioneers in virgin alluvia composed
of soft, silty soil. Proliferation of fibrous roots at the soil surface produces a
"felt-like" layer which entraps sediment and increases the rate of deposition
of both alluvium and leaf litter. Rosevear (1947)suggested that the fibrous
mat formed by R. racemosa prevents further establishment of that species
and conditions the soil for colonization :by Avicennia, which .requires a more
consolidated and elevated substratum (Jordan 1964). As the soil surface
becomes elevated, the rhizophoretum dies and A. germinans replaces R.
racemosa (Giglioli and Thornton 1965). The many pneumatophores of
Avicennia further accelerate deposition and the forest floor becomes even
more elevated. According to Giglioli and Thornton, if the amount of drainage
from higher ground is large and the swamp near the main river or a tributary,
a balance occurs between erosion and drainage of the swamp and land
elevation.
Zieman (in press) found that in Biscayne Bay, Florida, circular beds of
Thalassia testudiniim Koenig and Sims laid over depressions in the bedrock.
The depressions were over 5 m deep and filled with mangrove peat dated to
be 3,680 years old. Wharton (1883) suggested that living mangroves and their
peat produce organic acids that dissolve bedrock. Zieman suggested that
bedrock was dissolved under mangrove hammocks and hypothesized that as
the mangrove shoreline receded and sea level rose, Thalassia colonized the old
mangrove areas. Dodd and Siemers (1971) described a very similar, situation
on Bahia Honda and Big Pine Keys in the lower Florida Keys. They stated
that the topography developed during the lowered sea level of the Pleistocene
strongly controls Holocene sediment thickness and present biotic
distribution. They said, however, that the depressions were sinkholes and
thick sediment in underwater sinkholes promoted growth of Thalassia,
whereas depressions in shallow water or in the tidal zone supported growth of
R. mangle and A. nitida.
Schuster (1952) reported deposition of sediment on the mangrove forest
floor at every spring tide in Java, the processes of land elevation and soil
formation being accelerated by growth of beach thistles (Acanthus sp.) which
produced large quantities of organic matter.
Soil derived by sedimentation from river water is often poor in calcium
and potassium and, in mangrove swamps, tidal water is the main source of
68
-------
ECOLOGY OF HALOPHYTES
salt. In the early stages of mangal development, the clay particles absorb
calcium and potassium salts from seawater and a fine-grained soil, rich in
minerals, results. As evolution of the swamp continues, shelled animals invade
and grow on the trees and substratum. The organic content of the soil
increases and in the moist environment, decay processes are rapid and
calcareous particles are dissolved (Abel 1926). Wharton (1887) reported rapid
corrosion of CaCo3 in mangrove swamps at Aldabra. Fairbridge and Teichert
(1947) concluded that pools on reef flats at Low Isles (Marshall and Orr
1931) were caused by solution of CaCO3 by mangrove swamp acids. Reville
and Fairbridge (1957) suggested that the principal agent for destruction of
CaCC>3 in mangrove swamps is carbonic acid produced by decomposition of
organic matter. They also suggested that tannic acid from mangrove bark and
"humic acids" aid in decomposition.
Very little is known about the factors that form and condition mangrove
mud, which may lie in an unconsolidated state to a depth of ) .5 m. Schuster
(1952) discussed breakdown and modification of the substratum by bacteria,
fungi, actinomycetes, and myxomycetes. He mentioned the occurrence of the
bacteria Clostridium sp. and Azobacter sp. and the algae Nostoc sp. and
Anabena sp. in mangrove swamps and speculated that those organisms are
important in nitrogen fixation.
Most of the organic debris on and within mangrove soils is authochonous.
Because of the saline water, relatively high pH of surface soil water (often as
high as 7.8) and anaerobic conditions at low tide, plant detritus is only
partially broken down by bacteria, fungi, and algae. This causes formation of
peat, which is composed mainly of plant remains.
The role of birds in composition and fertility of mangrove soil has not
been investigated adequately. Birds in mangrove have been described by
Cawkell (1964), Haverschmidt (1965), Ffrench (1966), Parkes and
Dickerman (1967), Nisbet (1968), Field (1968), Dickerman and Gavino T.
(1969), Ffrench and Haverschmidt (1970), Dickerman and Juarez L. (1971),
and Ricklefs1 (1971). Large numbers of birds (Ffrench reported 94 species in
mangrove ir\ Trinidad), including egrets, ibis, herons, spoonbills, anhingas,
pelicans, storks, ospreys, and eagles, roost in mangrove trees but feed
elsewhere. In this way, nutrients are brought into the swamps and the
functions of such nutrients should be investigated.
Analyses of mangrove peat have been reported by several workers. Davis
(1940) gavei detailed accounts of soil profiles in the swamps of southern
Florida and, classified them according to their general composition, i.e.
homogeneous, heterogeneous, or layered'. He also classified them on the basis
of the type of vegetation which covered the soil and the probable types of
vegetation that formerly were present and contributed most to the
accumulated materials, Davis reported various types of soil profiles, some of
which indicated progressive soil accretion, while others did not, Giglioli and
Thornton (1965) gave soil profiles from the Gambia River basin ;n West
Africa. The profiles indicated typical alluvial soils in fhe process of silting.
69
-------
GERALD E. WALSH
The composition of mangrove swamp substratum is dependent upon its
source, age, position in the swamp, organisms present, and scouring by water
flow in the channels and over higher ground at high tide. Walsh (1967)
reported that up to 74.6% of the substratum in the center of the channels of
a swamp in Hawaii was composed of shells, pebbles, and gravel with diameter
greater than 3.35 mm. All of the alluvial particles, however, were less than
0.23 mm in diameter, and most sediments in mangrove swamps are of small
grain size.
Scholl (196-3) compared grain-size distribution of clastic sediments in two
mangrove swamps in southwestern Florida, where R. mangle was the
dominant species. In the swamp of the Ten Thousand Islands area, the
sediment was composed of fine to very fine calcareous-quartzitic sand and
coarse silt. The quartz content was approximately 70%, carbonate mineral
10-20%, and organic matter usually less than 10%. Isopleths of grain size
showed a zone of coarser-grained sediment (approximately 0.100-0.200 mm
diameter) along the shore flanking a belt of finer-grained sediment
(0.062-0.125 mm) inland. Another belt of coarser sediment (0.125-0.250
mm) lay landward of the finer-grained belt. In contrast, the sediments of the
Whitewater Bay area swamp were composed mainly of mollusc shells and
shell fragments. Less than 15% of the sediment was quartz and "little organic
detritus" was present. The grain sizes of surface sediments fell between 0.054
and 0.540 mm in diameter. Distribution of grain size was variable throughout
the swamp. Scholl attributed the differences in sediment characteristics
between the Ten Thousand Islands and the Whitewater Bay areas to
differences in patterns and strengths of the tidal currents. Strong tidal
currents which washed the former were lacking in the latter. Tables 2 and 3
give grain-size distributions in several swamps.
There is a paucity of data on physical and chemical characteristics of
mangrove soil. Values for physical and chemical factors from forests
dominated by different trees with different substrata overlap (Doyne 1933;
Davis 1940; Bharucha and Navalkar 1942; Chapman 1044a, b, c; Navalkar
and Bharucha 1948, 1949; Schuster 1952; Wyel 1953;He
-------
Table 2. Percentage grain-size distribution (mm diameter) in mangrove
swamp surface sediments.
El Salavador (Wyel 1953)
>1.0 mm 0.0-5.27.
1.0-0.5 0.0-4.2
0.5-0.4 0.0-8.0
0.4-0.3 0.0-6.0
0.3-0.2 0.0-18.0
0.2-0.1 0.0-18.0
0.1-0.06 0.0-16.6
0.06-0.03 22.0-43.8
0.03-0.017 7.0-32.0
0.017-0.007 3.0-27.0
0.007-0.003 0.5-8.0
<0.003 0.5-5.0
Brazil (Friese 1937)
>0.2 mm 4.7-7.0%
0.2-0.06
0.06-0.03
0.03-0.006
0.006-0.003
<0.003
7.2-11.8
6.7-10.1
6.6-10.4
22.1-24.5
43.6-46.6
India (Navalkar 1941)
2.0 3.9-4.0%
0.2 38.2-38.6
0.02 29.5-33.1
0.002 3.9-4.3
Java (Schuster 1952)
2 mm 0%
2.0-0.1 2
0.1-0.05 5
0.05-0.01 30
0.01 63
Florida (Scholl 1963)
Median grain-size from 10
stations varied between 0.006
and 0.700 mm.
Jamaica (Chapman 1944)
Coarse sand 39.9%
Fine sand 26.5
Clay 5.1
Silt 15.7
71
-------
GERALD E. WALSH
Table 3. Grain-size distribution in surface sediments of swamps dominated by
Avicennia alba (Navalkar 1941) and A. marina (Clarke and Hannon 1967).
Coarse sand = 2.0 mm diameter, fine sand = 0.2 mm, silt = 0.02 mm, clay =
0.002 mm.
Species Percentage of Dry Soil
A. alba
A. marina
Coarse sand
4.0
75.2
Fine sand
38.4
3.8
Silt
31.3
1.7
Clay
4.1
4.8
However, Avicennia marina and Arthrocnemum australasicum occurred only
where high salinity of the soil water endured for long periods of time or
where there were wide variations in salinity. Giglioli and King (1966) pointed
out that A. germinans grew in old soils of high salinity and that this high salt
content was a function of time. Avicennia was apparently able to exclude
Rhizophora racemosa because it was better adapted to high concentrations of
salt. Avicennia, unlike Rhizophora, absorbs large quantities of salt through its
roots and excretes them through the leaves (Scholander et al 1962). As shown
above, the fibrous nature of the substratum also appears to be important in
colonization, and it is most likely that combinations of factors, including soil
salinity, regulate species distribution.
Clarke and Hannon (1969) concluded that tidal action, as modified by
microtopography, was the major factor which affected soil salinity over long
periods of time, and that succession was mainly allogenic rather than
autogenic. This concept was shown to be true by Thorn (1967), who
demonstrated that although biotic and geomorphic processes are effective in
short-term changes on actively accreting shores, physiographic processes of
sedimentation and subsidence are more important over a long period of time.
Physiographic changes influence salinity of the soil, degree of water
saturation, soil type, and drainage, and therefore greatly influence the species
present.
Zonation and Succession
In general, in areas of large mangals, five geographic belts can be
distinguished (West 1956): (1) a belt of shore water and mudflats along the
coast, (2) a series of discontinuous sand beaches, variable in size, which are
interrupted by tidal inlets and mudflats, (3) a zone of mangrove forest,
usually one-half to three miles wide, (4) a fresh-water swamp, and (5)
equatorial rain forest. Although the beach zone is frequently absent, this
zonation was described in the Malay Peninsula (Watson 1928), western Africa
(Grew 1941), the Congo (Pynaert 1933), and in Guiana (Martyn 1934).
72
-------
ECOLOGY OF HALOPHYTES
Within the mangrove belt, there is usually a serai succession of vegetation in
relation to hydrological and climatic conditions. Day et al. (1953) held the
salinity gradient to be of great importance to distribution in South Africa,
and described correlations between rainfall, evaporation, upflow of salt water
from the sea, and serai change.
There have been several attempts to classify mangrove vegetation
according to physical characteristics of the environment. Watson (1928)
described two general classifications: (1) mangroves that grow on accretive
shores and (2) those that grow on sand. Watson also related species to the
tidal cycle and described five classes: (1) inundated by all high tides, (2)
inundated by medium high tides, (3) inundated by normal high tides, (4)
inundated by spring tides, and (5) occasionally inundated by exceptional or
equinoctial tides.
Stevenson and Tandy (1931), working at Low Isles in Australia, described
the mangrove habitat as (1) dense woodland, (2) muddy glades, and (3)
shingle tongues. At present, mangrove types are sometimes considered to be
related to the type of soil present. Troll and Dragendorff (1931) and Walter
and Steiner (1937) described mud and reef mangroves; Chapman (1944a)
added the categories of sand and peat mangroves. These four types are
generally recognized today.
Tansley, Watt, and Richards (1939) suggested that mangrove vegetation be
considered as a formation type on a world-wide basis. They recognized two
subformations: (1) the New World subformation, including western Africa,
and (2) the Old World subformation. Chapman (1944a) recommended that a
third subformation, the Australasian, be recognized because the species of
Avicennia are very distinct in their distribution and segregate into these three
geographical regions.
Davis (1940) said that the mangrove formation is composed of serai
communities. Although reef and sand communities appear to be climax
stages, the statement of Davis is generally true. The order of zonation varies
considerably-even in-geographically-related areasvPor example-, in the "Old
World subformation, Rhizophora is the pioneer species along river banks and
in the more protected regions along oceanic shores, whereas Avicennia or
Sonneratia pioneer on shores of greater wave and tidal action. In Jamaica, R.
mangle pioneers along protected shores, while Laguncularia racemosa
pioneers on sand spits where wave action is greater (Chapman 1944a).
Chapman (1940) pointed out that the presence of sea grass in submerged
areas accelerates the seaward extention of mangrove because it raises the
height of the sea bed, allowing R. mangle seedlings to grow.
The serai nature of mangrove vegetation in the Indo-West-Pacific region
was described in detail by Macnae (1968), who recognized succession in every
mangal he visited. Macnae stated that variation in development was often
found, succession being complete only where the amount of available fresh
water exceeded that lost through evaporation and transpiration. When losses
through evaporation and transpiration exceed income from rain and rivers,
73
-------
GERALD E. WALSH
the soil becomes hypersaline and zonation is interrupted.
Macnae described the effects of fresh-water imbalance in a zone ofCeriops
tagal (Perri) C. B. Rob. This zone was located between a seaward fringe of
Bruguiera gymnorhiza and a landward fringe of A. marina. Where fresh-water
loss exceeded gain, C. tagal became stunted. With increasing excess of
evaporation, the Ceriops bushes died, forming a bare area which expanded
both landward and seaward until only a few bushes grew near the Avicennia
and Bruguiera fringes.
In Florida, Davis (1940b) was the first investigator to give a detailed
account of succession in a mangal. He recognized seven principal
communities: (1) The pioneer Rhizophora mangle zone. This seaward stage
was composed of mangrove seedlings of various age growing in marl soil
below the level of low tide in shallow undisturbed water. Thalassia
testudinum Koenig and Sims and Cymadocea manatorum Aschers grew in
shoal areas near this zone, and Spartina alterniflora Loisel was present in
some parts of Florida. (2) Mature Rhizophora consocies. This stage was
composed of mature R. mangle with well-developed prop roots growing in
mangrove peat. (3) /Iwcermia-salt marsh consocies. This stage was composed
of the tree A. nitida and the salt marsh plants Satis maritima L., Salicornia
perennis Mill, Spartina alterniflora, S. spartinae (Trin.) Merv, Monanthochloe
littoralis Englem., and Sporobolus virginicus (L.) Kunth. This mangrove salt
marsh consocies grew on peaty soil and accumulated large amounts of organic
and inorganic detritus. During dry periods, soil salinity was very high, whereas
salinity was very low during rainy periods. In some places, Avicennia was
more than 30 cm in diameter, but in other places was a small gnarled bush.
(4) Mature mangrove associes. This stage consisted of large trees of R. mangle
and A. nitida growing together on peat soils in water of low salinity at
approximately the mean high tide mark. (5) Laguncularia racemosa consocies.
This stage did not occupy a specific habitat, but was found with both the
mature mangrove associes or between the Avicennia-sdt marsh associes and a
Conocarpus associes when the natural mangrove associes was not present. (6)
Conocarpus erectus transition associes. This was the final stage of mangrove
succession in Florida, as it was bordered on its landward edge by sand dunes,
upland tropical forest, or fresh-water marsh. Davis considered the Conocarpus
associes to be an ecotone, not a definite serai community. This was disputed
by Chapman (1944a) who reviewed the work of Borgeson (1909) and
concluded that Conocarpus formed a true serai stage. Chapman later (1970)
considered the Conocarpus community to be an ecotone between saline and
fresh-water communities. West (1956) described the final stage in serai
succession in Colombia as dominated by C. erectus in the drier and less saline
areas. (7) Dwarf-form mangroves. Davis recognized a scrub-mangrove facies of
dwarfed Rhizophora, Avicennia, and Laguncularia, which grew above the high
tide mark in fresh water. This dwarfed form was common in the Everglades
region.
74
-------
ECOLOGY OF HALOPHYTES
Thome (1954) listed many other plants in the mangals of Florida.
Holdridge (1940) gave an extensive description of the vegetative characters
and general characteristics of R. mangle, Langucularia racemosa, C. erectus,
and A. nitida, and reported Petrocarpus officinale Jacq., Anona glabra L.,
Bucida buceras L., and Drepanocarpus lunatus G.F.W. Meyer living in the
mangals of Puerto Rico.
Chapman (1944a) compared succession in the swamps of Jamaica with
that in Florida (Figs. 9 and 10). In both cases, Rhizophora was the pioneer
form, with Avicennia, Laguncularia, and Conocarpus inland. Asprey and
Robbins (1953) stated that there were few associates with mangrove in
Jamaica, a pattern similar to mangals in other parts of the world. Batis
maritima, Salicornia ambigua Michx., Acrostichium aureum L.,Alternanthera
ficoides, and S. virginicus occurred in the swamps of Jamaica. In other parts
of the world, other genenf and species occupy similar positions. For example,
Taylor (1959) described the mangals of Papua, New Guinea, as follows:
PROTECTED
Bays and cays
Thalassia-Cymodocea associes
Avicennia consocies
(sand, mud, or peat)
Laguncularia consocies
(sand, mud, or peat)
EXPOSED
Bays
Thalassia-Cymodocea associes
Laguncularia consocies
(consociation on cays)
Rhizophora locies
iools and wet depressions)
Dune-Strand vegetation
Reed-swamp consocies
(Typha domingensis)
Figure 9. Succession in the mangrove swamps of Jamaica (Chapman 1944a).
75
-------
GERALD E. WALSH
Open water
Beach associes
Dune associe
Marine 'grasses'
Rhlzophora family
V
Mature Rhizophora consocies
Avicennia, salt-marshVassocles
Scrub mangrove
fades
transition associes Marl prairie
associes
Rhizophora, brackish
marsh lociee
Rhizophora. fresh
marsh locies
4-
Fresh-water marsh
aasocies
Plneland, adaphlc
subcllntax association
Tropical Forest
Figure 10. Succession in the mangrove swamps of Florida (Chapman 1944a).
1. Salt water swamps
a. Tidal mangrove sequence
b. Mangrove marsh sequence
2. Brackish water swamps
a. Brackish swamp sequence
b. Estuarine sequence.
In the tidal mangrove sequence, each succeeding community occurred at
sites with successively longer periods of tidal inundation. The shore pioneer
species was C. tagal, followed by a broad zone of R. mucronata and B.
gymnorhiza. The final stage was dominated by Heritiera littoralis. The
boundary between the H. littoralis zone and the rain forest was dominated by
Intsia bijuga (Colebr.) O. Kunz. and was very sharp. All of the mangrove
species occurred as scattered individuals in all of the communities, although
the dominant species comprised over 50% of the number of species, and there
were sharp transitions between the zones.
In the mangrove marsh sequence, there was a fringing area of A. alba trees
up to 12m high, which changed gradually to a thicket of the same species up
to 6 m high. The thicket was bordered by swampy soil devoid of vegetation
and Taylor suggested that the sequence from tall trees to bare swampy
ground was regulated by soil salinity.
76
-------
ECOLOGY OF HALOPHYTES
Brackish water swamps occurred where mangrove forest was bordered
inland by fresh-water swamps. There were only two sharply-defined zones:
(1) a zone composed predominantly of A. alba and B. gymnorhiza, with a
dense ground cover of the fern Acrostichium speciosum Thunb.; and (2) a
zone dominated by Parinari corymbosum (Bl.) Mig. Hibiscus tiliaceus L. was
present in small numbers. Acrostichium speciosum was generally abundant in
this zone, but Acanthus ilicifolius L. sometimes made up 50% of the ground
cover.
The estuarine sequence was similar to the tidal mangrove sequence, except
the palm Nypa" fruticans Wurmb. dominated the zone which bordered the
fresh-water swamp.
In West Africa (Nigeria), Jackson (1964) recognized six groups of
mangrove on the dual basis of range of habitat and dispersal of seeds.
Group 1. Species restricted to the tidal areas and with specialized seed
habits. This group included R. racemosa, the pioneer species at the water's
edge and on the.storm beach, and A. nitida dominant along the inner edges of
closed lagoons. Boughey (1957) found Rhizophora only in open lagoons and
Avicennia only in closed lagoons in West Africa. Bews (1912) reported B.
gymnorhiza from lagoons in Natal. In the case of Rhizophora species, Keay
(1953) considered R. racemosa to be the pioneer species, with R. mangle
following on drier ground and R. harrisonii on wet ground. These were
followed inland by A. nitida, Laguncularia racemosa, and C. erectus. Gledhill
(1963) pointed out that the propagules of R. racemosa are between 30 and
65 cm long and, by virtue of their length, are suited for establishment in
flooded mud. The seedlings of R. mangle are approximately 20 cm long and
those of R. harrisonii 30 cm long. Gledhill felt these were adapted for
establishment on less heavily silted soil under more vigorous water current
conditions.
Group II. Species found normally in tidal areas and with specialized fruits
but normal seeds, or with buoyant seeds. Genera in this group were
Drepanocarpus, Dalbergia, Ormocarpum, and Hibiscus. The seeds of these
groups germinate in the water.
Group III. Species widely distributed along water courses and in swampy
areas, usually with unspecialized fruits. The genera here were Pterocarpus,
Cynometra, Lonchocarpus, Phyllanthus, and Phoenix. Seeds of the first four
are buoyant and are found floating in water. Seeds of Phoenix have never
been found in the drift.
Group IV. Species not restricted to water courses in the forest areas and
with no marked specializations. These species are associated with high or
well-distributed rainfall. The fruits and seeds are not associated witli dispersal
by water. This group included Anthocleista, Elaies, Conibretum, Alchornea,
and Paullinia.
77
-------
GERALD E. WALSH
Group V. Cultivated plants. These are plants which occur in strand
vegetation and have floating fruits which are dispersed by water. Genera
include Cocos, Terminalia, and Anacardium.
Group VI. Species whose seeds and seedlings are found in the swamp but
with few or no mature individuals present. These include Lonchocarpus,
Halomosia, Spondias, Cleistopholis, Dioclea, and Entada.
It is clear that mangrove swamps are not the simple communities some
writers thought them to be. Macnae (1966) described in detail a complicated
succession in the swamps of Queensland, Australia. There, the pioneer tree
was A. marina where there was a large amount of fresh-water inflow, or
Sonneratia alba where the influence of saline water was strong. The Avicennia
zone was composed of a row of mature trees, two or three trees deep, with
thickets of seedlings and saplings extending out onto a beach. Often, where
the influence of fresh water was strong, Aegiceras corniculatum Blanco
occurred in large numbers in the seedling and sapling thickets. On the other
hand, where S. alba fringed the shore, the pioneer belt was well-developed.
The alga Catenella nipa Zanard colonized the pneumatophores of both
Avicennia and Sonneratia. Macnae found no other algae there. This was
exceptional as large numbers of algae are present on the pneumatophores of
both genera in southern Australia and eastern Africa. The substrata of
shoreline fringes were considerably firmer than either the foreshore in front
or the Rhizophora forest behind because both/tw'cermw and Sonneratia have
a mass of intertwining absorptive roots which lie 20 to 40 cm below the
surface.
Behind the ocean fringe, there occurred an ocean shore sub-fringe
composed mainly of 7?. stylosa and occasionally of R. mucronata.
Rhizophora formed the fringing zone along creeks. Ding Hou (1958) and van
Steenis (1962) held that R. stylosa was found only on sandy shores and coral
terraces. Macnae stated that both R. styloas and R. mucronata grew in mud,
sand, and on coral debris, and speculated that the two forms are actually
variants of a single species. Whatever the taxonomic position may be, it is
clear that in contrast to the New World genera the southeastern-asiatic and
eastern Africa forms of Avicennia are better adapted for pioneering than
Rhizophorous forms (see Watson 1928; Macnae 1963, 1968).
In contrast to the pioneer fringe, the substratum in the well-developed
Rhizophora forest was always very soft and muddy due to entrapment of
sediments between the prop roots.
Landward of the Rhizophora forest lay broad areas of either (1) thickets
dominated by C. tagal, where the amount of rainfall was intermediate, or (2)
forests dominated by Bruguiera, where the amount of rainfall was large. In
the thickets, C. tagal was ordinarily the only species present, but Bruguiera
exaristata Ding Hou was sometimes subdominant. Occasionally, A. marina, B.
gymnorhiza, R. apiculata, R. stylosa, Xylocarpus granatum Konnig, and X.
78
-------
ECOLOGY OF HALOPHYTES
australasicum Ridley were present. In areas of much rainfall, the Ceriops
thickets were narrow and bordered by very dense forests dominated by either
B. parviflora or B. gymnorhiza. In B. gymnorhiza forests, scattered specimens
of X. australasicum occurred and the fern A. speciosum grew between the
trees. The Bruguiera forests were the tallest of the Australian mangrove.
Height of the trees appeared to depend upon the amount of fresh water
available, with tallest trees in areas of highest rainfall.
The landward fringe of the Queensland mangals was the most diverse of all
serai stages. Avicennia marina was the most abundant tree, but B. exaristata
Ding Hou was common, and C. tagal, C. decandra, Lumnitizera agallocha, L.
littorea, R. apiculata, and Exocoecaria agallocha L. were present. Xylocarpus
granatum and X. australasicum were present occassionally. Where the
landward fringe bordered a rain forest, many of the forest epiphytes grew on
the mangrove trees. These included the orchids Dischidia nummularia R. Br.
and Dendrobium sp., the ant plant Myrmecodia (beccari Hook.?), and the
ferns Drynaria rigidula Bedd., Platycerium sp., Polypodium acrostichoides
Forst., and P. quercifolium L.
The above description by Macnae of serai succession in a mangal is, in
general, typical of the large swamps of the Indo-Pacific region. This author
(1966, 1968) described characteristics of large mangals in detail, and
concluded that zonation of mangrove trees was due to the interaction of (1)
frequency of tidal flooding, (2) salinity of the soil water, and (3) water
logging Of the soil. All three are modified by the presence of creeks, gullies,
channels, and rivers. The second and third depend upon rainfall and/or the
supply of fresh water, evaporation, transpiration, and the nature and quality
of the soil. Chapman and Trevarthen (1953) stated that on muddy or sandy
shores, distribution of organisms is related to the nature of the substratum
which controls drainage, aeration, and penetrability.
The possible role of tidal flooding in relation to succession of Jamaican
mangroves was shown by Chapman (1944b), who related vegetation types to
the number of tidal submergences per year. His data showed decreasing tidal
influence between the seaward R. mangle stand and the landward C. erectus
stand (Table 4).
Tidal flooding alone does not determine species composition, zonation, or
succession in mangals. Clarke and Hannon (1967, 1969, 1970, 1971) studied
the physical habitat of mangroves in Australia in great detail. They concluded
that soil did not play a major role in control of plant distributional patterns
and that plant reaction on the soil did not regulate serai change. Microclimate
was important in providing conditions necessary for seedling development,
determining soil characteristics, and influencing competition between species.
Clark and Hannon showed that the holocoenotic complex (Fig. 11) was
intricate and that variation in any of the components affected species
distribution. The main factors were degree of tidal flooding, elevation of the
land, and salinity of the soil water. Plant zonation was associated closely with
elevation above mean sea level, seasonal patterns of soil salinity, and small
79
-------
GERALD E. WALSH
Table 4. Number of tidal inundations in a mangrove swamp in Jamaica (Chapman,
1944b).
Vegetation Inundations per year
Rhizophora swamp 700+
Rhizophora/Avicennia boundary 524
Avicennia swamp 432
Aeicennia/Laguncularia boundary 213
Center of salina 150
Laguncularia/Conocarpus boundary 4
differences in microtopography. Also, light and water-logging of the soil
were important to distribution, and a comparison of environmental
requirements of coastal halophytes was made (Fig. 12). The authors stated
that the sharpness of zonation depended upon the intensity of species
interaction at ecotones. Slight environmental changes related to topography
produced intense competition which made significant factors that were
normally of secondary importance. Generally, the severity of the
environment, including covering by mud and tidal scouring, determined the
success of a species in advancing seaward, whereas landward extension was
governed by ability to compete with other species in relation to salinity,
availability of fresh water, temperature, light, and humidity.
Macnae (1966) criticized the Watson (1928) scheme of classification based
on frequency of tidal flooding (described above) because it applied only to
ever-wet forests in Malaya. Instead, Macnae proposed that zonation be based
on the dominant tree. Dansereau (1947), in a phytosociological study of
mangrove in Brazil, described three natural associations: Rhizophoretum
manglei, Avicennietum tomentosae, and Laguncularietum racemosa.
Cuatresasas (1958) described the mangrove associations of South America as
Rhizophoretum brevistylae, Rhizophoretum mangleae, Brugieretum
gymnorrhizae, Sonneratietum albae, and Avicennietum nitidae. Schnell
(1952) described five "edaphic" associations in West Africa: Rhizophoretum
racemosae, Avicennietum nitidae, Drepanocarpeta-Rhizophoretum,
Ecastophylletum (Dalbergiaetum) brownei, and Cyperteum articulati.
Chapman (1970), in a very important paper on mangrove phytosociology,
compared succession in mangals throughout the tropics and gave eight
schemata that depicted zonation (Figs. 13-21). Chapman concluded that
there is great similarity in the vegetational communities and suggested an
extensive classification of natural associations according to the
Braun-Blanquet system. The classification consisted of 8 alliances, 15 orders,
and 40 associations, but must be considered tenuous at this time because of
lack of taxonomic and systematic data from many localities.
80
-------
Slope of land
Proximity to water source
Drainage and aeration
t
Biotic factor
Genotype
Tital inundation <
4-
Elevation above mean sea level
wind -
Salinity of soil water
t \\\
I \ \ B»lnf«ll
* \\
:ent and nature \ Evaporati<
plant cover \^
L^^«^\
^^* Nature of
Insolation _^_
1 Height of water table
Figure 11. The holocoenotic complex in mangrove swamps and salt marshes of the Sydney
District, Australia (Clarke and Hannon 1969).
Positive salt requirement
Mangroves
Small, if any, salt requirement
High light requirement
High salt Intermediate Low salt
tolerance salt tolerance tolerance
Requirement Indifferent to Intolerant of
for waterlogging waterlogging
waterlogging
Arthrocnemum Suaeda Triglochin Sporobolua, Jimcus
1 1
Seedlings tolerant Intolerant of
of 1
To 1 era
waterl
ow light low light
nt of Tol
ogging o
erant Intole
f waterlogging
rant
Figure 12. Environmental requirements of coastal halophytes of the Sydney District,
Australia (Clarke and Hannon 1971).
81
-------
Horth
Spartina j
Dlstichlis
~TJ
Avicennia nitida
Salt Marsh
Central
Avicennia nitida
Lasuncularia racemoaa
i
Salt Marsh
South
Rhizophora mangle
Rhi zophora
Avicennia ^ ^^
~T^
Avicennia nitida I
^ \
Laguncu lar ia I
^ \
Conocarpus A
^v
Rhizophofa-
Sawgrase
Figure 13. Succession in the mangrove swamps of the Gulf of Mexico and the Caribbean
Sea (Chapman 1970, Schema 1).
N. BRAZIL
Rhizophora racemosa
i
Avicennia nitida
Avicennia schaueriana
Lagunucularia
racemnsa
S. BRAZIL
Spartina
brasillensls
I
Rhizophora mangle
i
Avicennia nitida
Laguncularia
COLOMBIA .
(hard clay)
Pelliciera
rhizophorae
\
Avicennia
Lagunculai
ECUADOR
(mud)
Rhizophora
harrisonii
/;
nitida **
ria
S*
Rhizophora
mangle
/^
Conocarpue
Acres tic hum
(brackish)
Figure 14. Succession in the mangrove swamps of South America (Chapman 1970, Schema 2).
82
-------
Rhizophora racemosa
R. harrlsonli
R. harrjgonli
Figure IS. Succession in the mangrove swamps of West Africa (.Chapman 1970, Schema 3).
CENTRAL
SOUTH
UNHACA)
North
Avicennia marina
Arthrocnemum
(sand)
Avicennia marina
(seaward)
(mud)
Soimerati.
alba
South
Avicennia marina
Rhi zophora mucronata
Cerlops taga1
Bruguiera gyronorhiza
(rivers)
Rhizophora mucronata
I
Xylocarpus obovatus ^, Cerlopa tagal
Heritiera littoralia .
+ Acros tichum ^
Ayicgnnja. marina
(landward)
(Coast)
Avicennia marina ,
(Rivers)
RhizopJTpra roucronata
•estops tagal \
iru^uiera gymnorhiza \
Figure 16. Succession in the mangrove swamps of East Africa (Chapman 1970, Schema 4).
83
-------
BOMBAY GODAVAM
Avicennia _ ^^^^^ Sonneratia * Hyrroatachya
alba <
+ A. officinalis
Acanthus iltcifoltus
CEYLON
Rhizophora
+ R. apiculata
Bruguieri
Ceriops
Avlcennia
Excoecaria Mixed
(felling) Mangrove
|«"
^
Hyg fruttcajis
Acroatichum
aureum
Acanthua
ilicifolius
Figure 17. Succession in the mangrove swamps of India (Chapman 1970, Schema 5).
(Sand)
Ceriopa
Her 1 tier a minor
Mixed mangro
Rhizophora micronata
+ Rhizophora apiculata
Figure 18. Succession in the mangrove swamps of India (Chapman 1970, Schema 6).
84
-------
(Silt)
Open Coast
(Sand)
(Creeka, Bays, Lagoons)
Rhlzophora mucronata
thora apiculata
(brackish)
I
Sonneratia
caseolaris
Hypa fruttcans
Herltlera Uttoralia
Figure 19. Succession in the mangrove swamps of Malaysia, Indonesia, and Borneo (Chapman
1970, Schema 7).
S. W. & N. Key Zeal.
+ H. & N. East
Rhlzophora
mucronata
1
Avicennia
marina
Ar throcnemmn
Sonneratia
al
+ Camp
ba
ostemon
schultzli
1
/
/
/
i /
Avicennia
marina
;+ Aeglceraa
~
(Creeks)
ilzophora stylosa
ilzophora apiculata
t
Rhlzophora s tylosa /
Rhlzophora apiculata /
J
\
/
Brugulera gymnorhlza
Bruguiera parvi flora
+ Xylo carpus
^^^ australasicum
*T
Excoecaria
Xylocarpus
(felling) /
Lumnltzera
Cerlops (wet)
Aeglceras ^_
Avicennia
marina
Avicennia salt marsh
(arid)
Acrostlchum speciosum
Figure 20. Succession in the mangrove swamps of Australia (Chapman 1970, Schema 8)
85
-------
PAPUA/HEW GUINEA
(aand)
(estuaries)
Rhi zophora
—
i
"
f iz
Heritiera litoralis
Bruguiera
1 ^
Avicennia -^x^*^
Ceriops *
Aegiceras
^ corniculatum
Rhi zophora mucronata
* BruRuiera j^vmnorhi za
/
Lumni t zera
^^ raceme sti
Avlcennia SeSuvium
Excoecaria i
^^ "*¥
Excoecarja
Hibiscus
tiliaceus
1
Excoecaria
Melaleuca
Figure 21. Succession in the mangrove swamps of Papua, New Guinea, the Philippines, and
Oceania (Chapman 1970, Schema 9, part 1).
Ryukyu IB_.
Kandclla candel
(mud)
(sand)
Sonneratia alba
Aviccnnia marina
(+ Camp tostemon
"" philippincn^is)
(Viet Nam)
FIJI & TOHGA
Bruguiera gymnorhiza
Heritera litoralis
Figure 21. (Continued). Succession in the mangrove swamps of Papua, New Guinea, the
Philippines, and Oceania (Chapman 1970, Schema 9, part 2).
86
-------
ECOLOGY OF HALOPHYTES
ADAPTATIONS
Warming (1883) stated that mangroves have adapted to their environment
through (1) mechanical fixation in loose soil, (2) respiratory roots and
aerating devices, (3) viviparity, (4) specialized means of dispersal, and (5)
development of xerophytic structures in relation to soil salinity. Walter
(1931a, b; 1936a, b) and Walter and Steiner (1934) concluded from studies in
East Africa that zonation was related to the capacity of mangroves to
compete and survive in saline soils. Thus, they distinguished zones of
Rhizophora, Avicennia, and Sonneratia and stated that Rhizophora and
Avicennia bore great fluctuations in soil salinity, whereas Sonneratia required
a constant chloride content. It is clear, however, that zonation depends also
upon morphological and physiological adaptations. Wenzel (1925) gave
detailed descriptions of the anatomy of R. mucronata, R. mangle, B.
gymnorhiza, C. candolleana, A. officinalis, and X. granatum. Chapman
(1944c) described functional morphology of A. nitida in detail and presented
data on physiology of the pneumatophores. The gross morphology of most
species is described in many manuals of tropical trees.
Anatomical
Marco (1935) described the anatomy of the woods of rhizophoraceous
species from both mangals and upland forests of the Indo-West-Pacific region.
He divided the family into three groups and stated that the mangrove genera
formed a well-defined, natural aggregation that was readily separable from all
other members of the family. He suggested that Rhizophora, Bruguiera,
Ceriops, and Kandelia be placed in an independent family. Marco placed the
four genera in the anatomical division Rhizophoreae, characterized by (1)
heavily barred, exclusively scalariform perforation plates, (2) characteristic
scalariform intefvascular pitting, (3) little vasicentric parenchyma, (4)
numerous fine-celled multiseriate rays and very few uniseriate rays, (5)
libriform fibers with inconspicuous pits, and unilaterally and bilaterally
compound pitting between rays and vessels. These features segregated the
four genera from all other groups of the Rhizophoraceae, but their
significance as adaptive features has not been determined.
Reinders-Rouwentak (1953) stated that, in the Sonneratiaceae, the mature
wood of species from more saline environments contained a larger number of
smaller vessels than species from less saline areas. For example, S. griffithii
from the seashore of Bengal had 34-50 vessels/mm^ and the diameter range
was 85-100 u. Sonneratia apetala from the river had 18-32 vessels/mm^ with
diameter range of 135-150 u. Heiden (1893) gave a detailed account of
anatomy of the Combretaceae, including the genera Conocarpus,
Lumnitizera, and Laguncularia.
87
-------
GERALD E. WALSH
Macnae (1968) reviewed adaptations of mangroves with regard to growth
in ill-consolidated mud, specializations of stems and leaves, relationships
between root and shoot systems, and vivipary. Robyns (1971) considered
mangroves to be the only truly viviparous plants. He defined vivipary as the
process in which the seed remains attached in the fruit to the mother tree,
germinates into a protruding embryo with a long hypocotyl, and finally falls
from the tree. Genkel' (1962) speculated that mangroves evolved in the
ancient tropical forest from xerotic plants in which the seeds had no dormant
period and loss of fruit from the tree was delayed by chloride in the soil
water.
The roots of mangroves are not deep and tap roots are not present. For
descriptions of mangrove root systems see Goebel (1886), Schimper (1891),
Troll (1930), Troll and Dragendorff (1931), Uphoff (1941), and Macnae
(1968). Zieman (in press) found that height of R. mangle was related to root
length. In Rhizophora, the primary roots of the hypocotyl function for only
a short period of time and root functions are assumed by secondary roots
which extend from the main trunk. The cause of cessation of growth of
primary roots is not known, but Warming (1877), Johow (1884), and
Schimper (1891) suggested that they are injured mechanically by crabs and
snails. There are two kinds of roots in Rhizophora: (1) aerial roots that arise
from the main trunk and form arched stilts which penetrate the ground (prop
roots) and (2) subterranean roots that arise from the prop roots. Aerial roots
also arise adventitiously from the lower branches of trees. The prop and aerial
roots function in aeration and ventilation of the tree in general and of the
subterranean roots in particular. Most mangroves have schizogenous lacunae
in the cortex of the roots. The main function of the subterranean roots is
absorption of water and nutrients.
The anatomy of aerial roots has been described by Warming (1883),
Schenck (1889), Karsten (1891), Leibau (1914), Bowman (1917), Mullan
(1931, 1932, 1933), and Gill and Tomlinson (1971). According to Gill and
jrprrJjnsQnJ.aerial_rQots_first ap.pear.Qn theJiypocatyLor lower intejnades.af.
seedlings after 1 to 3 first-order branches have been produced. Later, they
arise on higher internodes and lower branches. Aerial roots also develop on
the high branches of mature trees. In general, the aerial roots originate on the
shoot in acropetal sequence. Gill and Tomlinson (1971) gave a detailed
account of root growth and anatomy.
When aerial roots reach and penetrate the ground, they undergo marked
changes which relate to subterranean function. According to Bowman (1917),
the absorptive subterranean roots are thick, spongy, and gas-filled due to'
great development of the primary cortex. The primary cortex of absorptive
roots is composed of large cells and very large intercellular spaces in which
idioblasts, trichoblasts, and root hairs are lacking (Bowman 1921). The
periderm of the absorptive root consists only of cork cells, whereas that of
the aerial root consists of both cork and "parenchymatic" tissue (Bowman
1921). Bowman also reported stone cells and idioblasts in all parts of R.
88
-------
ECOLOGY OF HALOPHYTES
mangle except the flower. These were trequently associated with tannin cells.
Sclerenchymatous tissue occupies a large portion of the stem and hypocotyl
of mangrove and makes anatomical study very difficult.
Two other rhizophoraceous genera, Bruguiera and Ceriops, do not have
aerial roots. Instead, they have subterranean cable roots which differentiate
into knee roots that penetrate the soil surface, and absorptive roots (Marco
1935).
Troll and Dragendorff (1931) gave an extensive account of the cable root
system of Sonneratia, and similar roots systems are present in some species of
Avicennia, Lumnitizera racemosa, X. australasicum, and X. moluccensis
Roem. For an extensive study of anatomy of respiratory roots of mangroves,
see Ernould( 1921).
Chapman (1944c) showed that the composition of gas in the roots of A.
nitida was similar to air and that there was no fundamental difference
between composition of gas in the pneumatophores and in the horizontal
roots. He stated that the large cortical air spaces allowed longitudinal gas flow
between organs. Scholander et al. (1955) studied respiratory gas exchange in
the roots of A. nitida and R. mangle. The radial roots of A. nitida send
numerous pneumatophores up to 30 cm above the ground. There is a direct
gas connection between the radial roots and the pneumatophores. When the
tide covered the pneumatophores, there was a decrease in the oxygen content
of the whole root system. At low tide, oxygen comprised between 15 and
18% of the gas content. At high tide, oxygen content was about 7%. At high
tide, the oxygen content dropped until the pneumatophores were again
exposed to air at low tide. There was little change in carbon dioxide content
of the roots over the tidal cycle.
Gas in the subterranean roots oof R. mangle contained 15 to 18% oxygen
and there was always a direct gas connection between these roots and
lenticels on the prop roots. The high oxygen tensions in the roots were
maintained by means of ventilation through the lenticels on the prop roots
(Scholander 1955).
Macnae (1968) gave diagrams of the cross sections of leaves of Rhizophora,
Avicennia, and Sonneratia. Schimper (1891, 1898) showed that the leaves of
most mangroves contain water storage tissue. This is initially in the form of a
hypodermis in Rhizophora and Avicennia and a centrally located layer of
cells in Sonneratia. Stace (1966) made a detailed study of leaf anatomy of
seven genera (Tables 5 and 6) and also the epidermal characteristics of
Bruguiera spp. and Avicennia spp. He concluded that the leaf and epidermal
characteristics of mangroves are similar to most xeromorphs. All species had
common epidermal features, notably a thick cuticular membrane, straight
epidermal cell walls, and the presence of water-storage tissue, hydathodes,
cork warts, and water stomata. See Artz (1936) for descriptions of the
cuticula of S.. alba, C. candolleana, R. mucronata, B. gymnorhiza,
Lumnitizera racemosa, X. obovatus, and A. officinalis. In the study of Stace,
almost all of the species studied had sunken stomata or stomata surrounded
89
-------
Table 5. Characteristics of the leaves
(Stace 1966).
of rhlzophoraceous mangroves
Venous system on
upper epidermis
Venous system on
lower epidermis
Rhizophora
Midrib only, very
broad and conspic-
uous; cells broader
than long
Midrib only, very
broad and conspic-
uous, or lateral
veins also discer-
Ceriops
Midrib only, nar-
row and inconspic-
uous; cells broader
than long
Midrib only, very
broad and conspic-
uous
Bruguiera and
Kandelia
Midrib only,
narrow but
conspicuous ;
cells broad-
er than long
Midrib only,
very broad
and conspic-
uous
nxMe
Epidermal cells Straight- or curved-
of non-venuous walled, not second
areas divided, mostly ca.
11-25 u across
Stomata
Subsldary
cells
Sunken, ca. 30-55
x 20-35 u; outer
stomatal ledge con-
spicuous, single or
with minute second
lip
5-8, cyclocytic
Con't on next page
Straight- or cur-
ved-walled, not
second divided,
mostly ca. 15-35
u across
Sunken, ca. 36-46
x 22-34 u; outer sto-
matal ledge conspic-
uous, conspicuously
two-lipped
6-8, cyclocytic
Mostly straight-
or curved-walled,
not second divi-
ded, mostly ca.
15-40 u across
Sunken, ca. 30-
44 x 16-28 u;
outer stomatal
ledge conspicuous
in some spp. con-
spicuously two-
lipped
4-6(8), cyclocytic
90
-------
Table 5 con't
Water stomata,
hydathodes and
cork-warts
Hypodermis, in-
eluding extra epi
dermal layers
Mesophyll
Water-storage
tissue
Large conspicuous
cork-warts on lower
epidermis, sometimes
also on upper epi-
dermis; water-like
structures on both
epidermides
Upper three-to five-
layered, sparsely
chloroplasted; lower
usually absent
One to three layers
of palisade and ca.
eight to ten layers
of spongy below
upper hypodermis
Upper hypodermis ?
Cork-warts + absent;
frequent water-sto-
mata-like structures
on both epidermides
All apparently
absent
Upper two-layered,
sparsely chloro-
plasted; lower
usually absent
Usually one layer
of palisade and
ca. eight to ten
layers of spongy
below upper hypo-
dermis
Upper hypodermis ?
Upper and lower
one-layered,
densely chloro-
plasted
Usually one layer
of palisade and
ca. eight to ten
layers of spongy
below upper
hypodermis
Absent, or ?
sometimes in
spongy mesophyll
91
-------
Table 6. Characteristics of leaves of combretaceous mangroves (Stace 1966).
Lumnitzera
Laguncularia
Venous system Absent, or midrib Midrib only, broad
on upper epider- only very inconspi- and conspicuous to
mis cuous; cells longer very inconspicuous;
than broad cells longer than
broad
Venous system on Midrib only, broad Midrib only, broad
lower epidermis and conspicuous and conspicuous
Epidermal cells Straight-walled, not Straight- or slight-
of non-venous second divided, most- ly curved-walled,
areas ly ca. 25-40 u across many second divided,
mostly ca. 15-30 p
Margin
Stomata
Of several regular Of small cells with
rows of rectangu- rounded lumina not
lar cells with arranged in rows
angular lumina
Sunken or not, not Scarcely sunken, not
protected by hairs, protected by hairs,
always more frequent usually more fre-
on upper epidermis, quent on upper epi-
absent only from dermis, absent pnly
margins, randomly from margins, orien-
Con't on next page
Conocarpus
At least midrib
and lateral
veins distinct;
cells longer than
broad
Midrib, lateral,
secondary and
lesser veins
distinct
Mostly curved
or straight-
walled, not se-
cond divided, most-
ly ca. 15-35 u
across
Of several regular
rows of rectangu-
lar cells with
angular lumina
Not sunken, pro-
tected by dense
hairs or not,
usually slightly
more frequent on
lower epidermis,
92
-------
Table 6 con't
oriented, ca. 24- tated at right
32 x 19.5-24.5 u.; angles to midrib on
outer stomatal ledge upper epidermis, ca.
conspicuous, single 25-35'x 20-26 u;
outer stomatal
ledge conspicuous,
single
Subsidiary cells (3)4-5(6), cyclo-
cytic
Trichomes
Water stomata,
hydathodes and
cork-warts
Domatia
(3-)4(-5), cyclo-
cytic
Compartmented hairs Compartmented hairs
only, often extreme- and apparently ses-
ly sparse to absent sile deeply sunken
on both epidermides glands on both
epidermides
Large water-stomata As in Lumnitzera
present on both but very sparse
epid.; hydathode-
like areas pre-
sent, mostly on mid-
rib of lower epidermis
and on margin
Shallow pits along Absent
margins may be
rudimentary domatia
Con't next page
absent from mar-
gin and lower
epidermal midrib,
randomly orienta-
ted, ca. 25-30 x
17-25 u; outer sto-
matal ledge fair-
ly conspicuous,
single
3-6, not differ-
enfei-afced
Compartmented
hairs and stalked
superficial glands
on both epidermides
Usually apparent-
ly absent, rare-
ly a few water-
stomata present
Large, primary-
axillary lebeti-
form domatia on
lower epidermis
93
-------
GERALD E.WALSH
Table 6 con't
Mesophyll Two layers of pali-
sade below each epi-
dermis; spongy absent
tissue layers of centrally
placed + isodiamet-
ric cells, not
chloroplas ted
Two layers of pali-
sade below upper
epidermis ; one to
two layers of
spongy, palisade
or mixed above
lower epidermis
lajers of centrally
placed + isodiamet-
ric cells, very
sparsely chloro-
plasted
One or two layers
of palisade below
upper epidermis,
one layer of pali'
sade above lower
epidermis;
spongy absent
layers of cen-
trally placed +
vertically elon-
gated cells,
sparsely chloro-
plasted
by dense trichomes. All genera, except Avicennia and Conocarpus, lacked
lateral and lesser epidermal veins, a condition associated with development of
water-storage tissue. Stack also gave a key to the genera based on epidermal
characters of the leaves.
Bowman (1921) observed that the water-storing hypodermis and
tannin-containing cells of R. mangle were much larger in trees that grew in
seawater than in trees from brackish water. Possible reasons for this will be
discussed under "Physiological" in this report.
Sidhu (1962, 1968) reported chromosome numbers of mangrove species
from India (Table 7). He concluded that most species from the mangrove
habitat possess higher chromosome numbers than other species of the same
genera from mesic habitats. However, among the mangroves, the size and
number of chromosomes did not show any correlation to habitat conditions.
Physiological
Most research on physiology of mangroves has stressed halophytic
adaptations. Halophytes are ordinarily distinguished from other plants by
their ability to grow in high concentrations of salt. They complete their entire
life cycles and compete successfully with other plants in saline environments.
Genkel' and Shakone (1946) classified halophytes as (1) Euhalophytes (salt
accumulating), (2) Crynohalophytes (salt excreting), (3) Glycohalophytes
94
-------
Table 7. Chromosome numbers (n) of mangroves and related species from India (Sidhu 1962, 1968).
Family and Species
Chromosome Number
Family and Species Chromosome Number
VC
Rhizophoraceae
R. mucronata 18
R. conlugata 18
13. parviflora 18
B_. gjymnorhiza 18
£. candolleana 18
C. roxburghiana 18
Sonneratiaceae
£. apetala 12
Duabanga sonneratloides 12
Myrsinaceae
Aegiceras corniculatum 24
Salvadoraceae
Salvadora persica 13
Acanthaceae
Acanthus illcifolius 24
Verbenaceae
Avicennla alba 16
Chenopodiaceae
Suaeda nudiflora 18
S_. monoica 9
S_. maritima 9
Euphorbiaceae
Exocoearia agallocha 65
Palmae
Sypa fruiticans 8
Sterculiaceae
Her!tiera littoralls 19
Meliaceae
Xylocarpus moluccensis 21
Xylocarpus gr ana turn 21
Fapilionaceae
Derrls uliginosa 10
Rubiaceae
Ixora parviflora 11
-------
GERALD E.WALSH
(salt impermeable), and (4) those in which salt is localized in special
structures. Depending on the species, mangroves may be placed in (1), (2), or
(4). It is doubtful that mangroves are intolerant or obligate halophytes,
although Stem and Voight (1959) and Connor (1969) have shown that R.
mangle and A. marina grow best when salt is present in the soil water. Some
mangroves (e.g. R. mangle, C. tagal, N. fruiticans) adapt to glycophytic
conditions and may be considered to be faculative halophytes. Mangroves
species have been reared in fresh water in the laboratory, and Stocker (1924,
1925) proposed the term "miohalophytes" for such plants.
Harbour (1970) suggested that ability to reproduce, rather than short-term
growth, should be the ultimate criterion of salt tolerance, but this has not
been studied with regard to mangroves. In the field, Bowman (1917), Davis
(1940), and Stern and Voight (1959) in Florida, and Pannier (1959) in
Venezuela reported that R* mangle grew and reproduced in fresh water, but
height of trees and area covered were greatest in brackish water.
In the laboratory, Winkler (1931) reported that Bruguiera eriopetala and
R. mangle grew and flowered in pots of sand watered only with fresh water.
Davis (1940) grew R. mangle in fresh water in the laboratory. Pannier (1959)
grew the same species in rain water and in salinities up to full strength
seawater. Although seedlings grew in the rain water, root growth was optimal
at 50% seawater and shoot growth was optimal in 25% seawater. Stern and
Voight (1959) reported that height, dry weight, and survival of R. mangal
increased with increasing salinity in the laboratory. They used artificial
seawater, and plant dry weight was approximately three times as great in the
highest salinity than in the lowest. Maximum growth occurred at salt
concentrations equivalent to seawater. Tatil (1964) grew R. mucronata, K.
candel, B. parviflora, C. tagal, A. ilicifolius, X. moluccensis, E. ,agallocha, and
Heritiera fames in salt concentrations between 0.3 and 1.2%. All species grew
at all salinities, but growth was best at 1.2%. Clarke and Hannon (1970)
found that A. marina at the 0-2 leaf stage maintained optimal growth in 20%
seawater. Concentrations above 60% seawater retarded growth. Seedlings at
the 2.4 leaf stage were more tolerant, and optimal growth occurred at 40%
seawater. Connor (1969) reported that the optimal concentration for
laboratory growth of A. marina from Australia was approximately 1.5%, or
half the concentration of seawater. Connor reported that suppression of
height by higher salt concentrations was more marked than suppression of
dry weight production. An important aspect of Connor's work was that while
growth appeared normal when sodium chloride was the main component of
the salt mixture, potassium chloride and calcium choloride suppressed growth.
Connor suggested that high concentrations of calcium caused nutrient
imbalance leading to iron deficiency and speculated that the responses of
mangroves to specific ions reflected the physiological ability of the plants to
adapt to concentrations in the root environment.
Temperature as a factor in seedling establishment of A. germinans was
shown by McMillan (1971). Exposure to temperatures of 39-40°C for 48
96
-------
ECOLOGY OF HALOPHYTES
hours was lethal to stemless seedlings, but not to seedlings with stems and
roots.
Bharucha and Navalkar (1942) reported the chloride content of leaf cell
sap of A. alba in relation to that of seawater and soisalinity (Table 9). They
concluded that seasonal variations in the chloride content of leaf cell sap were
dependent directly upon climatic conditions of temperature, rainfall, and
humidity. It will be shown later that such high sap concentrations are
common in mangroves that possess glands for salt excretion.
Table 9. Chloride content of seawater, soil water, and leaf cell sap of A. alba
(Bharucha and Navalkar 1942).
Percent Chloride
Seawater 0.77-3.24
Soil Water 0.55-3.47
Leaf cell sap 1.59-5.05
Blum (1941) reported osmotic pressures in the leaves of several mangrove
species from Java (Table 10). Avipennia had the highest osmotic pressure,
whereas Rhizophora, Bruguiera, 'and Sonneratia, genera which possess
mechanisms for salt exclusions and/or dilution, had relatively low osmotic
pressures.
Bole and Bharucha (1954) reported data on osmotic relationships in leaves
of A. alba (Table 11) and concluded that higher rates of transpiration brought
about higher accumulation of osmotically active substances in the older
leaves. The osmotic pressure did not vary directly with the water content, but
older leaves always had higher water contents and higher osmotic pressures
than younger leaves.
Chapman (1968) stressed that research on saline vegetation must
emphasize the roles of different ions upon plant metabolism. He stated that
the interrelations of sodium and potassium are particularly important because
the amount of potassium absorbed is influenced greatly by the presence of
s'odium. There is evidence that temperature and light affect the responses of
halophytes to salinity (Tsopa 1939), but tolerance of plants depends mainly
upon the type of soil salinity (Cl~, 804", etc.), the species or variety of plant,
and the stage of plant development (Chapman 1966).
97
-------
GERALD E. WALSH
Table 10. Osmotic pressures in the leaves and soil of mangroves from Java
(Blum 1941).
Species Osmotic Pressure, Atmos.
Leaf
A. officinalis, High tide 45
A. officinalis, Low tide 40
Sonneratia acida, High tide 27
Sonneratia acida, Freshwater 20
Rhizophora conjugata, Seawater 31
Bruguiera gymnorhiza, Freshwater 25
Soil
23
18,
6
0.3
23
0.3
Diff.
22
22
21
20
8
25
Table 11. Osmotic relationships in young and old leaves of A, alba (Bole and
Bharucha 1954).
Leaf
Young Old
Osmotic pressure, atmos. 38.8-47.7 51.5-57.4
Water content, percent 69.5-72.9 SI.5-62.1
Total carbon, percent 37.1-40.8 37.9-42.2
Total nitrogen, percent 1.1- 1.4 1.0- 1.6
Water Ioss/m2/hr, grams 0.56-0.74 0.92-1.00
Jennings (1968) demonstrated positive correlations between the sodium
and water contents and the phosphorus and water contents of halophytes.
Potassium had no appreciable relationship to succulence. Jennings stated that
increased succulence produced by high light intensity, aridity, and sodium
ions was brought about by essentially the same mechanism. Jennings also
suggested three mechanisms used by halophytes to cope with toxic
concentrations of ions. The first was export of the ions from the shoots and
leaves. This could occur in either of two ways: (a) transportation of ions
through the phloem to the roots ahd extrusion back to the soil or (b)
extrusion through specialized glands in the leaves. The former has not been
reported for mangrove, but salt excretion through epidermal glands occurrs in
A. alba (Walter and Steiner 1936), A. nitida (Biebl and Kinzel 1965),
Aegiceras (Areschog 1902a, b; Schmidt 1940a), and Aegiatilis (Ruhland
1915), Mullan (1931a, b, c) reported salt-excreting glands on the petioles and
upper and lower epidermis of A. alba, A. ilicifolius, and A. corniculatum. The
glands were most numerous on plants from hypersaline areas. They were
absent from A. ilicifolius from fresh water.
98
-------
ECOLOGY OF HALOPHYTES
The xylem sap of salt-excreting species is composed of approximately
0.2-0.5% sodium chloride. This concentration exceeds that of non-secreting
mangrove species by 10 times, and that of ordinary land plants by about 100
times (Scholander et. al. 1962)
Scholander et. ql. (1962) also reported salt excretion by A marina, A.
corniculatum, and Aegiatilis annulata. In Aegitalis, secretions from salt glands
contained between 1.8 and 4.9% sodium chloride, with highest values during
the day. Diel variation in rate of secretion occurred in Aegiceras, the average
sodium chloride content of salt gland secretions being 2.9% during the day
and 0.9% at night. The sodium chloride content of the xylem sap of
Avicennia was very high, ranging from 4 to 8 mg/ml; one sample of gland
secretion contained 4.1% sodium chloride. Jennings (1968) reported that the
secretion process selects sodium over potassium, the Na/K ratio being 13 in
the exudate, but only 3 in the leaves of Aegialitis. Atkinson et. al. (1967)
reported that in Aegiatilis, input of chloride to a mature leaf was
approximately 100 u-equiv./day and this was balanced by secretion, mainly
of sodium chloride, from the salt glands. Secretions from the salt glands
contained 450 u-equiv/ml of chloride, 355 u-equiv/ml of sodium, and 27
u-equiv/ml of potassium. Rate of secretion varied between 93
p-equiv/cm^/sec during the day and 3 p-equiv/cm^/sec in darkness. Atkinson
et. al. suggested that because the water potential of the secretion is similar to
that of the leaf, the secretory process involves active transport of salt
and movement of water by local osmosis. Atkinson also presented light and
electron microscope studies of the salt glands.
Rains and Epstein (1967) studied preferential absorption of potassium by
leaves of A. marina in the presence of high concentrations of sodium chloride.
They 'demonstrated that A. marina could (1) absorb and concentrate
potassium within its tissues in excess of the concentration in the substratum
and (2) preferentially select potassium when in the presence of high
concentrations of sodium, a closely-related ion. The ability to select
potassium over sodium is an extremely important adaptive character in the
marine environment.
Another significant adaptation is the ability to tolerate, without injury,
high internal concentrations of salt. Avicennia marina, unlike other
mangroves such as Rhizophora, Laguncularia, and Sonneratia, absorbs salt in
substantial amounts. The concentrations of ions in leaves examined by Rains
and Epstein (1967) were 30 mM potassium, 210 mM sodium, and 245 mM
chloride. The authors concluded that the effect of preferential absorption of
potassium was not to exclude sodium, but rather to raise the concentration
ratio of potassium to sodium from the value in seawater (1/40) to 1/7
within the tissue. The tissue did contain a high concentration of sodium
chloride (1.8 mM/g dry weight), but excretion by salt glands prevented higher
and possibly deleterious concentrations from developing.
Genkel' (1962) suggested that viviparous species of mangroves utilize
seedlings for exclusion of salt. He found that the chloride content of seedlings
99
-------
GERALD E. WALSH
increased in proportion to size and were adapted to high salt content in the
soil before dropping from the tree. This was shown to be true for R. mangle
by Lotschert (1968). Excess chloride in soils delayed loss of seedlings from
the tree and GenkeF concluded that vivipary is an adaptation to the salt
regime in tidal areas.
The second mechanism suggested by Jennings (1968) was limitation of
transfer of ions to the shoot by some mechanism located in the roots.
Scholander et. al (1966) showed that R. mucronata, Laguncularia racemosa,
and S. alba are efficient in salt exclusion. Atkinson et. al (1967) showed the
same for R. mucronata. Scholander et. al. (1966) stated that the
desalinization process in the root system produces a sap of fairly constant
concentration that is independent of rate of transpiration. Salt glands, when
present, eliminate salts left behind by transpiration.
Concentration of the soil solution, rainfall, tide, humidity, temperature,
transpiration, nature of the organisms, leaf age, water content, nitrogen
content, and carbon content have effects upon osmotic relationships of
mangroves (Blatter 1909; Cooper and Pasha 1935; Navalkar 1940, 1942,
1948; Bharucha and Navalkar 1942; Bole and Bharucha 1954). Gessner
(1967), however, found that water which passed from the stems to the leaves
of R. mangle was nearly salt-free.
Scholander et. al. (1965) reported that halophytes such as Rhizophora,
Osbornia, Salicornia, and Boris have strong negative sap pressures, ranging
from -35 to -60 atmospheres, whereas the osmotic potential of seawater is
approximately -25 atmospheres. The activity of water in the marine
environment is always higher than that of water in the roots, xylem sap, and
leaves. In R. mangle, Laguncularia racemosa, and C. erectus, the xylem sap
content of sodium chloride was only 1.2-1.5 mg/ml. At night, when
transpiration by Rhizophora and Osbornia was nil, sap tension was the same
as the osmotic potential of seawater but the solute pressure in the leaves was
10 to 20 atmospheres higher than seawater (Scholander 1971). In
Rhizophora, Laguncularia, and Conocarpus only about 50-70% of the
freezing point depression in leaf cells was produced by sodium and chloride
ions, most of the remaining solutes being organic (Scholander et. al 1966).
Benecke and Arnold (1931) demonstrated that osmotic pressure of S. alba
was lower under glycophytic than halophytic conditions. These pressure
differences give mangrove such as Rhizophora, Laguncularia, Sonneratia, and
Conocarpus, which do not possess salt-excreting organs, the ability to obtain
fresh water osmotically from seawater by transpiration and by diffusion at
the roots. Scholander (1968) concluded that Rhizophora, Sonneratia,
Avicennia, Osbornia, Bruguiera, Ceriops, Exocoecaria, Acrostichum,
Aegiceras, and Aegialitis separate fresh water from the sea by simple
nonmetabolic ultrafiltration of the seawater combined with ion transport.
The negative xylem pressure is produced by high salt concentration in the
cells, resulting in a solute pressure which exceeds that of seawater.
100
-------
ECOLOGY OF HALOPHYTES
The third mechanism proposed by Jennings (1968) for coping with toxic
concentrations of ions was production of increased succulence. High
concentrations of ions in the leaf may be prevented because of the dilution
effect brought about by increased water content of cells. Bowman (1921)
reported greater succulence in R. mangle from seawater than from fresh
water, a phenomenon which gives support to the third mechanism.
Reinders-Gouwentak (1953) found that succulent leaves were common in
Sonneratia and were due to the presence of a distinct hypodermal aqueous
tissue layer. In leaves that were immersed hi tidal water, the hypodermal layer
was three to five times as thick as leaves at higher levels of the same tree. The
same author stated that the hypodermal layer was almost absent in trees
grown in fresh water in botanical gardens. Reinders-Gouwentak believed that
succulence in Sonneratia was related to the chloride content of the water.
Jennings (1968) related succulence in halophytes to sodium metabolism.
He postulated that an outwardly-directed sodium pump exists in halophytes
and that this pump is related to cation-activated ATPase in the cell wall. This
same pump would drive potassium ions into the cell against an
electrochemical potential gradient. Jennings admitted that the evidence for
such a pump must be viewed with caution, but stated that there are no
reasonable arguments against its existence and suggested a relationship to
ATPases. In relation to succulence, Jennings proposed that sodium-activated
ATPases might be involved in the synthesis of new wall material or in
increasing cell wall extensibility. In a similar way, succulence is also induced
by increased amount of light which increases the rate of
photo-phosphorylation and production of ATP. Also, aridity causes
succulence because increased rate of transpiration causes the ration of
potassium to sodium in the shoot to change in favor of sodium. The
concentration of sodium in the xylem sap reaches such a level that the
sodium-activated ATPases in the plasmalemma bring about synthesis of ATP.
It should be stated that these proposals of Jennings are highly theoretical and
have yet to be tested.
Kylin and Gee '(1970) presented evidence that the leaves of A. nitida
possess ATPases that are dependent upon the ratio of sodium to potassium.
Enzyme activities were directly related to ionic strength of the growth
medium. Unlike animal systems in which synergistic effects of sodium and
potassium yield a peak at only one ratio, A. nitida yielded three peaks. At 50
mM total concentration (NaCl + KC1), activity peaks occurred at Na:K ratios
of 2:8, 5:5, and between 8:2 and 9:1. These results were interpreted as
indicating that either several enzymes functioned in the membrane system, or
else structural changes allowed more than one ion to activate a transport site.
Whatever the mechanism, the report of Kylin and Gee gives credence to the
hypothesis of Jennings that sodium-activated ATPases are present in
halophytes. Their role in succulence has yet to be established.
Salt exclusion, salt excretion, and succulence are not the only physiological
mechanisms whereby mangroves adapt to their saline environment. Metabolic
101
-------
GERALD E.WALSH
processes of photosynthesis, growth, and respiration are also important in
adaptation of mangrove, but little work has been reported. Chapman (1966)
pointed out that little is known about respiration and photosynthesis by
mature mangroves, but speculated that high concentration of salt in the soil
would cause slower rate of water uptake, slower rate of upward water
movement in the trunk, and slower transpirational loss as compared with
many other tropical trees. He suggested that the net result would contribute
toward a slow growth rate as compared with trees from mesophytic habitats.
Bharucha and Shirke (1947) stated that the respiratory activity of a
plant is influenced by food reserves. In the case of A. officinahs, the intensity
of respiration of seedlings increased from a minimal to a high rate and then
gradually declined. As the seedlings grew, there was an increase in water
content and fresh weight, but dry weight decreased, indicating that the
growing plant utilized reserve material for growth. Also, the authors showed
that in germinating seeds, respiration rate increased during the period of
absorption of water and gain in fresh weight.
Bharucha and Shirke also studied the respiration of seedlings of A.
officinalis from germination to the eighth day of growth in both air and
under seawater. Their data, some of which are given in Table 12, showed that
the rate of respiration increased with growth under both aerial and submerged
conditions. However, respiration rate was much slower under water and this
was ascribed to the limiting influence of oxygen in water. Chapman (1962b)
reported minimal respiration rates in "medium sized" seedlings of R. mangle.
He also reported that the cotyledonary body of R. mangle and the fruit wall
of A. marina (both structures function in transport of food from parent to
embryo) had very high respiration rates. On a dry weight basis, seedlings of
Avicennia had a higher respiration rate than those of Rhizophora. Chapman
suggested that this was related to differential development of aerenchyma.
Table 12. Respiration rates of A. officinalis in air and submerged in seawater
(after Bharucha and Shirke 1947).
Respiratory Index
Stage of Growth Air Submerged
1-Day seedlings 2.79 1.21
2-Day seedlings 2.85 1.48
3-Day seedlings 2.94 1.45
4-Day seedlings 3.09 1.46
5-Day seedlings 3.34 1.50
6-Day seedlings 3.67 1.51
7-Day seedlings 3.84 1.62
8-Day seedlings 3.92 1.66
102
-------
ECOLOGY OF HALOPHYTES
Bharucha and Shirke (1947) also reported increase in the respiration rate
of seedlings when the fruit wall was removed. Chapman (1962a, b; 1966b)
reported that the fruit wall caused a marked inhibition of respiration in A.
marina, A. nitida, R. mangle, R. apiculata, and B. gymnorhiza and speculated
that mangroves are capable of anaerobiosis, although the amount of energy
released under anaerobic conditions was inadequate for growth. Anaerobiosis
is apparently important during the periods when seedlings float in seawater
and no growth occurs. Brown et al. (1969) demonstrated anaerobic respiration
in A. marina, B. gymnorhiza, and R. apiculata.
Lotschert (1968) showed that chloride accumulates in the seedlings of R.
mangle before they fall from the tree. Conversely, Chapman (1944c) reported
that a salt exclusion mechanism operated in A. nitida. He stated that when
the seedlings fell from the tree, there was an immediate uptake of salt and a
sudden reduction in respiration rate. Successful colonization of Avicennia was
related, therefore, to the capacity of seedlings to respond to sudden changes
in internal salt content, whereas this was not necessary for Rhixophora
seedlings.
Arnold (1955) showed that the transpiration rates of mangroves are very
much lower than those of mesophytes. Because of this, Chapman (1962a)
suggested that mangroves are lacking in dry tropical areas, such as the west
coast of South America, because low humidity reduces respiration rate to the
point where seedlings cannot grow.
Lewis and Naidoo (1970) reported that the apparent transpiration rate of
A. marina in South Africa rose in the morning as light intensity increased and
humidity decreased. Maximum transpiration occurred at mid-morning, after
which the rate progressively decreased, regardless of atmospheric conditions.
Tidal inundation after the mid-morning maximum caused increase in
transpiration rate and a second maximum. The authors speculated that
decrease in rate at mid-morning was caused by incipient wilting following
excessive transpiration.
Chemical Composition
The chemical composition of mangrove trees has been studied by Sokoloff
et. al. (1950), Sidhu (1963), Morton (1965), and Golley (1969), but little is
known in relation to environmental factors and age of the trees. Sidhu (1963)
stated that concentrations of ash and sodium in leaves of species of
Rhizophora, Avicennia, zndAegiceras which grew near the sea were lower than
from species which inland, but gave data only for Avicennia (Table 13).
Table 13. Ash and sodium contents of three species of Avicennia (Sidhu 1963).
Species
Sea A. officinalis
A. alba
A. alba
Inland A. marina
Ash, percent
14.8
15.8
19.4
30.4
Sodium, percent
2.3
3.3
3.7
5.0
103
-------
GERALD E. WALSH
Sidhu (1963) also divided species into three catagories based on sodium
content of the leaf:
A. Species with more than 5% sodium: A. marina, Salvadora persica.
B. Species with 3-5% sodium: A. alba, Lumnitizera racemosa, Ceriops
candolleana, and A. ilicifolius.
C. Species with 1-3% sodium: A. officinalis, R. mucronata, S. Apetala, B.
Caryophylloides, A. Corniculatum, Eleopodendron inerme, Exocoecaria
agallocha, D. uliginosa.
Atkinson et. al (1967) reported concentrations of some ions in leaves of
various ages in R. mucronata and Aegiatilis annulata R.Br. Measurements
were made on successive leaf pairs of shoots (Tables 14 and 15).
Concentrations of sodium and chloride in the leaves of R. mucronata
increased with age, but in relation to amount of leaf water, chloride content
was constant, sodium concentration increased, and potassium concentration
deceased.
In contrast, there was a decrease in concentrations of sodium, potassium,
and chloride with age of A. annulata. Atkinson et. al. ascribed this to
excretion of .salt by epidermal glands, an adaptation lacking in Rhizophora.
Table 14. Concentrations of Na+, id", and Cl" in the leaves of R.
mucronata (after Atkinson et al. 1967). Leaves in Sample 1 were
youngest in the sequence of increasing age.
Sample Number
Dry weight (g)
Water (% fresh weight)
Na+ ((i-equiv/leaf)
Na+ (u-equiv/ml H20)
K+ (ji-equiv/leaf)
K+ (u-equiv/ml HO)
Cl" (u-equiv/leaf)
Cl~ (vi-equiv/-ml H20)
1
0.16
56
61
305
25
124
74
370
2
0.50
65
290
313
81
88
520
562
3
0.50
66
420
431
57
59
510
522
4
0.61
65
480
435
48
44
585
530
5
0.57
67
520
461
69
61
580
515
6
0.63
69
645
461
45
32
730
522
104
-------
ECOLOGY OF HALOPHYTES
Table 15. Concentrations of Na., K , Mg , and Cl in the leaves of
A. aimulata (after Atkinson et al. 1967). Leaves in Sample 1 were
youngest in the sequence of increasing age.
Sample Number
Dry weight (g)
Water (% fresh weight)
Na+ (u-equiv/leaf)
Na+ (u-equiv/ml HjO)
K+ (u-equiv/leaf)
K+ (ji-equiv/ml HjO)
Mg"1"1" (u-equiv/leaf)
Kg** (u-equiv/ml H20)
Cl (u-equiv/leaf)
Cl" (u-equiv/ml HZO)
1
0.32
72
420
518
155
191
108
133
415
512
2
0.45
60
325
480
106
157
330
488
290
429
3
0.45
60
275
411
87
130
440
659
270
405
4
0.50
59
280
388
93
129
590
819
361
361
5
0.44
60
235
356
70
106
530
802
386
386
There is a paucity of literature data concerning elemental composition of
mangroves. Most studies of which I am aware have not been published. Values
given in Tables 16 and 17 were personal communications from F.B. Golley
(Univeristy of Georgia, Athens), S. S. Sidhu (University of Western Ontario,
London, Canada), and T. F. Hollister (U. S. Environmental Protection
Agency, Gulf Breeze, Florida). The samples were collected in Panama
(Golley), India (Sidhu), and Florida (Hollister).
There are wide differences in concentrations of each element between
species. For example, Avicennia species contain relatively high concentrations
of sodium and potassium in all organs. Also, the roots of Laguncularia
racemosa contained very high concentrations of all elements, except
magnesium.
Sidhu (personal communication) found no correlation between the mineral
status of soils and the elemental content of 16 mangrove species. It is clear
that research which relates species and habitat to elemental composition is
needed.
105
-------
Table 16. Concentrations of various elements in mangrove leaves (personal communications: + Siddhu,
* Golley, # Hollister).
Species
Acanthus iltcifolius (+)
Aegiceras corniculatum (+)
Avicennia alba (+)
Avicennia marina (+)
Avicennia nitida (#)
Overstory
Understory
Avicennia officinalis (#)
Bruguiera caryophylloides (+)
Ceriops candolleana (+)
Conocarpus erectus (#)
Overstory
Understory
Derris ulginosa (+)
Exocaria agallocha (+)
Laguncularia racemosa (#)
Overstory
Understory
Lumnitizera racemosa
India (+)
Florida (#)
Rhizophora brevistyla (*)
Overstory
Understory
Rhizophora mangle
Hollister
Morton (1965)
Rhizophora mucronata (+)
Sonneratia apetala (+)
Concentration,
Ca
7800
6800
10400
11200
12430
6680
11600
24800
20400
13350
7600
17600
14200
6510
7600
24200
9100
12200
7800
10760
13500
19800
11200
Co
.
-
-
.
-
-
-
-
-
22
24
-
_
12
32
-
-
46
56
9
52
-
-
Fe
_
-
-
-
147
300
-
-
-
305
251
.
-
125
149
-
169
82
672
132
152
-
-
K
8200
8400
12100
15200
21000
29400
19100
6100
5300
4100
21600
25800
12900
17700
25000
22400
19900
8400
8500
16400
6500
21600
3100
Mg
8600
10200
10000
13500
3600
2320
12200
10000
11700
-
-
12400
14700
4440
4740
15600
3940
4700
5000
4320
8000
14500
10500
ppm dry weight
Mn
_
-
-
-
-
53
-
-
-
50
88
-
-
74
116
-
-
387
125
92
30
-
-
Na
30000
23100
35000
50600
4590
6500
23300
27700
36600
5500
4110
12300
4800
10520
8620
43100
9370
9800
8300
11130
-
22800
14900
P
3200
2500
2300
2300
-
-
4000
2400
2500
-
-
2600
4100
-
-
2500
-
9000
8000
-
1400
3300
3100
Si
30000"
4600
4000
5000
-
-
20800
17200
11000
-
-
18000
18800
.
-
5800
-
-
-
.
-
6000
4800
Zn
_
-
-
-
154
24
-
-
-
160
142
-
-
92
84
-
170
11
15
146
43
-
-
-------
Table 17. Concentrations of various elements in organs, other than leaves, of mangroves (personal
communications: * Golley, # Hollister).
Species
Concentration, ppm dry weight
Avicennia nitida (#)
Overstory steins
Under story stems
Roots
Conocarpus erectus (#)
Overstory stems
Understory stems
Roots
Laguncularia racemosa (#)
Overstory stems
Understory stems
Fruit .
Roots of seedlings
Lumnitizera racemosa (#)
Stems
Rhizophora brevistyla (*)
Overstory stems
Understory stems
Overstory fruits
Understory fruits
Rhizophora mangle (#)
Overstory stems
Fruit (2-10 cm long)
Fruit (11-20 cm long)
Roots of seedlings
Prop roots
Rhizophora mucronata (#)
Fruit
Ca
7430
7100
3930
3930
5600
21200
3100
6850
8590
19390
11000
12900
5700
5900
6900
8936
2350
1260
600
4850
932
Co
-
16
40
15
36
25
18
16
.
54
25
52
83
81
56
-
58
-
36
-
20
Cu
-
10
13
13
15
40
9
-
15
-
10
7
8
6
4
-
12
15
12
11
-
Fe
282
580
465
329
648
979
49
516
332
1000
99
36
1000
82
45
252
155
113
422
253
346
K
14700
27800
29200
4100
27300
-
10000
9200
35000
81000
13200
3000
3900
10100
7100
2200
4000
2990
13800
13800
13860
MB Mn
3080
3650
3700
3400
2400
7160
1880
1220
2920
1990
3405
1000
2900
2900
3400
2820
3050
2460
1070
2540
2620
Na
-
48
34
47
83
75
92
46
123
-
-
168
255
191
164
74
121
69
71
-
46
Zn
3730
9340
9000
412
1260
3560
4590
4120
4120
14100
618
5500
9500
9600
9700
8370
8890
10250
12840
10660
8620
227
28
121
180
112
162
89
38
79
514
25
11
12
11
7
120
2
152
12
74
6
-------
GERALD E. WALSH
Sokoloff et. al (1950) and Morton (1965) gave data from chemical analyses
of leaves of R. mangle from Florida (Table 18.) Their values for vitamin
content vary greatly but cannot be compared because methods of treatment
of leaves and assay procedures were not given. Sokoloff et. al. suggested that
leaf meal could replace alfalfa in chicken rations and Morton recommended
its use as cattle feed.
Golley (1969) compared the caloric content of R. brevistyla in Panama
with trees from tropical moist, premontane, and gallery forests. The energy
values of the mangrove (Table 19) were generally greater than those of the
other tropical trees.
Tannin is an important constituent of all parts of mangrove trees. Bowman
(1921) showed that tannin is usually stored in solid masses in cells, but is
frequently in solution in cytoplasm. He considered the tannin cells and water
storage tissue of R. mangle to constitute a true hypodermis.
The function of tannin in mangrove is unknown. Tannins have a strong
protein-binding capacity and, therefore, are able to inhibit enzymes. In the
living plant it is possible that tannins aid in resistance to fungi, as fungi have
been shown to cccur in large numbers in mangals (Swart 1958; Kohlmeyer
1965, 1966, 1968a, b, 1969a, b; Kohlmeyer and Kohlmeyer 1971; Ahern et
al. 1968; Rai et al. 1969, and Ulken 1970). Lee and Baker (1972a, b)
identified 52 species of soil fungi from a Hawaiian swamp. Swain (1965)
suggested that the presence of tannin causes resistance of dead organic matter
to attack by fungi and other decomposers. Crossland (1903) stated that the
Arabs of Zanzibar used mangrove wood for houses and furniture because it
was not attacked by termites, and suggested that the high tannin content
repelled the termites. The ability of tannins to inhibit enzymes probably
affects the rate of decay of plant detritus and, therefore, is important in
relationships within the detritus based food web. In most plants,
hydrolyzable tannins are usually present in leaves and fruit, whereas
condensed tannins occur in the bark or heartwood. This implies that leaves
and fruit of mangroves are less persistant as particulate detritus than woody
parts.
Most research on mangrove tannin has been done on samples of bark.
Drabble (1908) illustrated distribution of tannin in R. mangle and
Laguncularia racemosa. Trimble (1892) reported the empirical formula a
^25^25^11 f°r tannin fr°m tne bark of R. ^mangle. Baillaud (1912) found
that 30% of the dry weight of bark from Rhizophora andBruguiera in Africa
was comprised of tannin. Dry' bark of Xylocarpus contained 26% tannin.
Dried bark of R. mucronata from Africa contained between 41.3 and 42.8%
tannin (Anon. 1904). Brown and Fisher (1918) pointed out that tannin
content varied greatly between species from the Indo-West-Pacific region. In
Malaya, Buckley (1929) reported the following percentages of tannin in fresh
bark: R. Mucronata 20.7-30.8, R. congugata 7.9-17.6, B. gymnorhiza
14.5-25.6, B. eriopetala 17.3-23.0, B. caryophylloides 15.8, B. parviflora
4.7-7.6, C. candolleana 19.0-30.8, Carapa obovata 29.8-41.6. The fruits and
108
-------
Table 18. Chemical analysis of dry leaves of R. mangle from Florida (after
Sokoloff et al. 1950 and Morton 1965).
Total protein, percent
Crude ;fiber, percent
Crude fat, percent
Calcium, percent
Sulphur, percent
Ash, percent
Iodine, percent
Manganese, mg/kg
Thiamin, mg/kg
Riboflavin, mg/kg
Folic acid, mg/kg
Niacin, mg/kg
Pantothenic acid, mg/kg
12.1-14.3
13.9
2.9
1.6
0.6
6.7
0.8
0.3
1.56-2.03
4.5,5.6
0.60-0.67
20.3-28.0
4.0-4.5
7.5
13.9
3.6
1.4
-
10.1
0.5
0.03
130
190
320
2,400
53
Table 19. Mean caloric values and standard errors of R. brevistyla from Panama
(Golley 1969).
Energy, g cal/g dry weight
Compartment Mean SE
Canopy leaves 4182 22
.. Canopy stems 4337 11
Understory leaves 4299: 132
Understory stems 42'04 12
Canopy fruit 4298 29
Understory fruit 4360 20
Epiphytes 4585 11
Litter 4141 13
R)0ts 4034 48
109
-------
GERALD E. WALSH
leaves contained least tannin. Buckley concluded that R. mucronata was the
best source of tannin because the yield of bark per tree was good. Carapa
obovata contained the highest concentration of tannin, but its bark was thin
and the yield per tree was small. Drabble and Nierenstein (1907) reported
that older R. mangle trees contained more tannin than young trees.
Although mangroves are not used extensively as a source of dyes, when the
bark of R. mangle is treated with copper or iron salt, brown, olive, rust, and
slate-colored dyes are obtained (Fanshaws 1950, Morton 1965). According to
Morton, a boiled concentrate of the bark may be used for staining wood in
floors and furniture, dyes for textiles may be obtained from the roots, and a
red dye from the shoots may be used for coloring leather.
Energy Relationships
Except for a few reports on yield of wood (see "silviculture" section of
this report), little is known about production of organic matter in mangrove
swamps. The first detailed study of photosynthesis, respiration, biomass, and
export of organic matter was made by Golley et al. (1962) in a Puerto Rican
R. mangle forest in May. Gross photosynthesis was 8.23 g C/m^/day; total
respiration was 9.16 g C/m^/day. The greatest rates of photosynthesis (7.33 g
C/m^/dayO and respiration (4.31 g C/m^/day) occurred in the upper canopy
of leaves. Shaded leaves accounted for gross photo synthesis of only 0.40 g
C/m^/day and respiration of 0.48 g C/m^/day. Seedling photosynthesis was
0.12 g C/m^/day and respiration was 0.36 g C/m^/day. At the soil surface,
respiration by prop roots was 2.03 g C/m^/day. At and below the soil surface
respiration was 1.64 g C/m^/day. Gross photosynthesis and respiration above
ground was related to dry leaf biomass (1017 gm/m^), leaf area (4.4 m^/m^),
and chlorophyll a content of the leaves (1.19 g/m^). The trees were
approximately 8 m tall, and the factors measured attained their greatest
values at between 4 and 6 m height. Unfortunately, the subterranean algal
flora was not studied. This might have been important as Marathe (1965)
showed 12 algal species in the soil of mangals near Bombay. Another source
of primary production was algae on the roots and mud. Dawson (1954)
described many attached algal species from roots and mud in Vietnam. Golley
et al. stated the R. mangle community was more fertile than most marine and
terrestrial communities. It was not, however, as efficient as the montane rain
forest or coral reefs of Puerto Rico in conversion of sunlight into organic
matter under similar light regimes.
Miller (1972), using a model, calculated gross photosynthesis, net
photosynthesis, and respiration of R. mangle in Florida (Table 8). He
contrasted these data with those of Golley et al. (1962). Using Miller's model,
the estimates of Golley et al. corresponded to 9.4 g organic matter/m^/day
for gross photosynthesis, 3.4g organic matter/m^/day for net photosynthesis,
and 5.9 g organic matter/m^/day for respiration. Miller ascribed differences
between his data and Golley's to different leaf areas.
Miller's model predicted that maximum photosynthesis occurrs at a leaf
area index of approximately 2.5 if no acclimation to shade within the canopy
110
-------
Table 8. Gross primary production (P ), net primary production (P ), and
respiration (R) of R. mangle leaves in Florida (after Miller 1972). Data
are expressed as grams organic matter/m^/day.
Sunny
Height
1.75-2
1.50-1
1.25-1
1.00-1
0.75-1
0.50-0
0.25-0
0.00-0
Total
1.75-2
1.50-1
1.25-1
1.00-1
0.75-1
0.50-0
0.25-0
0.00-0
(m)
.00
.75
.50
.25
.00
.75
.50
.25
.00
.75
.50
.25
.00
.75
.50
.25
P
0.
0.
2.
30
3.
2.
0.
0.
12
0.
0.
2.
2.
2.
1.
0.
0.
g
08
31
17
.8
39
16
97
67
.83
09
34
14
43
15
31
57
39
P
n
0.03
0.12
0.93
1.48
1.64
0.87
0.31
0.17
5.55
0.05
0.21
1.32
1.33
0.91
0.36
0.10
0.03
June
R
0.05
0.
1.
1.
1.
1.
0.
0.
7.
0.
0.
0.
1.
1.
0.
0.
0.
19
24
61
75
33
66
51
34
January
03
13
82
10
24
95
47
37
Cloudy
P
_£
0.07
0.29
2.02
2.59
2.48
1.61
0.73
0.51
10.30
0.09
0.34
2.08
2.52
2.41
1.55
0.70
0.49
0
0
0
1
0
0
0
0
3
0
0
1
1
1
0
0
0
^n
.03
.12
.92
.13
.84
.35
.10
.02
.51
.05
.21
.28
.45
.19
.62
.23
.13
0
0
1
1
1
1
0
0
6
0
0
0
1
1
0
0
0
R
.04
.17
.09
.46
.65
.26
.63
.49
.79
.03
.12
.80
.07
.21
.93
.46
.36
Total
9.42 4.31
5.10
10.18
5.16 4.98
111
-------
GERALD E. WALSH
is present and predicted that production decreased with increase in leaf area
index and leaf width. Also, the environmental variables with the greatest
influence on primary production were air temperature and humidity.
Increase in solar radiation up to a point, increased primary production as did
increasing amounts of diffuse energy. Infrared variation decreased
production.
Gill and Tomlinson (1971) reviewed phenological phenomena associated
with growth of R. mangle in Florida. Although the general progression of
development appeared to be mediated endogenously, climatic factors were a
strong governing influence. Environmental control of growth was through
effects on development of the apical bud. Vegetative branches, inflorescences,
and axillary buds are developed within the apical bud of R. mangle. The
rates of leaf expansion and fall were highest in the summer when temperature
and radiation were maximal. Throughout the year, leaf fall was closely
correlated with leaf expansion so there was a fairly constant number of leaves
on a shoot. Flower buds appeared in greatest abundance between May and
July, open flowers between June and September, and fruit between
September and March. The hypocotyl appeared in March and greatest fall of
propagules was between June and October.
In some swamps, phytoplankton in the water contribute appreciably to
synthesis of organic matter. This was the subject of extensive studies by
Teixeira and Kutner (1962), Teixeira et al. (1965, 1967, 1969), Watanable
and Kutner (1965), Tundusi and Tundusi (1968), and Tundusi and Teixeira
(1964, 1968) in Brazil. Tundusi (1969) summarized the work in a Brazilian
mangal. Gross primary production of surface water ranged between 2.10 and
91.3 mg C/m^/hr. Respiration values were between 1.0 and 21.3 mg C/m^/hr.
Nannophytoplankton (size range 5-65 u) accounted for 61.8% of the total
carbon uptake. Diatoms were the numerically dominant unicells, a
phenomenon also reported by Mattox (1949) in Puerto Rico, Walsh (1967) in
Hawaii, and Bacon (1971) in Trinidad.
An important finding in the work of Golley et al. (1962) was that tidal
export of particulate matter was 1.1 g C/m^/day. Heald and Odum (1971)
reported production, consumption, and export of organic detritus in a R.
mangle stand in southern Florida. Heald and Odum pointed out that many
commercially important finfish and shellfish live in the mangrove
environment and that vascular plant detritus is the primary source of food for
many estuarine organisms.
Heald (1971) estimated that production of mangrove debris averaged 2.4 g
C/m^/day, oven dry weight. This was equivalent to almost nine tons/ha/yr.
Annually, plants other than mangrove accounted for less than 15% of the
total organic debris. Rate of degredation of mangrove detritus was related to
conditions of the environment. Breakdown was most rapid in brackish water
The amphipods Melita nitida- Smith and Corophium lacustre and the crab
Rithropanopeus harrisii Gould were important consumers of detritus in
brackish water.
112
-------
ECOLOGY OF HALOPHYTES
The actively photosynthesizing leaf of R. mangle was reported by Heald to
contain 6.1% protein, 1.2% fat, 67.8% carbohydrate, 15.7% crude fiber, and
9.2% ash. During abscission, protein and carbohydrate contents were 3.1 and
59.6%, respectively. After falling into brackish water, the carbohydrate
content of leaf detritus .fell to approximately 36%, but protein content rose
to approximately 22%. Heald speculated that increase in protein was due to
growth of bacteria and fungi on the detritus particles, and stated that the
food value of detritus, which was in the water for one year, was more
nutritious than that in the water for one or two months.
Odum (1971) studied the use of mangrove detritus as food by many animal
species. He concluded that vascular plant detritus which originated from R.
mangle leaves was the main source of food for the aquatic animal community.
As the detritus decomposed into finer particles, it became covered with
bacteria, fungi, and protozoans. The caloric content rose from 4.742 Kcal/g
in ash-free fresh leaves to 5.302 Kcal/g in ash-free remains of leaves which
were submerged for two months.
There were at least four pathways by which mangrove leaves were utilized
by heterotrophs: (1) dissolved organic substances from the leaves *
microorganisms •» higher consumers, (2) dissolved organic substances *
sorption on sediment and aged detritus particles * higher consumers, (3)
particulate leaf material •» higher consumers, and (4) particulate leaf material
** bacteria, fungi, and protozoans -»higher consumers. Odum believed the last
pathway to be the most important. He speculated that microorganisms
convert compunds such as cellulose and lignin into digestible protein utilized
by invertebrates and fishes.
SILVICULTURE
Mangrove is one of the most important sources of timber, fuel, posts,
poles, railroad ties, and tannin in the tropics. It also has resins which are used
as plywood adhesives, and the bark, leaves, shoots, and roots contain dyes.
Chatterjee (1958) gave the following uses for mangrove in India: Heritiera
(boat building, planking, fuel), Amoora cuculata (wooden pipes for hookahs
and wooden toys), Aegiatilis rotundiflora (extraction of high-grade salt after
burning), Avicennia (fuel wood for brick burning), Exocoecaria agattocha
(match boxes), Xylocarpus granatum (pencils), and Salicornia brachiata
(source of sodium carbonate).
Because of their many uses, silviculture of mangroves has been practiced
for many years in southeastern Asia. Banerji (1958) reported that R.
mucronata andB. gymnorhiza were grown successfully on a plantation of 685
acres in the Andaman Islands between 1898 and 1908. Banerji stated thatB.
gymnorhiza was an excellent source of poles for transmission lines and that
Rhizophora produced 30 cords of fuel wood per acre, whereas Bruguiera
produced 11 cords. The annual yield of firewood was estimated at 130,000
tons.
113
-------
GERALD E. WALSH
In the Andamans, clearfelling and planting of B. gymnorhiza was the best
silviculture method. Because this species grows slowly, a rotation to 100 years
was recommended for an exploitable breast-height girth of 27 inches. On
some plantations, Bruguiera attains a height of 30-35 feet (9.2-11.7 m) and a
girth of 9-12 inches (23-30 cm) in 15 years. The tree provides one of the
strongest timbers in India, and has a durability life of 10 years after treatment
with creosote.
In Thailand, Walker (1937, 1939) reported that R. conjugata and R.
mucronata were the mangrove of choice for poles and firewood and have
been used on a large scale for planting. There was an abundance of seedlings
at all times of the year. Seedlings of R. mucronata were less susceptible to
attack by crabs, and their long radicle was an advantage in competition with
growth of A. aureum and B. parvifloraa. Seedlings were deep-planted at
six-foot (1.8 m) intervals and the maximum felling girth was-eight inches (20
cm). The prescribed time for planting was two years after felling.
Unfortunately, Walker did not give production figures for the Thai
mangroves. He did point out that B. gymnorhiza was used for fuel, S.
griffithii for fishing stakes, and C. candolleana as fuel and tanbark.
Becking et al. (1922) divided the mangrove of southeastern Asia into three
classes based upon the diameters of mature trees: Class A, less than 20 cm, A.
corniculatum,, Scyphiphora hydrophyllacease Gaert., Ceriops spp.; Class B,
20-40 cm, A. marinaa, Lumnitzera racemosa, Bruguiera spp.; Class C, greater
than 40 cm., A. officinalis, S. alba, Rhizophora spp., Bruguiera spp.,
Xylocarpus spp. The authors showed that production of wood per unit area
by trees of larger diameter was greatest. Although Sonneratia was one of the
more productive genera, Backer and van Steenis (1954) stated that in Malaya
its economic usage was small. Small amounts were used for fuel and in boats
and houses. The young berries of Sonneratia can be consumed by humans and
pectin can be extracted from them.
According to Banijbatana (1958), approximately 133,400 ha of mangrove
forests were available for silviculture in Thailand. The shelterwood system
was judged best, and for young forest with trees of 20 cm girth and under,
clearing and thinning was recommended. For forests in which the majority of
trees were 20-50 cm, heavier thinning was recommended with seedling felling
foncontrol of C. roxburghiana and B. cylindricaca. The Rhizophoras reached
65-70 cm in girth between the ages of 39 to 43 years and a rotation system of
40 years was adopted. Yield was calculated at 50-60 cm per acre.
Approximate rates of growth in girth of several mangrove species in
Malaya were given by Durant (1941 (Table 22). The volume of wood per acre
of mixed Rhizophora forest increased from 1,375 cubic feet at 10 years to
5,600 cubic feet at 50 years. Durant recommended harvesting at 22-23 years
when the mean annual volume increment was at its maximum of 147.7. The
volume of wood at 22 years was 3,250 cubic feet per acre.
Noakes (1955) stated that the total area of mangrove forest in Malaya was
approximately 760 square miles. Of this, 460 square miles were under
114
-------
ECOLOGY OF HALOPHYTES
Table 22. Growth in girth of several mangrove species in Malaya (Durant 1941)
Age
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
£i
2
6
9
12
16
19
21
24
27
29
32
35
37
39
41
42
!k
5
10
13
16
18
20
22
24
26
28
30
32
34
36
B.
3
7
9
12
15
18
20
23
25
28
30
32
35
37
39
41
Girtl
B_._
halnesii
2
5
8
11
14
17
19
21
24
27
30
33
36
40
45
i , inches
B.
2
5
8
11
13
16
18
20
23
25
27
29
!L.
1
5
7
9
11
13
15
17
18
20
22
24
26
27
29
30
C.
2
5
a
10
13
15
18
20
22
24
sustained yield management. Rhizophora mucronata and R. conjugata were
the most important Malayan species, covering two-thirds or more of the total
area. Fuel was the main product of the mangrove forest, but poles for houses
and fish traps were also important.
According to Noakes, fruiting of Rhizophora occurred at the age of four
years, was annual and highly prolific. Full stocking by water-borne seedlings
occurred rapidly after clearcutting. As in Thailand, Rhizophora grew slowly.
The annual growth increment of boles was slightly greater than one inch in
the early stages of growth to just over one-half inch above 12 inches diameter.
The trees grew to a maximum height of 70-120 feet. Normal felling size was
50-60 feet in height and 1.5-2.5 feet in girth. Felling size was achieved in
20-30 years. The mean annual volume increment of mixed Rhizophora forest
culminated at approximately 25 years, allowing for a three-year regeneration
period. The yield was approximately 3,106 cubic feet of wood per acre.
Wadsworth (1959) reported silviculture of Laguncularia racetnosa in
Puerto Rico. Undisturbed stands 22 years old attained an average diameter at
breast height of 5.0 inches and 2,680 cubic feet of wood per acre. Natural
regeneration by water-borne seedlings occurred within two years after
clearfelling. Holdridge (1940) recommended a cutting cycle of five years and
a rotation* cycle of 25 years for exploitation of mangroves in Puerto Rico.
115
-------
GERALD E. WALSH
Golley et al. (1962) reported that in a R. mangle forest in Puerto Rico,
annual production of wood was 0.84 g/m^/day (0.42 g C/m^/day). This was
much less than that reported by Noakes in Malaya (14 g C/m^/day).
It should be pointed out here that in some areas, silviculture of mangrove is
practiced in the belief that the wood is resitant to marine boring organisms.
Southwell and Boltman (1971) tested resistance to marine borers by R.
mangle, R. brevistyla, A. marina, C. erectus, and Laguncularia racemose. Only
C. erectus showed natural resistance to teredo, pholad, and limnorid borers.
The Rhizophora species were almost completely destroyed after immersion in
Pacific Ocean water for 14 months.
HERBICIDES
Herbicides have been used for almost 20 years for control of mangrove. In
Africa (Sierra Leone), Ivens (1957) reported that application of the
auxin-type herbicides 2, 4-dichlorophenoxyacetic acid (2, 4-D), 2, 4,
5-trichlorophenoxyacetic acid (2, 4, 5-T), and 2-methyl,
4-chlorophenoxy-acetic acid (MCPA) were effective in eradication of both R.
racemosa and A. nitida when applied to the bases of trunks at concentrations
of 4-20% in diesel oil. Frilled R. racemosa were killed by the butyl ester of 2,
4-D at concentrations between 0.5 and 1.0%, whereas 4% was required to kill
unfrilled trees. Avicennia nitida was slightly more resistant to 2,4-D than/t.
racemosa,- concentrations between 2 and 4% being required to kill frilled
trees. 2, 4, 5-T and MCPA were not as effective as 2, 4-D. Recovery of trees
after treatment with 2, 4, 5-T was reported.
The first signs of herbicidal effect were noted approximately three weeks
after application, when the leaves turned yellow. Extensive defoliation
occurred by seven months after treatment, at which time many trees of both
genera were dead. Seedlings and young trees were more resistant than old
trees.
Ivens also reported that 3-(4-chlorophenyl)-l, 1-dimethylurea (CMU)
killed all trees when applied to the pneumatophores of A. nitida at the rate of
20 Ib/acre. Dalapon (2, 2-dichloropropionic acid) caused complete kill with
no regrowth at 40 and 80 Ib/acre. There was a small amount of regrowth after
application of dalopon at 20 Ib/acre.
Truman (1961) reported total kill of treated A. marina in Australia by 1%
2, 4-D applied to the basal bark. Only 54% were killed by treatment with 1%
2, 4, 5-T. Spotted gum (Eucalyptus maculata Hook.), an upland tree, was
only slightly affected by the same treatment. Truman concluded that A.
Marinna was very susceptible to auxin-type herbicides.
The concept of high susceptibility of mangrove to auxin-type herbicides
was extended by Tschirley (1969), Orians and Pfeiffer (1970), and Westing
(1971a, b), who stated that mangrove forests in Vietnam were destroyed after
•a single application of 6.72 kg/ha of the triisopropanolamine salt of 2, 4-D in
combination with 0.61 kg/ha of the triisopropanolamine salt of 4-amino-3, 5,
6-trichlorophcolinic acid (picloram). The forests were composed mainly of s'.
116
-------
ECOLOGY OF HALOPHYTES
alba, B. parviflora, B. gymnorhiza, A. marina, A. intermedia, R. conjugata, C.
candolleana, and N. fruiticans. Westing (197la) reported that treated areas in
Vietnam remained uncolonized by mangrove six years after treatment.
Westing (1971c) also published a list of references to effects of herbicides in
Vietnam. For reviews of herbicidal effects in Vietnam, see Boffey (1971) and
Aaronson(1971).
Westing (1971a) pointed out that application of 2, 4-D in combination
with picloram both defoliated and killed nearly all trees injhe sprayed areas.
He also stated that herbicides seemed to prevent recolonization by mangrove,
although he observed rapid recolonization of an area cleared by cutting. It is
true, however, that a large portion of the denuded mud flats of Vietnam are
only occasionally inundated by tidal water and that sufficient numbers of
seedlings for regeneration are not carried in. Another possible reason for lack
of recolonization may be related to texture of the denuded soil. Natural
regeneration of mangrove is greatly retarded when soil becomes too stiff or
hardens after exposure to the sun (Banijbatana 1958).
We (Walsh et al., in press) have studied affects of Tordon® 101 on
seedlings of R. mangle from Florida. Tordon 101 is a mixture of the
triisopropanolamine salts of 2, 4-D (39.6%) and picloram (14.3%). Seedlings
that had no leaves and one or two pairs of leaves were treated with 1.12,
11.2, and 112.0 kg/ha (1, 10, and 100 Ib/acre). These rates were equivalent to
0.44, 4.40, and 44.0 kg/ha 2, 4-D, and 0.16, and 16.0 kg/ha picloram. A
combination of 0.44 kg/ha 2, 4-D and 0.16 kg/ha picloram caused stunted
growth of seedlings without leaves, but had no permanent effects upon
seedlings with one or two pairs of leaves. Higher concentrations caused death
of all treated seedlings by 50 days after treatment.
We were never able to quantify tissue residues in seedlings without leaves
that had been treated at the lowest concentrations. The limits of
quantification were 0.02 ppm (parts per million) 2, 4-D and 0.01 ppm
picloram. Even though tissue residues were very low, seedling development
was greatly inhibited. In seedlings with leaves, greatest herbicidal residues
occurred in the highest leaves and hypocotyl. Table 20 shows distribution of
2, 4-D and picolinic acid in the organs of seedlings treated when two pairs of
leaves were present.
At the tissue level, symptoms of. herbicide poisoning were desiccation of
leaves, plugging of vessel elements, and destruction of root cortex. Root
destruction probably impared the ability of seedlings to regulate salt and
water balance. For example, concentrations of sodium and potassium in
seedlings were directly related to application rate and time (Table 21). No
changes were found in concentrations of magnesium, manganese, calcium,
iron, or zinc. Strogonov (1964) said that symptoms of salt poisoning in plants
include bleaching of chlorophyll accompanied by browning of the leaves.
Both symptoms were observed in our treated seedlings.
117
-------
GERALD E. WALSH
Table 20. Concentrations of 2,4-D and picollnic acid, in parts per mlHion (+ 20%), of wet tissue in
organs of R. mangle seedlings treated with Tordon 101 when two pairs of leaves were present. Residues
were detected in every analysis ot seedlings treated with 1.12 kg/ha but were below quantifiable
levels (0.01 for plcolinlc acid, 0.02 for 2,4-D) (Balsh et al. in press).
Treatment
kg /ha
11.2
112.0
Day
30
40
10
Roots
2.4-D
0.02
0.02
1.23
FA
0.01
0.01
0.39
Hypocotyl
2.4-D
0.10
0.23
1.68
PA
0.03
0.10
0.49
Stem
2.4-D
0.02
0.23
1.02
1st leaves
PA
0.01
0.10
0.43
2.4-D
0.02
0.29
0.63
PA
0.01
0.10
0.24
2nd leaves
2.4-D
0.13
0.35
0.87
PA
0.06
0.10
0.41
Table 21. Concentrations of sodium and potassium in the stems of R. mangle
seedlings treated with Tordon 101 when two pairs of leaves were present; 50
days after treatment (Walsh, unpubl.)
Treatment
Control
1.12 kg/ha
11. 2 kg/ha
112.0 kg/ha
Na
37,500
49,800
64,900
96,200
ppm Dry Weight
K
4,375
4,821
5,295
6,321
Acknowledgements
I thank Drs. Frank B. Golley and S. S. Sidhu and Mr. Terry A. Hollister for
data on elemental composition. Special thanks are given to Mrs. Ann Valmus,
Librarian at the Gulf Breeze Laboratory, for aid in obtaining copies of many
publications and for her patience in checking the references. The quotation
from 'The Night Country" was made with permission of Charles Scribner's
Sons, Publishers, New York. Thanks are also given to Mrs. Steven Foss for
making the illustrations.
® Registered trademark, Dow Chemical Co., Midland, Michigan. Reference to trade
names in this publication does not constitute endorsement by the Environmental
Protection Agency.
118
-------
ECOLOGY OF HALOPHYTES
REFERENCES
Aaronson, T. 1971. A tour of Vietnam. Environment 13: 3443.
Abe, N. 1937. Ecological survey of Iwayama Bay, Palao. Palao Trop. Biol.
Stn. Stud. (Tokyo) 1: 217-324.
Abe, N. 1942. Ecological observations on Melaraphe (Littorinopsis) scabra
(Linnaeus) inhabiting the mangrove tree. Palao Trop. Biol. stn. Stud.
(Tokyo) 2: 391-435.
Abel, 0. 1926. Amerikafahrt: Eindrucke, Beobachtungen und Studien eines
Naturforschers auf einer Reise nach Nordamerika und Westindien. Gustav
Fischer. Jena, 462 pp.
Abel, O. 1927. Fossile Mangrovesumpfe. Palaont. Z. 8: 130-140.
Abeywickrama, B. A. 1964. The estuarine vegetation of Ceylon, in Les
problemes scientifiques des deltas de la zone tropicale humide et leurs
implications. Colloque de Dacca. UNESCO, Paris, pp. 207-209.
Abreu, S. F. 1926. Notes on the tanning materials of Brazil. Am. Leather
Chem. Assoc. J.21: 357-358.
Acosta-Solis, M. 1947. Commercial possibilities of the forests of Ecuador —
mainly Esmeraldas Province. Trop. Woods Yale Univ. Sch. For. 89: 1-47.
Acosta Soils, M. 1959. Los manglares del Equador. Contrnes Inst. Ecuat.
Cienc. Nat. No. 29, 82 pp.
Acuna, R. A. 1953. Mangrove swamps. For. Leaves 6: 35-41.
Adriani, M. J. 1937. Sur la transpiration de quelques halophytes cultivies dans
les milieux different en comparison avec celle de quelques nonhalophytes.
Proc. K. ned. Akad. Wet. 40: 524-529.
Ahmad, N. 1967. Seasonal changes and availability of phosphorus in
swamp-rice soils of north Trinidad. Trop. Agric. Trinidad 44: 21-32.
Alexander, T. R. 1967. Effect of hurricane Betsy on the southeastern
Everglades. Q. J. Fla. Acad. Sci. 30: 10-24.
Almodovar, L. R. and R. Biebl. 1962. Osmotic resistance of mangrove algae
around La Parguera, Puerto Rico. Rev. Algol. 3: 203-208.
Almodovar, L. R. and F. Pagan. Notes on a mangrove lagoon and mangrove
channels at La Parguera, Puerto Rico. Nova Hedwigia in press.
Alston, A. H. G. 1925. Revision of the genus Cassipourea. Kew Bull. 6:
241-276.
Altrock, A. von. 1897. Uber Mangrpve-Nutzung in St. Catharina Brasilien.
Tropenpflanzer 1: 263.
Altevogt, R. 1955. Beobachten und Untersuchungen an Indischen
Winkerkrabben. Z. Morph. Oekol. Tiere. 43: 501.
Altevogt, R. 1957. Untersuchungen zur Biologic und Physiologic Indischer
Winkerkrabben. Z. Morph. Oekol. Tiere 46:1.
Anderson, J. A. R. 1964. The structure and development of the peat swamps
of Sarawak and Brunei. Malay. J. Trop. Geogr. 18: 7-16.
Anderson, J. A. R. 1964. Observations on climatic changes in peat swamp
forests in Sarawak. Comm. For. Rev. 43: 145-158.
119
-------
GERALD E. WALSH
Anon. 1904. Mangrove barks, and leather tanned with these barks, from
Pemba and Zanzibar. Bull. Imper. Instit., London, Sept., pp. 163-166.
Anon. 1951. Three hundred homes blackened by mangrove root gas. Miami
Herald, Nov. 14.
Anon. 1957. Establishment of mangroves. Rep. For. Admin., Malaya, No. 34.
Anon. 1958. Poisoning of Exoecaria agallocha. Rep. For. Dept., Fiji, No. 7.
Anoh. 1961. Mangrove gas kills two men in Fort Lauderdale ditch. Miami
News, July 28.
Anon. 1965. New fruit-fly found in mangroves. Aust. Nat. Hist. 15: 60.
Arbelaez, E. P. 1956. Plantas utiles de Colombia. Libreria Colombiana,
Bogota, pp. 642-643.
Arenes, J. 1954. Rhizophoraceae Madagascarienses nova. Not. Syst. 15: 14.
Areschoug, F. W. C. 1902a. Untersuchungen uber den Blattbau der
Mangrovepflanzen. Bibl. Bot. 56: 1-90.
Areschoug, F. W. C. 1902b. Om bladbyggnaden hos Mangrove-vaxterna. Bot.
Notiser, 1902, pp. 129-140.
Areschoug, F. W. C. 1904. Zur Frage der Salzausscheidung der
Mangrovepflanzen und anderer mit ihnen zusammen wachsender
Strandpflanzen. Flora, Marburg 93: 155-160.
Argo, V.' N. 1953. Root growth claims soil from the sea: mangrove
(Rhizophora and Avicennia) spreads by unique adaptation. Nat. Hist. 72:
52-55.
Armstrong, P. W. 1954. Shorelines and coasts of the Gulf of Mexico. U. S.
Dept. Int. Fish. Bull. 89: 39-65.
Arnott, G. W. 1838. On the Rhizophorae. Ann. Nat. Hist. 1: 359-374.
Artz, T. 1936. Die Kutikula einiger Afrikanischer Mangrove Pflanzen. Ber.
Dtsch. bot. Ges. 54: 247-260.
Ascherson, P. 1903. Der nordlichste Fundort der Mangrove in Agypten. Bot.
Z. 61, Abt.S, pp. 235-238.
Asenjo, C. F. and J. A. Goyco. 1942. El aceite del almendro tropical. Puerto
Rico Dept. Agr. Com. Bol. Mens. 1: 5-7.
Ashby, W. C. and N. C; Beadle. 1957. Studies in Halophytes, III. Salinity
factors in the growth of Australian saltbushes. Ecology 38: 344-352.
Ashton, P. S. 1969. Speciation among tropical forest trees: some deductions
in the light of recent evidence. Biol. J. Linn. Soc. London 1: 155-196.
Asprey, G. F. 1959. Vegetation in the Caribbean area. Caribb. Q. 5: 245-263.
Asprey, G. F. and R. G. Robbins. 1953. The vegetation of Jamaica. Ecol
Monogr. 23: 359-412.
Atkinson, M. R., G. P. Findlay, A. B. Hope, M. G. Pitman, H. D. W. Saddler,
and K. R. West. 1967. Salt regulation in the mangroves Rhizophora
mucronata Lam. and Aegialitis annulata R. Br. Aust. J. Biol. Sci. 589-599.
Aubert, G. 1954. Les sols hydromorphes d'Afrique Occidental Francaise.
Trans. 5th Int. Congr. Soil Sci., pp. 447-450.
Aubreville, A. 1964. Problems de la mangrove d'hier et d'aujourd'hui
Addisonia 4: 19-23.
120
-------
ECOLOGY OF HALOPHYTES
Ayers, A. D. 1951. Seed germination as affected by soil moisture and salinity.
Agron. J. 44: 82-84.
Axelrod, D. I. 1952. A theory of angiosperm evolution. Evolution 6: 29-60.
Backer, G. A. and C. G. G. J. van Steenis. 1951. Sonneratiaceae. Fl. Males. 1:
280-289.
Bacon, P. R. 1967. Life in the estuaries of the Caroni Swamp. J. Biol. Univ.
West Indies, Trinidad 2: 10-13.
Bacon, P. R. 1970. The ecology of Caroni Swamp, Trinidad. Spec. Publ.
Central Statistical Off., Trinidad, 68 pp.
Bacon, P. R. 1971c. Plankton studies in a Caribbean estuanne environment.
Caribb. J. Sci. ^: 81-89.
Bacon, P. R. 1971b. The maintenance of a resident population of Balanus
eburneus (Gould) in relation to salinity fluctuations in a Trinidad
mangrove swamp. J. Exp. Mar. Biol. Ecol. 6: 189-198.
Bacon, P. R. 1971C. Studies on the biology and cultivation of the mangrove
oyster in Trinidad with notes on other shellfish resources. Trop. Sci. 12:
265-278.
•Baillaud, E. 1912. La situation et la produktion des matieres tannates
tripicals. J. d'Agric. Trop. 12: 105-107.
Baillon, H. 1875. Combretacees. Hist. PI. 6: 260-283.
Baillon, H. 1875. Rhizophoracees. Hist. PI. 6: 284-304.
Baker, R. T. 1915. The australian grey mangrove. J. R. Soc. N.S.W. 49: 257.
Bakhuizen van der Brink, R. C. 1921. Revisio generis Avicenniae. Bull. Jard.
bot. Buitenz., Ser. 3, 3: 199-226.
Baltzer, F. 1969. Les formations vegetales associees au delta de la Dumbea
(Nouvelle Caledonie). Cah. ORSTOM, Ser. Geol. 1,1: 59-84.
Banerji, J. 1958a. The mangrove forests of the Andamans. Trop. Silvic. 20:
319-324.
Banerji, J. 1958b. The mangrove forests of the Andamans. World For. Congr.
3: 425-430.
Banijbatana, D. 1957. Mangrove forest in Thailand. Proc. 9th Pac. Sci. Congr.,
Bangkok, pp. 22-34.
Barbour, M. G. 1970. Is any angiosperm an obligate halophyte? Am. Midi.
Nat. 84: 105-120.
Barbour, W. R. 1942. Forest types of tropical America. Carrib. For. 3:
137-150.
Barnard, R. C. and G. G. K. Setten. 1947. Investigation scheme for growth
and increment studies in Jahore mangrove forest. Malay. For. 16: 40.
Barroweliff, M. 1912. Chemical notes on some malayan economic products.
Malay. Agric. J. 1: 178.
Barry, J. P., L. C. Kiet, and V. V. Cuong. 1961. La vegetation des plages
vasosablonneuses de la presquile de Cam Ranli. Ann. Fac. Sci. 1961, pp.
129-140.
Bascope, F., A. L. Bernardi, R. N. Jorgenson, K. Huek, H. Lamprecht, and P.
121
-------
GERALD E.WALSH
Martinez. 1959. Descripciones de arboles forestales No. 5. Los manglares
en America. Inst. for. lat. Invest. Capacit. Merida 5: 1-53.
Baxter, P. H. 1968. Vegetation notes on Eraser Island. Queensland Nat. 19:
11-20.
Baylis, G. T. S. 1940. Leaf anatomy of the New Zealand mangrove. Trans. R.
Soc. New Zealand 70: 164-170.
Beadle, N. C. W. 1952. Studies in halophytes. I. The germination and
establishment of the seedlings of five species of Atriplex in Australia.
Ecology 33: 49-62.
Beard, J. S. 1944. The natural vegetation of the island of Tabago, British West
Indies. Ecol. Monogr. 14: 135-163.
Beard, J. S. 1944. Climax vegetation in tropical America. Ecology 25:
127-158.
Beard, J. S. 1946. The Natural Vegitation of Trinidad. Clarendon Press,
Oxford, 152pp.
Beard, J. S. 1955. The classification of tropical American vegetation types.
Ecology 36: 89-100.
Beard, J. S. 1967a. An inland occurrence of mangrove. West Austr. Nat. 10:
112-115.
Beard, J. S. 1967b. Some vegetation types of tropical Australia in relation to
those of Africa and America. J. Ecol. 55: 271-290.
Becking, J. H., L. G. den Berger, and H. W. Meindersma. 1922. Vloed-of
mangrovebosschen in Ned.-Indie. Tectona. 15: 561-611.
Behrens Motta, A. 1960. Estudios preliminares para la produccion de extracto
curtiente de corteza de mangle (Rhizophora mangle). La Escuela de
Farmacia 20: 28-32.
Belcher, C. and G. P. Smooker. 1934. Birds of the colony of Trinidad and
Tobago. Ibis 4: 572-595.
Benecke, W. and A. Arnold. 1931. Kulturversuche mit Keimlingen von
Mangrovepflanzen.Planta(Berl.) 14: 471-481.
Bennett, H. and R. D. Coveney. 1959. Mangrove bark from Sarawak. Trop.
Sci. 1: 116-130.
Benson, A. A. and M. R. Atkinson. 1967. Choline sulphate and phosphate in
salt secreting plants. Fed. Proc. 26: 394.
Bentham, G. 1859. Synopsis of Legnotideae, a tribe of Rhizophoraceae. Proc.
Linnean Soc. Bot. 3: 65-80.'
Bentham, G. and J. D. Hooker. 1865a. Rhizophorae. Gen. PI. 1: 677-683.
Bentham, G. and J. D. Hooker. 1865b. Combretaceae. Gen. PI. 1: 683-690.
Bergman, H. F. 1954. Oxygen deficiency as a cause of disease in plants. Bot.
Rev. 25:417-485.
Bernstein, L. 1964. Salt tolerance of plants. U.S.D.A. Bull. 283. Govt. Print.
Off., Washington, D. C.
Bernstein, L. and H. E. Hayward. 1958. Physiology of salt tolerance. Ann.
Rev. Plant Physiol. 19: 25-46.
Berry, A. J. 1963. Faunal zonation in mangrove swamps. Bull. Singapore
122
-------
ECOLOGY OF HALOPHYTES
Natl. Mus. 32: 90-98.
Berry, A. J. 1968. Fluctuations in the reproductive condition of Cassidula
auris felis, a malayan mangrove ellobiid snail. (Pulmonata, Gastropoda). J.
Zool. 154! 377-390.
Berry, A. J., S. C. Loong, and H. H. Thum. 1967. Genital systems ofPythia,
Cassidula and Auricula (Ellobiidae, Pulmonata) from malayan mangrove
swamps. Malacol. Soc. London Proc. 375: 325.
Berry, E. W. 1913. A fossil flower from the Eocene. Proc. U. S. Natl. Mus.
45: 261-263.
Berry, E. W. 1924. The Middle and Upper Eocene floras of southeastern
North America. U. S. Geol. Surv. Prof. Pap. 92: 85-87 and 189-190.
Berry, E. W. 1930. Revision of the Lower Eocene Wilcox Flora of the
southeastern states. U. S. Geol. Surv. Prof. Pap. 156: 1-196.
Berry E. W. 1936. Miocene plants from Colombia, South America. Bull.
Torrey Bot. Club 63: 53-66.
Beschel, R. E. and P. J. Webber. 1962. Gradient analysis in swamp forests.
Nature 194: 207-209.
Bews, J. W. 1916. The vegetation of Natal. Ann. Natal Mus. 2: 253-331.
Bews, J. W. 1920. The plant ecology of the coast belt of Natal. Ann. Natal
Mus. 4: 368-469.
Bharucha, F. R. and B. S. Navalkar. 1942. Studies in the ecology of
mangroves. 3. The chloride-content of sea-water, soil-solution and the leaf
cell-sap of mangroves. J. Univ. Bombay 10: 97-106.
Bharucha, F. R. and V. S. Shirke. 1947. A study of the important katabolic
changes in the seedlings of Avicennia officinalis Linn. J. Univ. Bombay 15:
1-14.
Biebl, R. 1962. Protoplasmatisch-okologische Untersuchungen an
Mangrovealgen von Puerto Rico. Protoplasma 55: 572-606.
Biebl, R. 1964. Temperaturresistenz Tropischer Pflanzen auf Puerto Rico.
Protoplasma 59: 133-156.
Biebl, R. and H. Kinzel. 1965. Blattbau und Salzhaushalt von Laguncularia
mcemosa (L) Gaertn. f. und anderer Mangrovenbaume anf Puerto Rico.
Ost.bot. Z. 112: 56-93.
Bigarella, J. J. 1946. Contribuicao do estudo da planicie littornea do Estado
do Parana. Agric. de Biol. Tecnol. 1: 75-111.
Birch, W. R. 1963. Observations on the littoral and coral vegetation of the
Kenya coast. J. Ecol. 51: 603-615.
Biswas, K. 1927. Flora of the saltmarshes. J. Dept. Sci. Univ. Calcutta 8:
1-47.
Biswas, K. 1934. A comparative study of Indian species of Avicennia. Notes
Bot. Card. Edinburgh 18: 159-166.
Blake, S. T. 1940. The vegetation of Goat Island and Bird Island in Mareton
Bay. Old. Nat. 11:94-101.
Blake, S. T. 1968. The plants and plant communities of Fraser, Moreton, and
Stradbroke Islands. Old Nat. 19: 23-30.
123
-------
GERALD E. WALSH
Blatter, E. 1905. The mangrove of Bombay presidency and its biology. J.
Bombay Nat. Hist. Soc. 16: 644-656.
Blatter, E. 1908. On the flora of Cutch. I. J. Bombay Nat. Hist. Soc. 18:
756-777.
Blatter, E. 1909. On the flora of Cutch. II. Bombay Nat. Hist. Soc. 19:
157-176.
Blomquist, H. L. and L. R. Almodovar. 1961. The occurrence of Gelidiella
tenuissima Feldm. et Hamel in Puerto Rico. Nova Hedwiga 111: 67-69.
Blum, G. 1941. Uber osmotische Untersuchungen in der Mangrove. Ber.
Schweiz. bot. Ges. 51: 401-420.
Boaler, S. B. 1959. Conocarpus lancifolius Engler in Somaliland Protectorate.
Empire For. Rev. 38: 371-379.
Boer, N. P. de, Th. van der Hammen, and T. A. Wijmstra. 1965. A
palynological study on the age of some borehole smaples from the
Amazonas delta area, N. W. Brazil. Geol. Mijnb. 44: 254-258.
Boergesen, F. 1909. Notes on the shore vegetation of the Danish West Indian
Islands. Bot. Tidsskrift. 29: 201-259.
Boergesen, F. and O. Poulsen. 1900. La vegetation des Antilles danoises. Rev.
gen. deBot. 12: 99.
Boffey, P. M. 1971. Herbicides in Veitnam: AAAS study finds widespread
devastation. Science 171: 43-47.
Bole, P. V. and F. R. Bharucha. 1954. Osmotic relations of the leaves of
Avicennia alba Bl. J. Univ. Bombay 22: 50-54.
Bond, G. 1956. A feature of the root nodules of Casuarina. Nature 177: 192.
Booberg, G. 1933. Die malayische Strandflora-ein Revision der
Schimperschen Artenliste. Bot. Jb. 66: 1-38.
Bonfils, P. and J. Faure. 1961. Etude des sols due Bao Bolon. Agron. Trop.
(Maracay) 16: 127-147.
Boone, R. S., M. Chudnoff, and E. Goytia. 1969. Chemical control of
Bostrichidae during air drying of fence posts. U. S. For. Ser. Res. Pap. IFT,
No. 8: 1-8.
Boughey, A. S. 1957. Ecological studies of tropical coast-lines. I. The Gold
Coast, West Africa. J. Ecol. 45: 665-687.
Boughey, A. S. 1963. Dating of tropical coastal regression (based on roots of
mangrove (Rhizophora mucronata). Nature 200: 600.
Bouillene, R. 1930. Un voyage botanique dans le Bas-Amazone. In Une
Mission Biologique Beige au Bresil 2: 13-185. Imprimerie Medicale et
Scientifique, Bruxelles.
Bouillene, R. and F. Went. 1933. Reserches experimentales sur la
neoformation des racines les plantules et les boutures de plantes
superecures. Ann. Jard. Bot. Buitz. 43: 25.
Bournot, K. 1913. Gewinnung von Lapachol aus dem Kernholz von Avicennia
tomentosa. Archiv der Pharm. 1913: 351-356.
Bower, C. A. and C. H. Wadleight. 1948. Growth and cationic accumulation
by four species of plants as influenced by various levels of exchangeable
124
-------
ECOLOGY OF HALOPHYTES
sodium. Soil Sci. Am. Proc. 13: 218-223.
Bowman, H. H. M. 1915. Report on botanical work at the Tortugas
Laboratory for the season of 1915. Carnegie Inst. Wash. Yearb No. 14, p
200.
Bowman, H. H. M. 1916a. Report on botanical investigation at the Tortugas
Laboratory, season 1916. Carnegie Inst. Wash. Yearb. No. 15, pp. 188-192.
Bowman, H. H. M. 1916b. Physiological studies on Rhizophora. Proc. Natl.
Acad. U. S. 2: 685-688.
Bowman, H. H. M. 1917. Ecology and physiology of the Red Mangrove. Proc.
Am. Philos. Soc. 56: 589-672.
Bowman, H. H. M. 1918. Botanical ecology of the Dry Tortugas. Carnegie
Inst. Wash., Pub. No. 252, Pap. Dept. Mar. Biol., vol. 12, paper V, pp.
109-138.
Bowman, H. H. M. 1921. Histological variations in Rhizophora mangle. Pap.
Michigan Acad. Sci. 22: 129-134.
Brandis, D. 1898. Combretaceae. Nat. Pflanzenfam. III. 7: 106-130.
Brass, L. J. 1938. Botanical results of the Archibold Expeditions. IX. Notes
on the vegetaion of the Fly and Wassi Kussa Rivers, British New Guinea. J.
Arnold Arb. 19: 175-190.
Breen, C. M. and B. J. Hill. 1969. A mass mortality of mangroves in the Kosi
estuary. Trans. R. Soc. S. Africa 38: 285-303.
Breen, C. M. and I. D. Jones. 1969. Observations on the anatomy of foliar
nodes of young Bruguiera gymnorrhiza. J. S. Afr. Bot. 35: 211-218.
Brelie, G. v. d. and M. Teichmuller. 1953. Beitrage zur Geologie El Salvadors.
III. Mikroskopische Beobachtungen an Mangrove - Sedimenten aus El
Salvador. Neues Jb. Geol. Paleontol., Mh., No. 6, pp. 244-251.
Brenner, W. 1902. Ueber die Luftwurzeln von Avicennia tomentosa. Ber.
Dtsch. bot. Ges. 20: 175-189.
Briolle, C. E. 1969. Le cocotier sur les terres de mangrove au Cambodge:
amenagement de plantations familiales. Oleagineaux 24: 545-549.
Broekhuysen, G. J. and H. Taylor. 1959. The ecology of South African
Estuaries. Part viii: Kosi Bay system. Ann. S. Afr. Mus. 44: 279-296.
Bronnimann, P. and L. Zaninetti. 1965. Notes sur Lituola salsa (Gushman et
Bronnimann 1948), un Foraminifere da la mangrove de 1'ile de la Trinitie,
W. I. Arch. Sci. 18: 608-615.
Brown, D. S. 1971. Ecology of Gastropoda in a south African mangrove
swamp. Proc. Malacol. Soc. London. 39: 263-279.
Brown, J. M. A., H. A. Outred, and C. F. Hill. 1969. Respiratory metabolism
in mangrove seedlings. Plant Physiol. 44: 287-294.
Brown, R. 1814. In Matthew Flinders' A Voyage to Terra Australis, Vol. II,
Appendix No. III. G. and W. Nicol, London.
Brown, W. H. and A. F. Fischer. 1918. Philippine mangrove swamps.
Philippines Dept. Agrjc. Nat. Resources Bull. No. 17, pp. 1-132.
Brown, W. H. and A. F. Fischer. 1920. Philippine mangrove swamps.
125
-------
GERALD E. WALSH
Philippines Bur. For. Bull. No. 22, pp. 9-125.
Browne, F. G. 1955. Forest trees of Sarawak and Brunei and their products.
Sarawak Gov. Printer, Kuching.
Bruwnell, f. F. and J. G. Wood. 1957. Sodium, an essential micro-nutrient for
Atriplex vesicaria Reward. Nature. 179: 635-636.
Brunnich, J. C. and F. Smith. 1911. Some Queensland mangrove barks and
other tanning materials. Old Agric. 27: 86.
Buchanan, J. 1874. On the flowering plants and ferns of Chatham Islands.
Trans. N. Z. Inst. 7: 333-341.
Buckley, T. A. 1929. Mangrove bark as a tanning material. Malay. For. Rec.
7,40 pp.
Bunning, E. 1944. Botanische Beobachtungen in Sumatra. Flora 137:
334-344.
Burkill, I. H. 1935. A dictionary of the Economic products of the Malay
Peninsula. 2 vols., 2402 pp. Publ. by Governments of the Straits
Settlements and the Federated Malay States, London.
Burkholder, P. R. and L. M. Burkholder. 1958. Studies on B. vitamins in
relation to productivity of the Bahia Fosforescente, Puerto Rico. Bull. Mar.
Sci. Gulf Caribb. 8: 201-223.
Burkholder, P. R. and L. R. Almodovar. Species composition and
productivity of algal mangrove communities in Puerto Rico. In press Q. J.
Fla. Acad. Sci.
Burtt, Davy, J. 1938. The classification of woody vegetation types. Imperial
For. Inst. Pap. No. 13, Oxford.
Calderon, S. E. and P. C. Stanley. 1941. Flora Salvadorena, 2nd. Ed., El
Salvador.
Candolle, A. P. de. 1828a. Combretaceae, Prodr. Syst. Nat. 3: 9-24.
Candolle, A. P. de 1828b. Rhizophoraceae, Prodr. Syst. Nat. 3: 31-34.
Carey, G. 1934. Further investigations on the embryology of viviparous seeds.
Proc. Linn. Soc. N. S. W. 59: 392-410.
Carey, G. and L. Fraser. 1932. The embryology and seedling development of
Aegiceras majus Gaertn. Proc. Linn. Soc. N. S. W. 57: 341-360.
Carlquist, S. 1967. The biota of long distance dispersal. V. Plant dispersal to
Pacific islands Bull. Torrey Bot. Club 94: 129-162.
Carter, G. S. 1930. The fauna of the swamps of Paraguayan Chaco in relation
to its environment. I. Physicochemical nature of the environment. J. Linn.
Soc. London, Zool. 37: 206-258.
Carter, J. 1959. Mangrove succession and coastal change in South-West
Malaya. Trans. Inst. Br. Geogr. 26: 79-88.
Case, G. 0. 1938. The use of vegetation for coast protection. Agric. J. Br.
Guiana 9: 4-11.
Catala, R, L. A. 1957. Report on the Gilbert Islands: some aspects of human
ecology. Atoll Res. Bull. 59: Oct. 31.
Cawkell, E. M. 1964. The utilization of mangroves by African birds. Ibis 106:
251-253.
126
-------
ECOLOGY OF HALOPHYTES
Chandler, M. E. J. 1951. Notes on the occurrence of mangroves in the
London clay. Proc. Geol. Assoc. 62: 271-272.
Champion, H. G. 1936. A preliminary survey of the forest types of india and
Burma. Indian Forest Rev. Bot. 1, 265 pp.
Chang, Hung-ta, Chao-Chang Chang, and Paisun Wang. 1957. The mangrove
vegetation of the Liochow Peninsula. Sunyatsenia Nat. Sci. Ed. 1957:
122-143 (in Chinese).
Chapman, V. J.' 1936. The halophyte problem in the light of recent
investigations. Q. Rev. Biol. 11: 209-220.
Chapman, V. J. 1940. The botany of the Jamaica shoreline. Geogr. J. 96:
312-323.
Chapman, V. J. 1942. A new perspective in halophytes. Q. Rev. Biol. 17:
291-373.
Chapman, V. J. 1944a. 1939 Cambridge University Expedition to Jamaica. I.
A study of the botanical processes concerned in the development of the
Jamaican shore-line. J. Linn. Soc. London Bot. 52: 407-447.
Chaprnan, V. J. 1944b. 1939 Cambridge University Expedition to Jamaica. II.
A study of the environment of Avicennia nitida Jacq. in Jamica. J. Linn.
London Soc. Bot. 52: 448-486.
Chapman, V. J. 1944c. 1939 Cambridge University Expedition to Jamica. III.
The morphology of Avicennia nitida Jacq. and the function of its
pneumatophores. J. Linn. Soc. London Bot. 52: 487-533.
Chapman, V. J. 1962a. Respiration studies of mangrove seedlings. I. Material
and some preliminary experiments. Bull. Mar. Sci. Gulf Caribb. 12:
137-167.
Chapman, V. J. 1962b. Respiration studies of mangrove seedlings. II.
Respiration in air. Bull. Mar. Sci. Gulf Caribb. 12: 245-263.
Chapman, V. J. 1966a. Some factors involved in mangrove establishment. In
Les problemes scientifiques des deltas de la zone tropicale humide et leurs
implications. Colloque de Dacca. UNESCO, Paris, pp. 219-225.
Chapman, V. J. 1966b. Vegetation and salinity. In Salinity and Aridity, H.
Boyko (ed.), Junk Publishers, The Hague, Netherlands, pp. 23-42.
Chapman, V. J. 1969. Lagoons and mangrove vegetation, in Lagunas Costeras,'
un Simposio, Mem. Simp. Intern. Lagunas Costeras. UNAM-UNESCO, Nov.
28-30, 1967. Mexico, D. F., pp. 505-514.
Chapman, V. J. 1970. Mangrove phytosociology. Trop. Ecol. 11: 1-19.
Chapman, V. J. and J. W. Ronaldson. 1958. The mangrove and salt-grass flats
of the Auckland Isthmus. N. Z. Dept. Sci. Ind. Res., Bull. 125, 79 pp.
Chapman, V. J. and C. B. Trevarthen. 1953. General schemes of classification
in relation to marine coastal zonation. J. Ecol. 41: 198-204.
Chatelain, G. 1932. L'exploitation forestiere a la Guayane Francaise. Actes e
Comp. Rendus de 1'Association Colonies-Sciences, Paris 8: 217-222.
Chattaway, M. M. 1932. The wood of the Sterculiaceae. 1. Specialization of
the vertical wood parenchyma within the sub-family Sterculieae. New
127
-------
GERALD E. WALSH
Phytol. 31: 119-132.
Chattaway, M. M. 1938. The wood anatomy of the family Sterculiaceae. Phil.
Trans. R. Soc. B. 228: 313-365.
Chatterjee, D. 1958. Symposium on mangrove vegetation. Sci. Cult. 23:
329-335.
Chaudhri, 1.1.1967. The vegetation of Karachi. Vegetatio 10: 229-246.
Chayapongse, C. 1955. Yield of mangrove forest. Vanasarn For. J. 2, April
1955.
Cherrie, G. K. 1891. Notes on Costa Rican birds. Proc. U. S. Natl. Mus. 14:
517-537.
Chevalier, A. 1924. Exploitation des Paletuviers a tanin et leur valeur d'apres
quelques travaux recents. Rev. Bot. Appl. Agric, Colon 4: 320.
Chevalier, A. 1929. Possibilite de cultiver le Bananier nain sur les terrains a
Paletuviers ou Bresil et en Afrique occidentale. Rev. Bot. Appl. Agric.
Colon 9: 334-337.
Chevalier, A. 1931. Graines d'Avicennia
comme aliment de famine. Rev. Bot. Appl. Agric. Colon 11: 1000.
Chidester, G. H. and E. R. Schafer. 1959. Pulping of Latin American woods.
Products Lab. Rep. No. 2012, Madison, Wisconsin, 11 pp.
Chiovenda, E. 1929. Flora Somala. Rome, 436 pp.
Chipp, C. G. 1921. A list of the fungi of the Malay Peninsula. Bull. Straights
Settlements 11: 311-418.
Chipp, T. F. 1931. The vegetation of northern tropical Africa. Scot. Geog.
Mag. 47: 193-214.
Chowdhury, K. A. 1934. The so-called terminal parenchyma cells of the
wood of Terminalia tomentosa W. and A. Nature 133: 215.
Christian, C. S. and G. A. Stewart. 1953. General report on a survey of the
Katherine-Darwin region, 1946. CSIRO Austr. Land Res. Ser. 1.
Christophersen, E. 1935. Flowering plants of Samoa.-B. P. Bishop Museum
Bull. No. 128, Honolulu, Hawaii.
Ciferri, R. 1939. La associazioni de litorale marine della Somalia meridionale.
Riv. Biol. Colon No. 2.
Clarke, L. D. and N. J. Hannon. 1967. The mangrove swamp and salt marsh
communities of the Sydney district. I. Vegetation, soils and climate. J.
Ecol. 55:753-771.
Clarke, L. D. and N. J. Hannon. 1969. The mangrove swamp and salt marsh
communities of the Sydney district. II. The holocoenotic complex with
particular reference to physiography. J. Ecol. 57: 213-234.
Clarke, L. .D. and N. J. Hannon. 1970. The mangrove swamp and salt marsh
communities of the Sydney district. III. Plant growth in relation to salinity
and waterlogging. J. Ecol. 58: 351-369.
Clarke, L..P. and N. J. Hannon. 1971. The mangrove swamp and salt marsh
communities of the Sydney district. IV. The significance of species
interaction. J. Ecol. 59: 535-553.
Coaldrake, J. E. 1961. The ecosystem of the coastal'lowlands (Wallum) of
128
-------
ECOLOGY OF HALOPHYTES
southern Queesland. CSIRO Austr. Bull. No. 28, 138 pp.
Cockayne, L. 1958. The vegetation of New Zealand. Hafner Pub. Co., New
York, 456 ppb.
Coelho, P. A. 1965-1966. Os crustaceos decapodos de alguns manguezais
pernambucanos. Trab. Inst. Oceanogr., Univ. Fed. Pernambuco, Recief
(7/8): 71-89,
Cogger, H. G. 1959. Australain goannas. Aust. Mus. Mag. 13: 71-75.
Cohen, E. 1939. The marine angiosperms of Inhaca Island. S. Afr. J. Sci. 36:
246.
Collins, M. I. 1921. On the mangrove and salt marsh vegetation near Sydney,
N. S. W., with special reference to Cabbage Creek, Port Hacking. Proc.
Linn. Soc. N. S. W. 46: 376-392.
Colman, J. 1940. Zoology of the Jamaican shoreline. Geogr. J. 96: 323-327.
Compere, P. 1963. The correct name of the Afro-American black mangrove
(Avicenniagerminans L., Verbenaceae). Taxonomy 13: 150-152.
Connor, D. J. 1969. Growth of grey mangrove (Avicennia marina) in nutrient
culture, Biotropica 1: 36-40.
Cook, E. P. and R. C. Collins. 1903. Economic plants of Puerto Rico. Cont.
U.S.Natl. Herb. 8:2.229-230.
Cook, M. T. 1907. The embryology of Rhizophora mangle. Bull. Torrey Bot.
Club 34: 271-277.
Cook, M. T. 1908. The hypertrophied fruit of Bucida buceras. Bull. Torrey
Bot. Club 35: 305-306.
Cooper, R. E. and S. A. Pasha. 1935a. Osmotic and suction pressure of some
species of mangrove vegetation. J. Indian Bot. Soc. 14: 109-120.
Cooper, R. E. and S. A. Pasha. 1935b. Osmotic pressure and the H-ion
concentration of sea weeds in relation to those of sea-water. J. Indian Bot.
Soc. 14: 3.
Comer, E. J. H. 1940. Wayside trees of Malaya, Vol. 1. Government Printer,
Singapore, 772 pp.
Cotton, B. C. 1943. Australian Gastropoda of the families Hydrobiidae,
Assimineidae, and Acmaeidae. Trans. R. Soc. Austr._66: 124-129.
Cotton, B. C. 1950. An old mangrove mud-flat exposed by wave scouring at
Glenelg, South Australia. Trans. R. Soc. S. Austr. 73: 59-61.
Craighead, F. C. 1964. Land, mangroves and hurricanes. Fairchild Trop. Card.
Bull. 19: 5-32.
Craighead, F. C. 1968. The role of the alligator in shaping plant communities
and maintaining wildlife in southern Everglades. Florida Nat. 41: 2-7.
Craighead, F. C. 1969. Vegetation and recent sedimentation in Everglades
National Park. Fla. Nat. 42: 157-166.
Craighead, F. C. 1971. The Trees of South Florida. Vol. 1. The Natural
Environments and Their Succession. Univ. Miami Press, Coral Gables, 212
pp.
Craighead, F. C. and V. C. Gilbert. 1962. The effects of hurricane Donna on
129
-------
GERALD E. WALSH
the vegetation of southern Florida. Q. J. Fla. Acad. Sci. 25: 1-28.
Crane, J. 1941. Eastern Pacific expeditions of the New York Zoological
Society. XXVI. Crabs of the genus Uca from the west coast of Central
America. Zoologia, N. Y. 26: 145-208.
Crane, J. 1947. Intertidal brachygnathous crabs from the west coast of
tropical America with special reference to ecology. Zoologia, N. Y. 31:
69-95.
Creager, D. B. 1962. A new Cercospora ofRhizophora mangle. Mycologia 54:
536-539.
Crossland, C. 1903. Note on the dispersal of mangrove seedlings Ann. Bot.
Fenn. 17: 267-270.
Cruz, A. A. de la and J. F Banaag. 1967. The ecology of a small mangrove
patch in Matabung Kay Beach, Batangas Province. Univ. Philipp. Nat. Appl.
Sci. Bull. 20: 486-494.
Cuatrecasas, J. 1947. Vistazo a la vegetacion natural del calima. Acad.
Colombiana de Cient. Exact. Fis. y Nat. Rer. 7: 306-312.
Cuatrecasas, J. 1952. Mangroves of the Pacific coast of South America.
Abstract of a series of lectures presented before the Department of Botany
Seminar, Northwestern University, by the botanical staff of the- Chicago
Natural History Museum (cited in West, 1956).
Cuatrecasas, J. 1958a. Introduction al estudia de los manglares. Boln. Soc.
Bot. Mex. 23: 84-98.
Cuatrecasas, J. 1958b. Aspectos de la vegetacion natural de Colombia. Rev.
Acad. Colombiana Cienc. Exactas, Fis., Nat. 10: 221-264.
Curtis, A. H. 1888. How the mangrove forms islands. Card. For. 1: 100.
Daiber, F. C. 1960. Mangroves: The tidal marshes of the tropics. Univ.
Delaware Estuarine Bull. 5: 10-15.
Daiber, F. C. 1963. Tidal creeks and fish eggs. Univ. Delaware Estuarine Bull.
7: 6-14.
Dale, I. R. 1938. Kenya mangroves. Z. Weltforstwirtsch. 5: 413-421.
Danhof, G. N. 1946. Rotation and management of mangrove in Riouw-Singga
Archipelago. Tectona 36: 59-72.
Dabsereau, P. 1947. Zonation et succesion sur la restinga de Rio de Janeiro. I.
La holosere. Rev. Can. Biol. 6: 448-477.
Dansereau, P. 1957. Biogeography, an Ecological Perspective. Ronald Press.
New York, 394pp.
Darteville, E. 1949. Les mangroves d'Afrique equatorial. Atti. Acad. Ligure.
Sci. Lett. 6: 1-48.
Darteville, E. 1950. Les mangroves de Congo et les autres mangroves
d'Afrique occidentale. Bull. Seances Inst. R. Coll. Beige 21: 946-971.
Davies, J. B. 1967. The distribution of sand flies (Culicoides supp.) breeding
in a tidal mangrove swamp in Jamaica and the effects of tides on the
emergence of C. furens Poey and C. barbosai (Wirth and Blanton). West
Indies Med. J. 16: 39-48.
Davies, J. B. 1969. Effect of felling mangroves on emergence of Culicoides
130
-------
ECOLOGY OF HALOPHYTES
spp. in Jamaica. Mosq. News 29: 566-571.
Davis, C. C. 1966. Notes on the ecology and reproduction of Trichocorixa
reticulata in a Jamaican salt-water pool. Ecology 47: 850-852.
Davis, C. C. and R. H. Williams. 1950. Brackish water plankton of mangrove
areas in southern Florida. Ecology~31: 519-531.
Davis, J. H., Jr. 1938. Mangroves, makers of land. Nat. Mag. 31: 551-553.
Davis, J. H. 1939a. Vivipary and dispersal of mangrove seeds. J. Tennessee
Acad. Sci. 15: 415.
Davis, J. H. 1939b. The role of mangrove vegetation in land building in
southern Florida. Am. Phil. Soc. Yearb., 1938, pp. 162-164.
Davis, J. H. 1940a. Peat deposits of Florida. Fla. Geol. Sur. Bull. 30: 1-247.
Davis, J. H. 1940b. The ecology and geologic role of mangroves in Florida.
Pap. from the Tortugas Lab. Vol. 32. Carnegie Inst. Wash. Pub. No. 517,
pp. 303-412.
Davis, J. H. 1942. The ecology of the vegetation and topography of the sand
keys-of Florida. Pap. from the Tortugas Lab. Vol. 33. Carnegie Inst. Wash.
Pub. No. 524, pp. 113-195.
Davis, J. H. 1943. The natural features of southern Florida. Florida Dept.
Conserv. Geol. Bull. No. 25. 311 pp.
Davis, J. H. 1945. Jamaican shoreline ecology. Ecology 26: 312.
Davy, J. B. 1938. The classification of tropical woody vegetation-types.
Imperial For. Inst., Univ. Oxford, Inst. Pap. No. 13. 85 pp.
Dawson, E. Y. 1954. Marine plants in the vicinity of the Institut
Oceanographique de Nha Trang, Viet Nam. Pac. Sci. 8: 372-481.
Day, J. H. 1951. The ecology of South African estuaries, Part I. General
considerations. Trans. R. Soc. S. Afr. 33: 53-91.
Day, J. H., N. A. H. Millard, and A. D. Harrison. 1952. The ecology of South
African estuaries. Part VII. The biology of Durban Bay. Ann. Natal Mus.
13: 259.
Day, J. H., N. A. H. Millard, and G. J. Broekhuysen. 1953. The ecology of
South African estuaries. Part IV. The St. Lucia system. Trans. R. Soc.
South Africa 34: 129.
Day, J. H. and J. F. C. Morgans. 1956. The ecology of South African
estuaries. Part VIII. The biology of Durban Bay. Ann. Natal Mus. 13: 259.
Decaisne, J. 1835. Observations sur quelque nouveau generes et especes des
plantes de 1'Arabie-Hereuse. Rhizophoraceae. Ann. des Sci. Nat., Bot.,
Paris, pp. 75-77.
Degener, 0. 1946. Flora Hawaiiensis, Books 1-4. The Patton Col., Honolulu.
Deignan, H. G. 1961. Type specimens of birds in the United States National
Museum. U. S. Natl. Mus. Bull. 221, 718 pp.
Delevoy, G. 1945. Les mangroves africaines. Bull. Soc. Cient. For. Belg. 52:
84-89.
Delf, E. M. 1912. Transpiration in succulent plants. Ann. Bot. 26: 409-412.
Dent, J. M. 1947. Some soil problems in empoldered rice lands in Sierra
Leone. Empire J. Exp. Agric. 15: 206-212.
131
-------
GERALD E. WALSH
Derijard, R. 1965. Contribution a 1'etude du peuplement des sedments
sable-vaseux, et vaseux intertidaux, compactes ou fixes par la vegetation de
la region de Tulear (Madagascar). Rec. Trav. Stat. Mar. Endoume. Fasc. h.
s. suppl. No. 43in-8, 94 pp.
Detwiler, S. B. 1948. Some basic information regarding tannin and
tannin-yielding crops, prepared for use by tannin-research workers. U. S.
Off. For. Agric. Washington, D. C. 112 pp.
D'Hoore, J. 1963. Soil Map of Africa (5th revision). C.C.T.A./C.S.A., Joint
Project No. 11, Leopoldville,
Dickerman, R. W. and G. Gavino T. 1970. Studies of a nesting colony of
green herons at San Bias, Mexico. Living Bird 8: 95-111.
Dickerman, R. W. and C. Juarez L. 1971. Nesting studies of the boat-billed
heron Cochlearius cochlearius at San Bias, Nayarit, Mexico. Ardea 59:
1-16.
Dickey, D. R. and A. J. van Rossem. 1938. The birds of El Salvador. Field
Mus. Nat. Hist. Publ. Zool. Ser. 23.
Diels, L. 1915. Vegetationstypen vom untersten Kongo. Vegetations-bilder
12, VIII, 16pp.
Ding Hou. 1957. A conspectus of the genus Bru.gu.iera (Rhizophoraceae).
Nova Guinea (n.s.) 8: 163-171.
Ding Hou. 1958. Rhizophoraceae. F. Males. I. 5: 429-493.
Ding Hou. 1960. A review of the genus Rhizophora with special reference to
the Pacific species. Blumea 10: 625-634.
Ding Hou. 1965. Studies in the Flora of Thailand. Rhizophoraceae. Dansk.
Bot. Arkiv. 23: 187-190.
Dodd, J. R. and C. T. Siemers. 1971. Effect of Late Pleistocene karst
topography on Holocene sedimentation and biota, lower Florida Keys.
Geol. Soc. Am. Bull. 82: 211-218.
Doderlein, L. 1881. Botanische Mitteilungen aus Japan. Bot. Centrabl. 8:
27-31.
Dolianiti, E. 1955. Frutos de Nipa no Palaeocene de Pernambuco. Div. Geol.
e Mineral Brasil Bol. 158: 1-36.
Doyne, H. C. 1937. A note on the acidity of mangrove swamp soils. Trop.
Agric. Trinidad 14: 236-237.
Doyne, H. C. and R. R. Glanville. 1933. Some swamp rice growing soils Sierra
Leone. Trop. Agric. Trinidad 10: 132-138.
Drabble, E. 1908. The bark of the red and the white mangroves. Q. J. Instit.
Comm. Res. Tropics 3: 33-39.
Drabble, E. and L. Hilda. 1905. The osmotic strength of cell-sap in plants
growing under different conditions. New Phytol. 4: 8.
Drabble, E. and M. Nierenstein. 1907. A note on the West-African mangroves.
Q. J. Instit. Comm. Res. Tropics 2.
Drakenstein, H. A. Rheede tot. 1678-1703. Hortus indicus malabaricus. 12
vols. (Sonneratiaa, vol. 3; Aegiceras, Rhizohora, and Bruguiera, vol. 6,
Avicennia, vol. 4). Van Someren and van Dijk, Amsterdam.
132
-------
ECOLOGY OF HALOPHYTES
Drar, M. 1933. A note on some plants of Gebel Elba: Avicennia officinalis.
Hart. Rev. Egypt. Hart. Soc. 21: 7-10.
Dubois, J. 1954. Sur une classification de sols de delta soumis a des influences
salines appliquee au bas-Senegal. Proc. 2nd. Int. Afr. Soil Conf. 2:
1119-1124.
Dugros, M. 1937. Le domaine forestier inonde de la Cochine. Bull. Econ. de
PIndochine 40: 283-314.
Durand, J. H. 1964. La mise en valeur des mangroves de la cote N. W. de
Madagascar. Bull. AFES Versailles 5: 200-206.
Durant, C. C. L. 1941. The growth of mangrove species in Malaya. Malay.
For. 10:3-15.
Eddy, W. H. and B. Sokoloff. 1953. Mangrove meal as a cattle food
ingredient. Southern Bio-Research Lab., Fla. Southern Col., Lakeland,
Florida.
Edwall, P. W. 1877. Enseio para una synonimia dos nomes populares das
plantas indigenas do estado de Sao Paulo. Sao Paulo, Boletin No. 16.
Eggers, H. von. 1877. Rhizophora mangle L. Vidensk. Meddel. Dansk
Naturhist. Foren. Kjobenhaven 28: 177-181.
Eggers, H. von. 1892. Die Manglares in Ecuador. Bot. Centralb. 52: 49-52.
Eggers, H. von. 1894. Das Kustenbebiet von Ecuador. Geog. Gesell. Bremen
17: 265-289.
Egler, F. E. 1948. The dispersal and establishment of red mangrove in
Florida. Caribb. For. 9: 299-310.
Egler, F. E. 1952. Southeast saline Everglades vegetation, Florida, and its
management. Veg. Acta Geobot. 3: 213-265.
Egler, F. E. 1961. A cartographic guide to selected regional vegetation
literature — where plant communities have been described. II. Southeastern
United States. Sarracenia 6: 1-87.
Eisley, L. 1971. The Night Country, Charles Scribner's Sons, New York, 240
pp.
Ekman, S. 1953. Zoogeography of the Sea. Sidgewick and Jackson Ltd.,
London, 417 pp.
Emery, K. 0. and R. E. Stephenson. 1957. Estuaries and lagoons, in Treatise
on Marine Ecology and Paleoecology. Vol. 1, J. S. Hedgepeth (ed.). Geol.
Soc. Am. Mem. 67: 673-750.
Engler, A. 1876. Rhizophoraceae in Martius. Flora Brazil. 12: 426.
Engler, A. 1921. Die Pflanzenwelt Afrikas. Erster Band, I Heft;Driter Band,
II Heft. Leipzig.
Engler, A. and L. Diels. 1899. Monographieen Afrikanisher Pflanzenfamilien
und - gattungen. III. Combretaceae-Comfcretum. 116 pp., Leipzig.
Engler, A. and L. Diels. 1900. Monographieen Afrikanisher Pflanzenfamilien
und - gattungen. IV. Combretaceae excl. Combretum. 44 pp. Leipzig.
Enns, T. 1965. Tracer studies of water and sodium transport in mangroves
(Laguncularia racemosa). Havalradets Dkr. 48: 161-163.
Epstein, E., W. E. Schmid, and D. W. Rains. 1963. Significance and technique
133
-------
GERALD E. WALSH
of short-term experiments on solute absorption by plant tissue. Plant Cell
Physiol 4: 79-84.
Erichson, R. 1921. Die Mangrove-vegetation ihre Verbreitung und ihre
Bedeuting fur Schwemmland-bildungen. Ungerdrukte Diss.
(Maschinenschrift), Halle.
Erichson, R. 1925. Die Mangrove-Vegetation. Z. f. Naturfr. 16: 190-199.
Ernould, M. 1921. Recherches anatomiques et physiologiques sur les racines
respiratoires. Mem. Acad. R. de Bel. 6: 1-52.
Exell, A.W. 1931. The genera of Combretaceae. J. Bot. 69: 113-128.
Exell, A. W. 1954. Combretaceae. Fl. Males 1: 533-589.
Exell, A. W. 1958. Combretaceae. Ann. Mo. Bot. Gar. 45: 143-164.
Exell, A. W. and C. A. Stace. 1966. Revision of the Combretaceae. Bol. Soc.
Broteriana 40: 5-25.
Faber, F. C. von. 1913. Uber Transpiration und osmotischen Druch bei den
Mangroven. Ber. Dtsch. bot Ges. 31: 277-286.
Faber, F. C. von. 1923. Zur Physiologic der Mangroven. Ber. Dtsch. bot. Ges.
41:227-234.
Fairbridge, R. W. and C. Treichert. 1947. The rampart system at Low Isles.
Rep. Great Barrier Reef Comm. 6: 1016.
Fanshawe, D. 1948. Forest products of British Guiana. Part I. Principal
timbers. For. Bull. 1 NS, For. Dept. Brit. Guiana, 39 pp.
Fanshaw, D. 1950. Forest products of British Guiana. Part II. Minor forest
products. For. Bull. 2 NS, For. Dept. Brit. Guiana, pp. 16-18,49.
Fanshaw, D. 1952. The Vegetation of British Guiana, A Preliminary Review.
Pap. For. Inst., Oxford, 96 pp.
Feldman, J. and R. Lami. 1936. Sur la vegetation de la mangrove a la
Guadelope. C. R. Acad. Sci. (Paris) 203: 883-885.
Feliciano, C. 1962. Notes on the biology and economic importance of the
land crab Cardiosoma guanhumi Latreille of Puerto Rico. Contrib. Inst.
Mar. Biol. Univ. Puerto Rico, Mayaguez, 1-29.
Fenner, C. and J. B. Cleland. 1932. The geography and botany of the
Adelaide coast. S. Aust. Nat. 14: 45-48, 55-56, 109-120, 128-133.
Fernandos, D. A. 1934. Ueber Mangroven Cultuven. Tectona 27: 299-303.
Ferns, G. W. 1955a. Tidal forests in southeast Kalimantan. Indian J. For 4-
3-5.
Ferns, G. W. 1955b. Hutan pajan sekitar Kalimantantenggara (Tidal forests of
S. E. Borneo). Rimba Indonesia Penerb. pop. 4: 90-98.
Ffrench, R. P. 1966. The utilization of mangroves by birds in Trinidad Ibis
108: 423-424.
Ffrench, R. P. 1970. The scarlet Ibis in Surinam and Trinidad Living Bird 9'
147-165.
Field, G. D. 1968. Utilization of mangroves by birds on the Freetown
peninsula, Sierra Leone. Ibis 110: 354-357.
Finucane, J. H. 1965. Threadfish in Tampa Bay, Florida. Q. J. Fla Acad Sci
28: 267-270.
134
-------
ECOLOGY OF HALOPHYTES
Fischer, P. H. 1940. Notes sur les peuplements littoraux d'Australia. III. Sur
le faune de la Mangrove australienne. Mem. Soc. Biogeogr. 7: 315-329.
Fisher, C. E. C. 1927. Contributions to the flora of Burma, 2. Kew Bull.
1927: 81-94.
Flamm, B. R. and J. H. Cravens. 1971. Effects of war damage on the forest
resources of South Vietnam. J. For. 69: 784-790.
Fly, L. B. 1952. Preliminary pollen analysis of the Miami, Florida, area. J.
Allergy 23: 48-57.
Forattini, 0. P. 1957. Culicoides daaregiao neotropical (Diptera,
Ceratopogonidae). Arch. Hig. Sao Paulo 11, 526 pp.
Forattini, 0. P., E. X. Rabello, and D. Pattoli. 1958. Culicoides de regiao
neotropical. 2. Observacoes sobre biologia em conicoes naturais. Arch. Hig.
Sao Paulo 12: 1-52.
Fosberg, F. R. 1947. Micronesian mangroves. J. N. Y. Bot. Card. 48:
128-138.
Fosberg, F. R. 1953. Vegetation of central Pacific atolls. Atoll Res. Bull. 23,
Sept. 30.
Fosberg, F. R. 1960. The vegetation of Micronesia: 1. General description of
the vegetation of Guam. Bull. Am. Mus. Nat. Hist. 119: 1-75.
Fosberg, F. R. 1961. Vegetation-free zone on dry mangrove coasts. U. S.
Geol. Soc. Prof. Pap. No. 424D, pp. 216-218.
Fosberg, F. R. 1964. Vegetation as a geological agent in tropical deltas. In Les
problems scientifiques des deltas de la zone tropicale humide et leurs
implications. Colloque de Dacca. UNESCO, Paris, pp. 227-233.
Fourmanoir, P. 1953. Notes sur la faune de la mangrove dans la region de
Majunga: crabs, crevettes, poissons. Nat. Malgache 5: 87-92.
Foxworthy, F. W. 1910. Distribution and utilization of the mangrove-swamp
of Malaya. Ann. Jar. Bot. de Buitz., Suppl. 3, Part I, pp. 319-344.
Foxworthy, F. W. and D. M. Matthews. 1916. Mangrove and nipa swamps of
British North Borneo. For. Bull. British N. Borneo 1: 1-67.
Freise, F. W. 1932. Brasilianische Gerbstoffpflanzen und ihre Ausbeutung.
Tropenpflanzen 35: 70-74.
Freise, F. W. 1935. A importancia de conversazao dos mangues como viveiros
de peixes. Anais do 1° Congr. Nac. de Presca, Rio de Janeiro, 1934. pp.
315-319.
Freise, F. W. 1938. Untersuchungen am Schlick der Mangrovekuste Brasiliens.
ChemiederErde2: 333-355.
Freyberg, B. von. 1930. Zerstorung und Sedimentation and der
Mangrovekuste Brasiliens. Leopoldina 6: 69-118.
Fryckberg, M. 1945. Mangrove bark. Agric. Am. 5: 199.
Fuchs, H. P. 1970. Ecological and palynological notes on Pelliciem
rhizophoraee. Acta Bot. Need. 19: 884-894.
Ganapati, P. N. and M. V. L. Rao. 1959. Incidence of marine borers in the
mangroves of the Godavari estuary. Curr. Sci. 28: 332.
Gardner, C. A. 1923. Botanical notes from the Kimberly division of Western
135
-------
GERALD E. WALSH
Australia. W. Aust. For. Dept., Bull. No. 32, Government Printer, Perth,
Australia.
Gardner, C. A. 1942. The vegetation of Western Australia with special
reference to the climate and soils. J. Proc. R. Soc. West Austral. 28:
xi-lxxxvii.
Gehrmann, K. 1911. Zur Blutenbiologie der Rhizophoraceae. Ber. Dtsch. hot.
Ges. 29: 308-318.
Genkel, P. A. 1963. K. ecologii restanii mangrov. In Fizioligiya drevesnykh
rastenii. Akad. Nauk SSR, Moscow. Referat. Zh. Biol. No. 7V151, pp.
223-231
Genkel, P. A. and I. S. Fan. 1958. On the physiological significance of
viviparity in mangrove plants. (In Chinese: Russian summary). Acta Bot.
Sin. 7: 51-71.
Gerlach, S. A. 1958. Die Mangroveregion tropischer Kusten als Lebensraum.
Z. Morph. Okol. Tiere 46: 636-731.
Gerlach, S. A. 1963. Okologische Bedeutung der Kuste als Grenzraum
zwischen Land und Meer. Naturw. Rdsch. 16: 219-227.
Germeraad, J. H., C. A. Hopping, and J. Muller. 1965. Palynology of Tertiary
• sediments from tropical areas. Rev. Palaeobotan. Palynol. 6: 139-348.
Gessner, F. 1955. Die Mangroven. Hydrobotanik, Band I. Veb Deutscher
Verlag der Wissenchaften, Berlin^!? pp., pp. 249-262.
Gessner, F. 1967. Untersuchungen an der Mangrove in Ost-Venezuela. Int.
Rev. Ges. Hydrobiol. 52: 769-781.
Giglioli, M. E. C. and I. Thornton. 1965a. The mangrove swamps of Keneba,
Lower Gambia River basin. I. Descriptive notes on the climate, the
mangrove swamps and the physical composition of their soils. J. appl. Ecol.
2; 81-103.
Giglioli, M. E. C. and I. Thornton. 1965b. The mangrove swamps of Keneba,
Lower Gambia River basin. II. Sulphur and pH in the profiles of swamp
soils. J. appl. Ecol. 2: 257-269.
Giglioli, M. E. C. and D. F. King. 1966. The mangrove swamps of Keneba,
Lower Gambia River basin. HI. Seasonal variations in the chloride and
water content of swamp soils, with observations on the water level and
chloride concentration of free soil water under a barren mud flat during
the dry season. J. appl. Ecol. 3: 1-19.
Gill, A. M. 1969. Tidal trees: orient and Occident. Bull. Fairchild Trop. Card.
My 1969, pp. 7-10.
Gill, A. M. 1970. The mangrove fringe of the Eastern Pacific. Bull. Fairchild
Trop. Card. July 1970, pp. 7-11.
Gill, A. M. Endogenous control of growth-ring development in Avicennia.
For. Sci. in press.
Gill, A. M. and P. B. Tomlinson. 1969. Studies on the growth of red
mangrove (Rhizophora mangle L.). I. Habit and general morphology.
Biotropica 1: 1-9.
Gill, A. M. and P. B. Tomlinson. 1971. Studies on the growth of red
136
-------
ECOLOGY OF HALOPHYTES
mangrove (Rhizophora mangle L.). II. Growth and differentiation of aerial
roots. Biotropica 3: 63-77.
Gill, A. M. and P. B. Tomlinson. 1971. Studies on the growth of red
mangrove. (Rhizophora mangle L.). III. Phenology of the shoot.
Biotropica, 3: 109-124.
Gilmour, A. J. 1965. The implication of industrial development on the
ecology of a marine estuary. Victoria Fish. Wildl. Dept. Fish Contrib. 20:
1-12.
Gilroy, A. B. and L. J. Chwatt. 1945. Mosquito-control by swamp drainage in
the coastal belt of Nigeria. Ann. Trop. Med. Parasitol. 39: 19-40.
Glanville, R. R. and H. C. Doyne. 1933. Some swamp soils of Sierra Leone.
Trop. Agric. 10: 132.
Classman, S. R. 1952. Flora of Ponape. B. P. Bishop Mus. Bull. No. 209,
Honolulu, Hawaii.
Gleason, H. A. and M. T. Cook. 1927. Plant ecology of Puerto Rico:
Scientific survey of Puerto Rico and the Virgin Islands. Proc. N. Y. Acad.
Sci. 7: 1-96.
Gledhill, D. 1963. The ecology of the Aberdeen Creek mangrove swamp. J.
Ecol. 51:693-703.
Glynn, P. W. 1964. Common marine invertebrate animals of the shallow
waters of Puerto Rico. Historia Natural de Puerto Rico. Univ. Puerto Rico,
Mayaguez, pp. 12-20.
Glynn, P. W., L. R. Almodovar, and J. C. Gonzalez. 1964. Effects of
Hurricane Edith on marine life in La Parguera, Puerto Rico. Caribb. J. Sci.
4: 335-345.
Goebel, K. von. 1886. Ueber die Rhizophoren-vegetation. Sitz. ber. d. Naturf.
Ges. Rostock. December 1886.
Goebel, K. von. 1889. Ueber die Luftwurzeln von Sonneratia. Ber. Dtsch.
bot. Ges. 5: 249-255.
Gokhale, A. V. 1962. Control of mangroves. Poona Agric. Col. Mag. 53: 30.
Gola, G. 1905. Studi sui rapporti de la distribuzione della piante e la
constituzione fisico-chimica del suolo. Ann. di Bot. 3: 455-512.
Golley, F. B. 1969. Caloric value of wet tropical forest vegetation. Ecology
50: 517-519.
Golley, F., H. T. Odum, and R. F. Wilson. 1962. The structure and
metabolism of a Puerto Rican red mangrove forest in May. Ecology 43:
9-19.
Gomez-Pompa, A. 1966. Estudios botanicos en la region de Misantla,
Veracruz, Publ. Inst. Mex. Rec. Nat. Renov. Mexico, 173 pp.
Gonzales, J. G. and T. E. Bowman. 1965. Planktonic copepods from Bahia
Fosforescente, Puerto Rico, and adjacent waters. Proc. U. S. Natl. Mus.
117:241-304.
Gonzalez Ortega and J. Candelon. 1934. Rhizophora mangle L. Bol.
Pro-Cultura Regional, Mazatlan, Mexico 1: 14-16.
Gooch, W. L. 1944. Survey of tannin resources in Mexico. J. For. 43: 56-58.
137
-------
GERALD E. WALSH
Good, R. 1953. The Geography of Flowering Plants. 2nd Ed. Longmans,
Green and Co., London, xiv, 452 pp.
Gooding, E. G. B., A. R. Loveless, and G. R. Proctor. 1965. Flora of
Barbados. H.M.S.O., London, 486 pp.
Goossens, H. J. 1936. Tambakherverkaveling in het ressort legal de
Binnenvisserij. Landbouw 12: 191-200.
Gosh, D. 1967. Mangrove: A potential Indian tanning material. Tanner
(Bombay) 21.379-383.
Graham, B. M. 1929. Notes on the mangrove swamp of Kenya. J. E. Afr. and
Uganda Nat. Hist. Soc. 29: 157-164.
Graham, C. L. 1934. Flora of the Kratabo region, British Guiana. Ann.
Carnegie Mus. 22: 204.
Graham, J. 1929. Mangroves of East Africa. J. East Afr. Nat. Hist. Soc. 29:
157.
Graham, S. A. 1964. The genera of Rhizophoraceae and Combretaceae in the
southeastern United States. J. Arnold Arbor. 45: 285-301.
Grant, D. K. S. 1938. Mangrove woods of Tanganyika territory, their
silviculture and dependent industries. Tanganyika Notes and Rec. 1938
(April), pp. 5-15.
Grass, T. H. 1904. Forstatatistik fur die Waldungen des Ruflyideltas,
angefangen im Jahre 1902. Ber. uber Land-u. Forstwirtsch (Deutsch
Ostafrika)2: 165-186.
Gray, J. 1960. Temperate pollen genera in the Eocene (Clairborne) flora,
Alabana. Science 132: 808-810.
Greenway, H. 1962. Plant responses to saline substrates. III. The offsets of
nutrient concentration on the growth and ion uptake ofHordeum vulgare
during a sodium stress. Aust. J. Biol. Sci. 16: 616-628.
Greenway, H. and A. Rogers. 1963. Growth and ion uptake of Agrophyron
elongatum on saline substrates, as compaied with a salt-tolerant variety of
Hordeum vulgare. Plant and Soil 18: 21-30.
Gregory, D. P. 1958. Rhixophoraceae. Ann. Miss. Bot. Card. 45: 136-140.
Grewe, F. 1941. Afrikanische Mangrovelandschaften, Verbreitung und
wirtschaftsgeographische Bedeutung. Wiss. Veroff. D. Mus. F. Landerk. N.
F. 9: 103-177.
Griffith, A. L. 1950. Working scheme for the mangroves of the Zanzibar
Protectorate. Gov. Printer, Zanzibar, 42 pp.
Groom, P. and S. E. Wilson. 1925. On the pneumatophores of paludal species
of Amoora, Goaurapa and Heritiera. Ann. Bot. 39: 9-24.
Guillaumin, A. 1964a. Rhizophoraceae. Scientific Results of the French and
Swiss Botanical Mission to New Caldonia (1950-1952). HI. Mem. Mus. Nat.
Hist. Natur. Nouv. Ser., Ser. B. Bot. 15: 76-77.
Guillaumin, A. 1964b. Revision des Rhizophoracees. Not. Syst. 3: 55.
Guppy, H. B. 1906. Observations of a Naturalist in the Pacific Between 1896
and 1899. Vol. II, Plant Dispersal. Macmillan and Co., Ltd., London. 627
pp.
138
-------
ECOLOGY OF HALOPHYTES
Guppy, H. B. 1917. Plants, Seeds and Currents in the West Indies and Azores.
Macmillian and Co., Ltd., London. 531 pp.
Gurke, A. N. 1895/1897. Notizen uber die Verwertung der Mangroverinde als
Gerbmaterial. Not. Konigl. Bot. Card. Mus. Berlin, 1895/1897, No. 1.
Haan, J. H. De. 1931. Het een en auder over de Tjilatjapsche vloedbosschen.
Tectona 24: 39-76.
Haberlandt, G. 1895. Uber die Ernahrung der Keimlange und die Bedeutung
des Endosperms bei viviparen Mangrovepflanzen. Ann. Jar. Botan. Buiten.
12: 105-114.
Haberlandt, G. 1910. Botanische Tropenreise; Indo-Malayische
Vegetationsbilder und Reiseskizzen. Wilhelm Engelman, Leipzig, 296 pp.
Haden-Guest, S. J. K. Wright, and E. M. Teclaff. 1956. A World Geography of
Forest Resources. The Ronald Press Co., New York, 736 pp.
Hagen, B. 1890. Die Pflanzen-und Thierwalt von Deli auf der Ostkuste
Sumatra. Tidj. van het Kon. Nederl. Aardijks. Gen. 7: 1-240.
Hager, L. C. 1959. Anatomical study of Avicennia marina (Forsk.) Vieih.
(Formerly called A. officinalis). M. S. Botany Dept., Univ. Witwatersrand,
Johannesburg.
Hamilton, A. A. 1919. An ecological study of the salt marsh vegetation of the
Port Jackson District. Proc. Linn. Soc., N.S.W. 44: 463-513.
Hammen, T. van der. 1962. A palynological study on the Quaternary of
British Guiana. Leidse Geol. Meded. 29: 125-180.
Hammen, T. van der and T. A. Wijmstra. 1964. A palynological study on the
Tertiary and Upper Cretaceous of British Buiana. Leidse Geol. Meded. 30:
183-241.
Hammer, L. 1961. Sobre la ecologia de aqua de lus mangles. Bol. Inst.
Oceanogr. 1: 249-261.
Hammer, H. E. 1929. The chemical composition of Florida Everglades peat
soils, with special reference to their inorganic constituents. Soil Sci. 28:
1-14.
Hammond, W. 1971. A case for mangroves. Littoral Drift. 1: 2-5.
Harms, J. W. 1941. Beobachtungen in der Mangrove Sumatras, insbesondere
uber Lebensweise und Entwicklung der Solzlandturbellarie Stylochus sp.
und der Oncis stuxbergi. Z. wiss. Zool. 154: 389-408.
Harms, J. W. and 0. Dragendorff. 1933. Die Realisation von Genen und die
consecutive Adaption. 3. Mitt. Osmotische Untersuchungen an Phycosoma
lurco Sel. und DeMan aus den Mangrove-Vorl andern der Sumatra Inseln.
Z. wiss. Zool. 143: 263-322.
Harper, R. M. 1917. Geography of central Florida. Fla. St. Geol. Surv., 18th
Ann. Rep., pp. 27-276.
Harper, R. M. 1927. Natural resources of southern Florida. Fla. Geo. Surv.
Ann. Rep. 18: 27-192.
Harrer, F. 1939. Die Mangroven des Rufiji-Deltas in Deutsch-Ostafrika.
Kolonialforstl. Mitt. 2: 160.
Harris, J. A. and J. V. Lawrence. 1917. The osmotic concentration of the sap
139
-------
GERALD E. WALSH
of the leaves of mangrove trees. Biol. Bull. 32: 202-211.
Harrison, S. G. 1961. Notes on the vegetation of the coastal region of British
Guiana. J. R. Agric. Comm. Soc. B. G. 40: 93-104.
Harrisson, T. 1965. Some quantitative effects of vertebrates on the Borneo
flora, in Symp. Ecolo. Res. Humid Trop. Veg. UNESCO Sci. Coop. Of.
Southeast Asia, Kuching, Sarawak, pp. 164-169.
Harshberger, J. W. 1908. The comparative leaf structure of the sand dune
plants of Bermuda. Proc. Am. Phil. Soc. 47: 97-110.
Harshberger, J. W. 1914. The vegetation of south Florida, south of 27°30'
North, exclusive of the Florida Keys. Wagner Free Inst. Sci. Philadelphia
Trans. 7: 49-189.
Hart, M. G. R. 1959. Sulphur oxidation in tidal mangrove soils of Sierra
Leone. Plant and Soil 11: 215-236.
Hart, M. G. R. 1962. Observations on the source of acid in empoldered
mangrove soils. I. Formation of elemental sulphur. Plant and Soil 17:
87-98.
Hart, M. G. R. 1963. Observations on the source of acid in empoldered
mangrove soils. II. Oxidation of soil polysulphides. Plant and Soil 19:
106-114.
Hartmann, G. 1956a. Zur Kenntnis des Mangrove-Estero-Gebietes von El
Salvador und seiner Ostracoden-Fauna I. Meeresforsch. 12: 219-248.
Hartmann, G. 1956b. Zur Kenntnis des Mangrove-Estero-Gebietes von El
Salvador und seiner Ostracoden-Fauna. II. Meeresforsch. 13: 134-159.
Hartman-Schroder, G. 1959. Zur Oekologie der Polychaete des
Mangrove-Estero-Gebietes von El Salvador: Beitr. z. neotrop. Fauna 1:
69-183.
Hartnoll, R. G. 1965. Notes on the marine grapsoid crabs of Jamaica. Proc.
Linn. Soc. London 176: 113-147.
Hatheway, W. H. 1953. The land vegetation of Arno Atoll, Marshall Islands.
Attoll Res. Bull. 16, April 30.
Hanheway, W. H. 1955. The natural vegetation of Canton Island, an
equatorial Pacific atoll. Res. Bull. 43, Aug. 15.
Haug, T. A. 1909. Die Mangrove Deutsch-Ostafrikas. Naturw. Z. Forst-u.
Landwirt. 7:413-425.
Haverschmidt, F. 1962. Notes on some Surinam breeding birds. II. Ardea 50'
173-179.
Haverschmidt, T. 1965. The utilization of mangroves by South American
birds. Ibis 107: 540-542.
Haverschmidt, F. 1969. Notes on the Boat-billed Heron in Surinam. Auk 86:
130-131.
Hayata, B. 1912. Rhizophoraceae. Icones Plant. Formosarum 2: 15-16.
Heald, E. 1971. The production of organic detritus in a south Florida estuary.
Univ. Miami, Sea Grant Tech. Bull. No. 6, 110 pp.
Heald, E. 1970. The contribution of mangrove swamps to Florida Fisheries.
Proc. Gulf Caribb. Fish. Inst., 22nd Ann. Session, pp. 130-135.
140
-------
ECOLOGY OF HALOPHYTES
Heiden, H. 1893. Anatomische Characteristik der Combretaceen. Bot.
Centralbl. 55: 353-391.
Heiden, H. 1893. Anatomische Characteristik der Combretaceen. Bot.
Centralbl. 56: 1-12, 65-75,129-136, 163-170, 193-200, 225-230, 355-360,
385-391.
Hellmayer, C. E. and B Conover. 1948. Catalogue of birds of the Americas.
Field Mus. Nat. Hist. Publ. Zool. Ser. 8, Part 1, No. 2.
Hemsley, W. B. 1894. The flora of the Tonga or Fnendly Islands. J. Linn.
Soc. London Bot. 30: 176.
Henckel, P. A. 1963. On the ecology of the mangrove vegetation. Mitteil. d.
forist.-soziol. Srbeitsgem., N.F. 10: 201-206.
Henkel, J. S., S. St. C. Ballenden, and A. W. Bayer. 1936. Account of the
plant ecology of the Dukuduku forest reserve and adjoining areas of the
Zululand coast belt. Ann. Natal Mus. 8: 95-123.
Herbert, D. A. 1932. The relationships of the Queensland Flora. Proc. R. Soc.
Queensland 44: 2-22.
Herklots, G. A. C. 1961. The Birds of Trinidad and Tobago. Collins Press,
London, 287 pp.
Hesse, P. R. 1961a. Some differences between the soils of Rhizophora and
Avicennia mangrove swamps in Sierra Leone. Plant and Soil 14: 335-346.
Hesse, P. R. 1961b. The decomposition of organic matter is a mangrove
swamp soil. Plant and soil 14: 249-263.
Hesse, P. R. 1962. Phosphorus fixation in mangrove swamp muds. Nature
193: 295-296.
Hesse, P. R. 1963. Phosphorus in mangrove swamp mud with particular
reference to aluminum toxicity. Plant and Soil 19: 205-218.
Heylighers, P. C. 1965. Lands of the Port Moresby-Kairuka area, Papua,-New
Guinea. VIII. Vegetation and ecology of the Port Moresby-Kairuka area.
C.S.I.R.O. Austr. Land. Res. Ser. 14: 146-173.
Hicks, G. H. 1894. Nourishment of the embryo and the importance of the
endosperm in viviparous mangrove plants. Bot. Gaz. 19: 327-330.
Hill, B. J. 1966. A contribution to the ecology of the Umalalazi estuary.
Zool. Afr. 2: 1-24,
Hill, T. G. 1908. Observations on the osmotic properties of the root ahirs of
certain salt marsh plants. New Phytol. 7: 133-142.
Hillier, J. M. 1907. Anti-opium plants. Kew Bull. 1907: 198-199.
Hillier, J. M. 1908. The malayan anti-opium plant. Kew Bull. 1908: 235-236.
Hillis, W. E. 1956. The production of mangrove extract in the delta region of
Papua. Empire For. Rev. 35: 220-236.
Hirakawa, Y. 1933. Synoekologische Studien uber die Strandflora der Insel
Formosa. 1. Rep. Taihoku Bot. Gar. 3: 1-47.
Hirakawa, Y. 1936. Mangrove vegetation in Takao. J. Taiwan Mus. Ass. 4: 1-5
(in Japanese).
Ho.Ching 1957. Ecology of mangrove plants. Biol. News 8: 1-5.
Ho, R. 1962. Physical geography of the Indo-Australian tropics, m, Impact of
141
-------
GERALD E. WALSH
Man on Humid Tropics Vegetation. Goroka, New Guinea, pp. 19-35.
Ho, T. 1899. Rhizophorae in Japan. Ann. Bot. 456468.
Hodge, W. H. 1956. The trees that walk to the sea. Nat. Mag. 49: 456.
Hoffmeister, J. E. and H. G. Multer. 1965. Fossil mangrove reefs of Key
Biscayne, Florida. Geol. Assoc. Am. Bull. 76: 845-852.
Hofmeister, W. 1859. Neuere Beobachtungen uber Embryobildung bei
Phanerogamen. Pring. Jahrb. 1: 82.
Holdridge, L. R. 1940. Some notes on the mangrove swamps of Puerto Rico.
Caribb. For. 1: 19-29.
Holdridge, L. R. 1942. Trees of Puerto Rico, USDA, For. Serv., Trop. For.
Experiment. Stat. Occas. Pap. No. 1.
Holdridge, L. R., L. V. Teesdale, J. E. Meyer, E. Z. Little, E. F. Horn, and J.
Marrero. 1947. The forests of Central Equador. U. S. For. Ser. Bull. 1947,
134pp.
Hpnel, Fr. von. 1882. Beitrage zur Pflanzenanatomie und Physiologie V. Zur
Anatomic der Combretaceen. Bot. Z. 40: 177-182.
Hooker, W. J. 1830. Terminalia catappa L. Bot. Mag. 57.
Horn, C. L. 1945. Plant resources of Puerto Rico, in Plants and Plant Science
in Latin America, F. Verdoorn (ed.), Chronica Botanica Co., Waltham,
Mass., pp. 83-84.
Horn, E. F. 1946. Brazilian tanning materials. Trop. Woods 88: 33.
Horn, E. F. 1948. Durability of brazilian corssties. Trop. Woods 93: 30-35.
Hosakawa, T. 1957. Outline of the mangrove and strand forests of the
micronesian islands. Mem. Fac. Sci. Kyushu U.ni. Ser. E, 2: 101-11.8, .
How Koon-Chew and Ho Chun-Nien. 1953. Rhizophoraceae in the Chinese
Flora. Acta Phytotaxon. Sin., Vol. 2.
Howard, R. A. 1950. Vegetation of the Bimini Group, Bahamas. Ecol.
Monogr. 20: 317-349.
Howe, M. A. 1911. A little-known mangrove of Panama. J. New York Bot;
Card. 12: 61-72.
Howes, F. N. 1953. Vegetable tanning materials. Butterworth's Sci. Publ.,
London, pp. 77-79.
Howmiller, R. and A. Weiner. 1968. A limnological study of a mangrove
lagoon in the Galapagos. Ecology 49: 1184-1186.
Huberman, M. A. 1957. Mangrove silviculture. Trop. Silvic. 13: 188-195.
Huek, K. 1969. Los mangalares de America. Description de arboles forstales,
No. 5. Bol Inst. For. Lat. Amer., Merida, Venezuela.
Huggard, E. R. 1.960. Logging road construction. Proc. 5th World For Congr
3:1921-1926. '
Hummel, K. 1930. Tierfahrtenbilder vom Tropenstrand. Natur und Mus. 60.
Hunter, J. B. 1970. A survey of the oyster population of the Freetown
estuary, Sierra Leone, with notes on the ecology, cultivation, and possible
utilization of mangrove oysters. Trop. Sci. 11: ,276-285.
Ito, T. 1899a. Rhizophoraceae in Japan. Ann. Bot. 13: 465-466.
Ito, T. 1899b. Mangroves growing in Japan. Nature 60: 79.
142
-------
ECOLOGY OF HALOPHYTES
Ivens, G. W. 1957. Arboricides for killing mangroves. W. Afr. Rice Res. Stat.
Per. Sci. Rep. No. 8, 6 pp.
Jackson, C. R. 1952. Some topographic and edaphic factors affecting
distribution in a tidal marsh. Q. J. Fla. Acad. Sci. 15: 137-146.
Jackson, G. 1964. Notes on west African vegetation: I. Mangrove vegetation
at Ikorodu, western Nigeria. West Afr. Sci. Assoc. J. 9: 98-111.
Jackson, M. L. 1958. Soil Chemical Analysis. Prentice-Hall, Englewood Cliffs,
N. J. 498 pp.
Jacques-Felix, H. 1957. LesRhizophora de la mangrove atlantique d'Afrique.
J. Agric. Trop. Bot. Appl. 4: 343-347.
Jakobi, H. 1955. O genero Enhydrosoma no manguezal da costa S.
Paulo-Parana (Harpacticoidea-Crustacea). Dusenia 6: 89-96.
Janssonius, H. H. 1950. Vessels in the wood of Javan mangrove trees. Blumea
6: 465-469.
Jardine, F. 1928. The topography of the Townsville littoral. Rep. Great
Barrier Reef Comm. 2: 70-87.
Jenik, J. 1967. Root adaptations in west African trees. J. Linn. Soc. London
Bot. 60: 136-140.
Jenik, J. and G. W. Lawson. 1968. Zonation of microclimate and vegetation
on a tropical shore in Ghana. Oilos 19: 198-205.
Jennings, D. H. 1968. Halophytes, succulence, and socium-a unified theory.
NewPhytol. 67: 899-911.
Jennings, J. N. and C. F. Bird. 1967. . Regional geomorphological
characteristics of some Australian estuaries, in Estuaries, G. H. Lauff (ed.),
AAAS, Washington, pp. 121-128.
Johnson, I. M. 1949. The botany of San Jose Island (Gulf of Panama).
SargentiaS: 1-306.
Johow, F. 1884. Vegetationsbilder aus West-Indien und Venezuela. I. Die
Mangrove-Sumpfe. Kosmos, Z. ges. Entwicklungs. 14: 415-426.
Joly, A. B. and N. Yamaguishi-Tomita. 1967. Dawsoniella bostrychiae: a new
parasite of mangrove algae. Sellowia 19: 63-70.
Jong, B. de. 1934. Ueber Mangrove Cultiven. Tectona 27: 258-298.
Jonker, F. P. 1942. Rhizophoraceae in Pulle. Fl. Suriname 3: 36.
Jonker, F. P. 1959. The genus Rhizophora in Suriname. Acta Bot. Neerl. 8:
58-60.
Jonker, H. A. J. 1933. De vloedbosschen van den Riouw lingga Archipel.
Tectona 26: 717-741.
Jordan, H. D. 1954. The development of rice research in Sierra Leone. Trop.
Agri. Trinidad 31: 27-32.
Jordan, H. D. 1959. The utilization of saline mangrove soils for rice growing.
Trans. 3rd int. Afr. Soils Conf., Dalaba, pp. 327-331.
Jordan, H. D. 1964. The relation of vegetation and soil to the development of
mangrove swamps for rice growing in Sierra Leone. J. appl. Ecol. 1:
209-212.
Jost, L. 1887. Kenntnis der Athmungsorgane der Pflanzen. Bot. Z. 45:
143
-------
GERALD E. WALSH
601-6o6, 617-628,633-642.
Kalk, M. 1959. The zoogeographies! composition of the intertidal fauna at
Inhaca Island, Mozambique. S. Afr. J. Sci. 55: 178.
Kalk, M. 1959. The fauna of the shores of northern Moxambique. Rev. de
Biol., Lisbon 2: 1.
Kanehira, R. 1938. On the genus Sonneratia in Japan. J. Jap. Bot. 14:
421424.
Karny, H. 1928a. Signs of life in mangrove vegetation of Java. Palaeobiologica
1: 475-580.
Karny, H. 1928b. Lebensspuren in der mangroveformation Javas (Bin Beitrag
zur Losung des Flyschproblems-Aus einum Briefan O. Abel).
Paleobiologica 1: 475-480.
Karsten, G. 1891. Ueber die Mangrove-Vegetation im Malayschen Archipel.
Eine Morphologische-biologische Studie. Bibl. Bot. 22: 1-71.
Karsten, G. 1905. Die Mangrovevegetation. Vegetationsbilder 2: 7-12.
Karyone, T. 1927. The mangrove of the South Sea Islands. J. Jap. Bot.
116-120.
Kassas, M. and M. A. Zahran. 1967. On the ecology of the Red Sea littoral
salt marsh, Egypt. Ecol. Monogor. 37: 297-316.
Kato, K. 1966a. Geochemical studies on the mangrove region of Cananeia,
Brazil. I. Tidal variations of water properties. Bol. Inst. Oceanogr. 15:
13-20.
Kato, K. 1966b. Geochemical studies on the mangrove region of Cannaneia,
Brazil. II. Physico-chemical observations on the reduction states. Bol. Inst.
Oceanogr. 15: 21-24.
Kawano, Y., H. Matsumoto, and R. Hamilton. 1961. Plant products of
economic importance in Hawaii. II. Tannins. Tech. Prog. Rep. No. 130,
Univ. Hawaii, Agric. Exper. Stn., Honolulu, pp. 1-14.
Keast, A. 1959. Australian birds, their zoogeography and adaptations to an
arid climate, in Biogeogrphy and Ecology in Australia, A. Deast, R. L.
Cracker, and C. S. Christian (eds.), pp. 89-114.
Keay, R. W. J. 1953. Rhizophora in west Africa. Kew Bull. 1: 121-127.
Kehar, N. D. and S. S. Negi. 1953. Mangrove leaves as cattle feed. Sci. Cult.
18: 382-382.
Kehar, N. D. and S. S. Negir. 1954. Mangrove leaves as cattle feed. Sci. Cult.
19: 556-557.
Keller, B. 1925. Halophyten und Zerophyten-studien. J. Ecol. 13: 224-261.
Kellett, J. 1969. Looking back on the Florida freeze of '62. Principle 13:
23-35.
Keng, H. 1952. Mangrove in Taiwan. Taiwan Agric. For. Month. 6: 38-39.
Kerr, A. 1928. Kaw Tao, its physical features and vegetation. J. Siam Soc
Nat. Hist. Suppl. 7: 137-149.
Khan, S. A. 1956. Mangrove forests of Andhra State. Proc. Silvic. Conf.,
DehraDun9: 182-186.
144
-------
ECOLOGY OF HALOPHYTES
Khan, S. A. 1961. Regeneration of Avicennia officinalis in the coastal forests
of West Pakistan. Pak. J. For. 11: 43-45.
Kiener, A. 1966. Contributions a 1'etude ecologique et biologique des eaux
saumatres malagaches. Les poissons euryhalins et leur role dans le
developpement des peches. Vie Milieu 16: 1013-1149.
Keinholz, R. 1928. Environmental factors of Philippine beaches with
particular reference to the beach at Puerto Galera, Mindoro. Philipp. J. Sci.
36: 199-213.
Kint, A. 1934. De Luchtfoto en de topografische terreingsteldheid in de
Mangrove. De Trop. Natuur 23: 173-189.
Kipp-Goller, T. 1940. Uber Bau and Entwicklung der viviparen
Mangrovekeimlinge. Z. f. Bot. 35: 1-40.
Koerner, T. 1904. Encorces tannantes de mangliers. J. Agric. Trop. 4: 113.
Kohlmeyer, J. 1966. Neue Meirespilze an Mangroven. Ber. Dtsch. bot. Ges.
79: 27-37.
Kohlmeyer, J. 1968. A new Trematosphaeria from roots of Rhizophorra
racemosa. Mycopathol. Mycol. Appl. 34: 1-5.
Kohlmeyer, J. 1969. Ecological notes on fungi in mangrove forests. Trans.
Brit. Mycol. Soc. 53: 237-250.
Kohlmeyer, J. 1968. Marine fungi from the tropics. Mycologia 40: 252-270.
Kohlmeyer, J. 1969. Marine fungi of Hawaii including the new genus
Heliascus. Can. J. Bot. 47: 1469-1487.
KohJmeyer, J. and E. Kohlmeyer. 1965. New marine fungi from mangroves
and trees along eroding shorelines. Nova Hedwiga 9: 89-104.
Kohlmeyer, J. and E. Kohlmeyer. 1971. Marine fungi from tropical America
and Africa. Mycologia 63: 831-863.
Kolwitz, R. 1933. Zur Okologie der Pflanzenwelt Brasiliens. Ber. Dtsch. bot.
Ges. 51: 396-406.
Koriba, K. 1958. On the periodicity of tree growth in the tropics. Card. Bull.
Singapore 17: 11-81.
Korner, A. 1900. Bericht uber weiter Untersuchungen von
Mangrove-Extrakten. Jahresbericht der Deutschen Gerberschule Freiberg in
Sachsen. Ferb. Freiberg, 1900.
Kostermans, A. J. G. H. 1959. Monograph of the genus Heritera Aitron
(Stercul.). Reinwardtia 4: 465-583.
Kostermans, A. J. G. H. 1961. A monograph of the genus Brownlo-wia Roxb.
(Tiliaceae). Communs For. Res. Inst. Bogor, No. 73, 62 pp.
Krejci-Graf, K. 1935. Beobachtungen am Tropenstrand I — IV.
Senckenbergiana 17: 1-242.
Krejci-Graf, K. 1937. Uber Fahrten und Bauten tropischer Krabben. Geol.
Meere und Binnengewasser 1: 39-46.
Kudo, Y. 1932. The mangrove of Formosa. Bot. Mag. Tokyo 46: 147-156.
Kuenzler, E. J. 1969. Mangrove swamp systems, in Coastal Ecological
Systems of the United States. H. T. Odum, B. J. Copeland and E. A.
McMahon (eds.), Inst. Mar. Sci. Univ. North Carolina, Vol. 1, pp. 353-383.
145
-------
GERALD E. WALSH
Kumar, L. S. S. and W. V. Joshi. 1942. False polyembryony in viviparous
Rhizophora mucronata Lam. Curr. Sci. 11: 242.
Kunkel, G. 1965. Der Standort: Kompetenzfactor in der Stelzwurzelhildung.
Biol. Centrablatt 84: 641-651.
Kunkel, G. 1966. Uber die Struktur und Sukzession der Mangrove Liberias
und deren Randformationen. Ber. Schweiz. bot. Ges. 75: 20-40.
Kylin, A. and R. Gee. 1970. Adenosine triphosphatase activities in leaves of
the mangrove Avicennia nitida Jacq. PI. Physiol. 45: 169-172.
Laessle, A. M. and C. H. Wharton. 1959. Northern extensions in recorded
ranges of plants on Seahorse and associated keys, Levy County, Florida. Q.
J. Fla. Acad. Sci. 22: 105-113.
Lafond, J. 1957. Apercu sur la sedimentologie de 1'estuaire de la Betsiboka
(Madagascar). Rev. Inst. Fr. Ret. 12: 425-431.
Lagerwerff, J. V. 1958. Comparable effects of absorbed and dissolved cations
on plant growth. Soil Sci. 86: 63-69.
Lakhanpal, R. W. 1952. Nipa sahnii, a palm fruit in the Tertiary of Assam.
Paleobotanist 1: 289-294.
Lamb, F. B. 1959a. Prospects for forest land management in Panama. Trop.
Woods 110: 16-28.
Lamb, F. B. 1959b. The coastal swamp forests of Navico, Colombia. Caribb.
For. 20:79-89.
Lambert, F. and P. Polk. 1971. Observations sur 1'ostreculture a Cuba.
Hydrobiologia38: 9-14.
Lamberti, A. 1969. Contribuicao ao Conhecimento de Ecologic das Plantas
do Manguez al de Itanhaem. Bol. No. 317, Bot. No. 23, Fac. Fil.,Cien. e
Let. Univ. Sao Paulo, Brazil, 217 pp.
Landon, F. H. 1933. Planting in mangrove forests. Malay. For. 2: 131-133.
Landon, F. H. 1942. Mangrove volume tables. Malay. For. 11: 117-123.
Langenheim, J. H., B. L. Hackner, and A. Bartlett. 1967. Mangrove pollen at
the depositional site of Oligo-Miocene amber from Chiapas, Mexico. Bot.
Mus. Lean. Harv. Univ. 21: 289-324.
LaRue, C. D. and T. J. Musik. 1951a. Mangrove vivipary. Rep. Fed. Exp. Stn.
Puerto Rico, No. 13.
LaRue, C. D. and T. J. Musik. 1951b. Does the mangrove really plant its
seedlings? Science 114: 661-662.
LaRue, C. D. and T. Jm Musik. 1954. Growth, regeneration, and prevocious
rooting in Rhizophora mangle. Pap. Mich. Acad. Sci. Arts Lett. 39: 9-29.
Lawrence, D. B. 1949. Self-erecting habit of seedling red mangroves
(Rhizophora mangle L.). Am. J. Bot. 36: 426-427.
Lawson, G. W. 1966. The littoral ecology of West Africa. Ocean et Mar. biol.
Ann. Rev. 4: 405-448.
Le Cointe, P. 1947. Arvores e Plantas Uteis. Amazonia Brasiliera III. 2nd ed.
Companhia Editora Nacional, Sao Paulo, Brazil, p. 283.
Lee, B. K. H. and G. E. Baker. 1972. An ecological study of the soil
microfungi in a Hawaiian mangrove swamp. Pac. Sci. 26: 1-10.
146
-------
ECOLOGY OF HALOPHYTES
Lee, B. K. H. and G. E. Baker. 1972. Environment and the distribution of
microfungi in a Hawaiian mangrove swamp. Pac. Sci. 26: 11-19.
Leechman, A. 1908. The genus Rhizophora in British Guiana. Kew Bull.
1908: 4-8.
Leeuwen, W. D. von. 1911. Uber die Ursache der widerholten Verzweigung
der Stutzwurzeln von Rhizophora. Ber. Dtsch. hot. Ges. 29: 476-478.
Lendrum, N, G. 1961. Problems of coastal development of Florida. Shore and
Beach 29: 27-32.
Leopold, A. S. 1950. Vegetation zones of Mexico. Ecology 31: 507-518.
Lever, R. J. A. W. 1952. A malayan leaf tying caterpiller from mangroves
periodically submerged in sea water. Entomol. Mon. Mag. 88: 95-98.
Lewin, R. A. 1970. Toxin secretion and tail autotomy by irritated Oxynoe
panamensis (Opistobranchiata; Sacoglossa). Pac. Sci. 24: 356-358.
Lewis, J. 1956. Rhizophoraceae. Fl. Trop. E. Afr. Oct., 20 pp.
Lewis, O. A. M. and G. Naidoo. 1970. Tidal influence on the apparent
transpirational rhythms of the white mangrove. S. Afr. J. Sci. 66: 268-270.
Li, Shang-Kuei. 1948. Distribution of mangroves in Taiwan. Taiwan For. Exp.
Stn. Rep. 41-48:337-339.
Liebau, O. 1914. Beitrage zur Anatomie und Morphologic der
Mangrove-Pflanzen, insbesondere ihres Wurzelsysterns. Beitr. Biol. Pfl. 12:
181-213.
Lindeman, J. C. 1953. The vegetation of Suriname. Vol 1, Part 1. The
vegetation of the coastal region of Suriname. Paramaibo I - IV: 1-135.
Linke, 0. 1939. Die Biota des Jadebusenwattes. Helgol. wiss. Meeres. 1:
201-348.
Little, E. L., Jr. 1945. Miscellaneous notes on nomenclaure of United States
trees. Am. Midi. Nat. 33: 495-513.
Little, E. L., Jr. 1953. Check List of Native and Naturalized Trees of the
United States (Including Alaska). Agric. Handbook 41, U.S.D.A. For.
Serv., Washington, D. C. p. 365.
Little, E. L., Jr. 1961. Avicennia nitida. phytologia 8: 49-57.
Little, E. L., Jr. and F. H. Wadsworth. 1964. Common Trees of Puerto Rico
and the Virgin Islands. Agric. Handb. 249, U.S.D.A. For. Serv.,
Washington, D. C.
Liu, T. 1955. Notes on mangroves in Taiwan. Taiwan For. 1: 1-4.
Liu, t. 1956. Mangrove forests. For. Bull. (Natl. Taiwan Univ.) 2: 1-25.
Loder, J. W. and G. B. Russell. 1969. Tumor inhibitory plants. The alkaloids
of. Bruguiera sexdngula and Bruguiera exaristata. Aust. J. Chem. 22:
1271-1276.
Long, R. W. and O. Lakela. 1971. A Flora of Tropical Florida. Univ. Miami
Press, Coral Gables, Florida, 962 pp.
Lotschert, W. 1955. Die Mangrove-von El Salvador. Unsch. wiss. Tech. 55:
47-50.
Lotschert, W. 1960. Die Mangrove von El Salvador. Ber. D. Senckenberg
Naturforsch. Ges. 90: 213-224.
147
-------
GERALD E. WALSH
Lotschert, W. 1968. Speichern die keimlinge von Mangrovepflanzen Salz?
Umsch. wiss. Tech. 68: 20-21.
Lotschert, W. and F. Liemann. 1967. Die Salzspeicherung im Keimling von
Rhizophora mangle L. wahrend der Entwicklung auf den Mitterpflanze.
Planta77: 136-156.
Loveless, A. R. 1960. The vegetation of Antigua. J. Ecol. 48: 495-527.
Loveless, C. M. 1959. A study of the vegetation of the Florida Everglades.
Ecology 40: 1-9.
Lowe-McConnell, R. H. 1967. Notes on the nesting of the Boat-bill,
Cochlearius cochlearius. Ibis 109: 179.
Luederwaldt, H. 1919. Os manguesaes de Dantos. Rev. Mus. Paul. 11:
309-408. ,
Luytjes, A. 1923. De vloedbosschen van Atjeh. Tectonia 16: 575-601.
Macbride, J. F. 1960. Flora of Peru. Field Mus. Nat. Hist. Bot. Ser. 13: 72.
MacCaughey, V. 1917. The mangrove in the Hawaiian Islands. Hawaiian For.
Agr. 14: 361-366.
MacCaughey, V. 1918. The strand flora of the Hawaiian archipelago. Bull.
Torrey Bot. Club 45: 259-277.
MacKay, J. B. and V. J. Chapman. 19544. Some notes on Suaeda australis
Moq. var. nova zelandica var. nov. and Mesembryanthemum australe Sol.
ex Forst. Trans. R. Soc. N. Z. 82: 41-47.
Macluskie, H. 1952. The reclamation of mangrove swamp areas for rice
cultiviation. World Crops 2: 129-132.
Macnae, W. 1956. Aspects of life on muddy shores in South Africa. S. Afr. J.
Sci. 53:40-43.
Macnae, W. 1957. The ecology of the plants and animals in the intertidal
regions of the Zwartkops estuary, near Port Elizabeth, South Africa. J.
Ecol. 45: 113-131 and 361-387.
Macnae, W 1962. The fauna and flora of the eastern coasts of southern
Africa in relation to ocean currents. S. Afr. J. Sci. 58: 208-212.
Macnae, W. 1963. Mangrove swamps in South Africa. J. Ecol. 51: 1-25.
Macnae, W. 1966. Mangroves in eastern and southern Australia. Aust. J. Bot.
14: 67-104.
Macnae, W. 1967. Zonation within mangroves associated with estuaries in
north Queesland. in Estuaries, G. H. Lauff (ed.), AAAS Publ. 83: 432-441.
Macnae, W. 1968a. A general account of the fauna and flora of mangrove
swamps and forests in the Indo-West-Pacific region. Adv. Mar. Biol. 6:
73-270.
Macnae, W. 1968b. Mangroves and their fauna. Aust. nNat. Hist. 16: 17-21.
Macnae, W. and M. Kalk. 1962a. The mangrove swamps of Inhaca Island. J
Ecol. 50: 19-34.
Macnae, W. and M. Kalk. 1962b. The fauna and flora of sand flats at Inhaca
Island, Mozambique. J. Anim. Ecol. 31: 93-128.
Mahabale, T. S. and J. V. Deshpande. 1959. The genus Sonneratia and its
fossil aHies. Palaeobotanist 6: 51-64.
148
-------
ECOLOGY OF HALOPHYTES
Malaviya, M. 1963. On the distribution, structure and ontogeny of stone cells
in Avicennia officinalis L. Proc. Indian Acad. Sci., Sect. B 58: 45-50.
Malcolm, C. V. 1964. Effects of salt, temperature, and seed scarification on
germination of two varieties of Arthrocnemum halocnemoides. Jour. Proc.
R. Soc. W. Aust. 47: 72-74.
Many authors. 1959. Los Manglares en America. Inst. For. Latinoamer.,
Merida, Venezuela, 52 pp.
Marathe, K. V. 1965. A study of the subterranean algal flora of some
mangrove swamps. J. Indian Soc. Soil Sci. 13: 81084.
Marco, H. F. 1935. Systematic anatomy of the woods of the Rhizophoraceae.
Trop. Woods 44: 1-26.
Marcus, B. 1941. Over den slibbodem van zoutwatervijvers. Landbouw 17:
891-910.
Margolef, F. 1962. Comunidades naturales. Inst. Biol. Mar. Univ. Puerto Rico,
Spec. Pub.,499 pp.
Marshall, R. C. 1939. The Silviculture of the Trees of Trinidad and Tobago,
British West Indies. Oxford Univ. Press, London, 247 pp.
Martinez, M. 1959a. Plantas Medicinales de Mexico, 4th ed., Ediciones Botas,
Mexico, pp. 212-215.
Martinez, M. 1959b. Plantas Utiles de la Flora Mexicana. Revised edition,
Ediciones Botas, Mexico, pp. 397-401.
Martyn, E. B. 1934. A note on the foreshore vegetation in the neighborhood
of Georgetown, British Guiana, with special reference to Spartina
braziliensis. J. Ecol. 22: 292-298.
Masamune, G. 1930. Contributions to be phytogeography of Japan. I. Trans.
Nat. Hist. Soc. Formosa 20: 299-304 (in Japanese).
Mattox, N. T. 1949. Studies on the biology of the edible oyster, Ostrea
rhizophorae Guilding, in Puerto Rico. Ecol. Monogr. 19: 339-356.
Mauritzon, J. 1939. Contributions to the embryology of the orders Rosales
and Myratales. Lunds Univ. Arsskr. II. Sec. 2, 35: 1-120.
Mayr, E. 1944. Wallace's Line in the light of recent zoogeographical studies.
Q. Rev. Biol. 19: 1-14.
MaAdam, J. B. 1952. Forestry in New Guinea. Sci. Soc. Ann. Rep. Proc.,
New Guinea, pp. 41 -61.
McArthur, W. M. 1957. The plant ecology of Garden Island in relation to the
neighboring islands and the adjacent mainland. Proc. R. Soc. W. Aust. 40:
46-64.
McGill, J. T. 1958. Map of coastal landforms of the world. Geog. Rev. 48:
402-405.
McMillan, C. 1971. Environmental factors affecting seedling establishment of
the black mangrove on the central Texas Coast. Ecology 52: 927-930.
Mead, J. P. 1912. The mangrove forests of the west coast of the Federated
Malay States. Govt. Print. Office, Kuala Lumpur.
Meer Mohr, J. C. 1929. Abnormal fruits in Rhizophora conjugataa. Rec.
Trans. Bot. Need. 26: 15-18.
149
-------
GERALD E. WALSH
Meindersma, H. W., J. H. Becking, and L. G. den Berger. 1922. Vloed-of
mangrovebouschen in Ned-Indie. Tectona 15: 561-611.
Mell, C. D. 1919. The mangroves of tropical America. Sci. Am. Suppl. 2292:
388-389.
Merrill, A. C. 1905. A review of the identification of the species described in
Blanco's Flora de Filipinas. Dept. Int., Bur. Govt. Labs., Manila, Bull. No.
27.
Metcalf, C. R. 1931. The breathing roots of Sonneratia and Bruguieraa, a
review of recent work by Troll and Dragendorff. Kew Bull. 11: 465-467.
Millard, N. A. H. and A. D. Harrison. 1953. The ecology of South African
estuaries. Part V. Richards Bay. Trans. R. Soc. S. Afr. 34: 157-171.
Miller, A. H., H. Friedmann, L. Griscom, and R. T. Moore. 1957.
Distributional check-list of the birds of Mexico. Part 2. Pacific Coast
Avifauna, No. 33, 436 pp.
Miller, O. B. 1926. Mangroves at Sordivana Bay, N. Zululand. S. Afr. J. Nat.
Hist. 6: 54-55.
Miller, P. C. 1972. Bioclimate, leaf temperature, and primary production in
red mangrove canopies in south Florida. Ecology 53: 22-45.
Miller, R. C. 1957. Marine wood-boring molluscs of the Philippines. Pac. Sci.
Congr. 3A: 1575-1577.
Millspaugh, C. F. 1907. Flora of the sand keys of Florida. Field Mus. Publ.
No. 118. pp. 191-243.
Miquel, F. A. G. 1855-1859. Flora van Nederlandsch Indie. Flora Indiae
Batavae, vols. 1 and 2. Leipzig.
Miranda, F. 1958. Estuadios acerea de la vegetacion. in Los Recursos
Naturales del Surests y su Aprovechamiento, E. Beltran (ed.), Publ. Inst.
Mex. Rec. Nat. Renov. 2: 215-271.
Miranda, M. 1952. La vegetacion de Chiapas. Part II. Dept. de Prensa y
Turismo, Tuxtla Gutierrez, Chiapas, Mexico, pp. 119-120.
Miyata, L, N. Odani, and Y. Ono. 1963. An analysis of Jo-method of
dispersion of the population of Bruguiera conjugata (L.) Merrill. Rep.
Comm. For. Sci. Res., Kyushu Univ., No. 1, pp. 43-48.
Mogg, A. 0. D. 1963. A preliminary investigation of the significance of
salinity in the zonation of species in salt-marsh and mangrove swamp
associations. S. Afr. J. Sci. 59: 81-86.
Mokievskii, O. B. 1969. Biogeotsenotischeskaya sistemalitorali. Okeanologiya
9: 211-222.
Moldenke, H. N. 1958. Hybridity in the Verbenaceae. Am. Midi Nat 59-
333-370.
Moldenke, H. N. 1960a. Materials toward a monograph of the genus
Avicennia. I. Phytologia 7: 123-168.
Moldenke, H. N. 1960b. Materials toward a monograph of the genus
Avicennis. II. Phytologia 7: 179-232.
Moldenke, H. N. 1960c. Materials toward a monograph of the genus
Avicennia. III. Phytologia 7: 259-292.
150
-------
ECOLOGY OF HALOPHYTES
Moldenke, H. N. 1963. Avicenniaceae, Symphoremaceae and Verbenaceae.
Studies on the flora of Thailand, 1963. Dansk Bot. Ark. 23: 83-92.
Moldenke, H. N. 1967a. Additional notes on the genus Avicennia I.
Phytologia 14: 301-320.
Moldenke, H. N. 1967b. Additonal notes on the genus Avicennia II.
Phytologia 14: 326-336.
Moldenke, H. N. 1968a. Additional notes on the genus Avicennia. III.
Phytologia 15: 71-72.
Moldenke, H. N. 1968b. Additonal notes on the genus Avicennia. IV.
Phytologia 15:470478.
Monod, T. and R. Schnell. 1952. Les groupements et les unites geobotaniques
de la region Guineenne (Mangroves). Mem. De 1'Instit. Franc. d'Afrique
Noire 18: 197-218.
Montfort, C. and W. Brandrup. 1927. Physiologische und Pflanzen
geographische See Salzwirkungen. II. Okologische Studien uber Keimung
und erste Entwicklung bei Halophyten. Jahrb. Wiss. Bot. 66: 902-946.
Moore, H. B., L. T. Davies, T. H. Frazer, R. H. Gore, and N. R. Lopez. 1968.
Some biomass figures from a tidal flat in Biscayne Bay, Florida. Bull. Mar.
Sci. 18: 261-269.
Moreira Filho, H. and M. B. Kutner. 1962. Contribuicao para o conhecimento
das diatomaceas do manguesae de Alexandra. Bol. Univ. Parana Bot. 4:
1-24.
Morton, J. F. 1965. Can the red mangrove provide food, feed and fertilizer?
Econ. Bot. 19: 113-123.
Moul, E. T. 1957. Preliminary report on the flora of Onotoa Atoll. Atoll Res.
Bull. 57, Sept. 15.
Moss, C. E. 1954. The species of Arthrocnemum and Salicornia in southern
Africa. J. S. Afr. Bot. 20: 1-22.
Muir, J. 1937. The seed drift of South Africa, and some influences of ocean
currents on the strand vegetation. Mem. Bot. Sur. S. Afr. 16: 7-14.
Mullan, D. P. 1931. On the occurrence of glandular hairs (slat glands) on the
leaves of some Indian halophytes. J. Indian Bot. Soc. 10: 184-189.
Mullan, D. P. 1931. Observations on the water storing devices in the leaves of
some Indian halophytes. J. Indian Bot. Soc. 10: 126-133 and 184-191.
Mullan, D. P. 1932. Observations on the biology and physiological anatomy
of some Indian halophytes. I. J. Indian Bot. Soc. 11: 103-118 and
285-302.
Mullan, D. P. 1933a. Observations on the biology and physiological anatomy
of some Indian halophytes. II. J. Indian Bot. Soc. 12: 165-182.
Mullan, D. P. 1933b. Observations on the biology and physiological anatomy
of some Indian halophytes. III. J. Indian Bot. Soc. 12: 235-256.
Muller, J. 1961. A palynological contribution to the history of the mangrove
vegetation. Pac. Sci. Congr., 1960, pp. 146-147.
Muller, J. 1964. A palynological contribution to the history of the mangrove
vegetation in Borneo, in "Ancient Pacific Floras", Univ. Hawaii Press,
151
-------
GERALD E. WALSH
Honolulu, pp. 33-42.
Muller, J. 1968. Palynology of the Pedowan and Plateau sandstone
formations (Cretaceous-Eocene) in Savanak, Malaysia. Micropalaentology
14: 1-37.
Muller, J. and S. Y. Hou-Liu. 1966. Hybrids and chromosomes in the genus
Sonneratia (Sonneratiaceae). Blumea 14: 337-343.
Muller, J. and G. G. J. van Stennis. 1968. The genus Sonneratia in Australia.
North Queensland Not. 35: 6-8.
Murphy, R. C. 1938. The littoral of the Pacific coasts of Colombia and
Equador. Geogr. Rev. 29: 1-33.
Myers, J. G. 1933. Notes on the vegetation of the Venezuelan Llanos. J. Ecol.
21:335-349.
Myers, J. G. 1935. Zonation along river courses. J. Ecol. 23: 356-360.
Nakanishi, S. 1964. An epiphytic community on the mangrove tiee,Kandelia
kandel (Rhizophoraceae). Hikobia 4: 124.
Nash, G. V. 1908. The story of mangrove. Torreya 8: 73-78.
Navalkar, B. S. 1940. Studies in the ecology of mangroves. I. Determination
of the osmotic pressure of Avicennia alba Blume. J. Univ. Bombay 8: 1-19.
Navalkar, B. S. 1942. Studies in the ecology of mangroves. II. Physical factors
of the mangrove soil. J. Univ. Bombay 9: 97-106.
Navalkar, B. S. 1948. Studies in the ecology of mangroves III. J. Univ.
Bombay 16: 3545.
Navalkar, B. S. 1952. Succession of the mangrove vegetation of Bombay and
Salsette Island. J. Bombay Nat. Hist. Soc. 50: 157-161.
Navalkar, B. S. 1953. The analytical aspects of some of the marshy vegetation
of Bombay and Salsette islands. J. Bombay Nat. Hist. Soc. 51: 636-652.
Navalkar, B. S. 1956. Geographical distribution of halophytic plants of
Bombay and Salsette islands. J. Bombay Nat. Hist. Soc. 53: 335-343.
Navalkar, B. S. 1959. Studies in the ecology of mangroves. VII. Humus
content of mangrove soils of Bombay and Salsette islands. J. Univ. Bombay
28: 6-10.
Navalkar, B. S. and F. R. Bharucha. 1948. Studies in the ecology of
mangroves. IV. The hydrogen ion concentration of the seawater, soil
solution, and the leaf cell sap of mangroves. J. Univ. Bombay 17: 35-45.
Navalkar, B. S. and F. R. Bharucha. 1949. Studies in the ecology of
mangroves. V. Chemical factors of mangrove soils. J. Univ. Bombay 17:
17-35.
Navalkar, B. S. and F. R. Bharucha. 1950. Studies in the ecology of
mangroves. VI. Exchangeable bases of mangrove soils. J. Univ. Bombay 18'
7-16.
Newell, N., J. Imbrie, E. G. Purdy, and D. Thurber. 1959. Organism
communities and bottom facies, Great Bahama Bank. Bull. Am. Mus Nat
Hist. 117: 181-228.
Nicolic, M. and S. A. Melendez. 1968. El ostion del mangle, Crassostrea
rhizophorae Guilding 1928. Experiments indicales en el cultivo. Nota
152
-------
ECOLOGY OF HALOPHYTES
Invest Cent. Pesqueras Buta 7: 1-30.
Nisbet, I. C. T. 1968. The utilization of mangroves by malayan birds. Ibis
110:348-352.
Noakes, D. S. P. 1950. The mangrove charcoal industry. Malay. For. 24:
201-203.
Noakes, D. S. P. 1951. Notes on the silviculture of the mangrove forests of -
Matang, Perak. Malay. For. 14: 183-196.
Noakes, D. S. P. 1955. Methods of increasing growth and obtaining natural
regeneration of the mangrove tupe in Malaya. Malay. For. 18: 23-30.
Noakes, D. S. P. 1957. Mangrove. Trop. Silvic. 2: 309-318.
Noakes, D. S. P. 1958. Mangrove. Wld. For. Congr. 3: 415-424.
Noamesi, G. K. 1959. A revision of the Zylocarpeae (Meliaceae). Diss. Abstr.
19: 1531.
Noel, A. R. A. 1959. The vegetation of the fresh water swamps of Inhaca
Island. S. Afr. J. Bot. 25: 189-201.
Nudaliar, C. R. and H. Sunandar. 1952. Distribution of Rhizophora
mucronatd Lam. in the "backwater" of the west coast and its economic
importance. Madras Agric. J. 610-645.
Odani, N. 1964. An analysis of the mangrove communities of Iriomoti-jima, ^
Yaeyama Group, the Tyukyus. Rep. Comm. For. Sci. Res., Kyushu Univ., U)
No. 2, June. 3
Odum, E. P. 1971. Fundamentals of Ecology. 3rd ed., W. B. Saunders Co.,
Philadelphia, 574 pp.
Odum, H. T. 1971. Environment, Power and Society. John Wiley and Sons,
Inc., New York, 331 pp.
Odum, W. E. 1971. Pathways of energy flow in a south Florida estuary. Univ. —»
Miami, Sea Grant Tech. Bull. No. 7, 162 pp. -1
Ogura, Y. 1942. Mangroves in eastern Asia. Bot. and Zool. 10: 145-149 (in O
Japanese). Pj
Ogura, Y. 1958. Some problems on the mangrove. Bull. Yokohama Municip. QJ
Univ. Soc. 10: 545-578. «
Okuda, T., A. J. Garcia, and J. B. Alvarez. 1965. Variation estacional de los
elementos nutritives en el agua de la Laguna Y el Rio Unare. Bol. Inst.
Oceanogr. Univ. Oriente 4: 123-135.
Orians, G. H. And E. W. Pfeiffer. 1970. Ecological effects of the war in
Vietnam. Science 168: 544-554.
Orr, A. P. and F. W. Newhouse. 1933. Physical and chemical conditions in
mangrove swamps. Br. Mus. Nat. Hist. Sci. Rep., Great Barrier Reef Exped.
1928-1929.2: 102-110.
Ostenfeld, C. H. 1918. Stray notes from tropical west Australia. Dan. Bot.
Ark. 2: 1-29.
Outred, H. A. 1966. The effects of low oxygen tensions upon the intensity
and pattern of respiration in certain mangrove seedlings. M. Sc. Thesis,
Auckland Univ., Auckland, N. Z.
153
-------
GERALD E. WALSH
Oyama, K. 1950. Studies of fossil biocoenosis. No. 1. Biocoenological studies
on the mangrove swamps, with descriptions of new species from Yatuo
Group. Rep. Geol. Surv. Japan 132: 1-14.
Padmanabhan, D. 1960. The embryology of Avicennia officinalis L. I. Floral
Morphology and gametophytes. Proc Indian Acad. Sci. Sect. B, 52:
131-145.
Padmanabhan, D. 1962a. The embryology of Avicennis officinalis L. III. The
embryo. J. Madras Univ. 32B: 1-19.
Padmanabhan, D. 1962b. The embryology of Avicennia officinalis L. The
seedling. Proc. Indian Acad. Sci. 56: 114-122.
Paijmans, K. 1967. Lands of the Sofia-Pourgani area, Territory of Papua and
New Guinea. VI. Vegetation of the Sofia-Pourgani area. CSIRO Aust. Land
Res. Ser. 17: 142-167.
Paijmans, K. 1969. Lands of the Kerema-Vailala area, Papua-New Guinea.
VIII. Vegetation and ecology of the Kerema-Vailala area. CSIRO Aust.
Land Res. Ser. 23:95-116.
Pal, N. 1951. On the embryology of Terminalia catappa. Sci. Cult. 17:
178-179.
Pammel, L. H. and C. N. King. 1948. Gerination studies of some trees and
shrubs. Proc. Iowa Acad. Sci. 35: 184-197.
Pannier, P. 1959. El efecto de distintas concentrationes sotenas sobre el
desarrolo de Rhizophora mangle L. Acta cient. Venez. 10: 68-78.
Pannier, P. 1962, Estudio fisiologico sobre la viviparia de Rhizophora mangle
L. Acta cient. Venez. 13: 184-197.
Panshin, A. J. 1932. An anatomical study of the woods of the Philippine
mangrove swamps. Philipp. J. Sci. 48: 143-208.
Panzer, W. 1935. Sandkrabben-Spuren an der Kuste von Neu-Guinea. Nat.
Volk 65: 36-46.
Parham, J. W. 1964. Plants of the Fiju Islands. Govt. Press, .Suva, Fiji.
Parkes, K. C. and R. W. Dickerman. 1967. A new subspecies of mangrove
warbler (Dendroica petechia) from Mexico. Ann. Carnegie Mus. 39: 85-90.
Passler, J. 1912. Die untersuchgensergebnisse der Deutsch-Ostafrika
eingesandtern Mangroverinden. Pilanzer Jg. 8: 65.
Patil, R. P. 1961. A note on the vegetation at Sazina Khali in the Sunderbans.
Indian For. 87:481-483.
Patil, R. P. 1964. Cultivation of mangrove seedlings in pots at Allahabad U.P.
Sci. Cult. 30: 4344.
Patil, R. P. and S. S. Sidhu. 1961. Seedling anatomy of Bruguiera parviflora
W. and A. Proc. 48th Indian Sci. Congr. 3: 280.
Patton, R. T. 1942. Ecological studies in Victoria. VI. Salt marsh Proc R
Soc.Vict.54: 131-144.
Paulsen, 0. 1918. Chenopdiaceae from western Australia. Dan. Bot Ark 2-
56-66.
Payet, M., P. Pene, M. Sankale, G. Delorme, and H. Delatoure. 1962. Le
micro-climat de la presque ile du Cap-Bert: son determinisme
154
-------
ECOLOGY OF HALOPHYTES
physiologique. Bull. Soc. med. Afr. noire Lang. fr. 13: 40-58.
Paynter, R. A., Jr. 1955. The ornithogeography of the Yucatan Peninsula.
Peabody Mus. Nat. Hist., Yale Univ., Bull. No. 9, 374 pp.
Pearman, R. W. 1955. Tanning materials of the British Commonwealth.
Colonial Plant and Animal Products 5: 96.
Pearman, R. W. 1957. Mangrove bark - its value as a tanning material.
Leather Trades Rev. 125: 315-316.
Pearse, A. S. 1932. Animals in brackish water pools at Dry Tortugas. Pap
Tortugas Lab. 28: 125-142.
Pellegrin, F. 1952. Les Rhizophoracees de 1'Afrique Equatiriale Francaise.
Mus. Nat. Hist. Nat. Notulae Syst. 14: 292-300.
Penfound, W. T. 1952. Southern swamps and marshes. Bot. Rev. 18:
413-446.
Penfound, W. T. and E. S. Hathaway. 1938. Plant communities in the
marshlands of southeastern Louisiana. Ecol. Monogr. 8: 1-56.
Perrier de la Bathie, H. 1953. La mangrove (paletuviers) de Madagascar:
biologie et utilite. Rev. Int. Bot. Appl. Agric. Trop. 33: 373-374 and
581-582.
Petit-Thouars, A. du. 1813. Notice sur le manglier. J. Bot. 2: 27-41.
Phillips, J. 1959. Agriculture and ecology in Africa. Flora 131: 1-40.
Phillips, 0. P. 1903. How the mangrove tree adds new land to Florida. J.
Geogr. 2: 10-21.
Pidgeon, I. 1940. The ecology of the central coastal area of New South Wales.
III. Types of primary succession. Proc. Linn. Soc. N.S.W. 65: 221-249.
Pickening, C. 1879. Chronological History of Plants. Little, Brown and Co.,
Boston, 1222 pp.
Piexoto, A. R. 1959. Tropical-almound Terminalia; Fruit, oil, and tanning.
Bol. Agric. Minas Gerais Dept. Prod. Veg. 8: 69-71.
Fiji, L. van der. 1969. Evolutionary action of tropical animals on the
reproduction of plants. Biol. J. Linn. Soc. London 1: 85-96.
Pitot, A. 1958. Rhizophores et racines chez Rhizophora sp. Bull, Inst. Franc.
Afr. Noire 20: 1103-1138.
Pittier, H. 1898. Primitiae florae costaricensis. Anal. Inst. fis.-geogr. Nacl. 8:
1-26.
Pittier, H. 1908. Ensayo Sobre las Plantas Usuales de Costa Rica. H. L. and J.
B. McQueen, Washington, D. C., p. 112.
Pittier, H. 1926. Manual de las Plantas Usuales de Venezuela. Pub. by the
author, Caracas, p. 286.
Pollard, C. L. 1902. Plant agencies in the formation of the Florida keys. PI.
World 5: 8-10.
Pope, W. T. 1929. Manual of Wayside Plants of Hawaii. Advertiser Pub. Co.,
Honolulu, pp. 157-159.
Porsch, O. 1924. Vogel blumenstudien. I. Jb. wiss. Bot. 63: 553-706.
Post, E. 1963a. Systematische und pflanzen-Geographische Notizen zur
Bostrichia-Caloglassa Assoziation. Rev. Algel. 9: 1-84.
155
-------
GERALD E. WALSH
Post, E. 1963b. Zur Verbreitung und Okologie der Bostrychia-Caloglossa
Assoziation. Int. Rev. ges. Hydrobiol. Hydrogr. 48: 47-152.
Prain, D. 1903. Flora of the Sunderbans. Rec. Bot. Surv. India 2: 231.
Prakash, U. 1960. A Survey of the Deccan Intertrappean beds flora of India.
J. Palaentol. 34: 1027-1040.
Price, J. L. 1955. A survey of the freshwater fishes of the island of Trinidad.
J. Agric. Soc. Trinidad and Tobago, Pap. No. 863: 1-28.
Price, W. A. 1954. Shorelines and coasts of the Gulf of Mexico. U. S. Fish.
Bull. 89: 39-65.
Pustelnik, W. 1953. Analisis de la Corteza de Mangle Colorado (Rhizophora
mangle L.*) del oriente de Venezuela y su importancia en la elaboracion de
extractos curientes solidos. Agron. Trop. 3: 107-116 (also in Rev. Soc.
Venez. 5: 19-31).
Putnam, R. C. and A. V. Bowles. 1955. The chemistry of vegetable tannins.
XI. Mangrove (Borneo cutch). J. Am. Leather Chemists Assoc. 50: 42-46.
Pynaert, L. 1933. La mangrove congolaise. Bull. Agric. Congo Bel. 23:
184-207.
Quesnel, V. C. 1964. Suitability of sulphurous acid for hydrolysis of
condensed tannins. Tetrahedron Letters 48: 3699-3702.
Quinones, L. R. and J. F. Puncochar. 1943. Informe preliminar sobre la
utilizacion practica de la corteza del mangrove. Caribb. For. 5: 44-47.
Rai, J. N., J. P. Tewari, and K. G. Mukerji. 1969. Mycoflora of mangrove
mud. Mycopathol. Mycol. Appl. 38: 17-31.
Rains, D. W. and E. epstein. 1967. Prederential absorption of potassium by
leaf tissue of the mangrove, Avicennia marina: an aspect of halophytic
competence in coping with salt. Aust. J. Biol. Sci. 20: 847-857.
Rajagopalan, V. R. 1949. Cytological studies in Combretaceae I. Proc. Indian
Sci. Congr. 36: 137-138.
Rao, A. N. and L. C. Wan. 1969. A new record of folian sclerids in
Scyphiphora hydrophyllacea. Curr. Sci. 38: 594-595.
Rao, R. S. 1957. Observations on the mangrove vegetation of the Godavri
Estuary. Proc. Mangrove Swamp Symp., Ministry of Food and Agriculture,
Calcutta, India, pp. 36-44.
Raunkaiaer, C. 1934. The Life Forms of Plants and Statistical Plant
geography, Being the Collected Papers of C. Raunkiaer. Oxford Univ. Press,
Oxford, 632 pp.
Rawitscher, F. K. 1944. Algumas nocoes sobre a vegetacao do literal
brasileiro. Bol Assoc. dos Geogr. Bras. 5: 13-28.
Reinders-Gouwentak, C. A. 1953. Sonneratiaceae and other mangrove-swamp
families, anatomical structure and water relations. Fl. Males. 4: 513-515.
Reintjes, J. W. 1963. The importance of the occurrence of menhaden in the
coastal waters and estuaries of peninsular Florida. Proc. Gulf Caribb. Fish.
Inst. 16: 108-113.
Rendle, A. B. and S. LeM. Moore. 1921. A systematic account of the plants
156
-------
ECOLOGY OF HALOPHYTES
collected in New Caldonia and the Isle of Pines by Prof. R. H. Compton,
1914. I. Flowering plants (angiosperms). J. Linn. Soc. London 15:
245-418.
Revelle, R. and R. W. Fairbridge. 1957. Carbonates and carbon dioxide. Mem.
geol. Soc. Am. 67: 239-295.
Reyes, L. J. 1938. Philippine woods. Dept. Agric. Commerce, Manila, Tech.
Bull. No. 7.
Richards, P. W. 1952. The Tropical Rain Forest: an Ecological Study.
Cambridge Univ. Press. Cambridge, 299-312.
Ricklefs, R. E. 1971. Foraging behavior of mangrove swallows at Barro
Colorado Island. Auk 88: 635-651.
Ridgway, R. 1873. On some new forms of American birds. Am. Nat. 7:
602-619.
Ridgway, R. 1902. The Birds of North and Middle America, Part 2. U. S.
Natl. Mus. Bull. No. 50, 834 pp.
Ridley, H. N. 1920. New and rare plants from the Malay Peninsula. J. Fed.
Malay. St. Mus. 10: 128-156.
Ridley, H. N. 1922. The flora of the Malay Peninsula, Vol. 1, pp. 692-703, L.
Reeve and Co., London.
Ridley, H. N. 1930. The Dispersal of Plants Throughout the World. L. Reeve
and Co., London, 744 pp.
Rimbach, A. 1932. The forests of Equador. Trop. Woods. 31: 1-9.
Robas, A. K. 1970. South Florida's mangrove-bordered estuaries: their role in
sport and commercial fish production. Univ. Miami Sea Grant Inf. Bull.
No. 4, 28 pp.
Robertson, R. 1959. The mollusk fauna of Bahamian mangroves. Am.
Macacol. Union Ann. Rep. Bull. 1959: 22-23.
Robertson, W. B. 1962. Fire and vegetation in the Everglades. Proc. 1st Ann.
Tall Timbers Fire Ecol. Conf., pp. 67-80.
Robyns, W. 1971. Over viviparie en biotecnose bij planten. Meded. Kon.
Vlaamse. Acad. Wetensch. Lett. Schone Kusten Belgie 33: 3-14.
Rodriguez, G. 1959. The marine communities of Margarita Island, Venezuela.
Bull. Mar. Sci. Gulf Caribb. 9: 237-280.
Rodriguez, G. 1963. The intertidal estuarine communities of Lake Maracaibo,
Venezuela. Bull. Mar. Sci. Gulf Caribb. 13: 197-218.
Rodriguez, G. 1964. Physical parameters of the Maracaibo estuary and their
implications. Proc. Gulf. Caribb. Fish. Inst., 17th Ann. Sess., pp. 42-50.
Rogers, J. S. 1951. Native sources of tanning materials. U.S.D.A. Yearb.,
Wash., D.C., pp. 709-715.
Roig y Mesa, J. T. 1945. Plantas Medicinales, Aromaticas o Venenosas de
Cuba. Cultural, S. A., Habana, pp. 447449.
Roonwall, M. L. 1954. The marine borer, Bactronophorus thoracites (Gould)
Mollusca, Eulamellibranchiata, Teredinidae) as a pest of living trees in the
mangrove forests of the Sundurhaus, Bengal, India. Proc. Zool. Soc. Bengal
7:91-105.
157
-------
GERALD E. WALSH
Roos, P. J. 1964. The distribution of reef corals in Curacao. Natuurwetensch.
Studiekring Suriname Ned. Antillen Uitganen, 1-51.
Rosevear, D. R. 1947. Mangrove swamps. Farm For. 8: 23-30.
Russell, A. 1943. Conocarpus erecta (buttonwood, Zaragoza-mangrove), a
new domestic source of tannin. Chemurg. Dig. 2: 27-29.
Roth, I. 1965. Histogenese der Lentizellenam Hypocotyl von Rhizophora
mangle L. Ostero. Bot. Z. 112: 640-653.
Ruhland, W. 1915. Untersuchungen uber die Hautdrusender Plumbaginaceen.
J. Wiss. Bot. 55:409-498.
Russell, A. 1942. Natural tanning materials of the southeastern United States.
J. Am. Leather Chemists Assoc. 37: 340-356.
Putzler, K. 1969. The mangrove community, aspects of its structure,
faunistics and ecology, in Lagunas Costeros, Un Simposio, Mem. Simp.
Intern. Lagunas Costeras, UNAM-UNESCO, pp. 515-536.
Rutzler, K. 1970. Oil pollution: damage observed in tropical communities
along the Atlantic seabord of Panama. Bioscience 20: 222-224.
Rzedowski, J. and R. McVaugh. 1966. La vegetacion de Nuera Galicia. Con,
Univ. Mich. Herb. 9: 1-133.
Safford, W. E. 1905. The useful plants of the island of Guam. Contrib. U. S.
Natl. Herb., Vol. II.
Sagrario, C. D. del. 1957. Recovery of tannin from mangrove bark and its
conversion into a colorless tannin extract. Chem. Abstr. 53: 4792D.
Salvosa, F. M. 1936. Rhixophora. Nat. Appl. Sci. Bull., Univ. Philippines 5:
179-237.
Sawman-Wanakit, L. 1936. Forests in the south. Vanasarn For. J., Thailand,
Sp. Ed., No. 3, December.
Sanchez, R. and M. Elana. 1963. Datos relatives alos mangalares de Mexico.
Ann. Esc. Nac. Cienc. Biol. Mex. 12: 61-72.
Sandison, E. E. 1966. The effect of salinity fluctuations on the life cycle of
Balanus pallidus stutsburi Darwin in Lagos Harbour, Nigeria. J. Anim. Ecol.
35: 363-378.
Sandison, E. E. and M. B. Hill. 1966. The distribution of Balanus pallidus
stutsburi Darwin, Gryphaea gasar (Adanson) Dautzenberg, Mercierella
enigmatica Fauvel and Hydroides uncinata Philippi in relation to salinity in
Lagos Harbour and adjacent creeks. J. Anim. Ecol. 35: 235-250.
Santos, T. A. and I. B. Siapno. 1968. Cutting operations of mangrove forest
in Zamboanga peninsula (Mindanao). Philipp. Lumberman 14: 14-20.
Sargent, C. S. 1893. The mangrove tree. Card. For. 6: 97-98.
Sargent, C. S. 1903. Combretaceae. Silvic. N. Am. 5: 19-29.
Sargent, C. S. 1922. Manual of Trees of North America (Exclusive of
Mexico). 2nd ed., Houghton Mifflin Co., Boston, pp. 763-764.
Sargent, C. S. 1962. Effects of recent tropical cyclones on the coastal
vegetation of Mauritius. J. Ecol. 50: 275-290.
Sasaki, S. 1912. Plants of the Kojurin Forest, Formosa. Trans. Nat. Hist. Soc.
Formosa 2: 45-50.
158
-------
ECOLOGY OF HALOPHYTES
Sasaki, S. 1923. Kandelia rheedii is disappearing from the Taiwanian flora.
Trans. Nat. Hist. Soc. Formosa 13: 37-39.
Satayanaraynan, Y. 1959. Ecological studies of the Elephante Island. Inst.
Franc, de Pondicherry, Trav. Sec. Scien. Tech. 1: 99-114.
Sauer, J. D. 1961. Coastal plant geography of Mauritius. Coast. Stud. Ser.,
Louisiana St. Univ. 5: 1-153.
Sauer, J. D. 1967. Geographic reconnaissance of seashore vegetation along the
mexican gulf coast. Coastal St. Res. Inst. La. St. Univ., Tech. Rep. No. 56.
Saunders, J. B. 1958. Recent foraminifera of mangrove swamps and river
estuaries and their fossil counterparts in Trinidad. Micropaleontology 4:
79-92.
Saver, J. D. 1965. Geographic reconnaissance of western Australian seashore
vegetation. Aust. J. Bot. 13: 39-69.
Savory, H. J. 1953. A note on the ecology of Rhizophora in Nigeria. Kew
Bull. 1: 127-128.
Scheffen, W. 1937. Strandbeobachtungen im Malayischen Archipel. Geol.
Meere Binnengew. 1.
Scheffer, T. C. and C. G. Duncan. 1947. The decay resistance of certain
Central American'and Ecuadorian woods. Trop. Woods. 92: 1-24.
Schenck, H. 1889. Ueber die Luftwurzeln von Avicennia tomentosa und
Laguncularia racemosa. Flora 72: 83-88.
Schimper, A. F. W. 1891. Die Indo-Malayische Strandflora. Bot. Mit. Trop. 3:
1-204.
Schimper, A. F. W. 1892-1893. Rhizophoraceae. Nat.Pflanzenfam. 7: 42-56.
Schimper, A. F. W. 1903. Plant Geography on a Physiological Basis. Oxford
Univ. Press. Oxford, 839 pp.
Schimper, A. F. W. and F. C. von Faber. 1935. Pflanzengeographie auf
Physiologischer Grundlage. 2 vols., 3rd Ed., Jena.
Schmidt, J. 1902. Rhizophoraceae. Bot. Tidsskr. 24: 249-554.
Schmidt, J. 1905. Skuddene has den gamle verdens mangrovetraer. Nordiske
Forlag Econst. Bojeuen, 113 pp.
Schmidt, J. 1904. Zur Frage der Salzausscheidung der Mangrovepflanzen.
Flora 93: 260-261.
Schmidt, J. 1905. Bidrag til Kundskab om Skuddene hos den Gamle Verdens
Mangrove-traer. Bot. Tidsskr. 26: 1-113.
Schmidt, J. 1906. Die Kustenvegetation von Koh Chang. Vegetationsbilder 3:
78-91.
Schneider, E. E. 1916. Commercial woods of the Philippines: their
preparation and uses. Philipp. Bur. Forest., Bull. No. 14, pp. 179-182.
Schneider, J. 1921. Uber Mangrove auf den Philippinen. Gerger 47: 53-54.
Schnepper, W. C. R. 1933. Vloedbosch culturen. Tectona 26: 907-919.
Schnetter, M. 1969. Observaciones ecologicas en la isla de Salamanca
(Departmento del Magdalene) Colombia. Caldasia 10: 299-315.
Scholander, P. F. 1965. From the frozen forest to the tropical mangroves, in
Science in Alaska: Proc. 15th Alaskan Sci. Conf., College, Alaska, 31 Aug. -
159
-------
GERALD E. WALSH
4 Sept. 1964, AAAS, Washington, D. C.
Scholander, P. F. 1968. How mangroves desalinate seawater. Physiol.
Plantarum21: 258-268.
Scholander, P. F., E. D. Bradstreet, H. T. Hammel, and E. A. Hemmingsen.
1966. Sap concentrations in halophytes and some other plants. Plant
Physiol. 41: 529-532.
Scholander, P. F., L. van Dam, and S. I. Scholander. 1955. Gas exchange in
the roots of mangroves. Am. J. Bot. 42: 92-98.
Scholander, P. F., H. T. Hammel, E. A. Hemmingsen, and E. D. Bradstreet.
1964. Hydrostatic pressure and osmotic potential in leaves of mangroves
and some other plants. Proc. U. S. Natl. Acad. Sci. 52: 119-125.
Scholander, P. F., H. T. Hammel, E. Hemmingsen, and W. Carey. 1962. Salt
balance in mangroves. Plant Physiol. 37: 722-729.
Scholander, P. F., H. T. Hammel, E. D. Bradstreet, and E. A. Hemmingsen.
1965a. Sap pressure in vascular plants. Science 148: 339-346.
Scholander, P. F., H. T. Hammel, E. D. Bradstreet, and E. A. Hemmingsen.
1965b. Sap pressure in vascular plants (Addendum). Science 148: 1488.
Scholander, P. F. and M. de Oliveira Perez. 1968. Sap tension in flooded trees
and bushes of the Amazon. Plant Physiol. 42: 1870-1873.
Scholl, D. W. 1963. Sedimentation in modern coastal swamps, southwestern
Florida. Bull. Am. Assoc. Pet. Geol. 47: 1581-1603.
Scholl, D. W. 1964. Recent sedimentary record in mangrove swamps and rise
in sea level over the southwestern coast of Florida. I. Mar. Geol. 1:
344-366.
Scholl, D. W. 1965a. High interstitial water cholorinity in estuarine Mangrove
swamps. Florida. Nature 207: 284-285.
Scholl, D. W. 1965b. Recent sedimentary record in mangrove swamps and rise
in sea level over the southwestern part of Florida. II. Mar. Geol. 2:
343-364.
Scholl, D. W. Modern coastal swamp statigraphy and the ideal cyclotherm. i»
Environments of Coal Deposition, E. C. Dapples and M. B. Hopkins (eds.),
Geol. Soc. Amer. Spec. Pap., in press.
Scholl, D. W. and M. Stuijer. 1967. Recent submergence of southern Florida:
a comparison with adjacent coasts and other eustatic data. Geol. Soc Am
Bull. 78: 437-454.
Schone, H. 1963. Menotaktische Orientierung nach polarisiertem und
unpolarisierten Light bei der Mangrovekrabbe Goniopsis. Z. Vgl. Physiol
46:496-514.
Schone, H. and H. Schone. 1963. Balz und Verhaltensweisen der
Mangrovekrabbe Goniopsis cruentata Latr. und das Winkverhalten der
eulitoralen Brachyuren. Z. Tierpsychol. 20: 641-656.
Schratz, E. 1934. Beitrage zur Biologic der Halophyten. I. Zur
Keimungphysiologie. Jahrb. f. wiss. Bot. 80: 112-142.
Schuster, W. H. 1962. Das marine Littoral als Lebensraum Terrestrischer
Kleinarthropoden. Int. ges. rev. Ges. Hydrobiol. 47: 359-412.
16Q
-------
ECOLOGY OF HALOPHYTES
Schuster, W. H. 1952. Fish culture in brackish water ponds of Java.
Indo-Pacific Fish. Counc. Spec. Publ., No. 1, pp. 1-143.
Seifriz, W. 1943. The plant life of Cuba. Ecol. Monogr. 13: 375.426.
Sen Gupta, J. C. 1935. Die Osmotischen Verhaltnisse bei einigen Pflanzen in
Bengal (Indian). Ber. Dtsch. bot. Ges. 53: 783-795.
Sen Gupta, J. C. 1938a. Ueber die osmotische Werte und den Chloranteil in
Pflanzen einiger Salzgebietes Bengal. Ber. Dtsch. bot. Ges. 56: 474-485.
Sen Gupta, J. C. 1938b. Der histochemische Nachweis vor Natrium bei
einigen Pflanzen von Bengal. Ber. Dtsch. bot. Ges. 56: 486-494.
Sen Gupta, J. C. 1942. Investigation of the annual variation of the water
content, osmotic value and chloride fractions of some plants in Port
Canning, near Calcutta. 50th Ann. Vol R. Bot. Card. Calcutta, 1942.
Seshadri, T. R. and B. Venkataramani. 1959. Leucocyanin from mangroves.
Indian J. Sci. Res. 18B: 261-262.
Setchell, W. L. 1926. Phytogeographical notes on Tahiti. Univ. Cal. Publ. Bot.
12: 241-290.
Setten, G. G. K. 1947. A note on the growth and yield of Beruus (Bruguiera
cylindrica (L.) (BL). Malay. For. 16: 74.
Setten, G. G. K. 1955. A note on the growth and yield of Berus (B. cylindrica
(L.) Bl. syn. B. caryophylloides Bl.) in Selangor. Res. Pamphl. For. Res.
Instit. Fed. Malaya, No. 16,12 pp.
Shantz, H. L. and C. F. Marbut. 1923. The vegetation and soils of Africa. Am.
Geogr. Soc. Res. Ser. 13: 1-242.
Shah, J. J. and K. P. Sundarraj. 1965. Stipular sclerids in Rhizophora
mucronata L. Curr. Sci. 34: 155-156.
Shelford, V. E. 1963. The Ecology of North America. Univ. 111. Press, Urbana,
610pp.
Shier, D. E. 1969. Vermetid reefs and coastal development in the Ten
Thousand Islands, southwest Florida. Geol. Soc. Bull. 80: 485-508.
Shieve, F. 1937. The vegetation of the cape region of Baja California.
Madrona4: 105-113.
Shimada, Y. 1924. Investigations on Kandelia rheedii in Komoko, Taiwan.
Trans. Nat. Hist. Soc. Formosa 14: 25-29 (in Japanese).
Sidhu, S. S. 1960. Studies on mangroves. Natl. Acad. Sci. India, No. 1960,
pp. 111-112.
Sidhu, S. S. 1961. Vegetation of East Godavari. Proc. 48th Indian Sci. Congr.
3: 358-359.
Sidhu, S. S. 1961. Chromosomal studies on some mangrove species. Proc.
48th Indian Sci. Congr. 3: 302-304.
Sidhu, S. S. 1962. Chromosomal studies of some mangrove species. Indian
For. 88: 585-592.
Sidhu, S. S. 1963a. Studies on the mangroves of India. I. East Godavari
region. Indian For. 89: 337-351.
Sidhu, S. S. 1963b. Studies on mangroves. inSymposium OH Ecological
Problems in the Tropics. Proc. Natl. Acad. Scr. India, Sect. B. 33: 129-136.
161
-------
GERALD E. WALSH
Sidhu, S. S. 1968. Further studies on the cytology of mangrove species in
India. Caryologia 21: 353-357.
Simpson, C. T. 1932. Florida Wild Life. Macmillan, New York, pp. 9-11,118.
Simpson, J. G. and R. C. Griffiths. 1967. The fisheries resources of Venezuela
and their exploitation. Serie Recursos y Expl. Presqueros 1: 171-206.
Silveira, F. 1937. Mangrove. Rodriguesia 3: 131-154.
Simberloff, D. S. and E. 0. Wilson. 1969. Experimental zoogeography of
islands: the colonization of empty islands. Ecology 50: 278-296.
Singh. T. C. N. and A. T. Natarajan. 1953-1954. Morphology of the pollen
grains of the constituents of the mangrove vegetation (abstr.) Proc. Indian
Sci. Congr. 40: 99-100.
Slooten, D. V. van. 1937. Die Verbreitung von Lumnitzera und anderen
Mangrovegewachsen. Blumea Suppl. 1: 162-175.
Slooten, H. J. van der. 1960. Resina de Fenol-Formaldehido para
contrachapeado obtenida del tanio de Rhizophora mangle.. Inst. For.
Latino Amer. de Invest. Capacit., Merida, Venezuela, Bol. 6: 34-39.
Slud, P. 1964. The birds of Costa Rica, distribution and ecology. Bull. Am.
Mus. Nat. Hist. 128.
Small, J. K. 1914. Exploration in the Everglades and on the Florida keys. J.
N.Y.Bot. Card. 15:69-79.
Small, J. K. 1923. Mangroves, J. N. Y. Bot. Card. 24: 211.
Small, J. K. 1924. Mangroves. J. N. Y. Bot. Card. 25: 73-74.
Small, J. K. 1933. Manual of the Southeastern Flora. Univ. North Carolina
Press, Chapel Hill, pp. 938-939.
Smith, A. C. and I. M. Johnson. 1945. A phytogeographic sketch of Latin
America, in Plants and Plant Science in Latin America, F. Verdoorn, (ed.),
Chronica Botanica Co. Waltham, Massachusetts.
Smith, F. G. W., R. H. Williams, and C. C. Davis. 1950. An ecological survey
of the sub-tropical inshore waters adjacent to Miami. Ecology 31:119-146.
Smith, J. 1882. A Dictionary of Popular Names of the Plants Which Furnish
the Natural and Acquired Wants of Man in All Matters of Domestic and
General Economy. Macmillan, London, pp. 263-264.
Sodiro, R. P. L. 1901. El mangle rojo. Estudios de Medicina, Quito %: 3-18.
Sokoloff, B., J. B. Redd, and"R. Dutscher. 1950. Nutritive value of mangrove
leaves (Rhizophora mangle L. ). Q. J. Fla. Acad. Sci. 12: 191-194.
Solereder, H. 1885. Zur Anatomie und Systematik der Combretaceen. Bot.
Centralbl. 23: 161-166.
Solereder, H. 1908. Systematic Anatomy of the Dicotyledons. Clarendon
Press, Oxford, 423 pp.
Southwell, C. R. and J. D. Bultman. 1971. Marine borer resistances of
untreated woods over long periods of immersion in tropical waters.
Biotropica 3: 81-107.
Spackman, W., C. P. Dolsen, and W. Riegal. 1966. Phytogenic organic
sediments and sedimentary environments in the Everglades-mangrove
complex. Part I. Evidence of a transgressing sea and its effect on
162
-------
ECOLOGY OF HALOPHYTES
environments of the Shark River area of southwest Florida. Paleontrophica
117: 135-152.
Spackman, W., D. W. Scholl, and W. H. Taft. 1964. Field Guide Book to
Environments of Coal Formation in Southern Florida. Geol. Soc. Am., 67
pp.
Specht, R. L. and C. P. Mountford. 1958. Records of the
American-Australian Scientific Expedition to Arnhem Land. 3. Botany and
Plant Ecology. Melbourne Uni. Press, Melbourne.
Speck, N. H. 1960. Vegetation of the North Kimberly area. CSIRO Aust.
Land Res. Ser. 4: 41-63.
Speck, N. H. and M. Lazarides. 1964. Vegetation and pastures of the West
Kimberly area. CSIRO Aust. Land Res. Ser. 9: 140-174.
Spooner, G. M. and H. B. Moore. 1940. the ecology of the Tamar estuary. 6.
An account of the macroflora of the intertidal muds. J. mar. biol. Assoc.
U.K. 24: 283-291.
Sprague, T. A. and L. A. Boodle. 1909. Kokoti (Anopyxis ealaensis Sprague).
Bull. Misc. Inf., R. Bot. Card., Kew, London, pp. 309-312.
Stace, C. A. 1961. Cuticular characters as an aid to the taxonomy of the
South-West African species of Combretum. Mitt. Bot. Staatssam. Munchen
4: 9-17.
Stace, C. A. 1965. The significance of the leaf epidermis in the taxonomy of
the Combretaceae. J. Linn Soc. London Bot. 59: 229-252.
Stace, C. A. 1966. The use of epidermal characters in phylogenetic
considerations. NewPhytol. 65: 304-318.
Stamp, L. "D. 1925. The aerial survey of the Irrawaddy delta forests. J. Ecol.
13: 262-276.
Standley, P. C. 1924. Trees and shrubs of Mexico. Cont. No. 23., U. S. Natl.
Herb., Washington, D. C.
Standley, P. C. 1928. Flora of the Panama Canal Zone. Contrib. No. 27, U. S.
Natl. Herb., Washington, D. C. p. 283.
Standley, P. C. 1930. Flora of Yucatan. Field Mus. Nat. Hist. Bot. Ser. Ill,
No. 3, p. 371.
Standley, P. C. 1937. Flora of Costa Rica, Part II. Field Mus. Nat. Hist.,
Chicago, pp. 766-767.
Standley, P. C. and S. J. Record. 1936. The forests and flora of British
Honduras. Field Mus. Nat. Hist. Pub. No. 350, Bot. Ser. XII, pp. 275-276.
Steam, W. T. 1958. A key to the West Indian mangroves. Kew Bull. 13:
33-37.
Steenis, C. G. G. J. van. 1935. Maleische vegettieschetsen. Tijdschr. K. ned.
aardrijsk. Genoot. 52: 363-398.
Steenis, C. G. G. J. van. 1941. Kustaanwas en mangrove. Natuurwet. Tijdschr.
Ned. Ind. 101: 82-85.
Steenis, C. G. G. J. van. 1954a. Vegetatie en Flora. New Guinea 2: 218-275.
Steenis, C. G.G. J. van. 1954b. Plumbaginaceae. Fl. Males. 1: 107-112.
Steenis, C. G. G. J. van. 1958a. Rhizophoraceae. Flora Malesiana 5: 431-493.
163
-------
GERALD E.WALSH
Steenis, C. G. G. J. van. 1958b. Tropical shore formations. Proc. Symp.
Humid Trop. Veg., Tijiawi (Indonesia), pp. 215-217.
Steenis', C. G. G. J. van. 1962a. The distribution of mangrove plant genera
and its significance foe paleogeography. Kon. Neder. Akad. van Wetensch.
65: 164-169.
Steenis, C. G. G. J. van. 1962b. The land bridge theory in botany. Blumea 2:
235-372.
Steenis, C. G. G. J. van. 1963. Pacific Plant Areas. 1. Monogr. Natl. Inst. Sci.
Technol. Philipp., Manila, Monogr. No. 8, 279 pp.
Steenis, C. G. G. J. van. 1965. Concise plant geography of Java, in Flora Java,
C. G. G. J. van Steenis (ed.), Vol. 2, pp. 1-72.
Steenis, C. G. G. J. van. 1968. Do Sonneratia caseolaris and S. ovata occur in
Queensland or the Northern Territory? N. Queensland Nat. 35: 3-6.
Steenis, C. G. G. J. van and M. M. J. van Balgoog. 1966. Pacific Plant Areas.
2. Blumea, Suppl. 5, 312 pp.
Steers, J. A. 1929. The Queensland coast and the Great Barrier Reef. Geogr.
J. 74: 232-257.
Steers, J. A. 1937. The coral islands and associated features of the Great
Barrier Reef. Geogr. J. 89: 1-28 and 119-146.
Steers, J. A. 1940. Sand cays and mangroves in Jamaica. Geogr. J. 96:
,305-323.
Steers, J. A., V. J. Chapman, J. Colman, and J. A. Lofthouse. 1940. Sand
cays and mangroves in Jamaica. Geogr. J. 96: 305-328.
Stehle, H. 1945. Forest types of the Caribbean Islands, Part 1. Caribb. For.
Suppl. to Vol. 6, pp. 1-16.
Stephens, W. M. 1962. Trees that make land. Sea Front. 8: 219-230.
Stephens, W. M. 1963. Mangroves: trees that make land. Smithson. Inst. Rep.
1962, pp. 491-496.
Setphens, W. M. 1969. The mangrove. Oceans 2: 51-55.
Stephenson, T. A. 1939. The constitution of the intertidal fauna and flora of
South Africa. Part 1. J. Linn. Soc. London 40: 487-536.
Stephenson, T. A. and A. Stephenson. 1950. Life between tide marks in
North America. I. The Florida Keys. J. Ecol. 38: 354-402.
Stephenson, T. A., A. Stephenson, G. Tandy, and M. A. Spender. 1931. The
structure and ecology of Low Isles and other reefs. Br. Mus. Nat. Hist. Sci.
Rep..Great Barrier Reef Exped., 1928-1929.3: 17-112.
Stephenson, W., R. Endean, and I. Bennett. 1958. An ecological survey of the
marine"fauna of Low Isles, Queensland. Aust. J Mar. Freshwater Res 9'
261-318.
Stern, W. L. and G. K. Voight. 1959. Effect of salt concentration on growth
of red mangrove in culture. Bot. Gaz. 121: 36-39.
Stewart, A. 1916. Some observations concerning the botanical conditions of
the Galapagos Islands. Wis. Acad. Sci. Trans. 18: 272-340.
Steup, F. K. M. 1946. Boschbeheer in de vloedbosschen van riouw Tectona
36: 289-298.
164
-------
ECOLOGY OF HALOPHYTES
Stocker, 0. 1924. Beitrage zum Halophytenproblem. Okologische
untersuchungen der Strandund Dunenpflanzen des Bars (Vorpommern) Z
Bot. 16: 289-330.
Stocker, 0. 1925. Beitrage zum Halophytenproblem. Okologische
untersuchungen der Strandund Dunenpflanzen des Dars (Vorpommern). Z.
Bot. 17: 1-24.
Stocker, 0. 1935. Transpiration und Wasserhaushalt in verschieden Klima
Zonen. III. Jb. wiss. Bot. 81: 464-471.
Stocking, C. R. 1956. Osmotic pressure and osmotic value. Handb. Pflphysiol.
2: 63-64.
Stoddart, D. R. 1962. Three Caribbean atolls: Turneffe Islands, Lighthouse
Reef, and Glover's, British Honduras. Tech. Rep. Coastal Stud. Inst. La.
State Univ. 11,151pp.
Stokes, A. P. D. and R. C. Roberts. 1934. Saline peat profiles of Puerto Rico.
J. Wash. Acad. Sci. 24: 175-182.
Story, R. 1969. Vegetation of the Adelaide-Alligator area. CSIRO Aust. Land
REs. Ser. 25: 114-130.
Straatmans, W. 1954. Reclamation of tidal mud flats in Tonga. Tech. Pap.
South Pac. Comm., No. 53, 18 pp.
Strenzke, K. 1958. Axelsonia tubifera n. sp., ein neuer arthropleoner
Collembole mit Geschlechtsdimorphismus aus der brasilianischen
Mangrove. Acta. Zool. Cracov. 2: 607-619.
Strunk, L. 1906. Chemische untersuchungen von mangrovenrinden.
Tropenpflanzer 10: 116.
Studholme, W. P. and W. R. Philipson. 1966. A comparison of the cambium
in two woods with included phloem: Heimerliodendron brunonianum and
Avicennia vesinifera. N. Z. J. Bot. 4: 332-341.
Sundararaj, D. D. 1954. Mangrove (Avicennia officinalis L. ) leaves as cattle
feed. I. Sci. Cult. 19: 339.
Sukhamananda, M. 1951. Sutan mangrove Forest. Vanasarn For. J. 3: 1-16.
Suter, H. H. 1960. A General and Economic Geology of Trinidad and
Tobago. H.M.S.O., London, 145 pp.
Svenson, H. K. 1945. A brief review of the Galapagos flora, in Plants and
plant Science in Latin America. F. Verdoorn (ed.), Cronica Botanica Co.,
Waltham, Massachusetts.
Svenson, H. K. 1946. Vegetation of the coast of Equador and Peru and its
relation to the Galapagos Islands. Am. J. Bot. 33: 394-426.
Swain, T. 1965. The tannins, in Plant Biochemistry, J. Bonner and J. E.
Varner (eds.), Academic Press, New York, 1054 pp.
Swart, H. J. 1958. An investigation of the mycoflora in the soil of some
165
-------
GERALD E. WALSH
mangrove swamps. Acta Bot. Need. 7: 741-768.
Swart, H. J. 1963. Further investigations of the mycoflora in the soil of some
mangrove swamps. Acta Bot. Neerl. 12: 98-111.
Symington, C. F. 1940. Three malayan species of Bruguiera. Malay. For. 9:
131-139.
Tabb, D. C. and R. B. Manning. 1961. A checklist of the flora and fauna of
northern Florida Bay and adjacent brackish waters of the Florida mainland
collected during the period July 1957 through September 1960. Bull. Mar.
Sci. GulfCaribb. 11: 552-649.
Tabb, D. C., D. L. Bubrow, and R. B. Manning. 1962. The ecology of
northern Florida Bay and adjacent estuaries. Fla. State Board Conserv.
Tech. Ser. Publ. No. 39.
Tadeo, C. B. 1962. Plants with edible fruits in the Philippines. For. Leaves
14: 57.
Tanner, W. F. 1960. Florida coastal classification. Trans. Gulf Coast Assoc.
Geol. Soc. 10: 259-266.
Tansley, A. G. and F, E. Fritsch. 1905. The flora of the Ceylon littoral. New
phytol. 4: 27-36.
Taylor, B. W. 1959. The classification of lowland swamp communities in
northeastern Papua. Ecology 40: 703-711.
Taylor, J. S. 1931. A note on the flora of mangrove. Entomol. Rec. J. Var.,
N. 8.43:4142.
Taylor, W. R. 1925. The marine flora of the Dry Tortugas. Rev. Algol. 2:
113-135.
Taylor, W. R. 1928. The marine algae of Florida, with special reference to the
Dry Tortugas. Carnegie Inst. Wash., Publ. No. 379; Pap. from the Tortugas
Lab. No. 25, 219 pp.
Taylor, W. R. 1954. Sketch of the character of marine algae vegetation of the
shores of the Gulf of Mexico, in The Gulf of Mexico. It's Origin, waters,
and marine life. P.S. Galtsoff, Coordinator, U.S.D.I., Fish. Bull. 55:
177-192.
Teas, H. J. and F. Montgomery. 1968. Ecology of red mangrove seedling
establishment. Bull. Assoc. Southeast. Biol. 15: 56-57.
Teijsmann, J. E. 1957. Dagverhaal eener Botanische Reis over de Westkust
van Sumatra. Nat. Tijd. Ned. Ind. 14: 249- 376.
Teixerira, C. and M. B. Kutner. 1961. Contribuicao para o conhecimento das
diatomaceas da regiao de Cananeia. Bol. Inst. Oceanogr. Sao Paulo 11:
41-74.
Teixeira, C. and M. B. Kutner. 1963. Plankton studies in a mangrove
environment. I. First assessment of standing stock and principal ecological
factors. Bol. Inst. Oceanogr., Sao Paulo 12: 101-124.
Teixeira, C., M. B. Kutner, and F. M. S. Torgo. 1965. 0 efeito da respiracao
bacteriana no estudo da producao primaria. Rev. bras. Biol. 25: 287-294.
Teixeira, C., J. Tundisi, and M. B. Kutner. 1965. Plankton studies in a
mangrove environment. II. The standing stock and some ecological factors.
Biol. Inst. Oceanogr., Sao Paulo, Publ. 14: 1341.
166
-------
ECOLOGY OF HALOPHYTES
Teixeira, C., J. Tundisi and J. Santoro Ycaza. 1967. Plankton studies in a
mangrove environment. IV. Size fractionation of the phytoplankton. Biol.
Inst. Oceanogr. Sao Paulo 16: 39-42.
Teixeira, C., J. Tundisi, and J. Santoro Ycaza. 1969. Plankton studies in a
mangrove environment. VI. Primary production, zooplankton
standing-stock and some environmental factors. Int. rev. Ges. Hydrobiol.
54: 289-301.
Teng, K. Forests in the sea at Hainan Island. Chin. For. 2: 30-31.
Thorn, B. G. 1967. Mangrove ecology and deltaic geomorphology: Tobasco,
Mexico. J.Ecol. 301-343.
Thome, S. and A. F. Moller. 1901. Mangrove. Tropenpflanzen 5: 339.
Thompson, J. M. 1954. The genera of oysters and the Australian species.
Aust. J. Mar. Freshwater Res. 5: 132-168.
Thorne, R. F. 1954. Flowering plants of the waters and shores of the Gulf of
Mexico. U. S. Fish Wildl. Serv., Fish. Bull. 89: 193-202.
Thornton, I. and M. E. C. Giglioli. 1965. The mangrove swamps of Keneba,
Lower Gambia River Basin. II. Sulphur and pH in the profiles of swamp
soils. J. appl. Ecol. 2: 257-269.
Tieghem, Ph. van. 1898. Avicenniacees et Symporemacees. Place de ces deux
noubelles families dans la classification. J. Bot., Paris 12: 345-358.
Tinley, K. L. 1958. A preliminary report on the ecology of the Kosi lake
system. Unpublished report of the Natal Parks, Fish and Game Preservation
Board, Natal, 90 pp.
Tischler, G. 1910. Untersuchungen an Mangrove-und Orchideen-Wurzeln mit
specieller Bezichung auf die Statolithen des Geotropismus. Ann. Jard. Bot.
Buit.,Suppl. 3, pp. 131-186.
Tobler, F. 1914. Die Mangrove der Insel Ulenge (Deutsch-Ostafrika). Bot.
Jahrb. 50: 398-411.
Todd, R. and P. Bronnimann. 1957. Recent foraminifera and Thecamoebina
from the eastern Gulf of Paria. Cushman Found. Foram. Res. Spec. Publ.
No. 3.
Tokioka, T. 1942. Ascidians found on the mangrove trees in Iwayama Bay,
Palao. Palao Trop. Biol. Stn. Stud. (Tokyo) 2: 497-506.
Talken, H. R. 1967. The species of Arthroenemum and Salicornia
(Chenopodiaceae) in southern Africa. Bothalia 9: 255-307.
Tomlinson, T. E. 1957. Relationship between mangrove vegetation, soil
texture and reaction of surface soil after empoldering saline swamps in
Sierra Leone. Trop. Agric. Trinidad 34: 41-50.
Tomlinson, T. E. 1957. Changes in a sulphide-containing mangrove soil on
drying and their effect upon the suitability of the soil for the growth of
rice. Empire J. Exp. Agric. 25: 108-118.
Tralau, H. 1964. The genus Nypa van Wurmb. K. Svensk, Vetensk. Akad.
Handl.,Ser. 5, 10: 5-29.
Treub, M. 1883. Notes sur 1'embryon, le sac embryonaire et 1'ovule de
Avicennia officinalis. Ann. Jard. Bot. Buit. 3: 79-87.
167
-------
GERALD E. WALSH
Triana, I. and I. E. Planchon. 1862. Prodromus Florae Novo- Grandensis ou
enumeration des plantes de la Nouvelle-Grenade, avec descriptions des
especes nouvelles. Ann. Soc. Nat. Quart. Ser., Bot. 17: 1-382.
Trimble, H. 1892. Mangrove tannin. Contr. Bot. Lab. Univ. Penn. 1: 50-55.
Troll, W. 1930. Ueber die sogenannten Atemwurzeln der Mangroven. Ber.
Stsch. bot. Ges. 48: 81-99.
Troll, W. 1931. Botanische Mitteillungen aus den Tropen. III. Dictyotposis
paopagalifera W. Troll, eine neue Brackwasseralge ostindischer
Mangrovegebiete. Flora, N. F. 25: 474-502.
Troll, W. 1932. Uber die sogenannten Atemwurzelin der Mangroven. S. Br.
Ges. Morph. Physiol. 40: 46-48.
Troll, W. 1933a. Uber Acrostichum aurem und Acrostichum speciosum und
nestene Formen des letzeren. Flora, N. F. 28' 301-328.
Troll, W. 1933b. Camptostemon schultzii Mart, und C. philippensis Becc. als
neue Vertreter der australasiatischen Mangrove-vegetation. Flora, N. F. 28:
348-352.
Troll, W. 1942. Die Mangrovegewache. in "vergleichende Morphologie der
Hoheran Pflanzen" Erster Band, Dritter Teil, Berlin-Zehlendorf.
Troll, W. and 0. Dragendorff. 1931. Uber die Luftwurzeln von Sonneratia
Linn, und ilire biologische Bedeutung. Planta 13:311-473.
Truman, R. 1961. The eradication of mangroves. Aust. J. Sci. 24: 198-199.
Trumbull, S. 1954. Mangrove wood, leaves, may bar expansion of Everglades
Park. M.iami Herald, Apr. 14.
Tschirley, F. H. 1969. Defoliation in Vietnam. Science 163: 779-786.
Tsopa, E. 1939. La vegetation des halophytes du Nord de la Roumanie en
connexion avec celle du reste du pays S.I.G.M.A. 70: 1-22.
Tundisi, J. 1969. Plankton studies in a mangrove environment - its biology
and primary production, in Lagunas Costeras, un Simposio. Mem. Simp.
Intern. Lagunas Costeras. UNAM - UNESCO, Nov. 28-30, 1967, Mexico,
D. F., pp. 485494.
Tundisi, J. and C. Teixeira. 1965. Plankton studies in a mangrove
environment, in Simposio sobre a Oceanografic do Atlantico Sul
Occidental, Rio de Janiero, Brazil, 14-18 Se_tembro. 1964. Ann. Acad.
Brasil, Cienc. 37 (Suppl.): 192-193.
Tundisi, J. and C. Teixeira. 1968. Plankton studies in a mangrove
environment. VII. Size fractionation of the phytoplankton: some studies
on methods. Bol. Inst. Oceanogr. 17; 89-94.
Tundisi, J. and T. M. Tundisi. 1968. Plankton studies in a mangrove
environment. V. Salinity tolerances of some planktonic crustaceans. Bol.
Inst. Oceanogr., Sao Paulo 17: 57-65.
Turrill, W. B. and E. Milne-Redhead. 1956. Flora of tropical East Africa:
Rhizophoraceae. Crown Agents, London, 311 pp.
Uhvits, R. 1946. Effect of osmotic pressure on water absorption and
germination of alfalfa seed. J. Bot. 33: 278-285.
168
-------
ECOLOGY OF HALOPHYTES
Ule, E. 1901. Die vegetation von Cabo Frio an der Kuste von Brasilien. Bot.
Jb. 28:511-528.
Ulken, A. 1970. Phycomyceten aus der Mangrove bei Cananeia (Sao Paulo,
Brazilien). Veroff. Inst. Meeresforsch. Bremerhaven 12: 313-319.
Unwin, A. H. 1920. West african forests and forestry. E. P. Dutton and Co.,
New York, 416 pp.
Uphof, J. C. T. 1935. Die holzigen Halophyten-vereinen des Kustengebietes
Floridas. Mitt. Dut. Dendrol. Ges. 47: 39-53.
Uphof, J. C. T. 1937. Die nordliche Verbreitung der Mangroven im
atlantischen Gebiet. Arch. Hydrobiol. 31: 141-144.
Vahrmeijer, J. 1966. Notes on the vegetation of north Zululand. Afr. Wild!.
20: 151-161.
Vann, J. H. 1959a. The physical geography of the lower coastal plain of the
Guiana coast. Geogr. Branch, O.N.R., Washington, D.C., Project NR.
388-028, Tech. Rep. 1,91 pp.
Vann, J. H. 1959b. Landform-vegetation relationships in the Atrato delta.
Ann. Assoc. Am. Geogr. 49: 345-360.
Vanwijk, C. L. 1951. Soil survey of the tidal swamps of South Borneo in
connection with agricultural practices. Tectona 41: 75-110.
Vaughan, R. E. and P. 0. Wiehe. 1937. Studies on the vegetation of
Mauritius. I. J. Ecol. 25: 301-311.
Vaughan, T. W. 1909. The geologic work of mangroves in southern Florida.
Smithson. Misc. Collect. 52: 461-464.
Vazquez Bota, J. 1963. Classification de las melsas forestales de campeche.
Bol. Tech. Inst. Macl. For. Mex., Vol 10.
Venkateswarlu, J. 1935. A contribution to the embryology of
Sonneratiaceae. Proc. Indian Acad. Sci. 5: 23-29.
Venkateswarlu. J. 1944. The estuarial flora of the Godavari. J. Bombay Nat.
Hist. Soc. 44:431435.
Benkateswarlu J. and R. S. P. Rao. 1964. The wood anatomy and taxonomic
position of Sonneratiaceae. Curr. Sci. 33: 6-9.
Versteegh, F. 1952. Problems of silviculture and management of mangrove
forests in Indonesia. Doc. FAO Asia-Pac. For. Comm. 77, 5 pp.
Verwey, J. 1930. Einiges uber die Biologic Ost-Indisher Mangrovekrabben.
Terubial2: 167-261.
Vibul Vanakij, L. 1937. Mangrove forest. Vanasarn For. J., No. 6.
Vieillard, E. 1862. Plantes utiles de la Navelle-Caledonie. Ann. Sci. Nat. Paris,
ser. IV, vol. 16.
Visser, S. A. 1963. The occurrence of microorganisms in tropical swamps.
Biochem. J.89: 83-84.
Vu Van Cuong Humbert. 1964a. Flore et vegetation de la mangrove de la
region de Saigon-Cap Saint Jacques, Sud Viet-Nam. These Doct., 3rd Cycle,
spec. Sci. Biol., Paris, 199 pp.
Vu Van Cuong Humbert 1964b. Nouveautes pour la Flore du Gamboge, du
Laos, et de Vietnam (Rhizophoraceae, Sonneratiaceae, Myrtacae).
169
-------
GERALD E. WALSH
Addisonia4: 343-347.
Wadsworth, F. H. 1959. Growth and regeneration of white mangrove in
Puerto Rico. Caribb. For. 20: 59-71.
Wadsworth, F. H. and G. H. Englerth. 1959. Effects of the 1956 hurricane on
forests in Puerto Rico. Caribb. For. 20: 38-57.
Waisel, Y. 1958. Germination behavior of some halophytes. Bull. Res. Coun.
Israel (Botany) 60: 187-189.
Walker, F. S. 1937. The management and exploitation of the Klang mangrove
forests. Malay. For. 6: 71-78.
Walker, F. S. 1938. Regeneration of Klang mangroves. Malay. For. 7: 71-76.
Walker-Arnott, G. A. 1938. On the Rhizophoraceae. Ann. Mag. Nat. Hist. 1:
359-374.
Walsh, G. E. 1967. An ecological study of a Hawaiian mangrove swamp, in
Estuaries, G. H. Lauff (ed.)( AAAS Publ. 83, Washington, pp. 420-431.
Walter, H. 1936. Die Okologischen Verhaltnisse in der Nabel Wuste, Namib
(Deutsch Sudwestafrika). Ber. Dtsch. bot. Ges. 54: 17-32.
Walter, H. 1936a. Der wasser-und Salzgehalt der Ost-Afrikanischen
Mangroven. Ber. Dtsch. bot. Ges. 54: 33-49.
Walter, H. 1936b. Uber den Wasserhaushalt der Mangrovpflanzen. Ber.
Schweiz. bot. Ges. 46: 217-228.
Walter, H. and M. Steiner. 1936. Die Okologie der Ost-Afrikanischen
Mangroven. Z. Bot. 30: 65-193.
Warburg, 0. 1893. Rhizophora mangle, tropische Fragmente. Bot. Jb. 40:
517-548.
Ward, J. M. 1967. Studies in ecology on a shell barrier beach. I. Vegetatio 14:
241.
Waring, G. A. 1926. Geology of the Islands of Trinidad and Tobago, B.W.I.
John Hopkins Press, Baltimore, 180 pp.
Warming, E. 1877. Om Rhizophora mangle L. Bot. Notes 1877: 14-21.
Warming, E. 1883. Tropische Fragmente. II. Rhizophora mangle L. Bot. Jb.
4: 519-548.
Warming, E. and M. Vahl. 1925. Oecology of Plants. Oxford Univ. Press,
London, 422 pp.
Warner, G. F. 1967. The life history of the mangrove tree crab, Aratus pisoni
L. Zool. 153: 321-335.
Warner, G. F. 1969. The occurrence and distribution of crabs in a Jamaican
mangrove swamp. J. Anim. Ecol. 38: 379-389.
Watanabe, K. and M. Kutner. 1965. Bacteriological analysis of mangrove
waters at Cananeia. in Simposio Sobre a Oceanografia do Atlantico Sul
Occidental, Rio de Janeiro, Brazil, 14-18 Septembro, 1964. Ann. Acad.
Brazil Cienc. 37 (Suppl.): 3.
Watanabe, K. and M. Kutner. 1965. Plankton studies in a mangrove
environment. III. Bacterial analysis of waters in Cananeia. Bol. Inst.
Oceanogr. 14: 43-51.
Waterman, A. M. 1946. The effect of water soluble extracts from the
170
-------
ECOLOGY OF HALOPHYTES
heartwood of tropical American woods on the growth of two wood-decay
fungi. Trop. Woods. 88:3
Watson, J. G. 1928. Mangrove forests of the Malay Peninsula. Malay. For.
Rec. 6: 1-275.
Watson, J. G. 1934. Stick thinnings. Malay. For. 3: 12-17.
Webb, K. L. 1966. NaCl effects on growth and transpiration in Salicornia
bigelovii salt marsh halophyte. Plant and Soil 24: 261-268.
Webb, L. J. 1966. The identification and conservation of habitat types in the
wet tropical lowlands of North Queensland. Proc. R. Soc. Queensland. 78:
59-86.
Webber, J. H. 1898. Strandflora of Florida. Science (N.S.) 8: 658-671.
Weberling, F. 1960. Weitere Untersuchungen uber das Vorkommen
rudimentarer Stipeln bei den Myrtales (Combretaceae, Melastomataceae).
Flora 149: 189-205.
Webster, L. 1967. Distribution of mangroves in South Australia. A.A. (hon.)
thesis, Geogr. Dept., Univ. Adelaide, Adelaide, Australia.
Wei, S. 1947. The relation of typhoons to plant distribution in Taiwan. Bull.
Taiwan For. Res. Inst. 6: 1-14.
Weiss, M. H. 1966a. Apercu preliminaire sur les rapports entre le milieu et la
distribution des paletuviers le long d'une mangrove littorale proche de
Tulear. Rec. Trav. Stn. Mar. Endoume, Marseille, Suppl. 5: 165-173.
Weiss, M. H. 1966b. Apercu preliminaire sur une mangrove naturelle a
1'interieur des terres au Sud de Tulear. Rec. Trav. Stn. Mar. Endoume,
Marseille, Suppl. 5: 175-178.
Weiss, M. H. and A. Kiener. 1971. Observations relatives a la nature chimique
des eaux de la region de Tulear. Diversite, variations, relations avec les
zonations biocenotiques. Tethys (Suppl.) 1: 215-236.
Welch, B. L. 1963. From coral reef to tropical island via Thalassia and
mangrove. Va. J. Sci. 14: 213-214.
Wenzel, H. 1925. Die Mangroverinden als Gerbmaterialen. Anatomische
Untersuchungen der gerbstoffreichsten Mangroverinden. Bot. Arch. 59-96.
West, E. and L. E. Arnold. 1956. Native Trees of Florida. 2nd ed., Univ.
Florida Press, Gainesville, p. 162.
West, 0. 1945. Distribution of mangroves in the eastern Cape Provence. S.
Afr. J. Sci. 41: 238-242.
West, R. C. 1954. The mangrove swamp of the Pacific littoral of Colombia.
Coast. Geogr. Conf., Feb. 1954, OWR-NRC, Washington, D. C., pp. 45-50.
West, R. C. 1956. Mangrove swamps of the Pacific coast of Colombia. Ann.
Assoc. Am. Geogr. 46: 98-121.
West, R. C. 1957. The Pacific lowlands of Colombia, a negroid area of the
American tropics. La. St. Univ. Press, Baton Rouge, 278 pp.
Westermaier, M. 1900. Zur Kenntnis der Pneumatophoren. Bot. Unters. im
Anaschluss an eine Tropenreise, vol. 1.
Westing, A. H. 197la. Ecological effects of military defoliation on the forests
of South Vietnam. Bioscience 21: 893-898.
171
-------
GERALD E. WALSH
Westing, A. H. 1971b. Forestry and the war in South Vietnam. J. For. 69:
777-784.
Westing, A. H. 1971c. Herbicides as weapons in South Vietnam, a
bibliography. Bioscience 21: 1225-1227.
Wetmore, A. 1965. The birds of the Republic of Panama. I. Tinamidae
(Tinamous) to the Rhynchopidae (Skimmers). Smithson. Misc. Coll., No.
150.
Weyl, R. 1953a. Lithogenetische Studien in den Mangroven der Pazifik-Kuste.
Neues Jb. Beol. Palaontol., Mh, No. 5, pp. 202-218.
Weyl, R. 1953b. In den Mangroven El Salvadors. Nat. Volk 83: 26-30.
Wharton, W. J. L. 1883. Mangrove as a destructive agent. Nature 29: 76-77.
Wherry, E. T. 1920. Plant distribution around salt marshes. Ecology 1: 42-48.
White, C. T. 1926. A variety of Ceriops tagal C. B. Rob. (=C. candolleana W.
and A.). J. Bot. London 64: 220-221.
Whiteleather, R. T. and H. H. Brown. 1945. An Experimental Fishery Survey
in Trinidad, Tobago and British Guiana. U. S. Govt. Printing Office,
Washington, D. C. 130pp.
Whitford, H. 1911. The forests of the Philippines. Bur. For. Bull. 10 (1):
1-94.
Whitford, H. 1911. The forests of the Philippines. Bur. For. Bull. 10 (2):
1-113.
Whitlo, C. K. 1947. Animal life in mangroves. W. Aust. Nat. 1: 53-56.
Whittaker, R. H. 1953. A consideration of climax theory: the climax as a
population and pattern. Ecol. Monogr. 23: 41-78.
Wiehe, P. O. 1935. A quantitative study on the influence of tide upon
populations of Salicornia europaea. J. Ecol. 23: 323-333.
Wiele, N. 1882. Om Stammens og Bladets Byning hosAvicennia nitida L. Bot.
Tidsskr. 13: 156.
Wieler, A. 1898. Die function der Pneumathoden und das Aerenchym. Jb.
wiss. Bot. 32: 503-524.
Wiens, H. J. 1956. The geography of Kapingamarangi Atoll in the eastern
Carolines. Atoll Res. Bull. 48, June 1956.
Wiens, H. J. 1962. Atoll Environment and Ecology. Yale Univ. Press, New
Haven, 532 pp.
Wijk, C. L. van. 1953. Soil survey of the tidal swamps of South Borneo in
connection with the agricultural possibilities. Contrib. Cent. Agr. Res.
Stat., Bogor, Indonesia, No. 123.
Williams, R. R. 1911. The economic possibilities of the mangrove swamps of
the Philippines. Philipp. J. Sci. A. 6: 45-61.
Willis, J. H. 1944. Excursion to Seaholm salt marsh flora and mangroves
Viet. Nat. 61:40-41.
Wilson, E. 0. and D. S. Simberloff. 1969. Experimental zoo-geography of
islands: defaunation and monitoring techniques. Ecology 50: 267-278.
Wilson, 0. 1924. Latin America as a source of tanning materials. Chem
Metall. engin. 30: 303-305, 344-346, 398-399.
172
-------
ECOLOGY OF HALOPHYTES
Wilson, W. P. 1889. The production of aerating organs on the roots of swamp
and other plants. Proc. Acad. Nat. Sci. Philadelphia, pp. 67-69.
Winkler, H. 1931. Einige Bemerkungen uber Mangrove-Pflanzen und den
Amorphophallus titanum im Hamburger Botanischer Garten. Ber. Dtsch.
hot. Ges. 49: 87-102.
Womersley, H. B. S. and S. J. Edmonds. 1952. Marine coastal zonation in
southern Australia in relation to a general scheme of classification. J. Ecol.
40: 89-90.
Womersley, H. B. S. and S. J. Edmonds. 1958. A general account of the
intertidal ecology of south Australian coasts. Aust. J. Mar. Freshwater Res
9: 217-260.
Wyatt-Smith, J. 1953a. The malayan species of Bruguiera. Malay. For. 16:
156-161.
Wyatt-Smith, J. 1953b. The malayan species of Sonneratia. Malay. For. 16:
213-216.
Wyatt-Smith, J. 1954a. The malayan species of Avicennia. Malay. For. 17:
21-25.
Wyatt-Smith, J. 1954b. Mangrove flora replaced by fresh water forest. Malay.
For. 17: 25-26.
Wyatt-Smith, J. 1960. Field key to the trees of mangrove forests in Malaya.
Malay. For. 23: 126-132.
Wyatt-Smith, J. and A. J. Vincent. 1962. Progressive development in the
management of tropical lowland evergreen rain forest and mangrove forest
in Malaya. Malay. For. 25: 199-223.
Yamamoto, Y. 1940. Enumeration of the mangrove plants, including herbs,
climers, epiphytes, and the lower forms of plant life from the Dutch East
Indies. Trop. Agric. 12: 157.
Yamanaka, T. 1957. The forest vegetation of the Amani islands with special
reference to the Shiia sieboldii forest. Rep. U.S.A. Biol. Stat. Kochi Univ.
4, 14pp.
Yamashiro, M. 1961. Ecological study on Kandelia candel (L.) Druce, with
special reference to the structure and falling of seedlings. Hikobia 2:
209-214.
Young, C. M. 1930. A Year on the Great Barrier Reef. Putnam, New York,
246 pp.
Yonge, C. M. 1953. Aspects of life in muddy shores, in Essays in Marine
Biology, S. M. Marshall and A. P. Orr (eds.), Oliver and Boyd, Edinburgh,
pp. 29-49.
Zieman, J. C. Origin of circular beds of Thallasia (Spermatophyta:
Hydrocharitaceae) in south biscayne Bay, Florida and their relationship to
mangrove hammocks. Bui. Inst. Mar. Sci. in press.
Zilch, A. 1954. Moluscos de los manglares de El Salvador. Com. Inst. Trop.
Invest. Cient. (El Salvador) 3: 77-87.
Zinderen Bakker, E. M. van. 1965. On swamp vegetation and structure of
173
-------
GERALD E. WALSH
swamps in south and east Africa. Bot. Jb. Syst. Pflanzengeogr. 84:
215-231.
Zullo, V. A. 1969. Additional records of Tetrabalanus polygenus Cornwall,
1941 (Cirripedia, Thoracica). Occas. Pap. Calif. Acad. Sci. 74: 1-8.
174
-------
CONTRIBUTION NO. 155
-------
RESIDUES IN FISH, WILDLIFE,
AND ESTUARIES
Reprinted by
U.S. Environmental Protection Agency
from
Pesticides Monitoring Journal, Volume 6, Number 4, March 1973
Organochlorine Residues in Estuarine Mollusks,
1965-72—National Pesticide Monitoring Program1
Philip A. Butler3
Part I. General Summary and Conclusions
Part II. Residue Data—Individual States
SECTION A:
SECTION B:
SECTION C:
SECTION D:
SECTION E:
SECTION F:
SECTION G:
SECTION H:
SECTION I:
SECTION J:
SECTION K:
SECTION L:
SECTION M:
SECTION N:
SECTION O:
ALABAMA
CALIFORNIA
DELAWARE
FLORIDA
GEORGIA
MAINE
MARYLAND
MISSISSIPPI
NEW JERSEY
NEW YORK
NORTH CAROLINA
SOUTH CAROLINA
TEXAS
VIRGINIA
WASHINGTON
ABSTRACT
This paper describes the development of the national
program for monitoring estuarine mollusks in 15 coastal
States and reports the findings for the period 1965-72. The
report is presented in two parts: Part I. General Summary
ind Conclusions, and Part II. Residue Data—Individual
States.
1 Contribution No. 155 from the Gulf Breeze Environmental Research
Laboratory, U.S. Environmental Protection Agency, Gulf Breeze, Fla.
32561, an Associate Laboratory of the National Environmental
Research Center, Corvallis, Oreg.
3 Ecological Monitoring Branch, Technical Services Division, Office
of Pesticide Programs, U.S. Environmental Protection Agency, Gulf
Breeze, Fla. 32561.
238
Analyses of the 8,095 samples for 15 persistent organo-
chlorine compounds showed that DDT residues were ubiqui-
tous; the maximum DDT residue detected was 5.39 ppm.
Dieldrin was the second most commonly detected compound
with a maximum residue of 0.23 ppm. Endrin, mirex, toxa-
phene, and polychlorinated biphenyls were found only oc-
casionally. Results indicate a clearly defined trend towards
decreased levels of DDT residues, beginning in 1969-70. At
no time were residues observed of such a magnitude as to
imply damage to mollusks; however, residues were large
enough to pose a threat to other elements of the biota
through the processes of recycling and magnification.
PESTICIDES MONITORING JOURNAL
-------
Part I. General Summary and Conclusions
Introduction
Initial investigations of the effects of pesticides on
estuarine fauna were undertaken at the Gulf Breeze
Laboratory in 1958 to determine if the pesticide lindane
might be safely used directly in estuarine waters to con-
trol crabs preying on shellfish populations. The un-
expected acute toxicity of this chemical, not only to
crabs but also to nontarget organisms, revealed by these
early experiments prompted a broad investigation of both
the direct and indirect effects of persistent synthetic
pesticides. The extent of the problem was not known,
and the investigators were concerned about the potential
harm to estuarine fauna exposed to drainage waters from
large river basins where significant quantities of pesticides
were used. Marine commercial fisheries were recognized
as being especially vulnerable since a major portion of
their catch, both in tonnage and dollar value, is made up
of estuarine-dependent species.
The acute toxicity of a broad spectrum of pesticides was
determined under laboratory conditions (14-17). These
data, however, could be most useful only if there were
information on the actual levels of organochlorines
reaching the estuarine environment. Accordingly, a
search was undertaken for meaningful tools to measure
this type of pollution (6).
The decision to use mollusks as bioassay t6ols was based
on the findings of laboratory experiments designed to
measure the uptake and flushing rates of an array of
organochlorine pesticides under controlled conditions.
Various species of mollusks, but primarily the eastern
oyster, Crassostrea virginica, were exposed to appro-
priate concentrations of pesticides added continuously
to a flowing seawater system. Results indicated that
oysters detect DDT in the ambient water supply at levels
as low as 10 parts per trillion (lO"11). By the process
of biomagnification, residues of DDT as high as 25
ppm accumulate in oyster tissues within 96 hours at a
level of environmental contamination of only 1.0 ppb
(1). Oysters tolerate tissue residues of DDT at least as
high as 150 ppm without apparent ill effect provided
residues are accumulated slowly. However, as little as
0.1 ppm of DDT in the oyster's water supply terminates
feeding activities and at summer water temperatures
(31°C) will cause death.
Organochlorine residues are flushed rapidly from mol-
luscan tissues when the water supply is no longer
contaminated. In one experimental series, for example.
DDT residues of about 25 ppb in oysters and soft
VOL. 6, No. 4, MARCH 1973
clams, My a arenaria, diminished by 50-90% after a
week of flushing in clean water. Consequently, it is
possible to learn much about the periodicity of organo-
chlorine pollution in estuaries from samples of sedentary
species collected at appropriately brief intervals.
As a result of these studies, it was possible for the
Bureau of Commercial Fisheries to undertake a program
for monitoring pesticide residues in estuarine mollusks
to determine the extent of organochlorine pollution.
The collection of samples was not begun immediately in
some areas, while in others, sample collection was
terminated at an early date. The program was continu-
ously operative, however, from July 1965 through June
1972. In 1971, the Gulf Breeze Laboratory and the
monitoring program became a part of the U.S. Environ-
mental Protection Agency.
The following report describes the 7-year (1965-72) data
collection and discusses, specifically, the well-defined
trends in the magnitude of DDT residues in estuarine
mollusks. Except where noted, the term DDT includes
the metabolites TDE and DDE. All residue analyses
are presented, by State, in Part II of this report. A report
summarizing the first 3 years of this program was pub-
lished in 1969 (3).
Data Interpretation,
Although the eastern oyster has a wide distribution, it
was obvious that some other species might be more
available for monitoring in different geographical areas
or salinity regimes; thus, different species of mollusks
were tested in the laboratory to determine their relative
capabilities in the uptake and retention of organochlorine
pollutants (2). Such information is all important for
the understanding of these monitoring data.
In the tests, all species were exposed to the same
hydrographic conditions with low turbidity and a salinity
level about 80% that of seawater. It is probable that
species accustomed to different ecological conditions
would react more efficiently in nature than in the
Laboratory. Caution must be exercised in the extrapola-
tion of laboratory results to field conditions, and, at best,
such data serve only as guidelines for the interpretation
of residue levels in monitored samples.
In general, any of three species of oysters, four species
of mussels and two species of clams were found to be
reliable indicators of the magnitude of organochlorine
pollution (Table 1). In some areas it was necessary to
use the hard clam, M. mercenaria, although it is the least
satisfactory of the species evaluated. Under similar
239
-------
laboratory conditions, for example, hard clams ac-
cumulated pesticide residues less than half as large as
those in oysters. Moreover, the residues were flushed
from the clam much more quickly than from the oyster
when clean water was restored.
Sample Collection and Preparation
The management of estuarine molluscan resources is the
responsibility of the individual States; therefore, in
each coastal area there is a cadre of specialists who are
not only interested in estuarine pollution but who also
have the knowledge and equipment necessary to collect
shellfish samples. Without the continuing cooperation
TABLE 1.—Pelecypod mollusks used to monitor organo-
chlorine residues in 15 States—1965-72
SCIENTIFIC AND COMMON NAMES OP MOLLUSKS
Crassostrea glgas
Crassostrca vlrglnica
Oslrea lurlda
Modtolus demtssus
Modiolia modlolus
Mytilus californianus
Mytilus edulis
Mercenaria mercenaria
Mya arenarla
Corbicula flumtnea
STATE
Alabama
California
Delaware
Florida
Georgia
Maine
Maryland
Mississippi
New Jersey
New York
North Carolina
South Carolina
Texas
Virginia
Washington
Pacific oyster
eastern oyster
Olympia oyster
ribbed mussel
northern horse mussel
Californian mussel
blue mussel
hard clam
soft clam
Asiatic clam, fresh water
SPECIES COLLECTED
C, vlrglnica
C. gigas
O. lurida
M. demtssus
M. californianus
M. edulis
C. flumlnea
C. vlrglnica
M. demissus
M. mercenaria
C. virglntca
C. virglnlca
M. modlolus
M. edulis
M. arenarla
C. vlrginica
C. vlrginica
C. vlrginica
C. vlrginica
M. demissus
M. edulis
M. mercenaria
M. arenarla
C. vlrginica
C. vlrginica
C. vlrginica
C. virgintca
C. glgas
240
of these agencies (see Acknowledgment), this program
could not have achieved its objectives.
Estuaries with well denned drainage basins and bays that
could be considered "nursery areas" for estuarine fauna
were selected for monitoring.
Some sites were monitored because of suspected pollu-
tion problems. To insure continuity of data, permitting
detection of not only seasonal but yearly trends in
pesticide pollution levels, it was essential, too, that the
stations selected have shellfish populations large enough
for monthly collections over a number of years. Dupli-
cate samples of 15 or more mature mollusks were
collected and prepared for shipment at about 30-day
intervals. About 10% of all samples were analyzed in
replicate; the remaining duplicates were discarded after
satisfactory analysis of the sample. Sample collections
were interrupted by the loss of shellfish populations to
vandals, floods, and hurricanes, but the overall continuity
of the data was good.
Coverage of coastal estuaries was incomplete in this
program because other agencies were monitoring shell-
fish in some states, notably Alabama, Louisiana, and
Massachusetts. The number of sample collections by
State and year is tabulated in Table 2. The original plan
was to monitor each area for 5 years so that trends in
pesticide residue levels could be detected. The general
absence of residues in Washington estuaries, however,
prompted an earlier termination of monitoring in that
State. In addition to the samples tabulated, about 2,000
miscellaneous samples of other species of vertebrates and
invertebrates were collected and analyzed. These fre-
quently had more varied pesticide residues and at higher
levels than mollusks but are omitted from this report
because of difficulty in determining their source.
The analysis of all samples by a single laboratory to
insure uniformity seemed important in planning the
program. Various potential preservatives were examined
to find a method for shipping samples without resorting
to refrigeration. Eventually, it was discovered that by
dehydrating the homogenized tissues of mollusks or
other marine animals with a desiccant mixture, the dry
samples could be wrapped in aluminum foil and held
without refrigeration for 15 or more days without
degradation or loss of organochlorine residues (2). This
made it possible to ship the samples by regular mail.
In practice, samples of 15 or more mature oysters or
other mollusks were collected and taken to the cooperat-
ing agency's laboratory. Samples not to be processed
immediately could be refrigerated for 2 or 3 days in the
shell. If longer storage was necessary, animals were
shucked and the undrained meats frozen in mason jars.
The shucked meats were homogenized in an electric
blender, and approximately 25-g aliquots were blended
PESTICIDES MONITORING JOURNAL
-------
with precisely three times their weight of desiccant to
yield a total sample weight of about 100 g. Alternate
blending and chilling (not freezing) of sample is re-
quired to achieve a dry, free-flowing product. The
amount of desiccant used depends on the moisture con-
tent of the sample. Less desiccant is required for fish
(two times body weight), while up to nine times as much
desiccant may be used with small samples, plankton
for example, to achieve a 100-g final weight of the
sample to be processed. The desiccant is made up of
about 90% sodium sulfate and 10% Quso (Quso G30,
manufactured by Philadelphia Quartz Co., Philadelphia,
Pa.), a micro fine precipitated silica.
Analytical Procedures
Throughout the monitoring program samples were
routinely screened for the following substances: aldrin,
chlordane; p,p'-DDT, p.p'-TDE (ODD), p.p'-DDE,
dieldrin, endrin, heptachlor, heptachlor epoxide, lindane,
methoxychlor, mirex, and toxaphene. On the few oc-
casions when the o,p' isomers of DDT were detected in
quantifiable amounts, they were included with the p,p'
residues.
On receipt in the laboratory, samples were extracted
ftir 4 hours with petroleum ether in a Soxhlet apparatus.
Extracts were concentrated and transferred to 250-ml
separatory funnels. The extracts were diluted to 25 ml
with petroleum ether and partitioned with two, 50-ml
portions of acetonitrile previously saturated with petro-
leum ether. The acetonitrile was evaporated just to dry-
ness, and the residue eluted from a Florisil column
(12). The sample was then identified and quantitated
by electron capture gas chromatography. Three columns
of different polarity (DC-200, QF-1, and mixed
DC-200/QF-1) were used to confirm identification
Operating parameters on Varian Aerograph gas chrom-
atographs were as follows:
Columns: Pyrex glass 6' x W (o.d.) packed with 3% DC-200,
5% QF-1, and a 1:1 ratio of 3% DC-200 and 5%
QF-1, all on 80/100 mesh Gas Chrom Q
Temperatures: Detector—210° C
Injector—210° C
Oven—190° C
Carrier gas: Prepurified nitrogen at a flow rate of 40 ml/mln
A few samples were analyzed by thin layer chromatog-
raphy. All residues are reported on a wet-weight basis.
The lower limit of quantification was 10 ppb. Laboratory
tests conducted during the sampling period gave the
following recovery rates: p,/-DDE, 80-85%; p.p'-TDE,
92-95%; p,//-DDT, 91-95%. Data in this report do not
include a correction factor for percent recovery.
Toxaphene sometimes interfered with the quantification
of DDT residues which, in these cases, are reported as
approximate values. The lower limit of quantification of
toxaphene was 250 ppb. The presence of polychlorinated
biphenyls (PCB's) also interfered with the quantifica-
tion of DDT residues. In most instances, DDT was
calculated as though PCB's were not present. Acquisition
TABLE 2.—Summary of sample collections by State and year—1965-72
STATB
Alabama
California
Delaware
Florida
Georgia
Maine
Maryland
Mississippi
New Jersey
New York
North Carolina
South Carolina
Texas
Virginia
Washington
PRINCIPAL
SPECIES
MONITORED
C. virginlca
C. glgas
M. mercenaria
C. virginlca
C. virgin/Co
M. arenaria
C. virginlca
C. virglnlca
C. virginlca
M. mercenaria
C. virginica
C. virginica
C. virginlca
C. virginica
C. glgas
Total
NUMBER OF SAMPLE COLLECTIONS
1965
6
6
30
72
53
56
40
263
1966
136
16
80
95
18
71
23
148
96
142
133
117
218
1,293
1967
180
101
102
112
89
20
72
44
183
201
143
125
123
223
1,718
1968
13
167
99
82
127
79
26
72
45
175
204
145
93
120
214
1,661
1969
20
139
71
44
124
83
9
63
39
174
204
108
97
112
1,287
1970
45
35
121
44
15
66
33
148
124
103
105
839
1971
75
19
120
60
27
143
136
95
27
702
1972
30
6
60
36
8
88
66
29
9
332
TOTALS
33
772
287
374
664
396
88
470
219
1,059
1,031
610
728
669
695
8,095
VOL. 6, No. 4, MARCH 1973
241
-------
of the appropriate standards permitted the identification
of Aroclor 1254® in samples from California, Florida,
Georgia, Texas, and Virginia, and Aroclor 1242®
in samples from Virginia. Since 1970, PCS residues
have been approximately quantified in samples from
Florida and, more recently, from Virginia.
There is some question as to how much interference by
PCB's exists in the sample analyses reported in the early
years of the monitoring program. At this time there is
no way of knowing with certainty. It is considered sig-
nificant that in the period 1965-70 there was a 3-8%
annual increase in the domestic sale of these chemicals,
and total domestic sales in 1970 were more than double
sales in 1960; however, PCB residues were identified in
samples fron relatively few estuaries in 1971.
During the course of the program, several States ex-
tended the monitoring of their estuaries and collected
more samples than the Gulf Breeze Laboratory was
equipped to process. Analytical equipment similar to
that used at Gulf Breeze was provided to these agencies
as well as a manual of operations (Prepared by A. J.
Wilson, Jr., Research Chemist, Gulf Breeze Laboratory).
to insure similar methodology in analytical techniques.
For the first few collections under the new arrangement,
samples were split and analyses made by both the State
and Federal laboratories. Excellent comparability in data
was obtained (13) and thereafter, the State agency sub-
mitted only the monthly data reports to the Gulf Breeze
Laboratory. Such arrangements were in effect during
portions of the monitoring program in California,
Georgia, Maine, New York, and Virginia.
Data Summaries and Discussion
DDT with its analogs was the most commonly identified
pesticide and occurred in 63% of all samples analyzed
(Table 3). Dieldrin was the second most commonly
detected residue with an incidence of 15%. DDT and
dieldrin were detected in some samples from all States
monitored (Tables 4 and 5). Other organochlorine
residues were encountered infrequently and generally at
low levels, with the exception of toxaphene. The large
number of Georgia samples containing toxaphene re-
flects the direct contamination of the marine environ-
ment by the effluent from a single manufacturing plant.
The incidence of DDT residues varied markedly from
one drainage basin to another and was not correlated
with the magnitude of the residues. Only in New Jersey
and Alabama, for example, did all samples contain
detectable residues of DDT, but the size of DDT resi-
dues was greater in several other States (Table 4). It
is true that in both Alabama and New Jersey, monitored
oyster populations were exposed primarily to the runoff
from a single, although complex, drainage basin. In
other States, samples were collected from several distinct
drainage basins.
TABLE 3 —Summary of organochlorine residues detected in estuarine mollusks by Stale—1965-72
STATE
Alabama
California
Delaware
Florida
Georgia
Maine
Maryland
Mississippi
New Jersey
New York
North Carolina
South Carolina
Texas
Virginia
Washington
TOTAL
NUMBER
OF
SAMPLES
33
772
287
374
664
396
88
470
219
1,059
1,031
610
728
669
695
NUMBER OF SAMPLES WITH RESIDUES >5 PPB (JIG/KG) AND MAXIMUM
RESIDUE ( ) DETECTED IN PPB (AG/KG) ,.
DDT
33 (616)
712 (3,970)
216 (205)
230 (5,390)
96 (96)
72 (359)
71 (70)
285 (135)
219 (278)
858 (596)
768 (566)
332 (154)
530 (1,249)
585 (678)
78 (176)
DIELDRIN
6 (21)
194 (57)
37 (25)
27 (28)
141 (230)
14 (38)
11 (22)
19 (20)
52 (26)
456 (132)
12 (19)
24 (154)
134 (87)
112 (40)
1 (120)
ENDRIN
14 (19)
22 (32)
MlREX
12 (540)
TOXAPHENE
4 (11,000)
128 (54,000)
' 1
PCB's
121
25 (390)
1 16
'6
15
19 (2,800)
1 Present but not quantified.
242
PESTICIDES MONITORING JOURNAL
-------
TABLE 4.—Listing of States in order of frequency and
maximum value of DDT residues in mollusks
TABLE 5.—Listing of States in order of frequency and
maximum value of dieldrin residues in mollusks
STATE
Alabama
New Jersey
California
Virginia
New York
Maryland
Delaware
North Carolina
Texas
Florida
Mississippi
South Carolina
Maine
Georgia
Washington
FREQUENCY
OF RESI-
DUES (%)
100
100
92
87
81
81
75
75
73
62
61
54
18
15
11
STATE
Florida
California
Texas
Virginia
Alabama
New York
North Carolina
Maine
New Jersey
Delaware
Washington
South Carolina
Mississippi
Georgia
Maryland
MAXIMUM
VALUE
IN PPB
5,390
3,970
1,249
678
616
596
566
359
278
205
176
154
135
96
70
STATE
New York
California
New Jersey
Georgia
Alabama
Texas
Virginia
Delaware
Maryland
Florida
Mississippi
South Carolina
Maine
North Carolina
Washington
FREQUENCY
OF RESI-
DUES (%)
43
25
24
21
IS
18
17
13
13
7
4
4
4
1
<1
STATE
Georgia
South Carolina
New York
Washington
Texas
California
Virginia
Maine
Florida
New Jersey
Delaware
Maryland
Alabama
Mississippi
North Carolina
MAXIMUM
VALUE
IN PPB
230
154
132
120
87
57
40
38
28
26
25
22
21
20
19
NOTE: These comparisons are limited in that the number of samples,
number of sampling stations, periods (years) of sampling, and
species of mollusks differ for each State.
The magnitude of all DDT residues was low compared
to residues reported in carnivores such as fish-eating
birds. By extrapolation from laboratory experiments, the
monitoring data indicate that, in most cases, estuarine
pollution with DDT was intermittent and at levels in the
low parts-per-trillion range. In only 38 samples (0.5%)
did the residue exceed 1.0 ppm. These samples were
collected in California, Florida, and Texas in drainage
basins having intensive agricultural development. The
single highest residue of 5.39 ppm (DDT-3.70 ppm.
TDE-0.76 ppm, DDE-0.93 ppm) was observed in
oysters from the Caloosahatchee River drainage basin in
Florida where the seasonal pattern of residue fluctuations
indicated an agricultural or at least a scheduled use of
the pesticide (Fig. 1). It is significant that extensive
acreage in this drainage basin was devoted to sugarcane
and sweetcorn that would be maturing and receiving
fairly heavy applications of pesticides during the peak
residue periods indicated in Fig. 1 (R. G. Curtis, 1972,
Florida Cooperative Extension Service, personal com-
munication) . In controlled feeding experiments in the
laboratory, from 50 to 100% mortality was observed in
small populations of commercial species of shrimp.
crabs, and fish fed exclusively diets containing less than
3.0 ppm of p,p'-DDT (4).
In a survey of 7,000 analyses of mollusk samples com-
pleted in the period 1965-71, the mean residue com-
pc-. ::™ was 24% DDT, 39% TDE, and 37%. DDE.
Exc."r.;;ons to this average picture were Station 2 in
Nev I.'f'' where DDT comprised only 4% (mean of
47 -... .j in 5 years) and Station 18 in Washington
where DDT made up 75% of the residues (mean of
VOL. 6, No. 4, MARCH 1973
NOTE: These comparisons are limited in that the number of samples,
number of sampling stations, periods (years) of sampling,
and species of mollusks differ for each State.
36 samples in 3 years). Biotic recycling of persistent
residues is usually associated with the high percentages
of DDT metabolites found in dominant carnivores. It
is of interest that the metabolites were the only residues
detected in many of these analyses of filter-feeding
mollusks. Results of a study by Johnson et al. (10)
indicated that there are some animals, however, such
as aquatic insects, in which direct exposures to DDT
result in tissue residues that are more than 80% DDE.
The large percentage of the parent compound DDT in
residues from Washington mollusks does imply a direct
contamination of the estuarine environment, perhaps,
for insect control. But in general, the percentage distri-
bution of DDT metabolites in these samples revealed
little about the kinetics of DDT in the estuary.
M A M j JASOND
FIGURE 1.—DDT residues in the eastern oyster from the
Caloosahatchee River Basin, Lee County, Fla., by month of
collection—1967 and 1968
243
-------
Trends in DDT Residues
Many of the estuaries were monitored over a sufficient
period of time to permit detection of clearly defined
trends in DDT residue patterns. Average DDT residue
levels detected in, the first 2 to 5 years and average levels
in the final year of monitoring in each State are pre-
sented in Table 6. The overall picture is that of a
pronounced decline, about 55%, in the number of
samples containing DDT residues in excess of 100 ppb.
There was a 20% decrease in the 10-100 ppb range, and
a concomitant 44% increase in the number of samples
having negligible or undetectable DDT residues.
There are important exceptions to this average picture.
In California, New York, and Virginia, for example,
more samples had residues in excess of 10 ppb in 1971
than in earlier years. On the other hand, the percent of
samples having residues higher than 100 ppb declined
in these States. It would appear that in some areas,
DDT pollution has become more widespread, but has
resulted in residues of lower magnitude in the estuarine
food web.
Since organochlorine residues in mollusks showed a
continuing decline in most areas during the years that
domestic sales and presumably usage of PCB compounds
were increasing, PCB's were not considered to be a
significant factor in the early monitoring data.
Too few samples from Alabama were analyzed in this
program to indicate any trend in residue magnitude.
The mean value of 88 ppb of DDT in 33 samples col-
lected in 1969-70 may be compared, however, with
a mean residue level of 330 ppb in a series of 82 samples
of oysters collected in 1965-66 (7).
Exact comparisons by States of the data in Table 6 are
not valid since in succeeding years there were different
numbers of samples and occasionally different species of
mollusks collected at the same station. A more critical
review of data on DDT residues is possible for 10 sta-
tions in North Carolina. These stations were selected for
the continuity of sampling of the eastern oyster at
monthly intervals for more than 5 years. The number of
samples containing less than 11 ppb of DDT increased
steadily until, in 1971, 76% of all residues were in this
category as compared to only 8% in 1966 and 1967.
The corresponding decrease in the number of samples
containing larger residues is shown in Fig. 2 and Table 7.
TABLE 6.—Percent distribution of DDT residues of different magnitude in estuarine mollusks by State—1965-71
(7,000 samples)
STATE
Alabama
California
Delaware
Florida (1 station)
Georgia
Maine
Maryland
Mississippi
New Jersey
New York
North Carolina
South Carolina
Texas
Virginia
Washington (1 station)
Mean
PERCENT
<11 PPB
FIRST 2 TO
5 YEARS
14
23
43
85
82
19
42
26
22
52
34
18
92
39
1971
7
30
100
96
98
50
72
7
22
76
82
52
94
56
DISTRIBUTION OF SAMPLES
11-100 PPB
FIRST 2 TO
5 YEARS
69
30
62
57
15
17
81
56
69
60
68
47
53
67
8
49
1971
64
67
4
2
50
27
74
74
24
18
45
95
6
39
101-1,000 PPB
FIRST 2 TO
5 YEARS 1971
31
51 28
15 3
1
2 1
31 19
14 4
10
1
13 3
15 5
11 5
>1,OOOPPB
FIRST 2 TO
5 YEARS 1971
5 1
<1
<0.5
244
PESTICIDES MONITORING JOURNAL
-------
TABLE 7.—Trends in magnitude of DDT residues in oysters, 10 stations, North Carolina
YEAR
1966
1967
1968
1969
1970
Subtotal
1971
TOTAL
NUMBER OF
SAMPLES
60
119
120
120
109
528
115
Percent change in 1971
from average for
1966-70
<11 PPB
NUMBER OF PERCENT
SAMPLES DISTRIBUTION
5
9
26
29
61
130
87
8
8
22
24
56
25
76
+204%
11-100 PPB
NUMBER OF PERCENT
SAMPLES DISTRIBUTION
45
90
70
77
46
328
26
75
76
58
64
42
62
22
-65%
101-1,000 PPB
NUMBER OP
SAMPLES
10
20
24
14
2
70
2
PERCENT
DISTRIBUTION
17
16
20
12
2
13
2
-83%
80
60
c
0)
u
OJ
0.
40
20
119
120
120
103
115
1967 1968 1969 1970 1971
FIGURE 2.—Percent of eastern oyster samples containing
more than 10 ppb of DDT, average of monthly samples
collected at 10 stations in North Carolina (Numbers in bars
indicate total number of samples)
Conclusions
The data demonstrate that in most estuaries monitored,
detectable DDT residues have declined in both number
and magnitude in several species of estuarine mollusks
in recent years. DDT pollution in many estuaries, as
judged by the magnitude of the residues in mollusks,
peaked in 1968 and has been declining markedly since
1970.
The'sensitivity of mollusks to organochlorine pollutants
plus the fact that they are filter-feeders warrant the as-
sumption that the contribution of particulate DDT to
estuaries from one or more primary sources such as
drainage basin runoff waters, atmospheric fallout, and
persistent reservoirs in bottom sediments, has declined
significantly.
VOL. 6, No. 4, MARCH 1973
In view of the efficiency of mollusks in detecting and
storing residues of the persistent organochlorines, it is
clear that relatively low levels ofx this type of pollution
were present in the monitored areas during the period
1965 to 1972.
Appropriate correlations of the residue data reported
here with available records of drainage-basin discharge
rates, precipitation, and hydrographic factors in the
various types of estuaries should provide a useful model
for predicting the effects of future introductions of un-
specified synthetic substances chemically similar to
DDT.
See Appendix for chemical names of compounds discussed in
this paper.
LITERATURE CITED
(1) Butler, P. A. 1966. Pesticides in the marine environ-
ment. J. Appl. Ecol. 3(Suppl.): 253-259.
(2) . 1968. Pesticide residues in estuarine
mollusks. Proc. Natl. Symp. Estuarine Pollut. Stanford
University, Stanford, Calif., 1967. p. 107-121.
(3) . 1969. Monitoring pesticide pol-
lution. BioScience 19(10): 889-891.
(4) . 1969. Significance of DDT residues
in estuarine fauna, p. 205-220. In Chemical Fallout
Charles C. Thomas, Springfield, 111.
(5) Butler, P. A., A. J. Wilson, Jr., and R. Children. 1972.
The association of DDT residues with losses in marine
productivity, p. 262-266. In Marine pollution and sea
life. Fishing News (Books) Ltd., London, Eng.
(6) Butler, P. A., A. J. Wilson, Jr., and A. J. Rick. 1960.
Effect of pesticides on oysters. Proc. Natl. Shellfish.
Assoc. 51:23-32.
(7) Casper, V. L., R. J. Hammerstrom, E. A. Robertson,
Jr., J. C. Bugg, Jr., and J. L. Gaines. 1969. Study of
chlorinated pesticides in oysters and estuarine environ-
ment of the Mobile Bay area. USDHEW, Consum
Prot. Environ. Health Serv. 47 p.
(8) Duke, T. W., J. I. Lowe, and A. J. Wilson, Jr. 1970. A
polychlorinated biphenyl (Aroclor 1254®) in the water,
sediment, and biota of Escambia Bay, Florida. Bull
Environ. Contain. Toxicol. 5(2): 171-180.
245
-------
(9) Foehrenbach. ]., G. Mahmood, and D. Sullivan. 1971.
Chlorinated hydrocarbon residues in shellfish (Pele-
cypoda) from estuaries of Long Island, New York.
Pestic. Monit. J. 5(3): 242-247.
(10) Johnson, B. T., C. R. Sounders, H. O. Sanders, and R.
S. Campbell. 1971. Biological magnification and deg-
radation of DDT and aldrin by freshwater inverte-
brates. J. Fish. Res. Board Can. 28(5): 705-709.
(11) May, E. B. 1971. A survey of the oyster and oyster shell
resources of Alabama. Ala. Mar. Resourc. Bull. 4:1-53.
(12) Mills, P. A., J. H. Onley, and R. A. Gaither. 1963.
Rapid method for chlorinated pesticide residues in
nonfatty foods. J. Assoc. Off. Agric. Chem. 46(2): 186-
191.
(13) Modin, J. C. 1969. Chlorinated hydrocarbon pesticides
in California bays and estuaries. Pestic. Monit. J. 3(1):
1-7.
(14) U.S. Department of the Interior. 1962. Effects of pesti-
cides on fish and wildlife in 1960. Fish Wildl. Serv. Circ.
143. Washington, D.C. 52p.
(IS) _. 1963. Pesticide-wildlife
studies: a review of Fish and Wildlife Service investiga-
tions during 1961 and 1962. Fish Wildl. Serv. Circ. 167.
Washington, D.C. 109p.
(16) 7954. Pesticide-
wildlife studies 1963: a review of Fish and Wildlife
Service investigations during the calendar year. Fish
Wildl. Serv. Circ. 199. Washington, D.C. 129p.
(17) 7965. The effects of pesti-
cides on fish and wildlife. Fish Wildl. Serv. Circ. 226.
Washington, D.C. 77p.
A cknowledgments
I should like to but cannot acknowledge individually the
many people in administrative and technical positions
whose interest in this program made its efficient conduct
possible. It is my pleasure to thank especially Louis D.
Stringer, Thomas C. Carver, Chester E. Danes, Anne
Gibson, now of the National Marine Fisheries Service,
and my secretary, Madeleine Brown, for their continuing
cooperation and assistance.
We are greatly indebted to the graduate students and
technicians whose diligence in the collection and proces-
sing of samples made the program a reality. I trust that
the results will make them pleased with their participa-
tion.
The program could not have been developed without
the interest and skills of Alfred J. Wilson, Jr., Research
Chemist at the Gulf Breeze Laboratory.
Lastly, I thank the administrators and professional staffs
of the cooperating agencies who kindly let me think
that the monitoring program had the number one
priority on their busy schedules.
In view of the volume of data in this report, it is in-
evitable that there are sins of both omission and com-
mission. The writer would be most grateful to have
these called to his attention so that the record can be
appropriately emended.
246
COOPERATING AGENCIES— This alphabetical listing by States in-
cludes the names of investigators and, where appropriate, chemists and
their titles at the time they were participating in the program. Where
chemists are not listed, the samples were analyzed at the Gulf Breeze
Laboratory under, the supervision of Alfred J. Wilson, Jr., with the
assistance of Jerrold Forrester and Johnny Knight. The listing of more
than one principal investigator or agency in any one State reflects
changes taking place during the monitoring period 1965-72. Operational
funds were provided by the U.S.F.W.S., Bureau of Commercial Fish-
eries (BCF) for the collection of samples and for analytical equipment
where contracts are indicated. In States participating by agreement,
the BCF provided equipment and chemicals. In 1971-72, the program
was jointly funded by the National Marine Fisheries Service (NMFS)
and the Environmental Protection Agency.
ALABAMA
CALIFORNIA
DELAWARE
FLORIDA
GEORGIA
MAINE
MARYLAND
MISSISSIPPI
NEW JERSEY
NEW YORK
NORTH
CAROLINA
SOUTH
CAROLINA
TEXAS
VIRGINIA
WASHINGTON
Alabama Marine Resources Laboratory
Johnie H. Crance, Director; E. B. May,
Principal Investigator. Agreement.
California Dept. of Fish and Game, Marine
Resources Operations
Dr. H. C. Orcutt, Laboratory Supervisor; John
Modin, Chemist. Contracts. BCF: 14-17-0007-
332; 14-17-0002-211; -265; -337; -532.
California Department of Fish and Game,
Resources Agency
W. H. Griffith, Principal Investigator. Contract,
NMFS: N-042-10-72(N).
University of Delaware
Dr. F. C. Daiber, Principal Investigator. Con-
tracts, BCF: 14-17-0002-117; -261; -326.
State Board of Conservation Marine Laboratory
R. M. Ingle, Director of Research. Agreement.
Bureau of Commercial Fisheries — Environmental
Protection Agency, Gulf Breeze Laboratory.
Dr. T. W. Duke, Director. Agreement.
The University of Georgia
Dr. T. L. Linton, Principal Investigator. Con-
tracts, BFC: 14-17-0002-220; -267.
C. J. Durant, Principal Investigator and Chemist.
Contracts, BFC: 14-17-0002-344; -454.
Dr. R. J. Reimold, Principal Investigator. Con-
tract, NMFS: N-042-12-7KN).
Department of Sea and Shore Fisheries
L. Varney, Principal Investigator; John Hurst,
Laboratory Director and Chemist. Contracts,
BCF: 14-17-0007-333; 14-17-0002-206; -263;
-332; -434.
BCF Biological Laboratory
Dr. A. Rosenfield, Principal Investigator. Agree-
ment.
Gulf Coast Research Laboratory
Dr. W. P. Abbott, Principal Investigator. Con-
tracts, BCF: 14-17-0002-133; -172; -235; -341.
Dr. G. Gunter, Laboratory Director. Contract,
NMFS: N-042-11-7KN).
Rutgers— The State University, Oyster Research
Laboratory
Dr. H. H. Haskin and D. £. Kunkle, Principal
Investigators. Agreement.
New York State Department of Environmental
Conservation
D. H. Wallace, Director of Marine Fisheries;
J. Foehrenbach, Chemist. Contracts, BCF: 14-17-
0002-163; -219; -268; -345; -455; NMFS: N-042-
University of North Carolina, Institute for
Marine Sciences
Dr. A. F. Chestnut, Principal Investigator. Con-
tracts, BCF: 14-17-0002-182; -239; -343; NMFS:
N-042-15-71(N).
Bears Bluff Laboratories, Inc.
Dr. G. R. Lunz, Director (deceased). Contracts,
BCF: 14-17-0002-130; -171; -234; -340; -426.
State of Texas, Parks and Wildlife Department
T. R. Leary, Coastal Fisheries Coordinator;
R. Childress, Principal Investigator. Agreement.
Virginia Institute of Marine Science
Dr. M. L. Brehmer, Principal Investigator;
Dr. R. J. Huggett, Principal Investigator and
Chemist. Contracts, BCF: 14-17-0002-138; -174;
-237; -342; -452; NMFS: N-042-13-71(N).
State of Washington, Department of Fisheries
C. Lindsay, R. E. Westley, Principal Investi-
gators. Contracts, BCF: 14-17-0002-134; -1"3;
-236.
PESTICIDES MONITORING JOURNAL
-------
Part II. Residue Data—Individual States
The following sections present residue data for the 15
coastal States where estuarine mollusks were monitored
for organochlorine residues. A map showing sampling
sites in the respective States together with a discussion
of the findings are included in each section.
SECTION A.—ALABAMA
Samples of the eastern oyster, Crassostrea virginica, were
collected in Alabama at 3-month intervals during 1968-
69 from four commercial reefs in or near Mobile Bay.
Samples were processed at the Alabama Marine Re-
sources Laboratory and mailed to the Gulf Breeze
Laboratory for chemical analysis.
Approximate station locations are shown in Fig. A-l.
Stations 1 and 2 on the eastern shore of Mobile Bay are
influenced more by the presumably cleaner Gulf of
Mexico waters than Stations 3 and 4 which are more
exposed to drainage waters from the Alabama-Tombig-
bee River Basin. Both Stations 1 and 4 are influenced to
an unknown extent by small drainage basins in the
coastal areas of Alabama. A summary of data on or-
ganochlorine residues in the monitored species, C.
virginica, is presented in Table A-l, and the distribution
of residues in this species for each sampling station by
date of collection in Table A-2. Many of these data
have already been published by the cooperating agency
(10).
All 33 samples contained detectable amounts of DDT,
but the sampling series was conducted in Alabama for
too few years to indicate annual trends in pollution
levels. An earlier study of pesticide residues in Mobile
Bay oysters (7) also reported a 100% incidence of
DDT in 82 samples analyzed; however, maximum DDT
residues at Shell Bank and Cedar Point reefs were 13
and 25 times higher in 1965 than those observed in
this study in 1969. Because of differences in sample
preparation in the two studies, 1965 residues could be
expected to be only about 10% higher than the 1969
data had there been no change in DDT pollution
levels in the bay. Alabama and New Jersey were the
only States of the 15 monitored in which 100% of the
samples contained detectable residues of DDT. The
maximum level of DDT in Alabama oysters (616 ppb)
was lower than residues found in four other States.
Dieldrin residues were small, but the 18% incidence
was significantly higher than the average incidence for
all States of 15%. The incidence and magnitude of
dieldrin residues in the 1965 study (7) were significantly
higher.
GULF OF MEXICO
10 mi
FIGURE A-l.—Diagram of coastal Alabama showing
approximate location of monitoring stations
1. Sbellbank—Bon Secour Bay
2. Klondike—Mobile Bay
3. Whitehouse—Mobile Bay
4. Cedar Point Reef—Mississippi Sound
TABLE A-l.—Summary of data on organochlorine residues in the monitored species (C. virginica), 1968-69—Alabama
STATION
NUMBER
1
2
3
4
LOCATION
Shellbank
Klondike
White House
Cedar Point
Occasional stations (2)
Total number of samples
Percent of samples positive for indicated compound
MONITORING
PEKIOD
1968-69
1968-69
1968-69
1968-69
1968-69
NUMBER OF
SAMPLES '
8
8
1
8
2
33
NUMBER OF POSITIVE SAMPLES AND
MAXIMUM RESIDUE ( ) DETECTED
IN PPB (0G/KO)
DDT
8 (214)
8 (445)
1 (616)
8 (372)
2 (237)
100
DIELDRIN
1 (14)
1 (14)
2 (21)
2 (13)
18
1 Each sample represents 15 or more mature mollusks.
VOL. 6, No. 4, MARCH 1973
247
-------
TABLE A-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of collection—
Alabama
[Blank = no sample collected; — = no residue detected above 5 ppb; T = >5 but <10 ppb]
YEAR COMPOUND
RESIDUES IN PPB (AG/KO)
JAN. FEB. MAR. APR.
STATION 1.— SHELL
1968 DDE
TDE
DDT
1969 DDE
TDE
DDT
Dieldrin
110
88
16
48 94
31 70
— 14
MAY JUNE JULY AUG. SEPT. OCT. Nov. DEC.
BANK— 8 SAMPLES1
110 33
52 15
12 —
48 26 T
25 17 T
— 13 —
STATION 2.— KLONDIKE— 8 SAMPLES 1
1968 DDE
TDE
DDT
1969 DDE
TDE
DDT
Dieldrin
230
180
35
120 18
100 94
— 44
— 14
STATION 3.— WHITE
1968 DDE
TDE
DDT
Dieldrin
1969 DDE
TDE
DDT
Dieldrin
320
240
56
20
110 15
83 98
— 36
— 21
210 45
130 37
34 12
170 73 22
110 53 18
23 62 —
HOUSE— 7 SAMPLES'
120 32
57 20
11 —
56 46
37 40
— 36
STATION 4.— CEDAR POINT— 8 SAMPLES i
1968 DDE
TDE
DDT
1969 DDE
TDE
DDT
Dieldrin
180
160
32
84 77
55 71
— 30
- 13
86 41
51 23
17 23
110 26 30
78 22 23
— 26 T
"J1
1 Each sample represents 15 or more mature moUusks.
248
PESTICIDES MONITORING JOURNAL
-------
SECTION B.—CALIFORNIA
The monthly collection of mollusks to monitor pesticide
pollution in 12 estuaries in California was initiated in
January 1966. Some of these stations were terminated
and other estuaries were added during the course of the
program. Samples were analyzed at the Gulf Breeze
Laboratory until May 1968; from then until May 1970
they were analyzed at the Marine Resources Operations
Laboratory of the Department of Fish and Game, Menlo
Park, Calif. During the period July 1970 June 1972,
samples were collected and analyzed at approximately
3-month intervals by the Department of Fish and
Game, Pesticides Investigations at Sacramento, Calif.
Six different mollusks (Crassostrea gigas, Corbicula
fluminea, Modiolus denissus, Mytilus californianus,
Mytilus edulis, and Ostrea lurida) were utilized for
monitoring; for the most part, a single species was
collected at each station. The relative ability of these
different mollusks to store organochlorine residues ap-
pears to be reasonably similar and, thus, comparisons of
the magnitude of residues in different estuaries can be
made with some confidence. In general, residue levels at
different stations followed patterns of suspected pollu-
tion loading in the associated drainage basin, regardless
of the species monitored.
The approximate station locations are shown in Fig.
B-l. A summary of data on organochlorine residues in
the monitored species is presented in Table B-l, and the
distribution of residues in these species for each sampling
station by date of collection in Table B-2. Results of
some of the analyses conducted by the Gulf Breeze
Laboratory during the period January 1966 December
1967 have been published by the cooperating agency
(13).
DDT residues in mollusks were consistently larger in
California than in any other area monitored with the
exception of a single station in south Florida. There is
a clear pattern of maximum pesticide residues being
correlated with proximity of the monitoring station to
runoff from agricultural lands. In southern California,
where most samples contained typically large residues.
residues were consistently higher at Hedionda and Mugu
Lagoons, the recipients of agricultural runoff waters,
than at Anaheim Slough which receives intermittent
runoff from the urban and industrialized sections of Los
Angeles. Residues in samples from estuaries draining
the intensely cultivated central and southern parts of the
State were larger, by one order of magnitude usually,
than those in samples collected from watersheds north
of San Francisco Bay where dairy land predominates.
The incidence of dieldrin residues (25%) was second
only to New York samples although residues were lower
in magnitude than in five other States. California and
Texas were the only States where endrin and toxaphene
VOL. 6, No. 4, MARCH 1973
11
PACIFIC OCEAN
0 60 n
FIGURE B-l.—Diagram of coastal California and the San
Francisco Bay area showing approximate location of
monitoring stations
1. Hedionda Lagoon
2. Anaheim Slough
3. Point Mugu
4. Baywood Park—Morro Bay
5. Los Osos Creek—Morro Bay
6. Elkhorn Slough
7. Coyote Point—San Francisco Bay, South
8. Guadalupe Slough—San Francisco Bay, South
9. Alviso Slough—San Francisco Bay, South
10. West Island—Sacramento-San Joaquin River Basin
11. False River—Sacramento-San Joaquin River Basin
12. Napa River—San Pablo Bay
13. Petaluma River—San Pablo Bay
14. Point San Quentin—San Francisco Bay, North
15. Bolinas Lagoon
16. Schooner Bay—Drakes Estero
17. Berries Bay—Drakes Estero
18. Tomales Bay—Tomales Bay
19. Nicks Cove—Tomales Bay
20. Gunther Island—Humboldt Bay
21. Bird Island—Humboldt Bay
from presumably agricultural sources were detected.
Polychlorinated biphenyl compounds were detected in
samples beginning in 1971, but were not quantified.
They occurred in a few samples from nearly all drainage
basins monitored.
Late in 1970 or early 1971, there was a sharp decline in
DDT residues in samples collected in estuaries draining
predominantly agricultural areas, i.e., San Francisco Bay
and the southern parts of the State. Decreased frequency
249
-------
of sample collection in 1970-71 makes it impossible
to pinpoint when this decline in DDT pollution oc-
curred. The typically small DDT residues in samples
from drainage basins north of San Francisco Bay
remained about the same throughout the monitoring
period.
TABLE B-l.—Summary of data on organochlorine residues in the monitored species, 1966-72—California
STATION
NUMBER
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
LOCATION
Hedionda Lagoon
Anaheim Slough
Point Mugu
Baywood Park
Los Osos Creek
Elkhorn Slough
Coyote Point
Guadalupe Slough
Alviso Slough
West Island
False River
Napa River
Petal uma River
Point San Quentin
Bolinas Lagoon
Schooner Bay
Berries Bar
Tomales Bay
Nicks Cove
Gunther Island
Bird Island
Occasional stations (15)
MONITORINC
PERIOD
1967-72
1967-72
1967-72
1966-72
1966-72
1966-72
1966-72
1968-72
1968-72
1967-72
1967-71
1968-72
1968-72
1966-70
1966-68
1966-72
1966-68
1966-72
1966-68
1966-72
1966-68
1966-72
PRINCIPAL
MOM ITORED
SPECIES
M. edulls
M, edulis
M. edulis
C. glgas
C. glgas
C. gigas
O. lurida
M. dtmtssus
M. demlssus
C. flumlnea
C. fluminea
M, demlssus
M. demlssus
C. glgas
C. glgas
C. gigas
C. gigas
C. glgas
C. glgas
C. glgas
C. glgas
Mixed
Total number of samples
Percent of samples positive for indicated compound
NUMBER OF
SAMPLES '
31
33
29
52
52
57
55
27
28
28
26
28
28
50
17
33
27
34
25
33
25
54
772
NUMBER OF POSITIVE SAMPLES AND MAXIMUM
RESIDUE ( ) DETECTED IN PPB (0o/Ko)
DDT
31 (3,970)
33 (833)
29 (1,758)
52 (601)
52 (412)
57 (2,305)
54 (362)
25 (407)
28 (328)
28 (2,280)
26 (1,850)
26 (210)
25 (268)
49 (440)
14 (45)
25 (43)
25 (44)
28 (45)
20 (37)
31 (78)
3 (T)
51 (1,144)
92
DlELDRIN
4 (T)
10 (31)
9 (16)
3 (24)
4 (27)
24 (57)
26 (43)
9 (37)
6 (25)
23 (22)
12 (24)
5 (T)
4 (10)
22 (23)
2 (T)
2 (T)
5 (T)
25 (26)
25
ENDRIN
I (T)
1 (T)
2 (19)
2 (19)
3 (T)
1 (18)
2 (T)
1 (19)
1 (T)
2
TOXAPHENE
2 (11,000)
2 (1,000)
<1
PCB'S »
2
2
2
1
2
1
I
1
1
I
1
1
1
4
3
NOTE: T = >5 but <10 ppb.
1 Each sample represents 15 or more mature mollusks.
1 Present but not quantified.
TABLE fl-2.—Distribution of organochlorine residues in the monitored species for each sampling station by date of
collection—California
[Blank = no sample collected; — = no residue detected above 5 ppb or no residue detected (PCB's); T = >5 but <10 ppb]
RESIDUES IN PPB (iia/no)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT.
STATION 1.— HEDIONDA LAGOON— M. EDULIS UNLESS OTHERWISE INDICATED— 31 SAMPLES
1967
DDE
TDE
DDT
Toxaphene
100
72
130
—
Nov.
1
= 130
240
3,600
11,000
DEC.
'90
84
740
970
250
PESTICIDES MONITORING JOURNAL
-------
TABLE B-2.—Distribution of organochlorine residues in the monitored species for each sampling station by date of
collection—California—Continued
YEAR
COMPOUND
RESIDUES IN PPB (IIO/KO)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG.
SEP.T. OCT. Nov. DEC.
STATION 1.—HEDIONDA LAGOON—M. EDULIS, UNLESS OTHERWISE INDICATED—31 SAMPLES »—Continued
1968
1969
1970
1971
1972
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
PCB's
DDE
TDE
DDT
Dieldrin
PCB's
130 52 200 210 91 130 120 168 » 105 120 136
73 31 88 220 103 154 80 171 74 73 58
920 200 440 300 42 86 59 129 63 120 164
52 211 • 242 118 227 95 139 347 466 76
_ 207 101 172 124 53 35 99 115 64
123 291 486 214 99 91 34 61 68 108
114
102
54
T
19 36 54 18
11 58 56 13
16 T 285 10
•j-
«)
14 31
T 31
10 10
T T
— «>
STATION 2.—ANAHEIM SLOUGH—M. EDVL1S, UNLESS OTHERWISE INDICATED—33 SAMPLES"
1967
1968
1969
1970
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
Endrin
DDE
TDE
DDT
DDE
TDE
DDT
270 6 HO a 170 310 464 203
91 45 62 110 186 102
160 43 110 77 108 52
— 31 — — — T
______
157 273 127 51 388 547
37 55 — 136 172 189
123 217 94 222 131 97
157
49
38
360
100
85
265 432 M64 440
68 109 127 170
33 51 65 110
T - - 12
T _ _ _
323 466 451 168
107 115 107 129
37 60 64 282
330
150
120
354
118
70
—
—
494
130
88
305
126
10
200
87
120
VOL. 6, No. 4, MARCH 1973
251
-------
TABLE B-2.—Distribution of organochlorine residues in the monitored species for each sampling station by date of
collection—California—Continued
YEAR
COMPOUND
RESIDUES IN PPB (AC/KG)
JAN. FEB. MAR. APR. MAY JUNE JULY Auo.
SEPT. OCT. Nov. DEC.
STATION 2.—ANAHEIM SLOUGH—M. EDULIS, UNLESS OTHERWISE INDICATED—33 SAMPLES1—Continued
1971
1972
DDE
TDE
DDT
Dieldrin
PCB's
DDE
TDE
DDT
Dieldrin
PCB's
75 103 185
53 164 101
23 T 22
T T T
— — —
64 80
24 53
18 10
T T
(i)
92
41
10
T
<»
STATION 3.—POINT MUGU—M. EDULIS, UNLESS OTHERWISE INDICATED—29 SAMPLES'
1967
1968
1969
1970
1971
1972
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
Endrin
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
PCB's
DDE
TDE
DDT
Dieldrin
PCB's
130 160 220
150 230 280
270 440 650
— — T
250 200 »370 170 366 207 255 465 » 269 360
230 210 350 180 494 168 65 388 278 443
460 340 790 430 749 363 32 432 566 955
— — 16 — — — T — — T
--------- *
226 560 334 365 '273 "298 • 918 « 349 112
121 — 301 63 31 116 117 34 146
161 391 248 120 92 40 580 176 185
•238
141
56
« 49 '65 "22 • 1 12
24 73 T 50
45 11 — 20
T T — T
-_ (4)
» 37 ' 24
12 10
T 10
T T
(I)
252
PESTICIDES MONITORING JOURNAL
-------
TABLE B-2.—Distribution of organochlorine residues in the monitored species for each sampling station by date of
collection—California—Continued
YEAR
COMPOUND
RESIDUES IN PPB (JIC/KO)
JAN. FEB. MAR. APR. MAY JUNE JULY Auo. SEPT. OCT.
Nov. DEC.
STATION 4.—BAYWOOD PARK—C. GIGAS—52 SAMPLES »
1966
1967
1968
1969
1970
1971
1972
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
54
26
25
—
110
29
58
96
24
25
123
23
24
33
12
T
T
74
34
25
—
110
42
73
43
13
13
111
38
164
220
74
69
82
35
26
—
62
50
96
40
T
30
139
—
131
226
87
64
75
25
20
24
130
47
130
160
40
61
180
70
351
215
58
46
76 52 55 62
32 19 22 25
24 14 — 18
16 — — —
80 120 51 82
34 49 29 40
67 70 37 49
48 49 48 48
17 19 — 13
— T T T
148 119 110
57 40 57
189 150 43
56
21
—
22
11
—
59 69 69
35 34 33
26 24 25
— — —
55 48 35
23 21 10
46 26 15
44 74
— —
— —
97 165 184
31 53 70
31 58 64
21
16
10
100
37
46
—
46
13
10
162
75
69
STATION 5.—LOS OSOS CREEK—C. GIGAS, UNLESS OTHERWISE INDICATED—52 SAMPLES1
1966 DDE
TDE
DDT
Dieldrin
1967 DDE
TDE
DDT
1968 DDE
TDE
DDT
1969 DDE
TDE
DDT
83 -
33
23
—
62 ;
29
41
100
32
37
66
T
T
58
27
21
—
120
47
96
61
21
21
70
37
72
43
17
14
—
63
43
130
42
13
T
126
—
131
88
39
30
27
110
42
120
70
24
36
104
56
239
65
25
20
10
93
57
92
'65
T
T
155
61
183
40
16
—
—
130
56
80
42
T
—
144
80
188
43
16
—
—
64
46
52
31
—
T
201
83
128
53
22
14
—
81
44
49
25
11
—
73
34
23
—
56
33
72
55
T
—
115
43
34
10
27
23
—
43
20
25
223
72
93
71 72
33 31
25 37
_ _
37 29
14 10
20 12
69
—
137
51
35
VOL. 6, No. 4, MARCH 1973
253
-------
TABLE B-2.—Distribution of organochlorine residues in the monitored species for each sampling station by date of
collection—California—Continued
YEAR
COMPOUND
RESIDUES IN PPB (AG/KO)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT.
OCT. Nov.
DEC.
STATION 5.—LOS OSOS CREEK—C. G1GAS, UNLESS OTHERWISE INDICATED—52 SAMPLES *—Continued
1970
1971
1972
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
PCB's
186 182 221 63
60 54 70 13
53 50 41 —
29 25
15 21
T 10
30 12
12 T
T 10
T T
— «>
STATION 6.—ELKHORN SLOUGH—C. GIG AS, UNLESS OTHERWISE INDICATED—57 SAMPLES'
1966
1967
1968
1969
1970
1971
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
En drill
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
Endrin
DDE
TDE
DDT
Dieldrin
PCB's
160
160
250
—
200
160
260
26
260
160
250
13
—
178
102
120
208
173
204
—
—
220
220
290
19
220
230
440
25
130
85
97
—
—
191
338
441
230
300
444
—
—
'67
37
72
—
—
96
120
110
11
200
200
390
29
120
92
61
—
—
126
156
346
445
582
808
—
—
96
110
96
20
230
260
690
30
170
160
230
—
—
280
393
808
270
285
491
—
—
89 88
95 82
85 65
18 —
210 300
340 390
860 920
39 33
214
212
411
—
—
215 324
223 253
304 96
353 325
276 236
411 375
— —
— —
'29
24
10
T
—
86 72
79 77
64 55
— —
160 200
200 260
390 500
10 10
173 122
129 70
237 159
27 —
19 —
424
358
704
7 43
7i
—
—
—
79 84 130
66 65 77
41 56 76
— 10 —
190 62 190
210 55 150
390 110 340
15 — 14
168 95
100 71
200 113
— —
— —
1413 237
502 171
630 284
'31
19
26
T
T
'28
37
17
11
u>
190
160
210
30
250
230
370
17
65
63
110
—
—
191
117
189
254
PESTICIDES MONITORING JOURNAL
-------
TABLE B-2.—Distribution of organochlorine residues in the monitored species for each sampling station by date of
collection—California—Continued
YPAB
RESIDUES IN PPB (/IO/KO)
JAN. FEB. MAR.
APR. MAY JUNE JULY Auo. SEPT. OCT. Nov. DEC.
STATION 6.— ELKHORN SLOUGH— C. GIGAS, UNLESS OTHERWISE INDICATED— 57 SAMPLES i— Continued
1972
DDE
TDE
DDT
Dieldrin
PCB's
'42
25
36
T
—
TT
T
10
57
u>
STATION 7.—COYOTE POINT—O. LURWA—S5 SAMPLES >
1966
1967
1968
1969
1*70
1971
1972
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
Endrin
DDE
TDE
DDT
Dieldrin
Endrin
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
PCB's
54 93 42
74 120 54
63 100 43
— 27 20
41 49 51
60 68 74
58 72 79
26 26 28
— _ _
44 59 47
57 95 69
53 100 84
13 — 18
— — —
81 52 172
46 — —
48 158 171
18 33 102
33 55 102
33 42 87
17
67
47
T
15
32
53
T
—
71 71 47
88 91 66
70 74 4«
29 23 —
61 65 52
82 76 78
89 69 70
23 21 25
— — 19
47 46 27
71 103 41
89 103 45
20 — —
10 — —
25 — 65
— 89 87
— 176 88
93 37 33
100 — 50
76 42 24
T
T
T
T
U)
30 35 50 42 33
38 46 65 71 51
39 42 55 62 40
— — — 21 15
51 47 39 33 46
84 58 58 37 86
80 50 50 43 110
17 43 13 — 16
_____
42 — 30 33
57 — 57 69
60 — 56 58
10 — — —
— — — _
34 163 36 52
38 99 T 78
33 100 39 59
14
24
24
T
63
79
75
21
46
84
51
16
—
24
30
26
VOL. 6, No. 4, MARCH 1973
255
-------
TABLE B-2.—Distribution of organochlorine residues in the monitored species for each sampling station by date of
collection—California—Continued
YEAR
COMPOUND
RESIDUES IN PPB (/io/ro)
JAN. FEB. MAR. APR. MAY JUNE JULY Auo. SEPT. OCT.
Nov.
DEC.
STATION 8.—GUADALUPE SLOUGH—M. DEMISSUS—21 SAMPLES1
1968 DDE
TDE
DDT
Dieldrin
1969 DDE
TDE
DDT
1970
1971 DDE
TDE
DDT
Dieldrin
1972 DDE
TDE
DDT
Dieldrin
PCB's
36 77
90 180
110 150
18 23
— — 34
42 — —
— — —
T
40
28
T
11
12
10
T
—
48 74 67 34
100 185 140 68
60 91 130 34
14 — 14 —
70 26 11 10
— 27 108 22
— — 204 T
No Samples Collected
T
26
10
T
T
T
10
T
(i)
24 19 24 34 T
53 57 58" T 24
T 40 26 — T
— — — — —
24 — 29
34 48 50
T T T
— T
— T
— 10
— 37
STATION 9.—ALVISO SLOUGH—M. DEM1SSUS—28 SAMPLES '
1968
1969
1970
1971
1972
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
PCB's
43
140
93
18
38 —
59 61
— —
42
76
33
13
38
17
—
T
T
10
T
—
46 69 74 47 26 28 18 11 — 13
59 170 169 95 72 80 45 79 T 30
78 77 85 66 35 34 35 11 T 27
1225 — — — — — — — —
55 98 52 12 17 30 — 39
55 — 73 161 42 45 56 27
88 — 108 111 T 20 T T
T - T
15 T T
T - 10
T — —
T
T
10
T
<«
256
PESTICIDES MONITORING JOURNAL
-------
TABLE B-2.—Distribution of organochlorine residues in the monitored species for each sampling station by date of
collection—California—Continued
YEA*
COMPOUND
RESIDUES IN PPB (/JO/KO)
JAN. FEB. MAX. APX. MAY JUNE JULY Auo. SEPT. OCT.
Nov. DEC.
STATION 10.—WEST ISLAND—C. FLUMINEA—28 SAMPLES »
1967 DDE
TDE
DDT
Dieldrin
Endrin
1968 DDE
TDE
DDT
Dieldrin
1969 DDE
TDE
DDT
1Q7fl
jy /u
1971 DDE
TDE
DDT
Dieldrin
1972 DDE
TDE
DDT
Dieldrin
PCB's
280 330 320 270 320 230
250 370 350 250 250 210
210 300 310 250 260 270
20 20 22 17 12 18
T T T — — —
390 500 280 370 251 196
290 400 200 220 224 183
240 290 190 210 320 223
16 22 15 16 — 21
177
—
168
198
126
173
T
11 T
10 T
10 10
— T
(4)
170 140 170 180 690 390
130 93 150 150 490 310
150 130 230 270 1,100 770
T 15 20 10 20 18
______
134 104 41 71
160 182 97 138
150 235 150 184
19 13 — —
||__i—-I
ucctcu •-
91 15
71 T
111 —
T T
*
STATION 11.—FALSE RIVER—C. FLVMINEA—26 SAMPLES1
1967
1968
1969
1970
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
Endrin
DDE
TDE
DDT
470
410
970
24
470 500 340 330
400 590 230 230
220 420 200 190
17 22 19 23
18 — — —
151 54
152 66
378 167
460
320
910
19
315
281
312
—
—
93
75
214
No ;
320
200
640
20
199
167
225
—
—
41
47
46
-------
TABLE B-2.—Distribution of organochlorine residues in the monitored species for each sampling station by date of
collection—California—Continued
YEA*
COMPOUND
RESIDUES IN PPB (/to/xo)
JAN. FEB. MAX. AM. MAY JUNE JULY Auo. SEPT. OCT. Nov. DEC.
STATION 11.— FALSE RIVER— C. FLUMINEA—26 SAMPLES >— Continued
1971
DDE
TDE
DDT
Dieldrin
21
41
20
T
STATION 12.—NAPA RIVER—M. DEMISSUS—2S SAMPLES»
1968
1969
1970
1971
1972
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
Endrin
DDE
TDE
DDT
Dieldrin
Endrin
DDE
TDE
DDT
Dieldrin
PCB's
T 25
22 46
T 26
10 — 100
30 — 48
T — —
11
33
T
—
—
27
143
41
T
T
T
21
23
T
—
21 24 23 IS 21 18 T
72 100 83 38 42 62 24
39 45 47 — 23 18 T
62 — 10 13 T — T 12
— — 24 45 30 58 24 37
— — T T T — T T
16
93
46
T
T
13 T T
68 T T
10 T T
T — —
_ _ —
T
T
10
T
«>
STATION 13.—PETALUMA RIVER—M. DEMISSVS—28 SAMPLES»
1968 DDE
TDE
DDT
1969 DDE
TDE
DDT
1970 DDE
TDE
DDT
Dieldrin
— 27 27 26 47 19
— 58 63 72 104 35
— 15 19 31 41 T
T — 124 49 12 T 17
T — 37 — 28 T 38
_ — — — — T T
T
T
T
—
— — 92
T 13 68
— — 108
— — 22 10
— 57 37 22
— — T T
28
71
26
10
258
PESTICIDES MONITORING JOURNAL
-------
TABLE B-2.—Distribution of organochlorine residues in the monitored species for each sampling station by date of
collection—California—Continued
YEAR
COMPOUND
RESIDUES IN PPB (/UJ/KO)
JAN. FEB. MAR.
STATION 13— PETALUMA
1971
1972
DDE
TDE
DDT
Dieldiin
DDE
TDE
DDT
Dieldrin
PCB's
T
18
10
T
T
T
10
T
—
APR. MAY JUNE JULY AUG. SEPT. OCT.
RIVER— M. DEMISSVS—2S SAMPLES "—Continued
T —
24 T
T —
— —
T
T
10
T
U)
Nov. DEC.
T
T
—
—
STATION 14.—POINT SAN QUENTIN—C. GIG AS—50 SAMPLES'
1966
1967
1968
1969
1970
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
DDE
TDE
DDT
12
20
14
—
52
130
88
23
43
79
44
12
74
80
130
18
18
T
30
37
12
—
34
65
49
11
44
96
89
17
53
149
193
19
T
T
47
60
19
—
30
59
49
15
43
78
67
17
62
134
76
54
31
37
52
83
23
14
42
75
70
19
43
97
63
12
47
—
182
66
64
45
69
120
45
20
23
55
34
13
36
95
69
—
143
143
154
51
39
23
59
92
38
—
39
85
64
21
59
110
100
—
25
54
24
37
47
24
—
45
120
89
19
59
110
100
12
13
23
T
52
82
43
—
53
130
63
17
25
60
T
—
30
41
T
57 51 55
90 88 84
49 33 40
— 11 15
30 31 100
74 68 50
38 36 85
11 — 10
38
86
52
—
— —
— 55
— 32
55
110
98
20
45
84
45
11
40
120
82
18
31
51
26
STATION 15.—BOLINAS LAGOON—C. GIGAS—17 SAMPLES1
1966
1967
DDE
TDE
DDT
DDE
TDE
DDT
10 T
— T
— —
T T 10 10 11 T 11 13 — T
11 13 16 17 16 14 21 20 — 15
T T T 11 14 12 13 U — n
T
10
—
T
11
—
—
—
T
T
—
VOL. 6, No. 4, MARCH 1973
259
-------
TABLE B-2.—Distribution of organochlorine residues in the monitored species for each sampling station by date of
collection—California—Continued
YEA»
COMPOUND
RESIDUES IN PPB (po/m)
JAN. FEB. MA». Aw. MAY JUNE JULY Auo. SEPT. OCT. Nov. DEC.
STATION 15.— BOLINAS LAGOON— C. GIGAS—ll SAMPLES *— Continued
1968
DDE
TDE
DDT
—
—
—
STATION 16.—SCHOONER BAY—C. G1GAS—33 SAMPLES»
1966
1967
1968
1OJGQ
i"oy
1970
1971
1972
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
PCB's
— T T — T T T T — T T —
— — T — T — — — — T T —
— — — — — — ______
T T 10 11 T 11 T IS — — T —
— — T 13 13 13 T 18 — — — —
_ — — 10 — T — 10 — — — —
—
—
—
11 10
T 11
T no
14 T T T
16 T T T
10 T T 10
T — — —
T T
T T
10 10
— T
«>
STATION 17.—BERRIES BAR—C. GIGA5—Z1 SAMPLES »
1966
1967
DDE
TDE
DDT
DDE
TDE
DDT
— 13
— 15
— T
T 11
T 14
_ —
T
T
—
17
20
T
10
17
—
14
18
10
13
16
T
16
17
11
10
10
—
12
14
10
17
13
—
13
15
T
II
11
—
T
12
—
12
T
—
13
16
10
T
T
—
15
18
T
T 11
T 10
— —
14 T
17 T
T —
260
PESTICIDES MONITORING JOURNAL
-------
TABLE B-2,—Distribution of organochlorine residues in the monitored species for each sampling station by date of
collection—California—Continued
RESIDUES IN PPB (/US/KG)
JAN. FEB. MAR. APR.
STATION 17— BERRIES BAR— C.
1968 DDE
TDE
DDT
Endrin
— 12
— 13
— T
19 —
MAY JUNE JULY AUG. SEPT. OCT. Nov. DEC.
GIG AS— 21 SAMPLES >— Continued
17
14
—
—
STATION 18.—TOMALES BAY—C. G1GAS—34 SAMPLES >
1966
1967
1968
1969
1970
1971
1972
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
Endrin
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
T T — — T — 14 11 14 14 T T
— T — — 11 — T T 11 12 — —
____T-------
T 12 11 11 T 11 T T T — 11 —
— T T 12 T 14 T — — — T —
T 10 TUT 13 T — — — T —
— 11 T
— — T
— 10 —
22 T
T T
18 T
T
T
10
T
T
12 T T
T T T
10 10 10
T
T
10
T
STATION 19.—NICKS COVE—C. GIGAS—25 SAMPLES'
1966
1967
DDE
TDE
DDT
DDE
TDE
DDT
— 12
T
— T
12 13
T 10
T 11
11 —
— —
— —
13 14
12 12
T 11
T
—
—
10
T
T
T
—
—
T
T
T
11 11
— T
— —
11 T
T —
T —
T T T
__. _^ «•*
— — —
T - 14
T — _
— — —
T
—
_
—
VOL. 6, No. 4, MARCH 1973
261
-------
TABLE B-2.—Distribution of organochlorine residues in the monitored species for each sampling station by date of
collection—California—Continued.
YEAR
RESIDUES IN PPB (/m/io)
JAN. FEB. MA*. Am. MAY JUNE JULY Auo. SEPT. OCT. Nov. DEC.
STATION 19— NICKS COVE— C. G1GAS—25 SAMPLES »— Continued
1968
DDE
TDE
DDT
—
—
—
STATION 20.—OUNTHER ISLAND—C. GIG AS—33 SAMPLES »
1966
1967
•
1968
1OAQ
lyW
1970
1971
1972
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
PCB's
— — — — — T 11TTTTT
— — — — — T 17 — 21TTT
T 10 — 47 11 14 — 11 — 18 14 20
10TTTTT T T — T T T
T — — T — T T — T T T T
30 28 12 19 19 19 24 12 16 15 21 22
— 13
— 11
— 54
- -- No Samples Collected •
11
11
10
T
T T T T
13 T — T
17 10 — 10
T T - T
T T
T T
10 10
— I
(4)
1966
1967
DDE
TDE
DDT
DDE
TDE
DDT
STATION 21.—BIRD ISLAND—C. GIG AS—2! SAMPLES'
262
PESTICIDES MONITORING JOURNAL
-------
TABLE B-2.—Distribution of organochlorine residues in the monitored species for each sampling station by date of
collection—California—Continued
YEAR
COMPOUND
RESIDUES IN PPB (^G/KO)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT.
Nov. DEC.
STATION 21—BIRD ISLAND—C. GIG AS—25 SAMPLES '—Continued
1968
DDE
TDE
DDT
—
—
—
' Each sample represents 15 or more mature mollusks.
1 DDE, TDE, and DDT values approximated because of presence of toxaphene.
•' C. gigas.
' Present but not quantified.
- M. demissus.
« M. calijornianus.
1 M. edulis.
SECTION C.—DELAWARE
Samples were collected at nine stations at monthly
intervals during the period October 1966 August
1969. The eastern oyster (Crassostrea rirginica)
ribbed mussel (Modiolus demissus), and hard clam
(Mercenaria mercenaria) were each collected at three
stations. All samples were analyzed at the Gulf Breeze
Laboratory. The approximate locations of the stations
are shown in Fig.-C-l. The Cape Henlopen station was
in Delaware Bay; the other stations were adjacent to the
Bay but exposed primarily to the runoff from large
agricultural areas in separate drainage basins. A sum-
mary of data on organochlorine residues in the moni-
tored species is presented in Table C-l, and the distribu-
tion of residues in these species for each sampling
station by date of collection in Table C-2.
The use of three different species for monitoring ob-
scured pollution patterns in Delaware estuaries to some
extent. The relative inefficiency of hard clams in storing
organochlorine residues makes Rehoboth Bay (Stations
7 and 8) appear to be generally free from this type of
pollution. The first samples of clams collected in
adjacent Indian River Bay (Station 9) also were free
of detectable residues; however, subsequent monitoring
using the ribbed mussel, showed Indian River Bay to be
moderately but continuously polluted. It is probable
that Rehoboth Bay was similarly polluted during the
monitorjng period. This same reasoning suggests that the
waters at Cape Henlopen were continually more polluted
with DDT than the small residues in the hard clams
would imply.
The magnitude of DDT residues in clams and oysters
showed no trend towards increased or decreased levels
during the 3-year monitoring period. In ribbed mussels.
however, there was a marked decline in the average level
VOL. 6, No. 4, MARCH 1973
of residues in the final year at Stations 1 and 2 as well
as Station 9. Delaware monitoring samples ranked 6th
in frequency and 10th in magnitude of DDT residues
in comparison with the other 14 States. The 13% inci-
dence of dieldrin residues was about the average for all
States.
0 10 mi
FIGURE C-l.—Diagram of coastal Delaware showing
approximate location of monitoring stations
I Leipsic River
2. Simons River
3. Bowers Beach—Murderkill River
4. Mispillion River
5. Broadkill River
6. Cape Henlopen—Delaware Bay
7. Thompson Island—Rehoboth Bay
8. Arrowhead Point—Rehoboth Bay
9. West Gables—Indian River Bay
263
-------
TABLE C-l.—Summary of data on organochlorine residues in the monitored species, 1966-69—Delaware
STATION
NUMBER
1
2
3
4
5
6
7
8
9
LOCATION
Leipsic River
Simons River
Bowers Beach
Mispillion River
Broadkill River
Cape Henlopen
Thompson Island
Arrowhead Point
West Gables
MONITORING
PERIOD
1967-69
1967-69
1966-69
1966-69
1966-69
1966-69
1966-69
1966-69
1966-69
Total number of samples
Percent positive for indicated compound
PRINCIPAL
MONITORED
SPECIES
M. demissus
M. demissus
C. virginica
C. virginica
C. virginica
M. mercenaria
M. mercenaria
M. mercenaria
M. demissus
NUMBER OF
SAMPLES '
27
25
34
35
34
32
33
34
33
287
NUMBER OF POSITIVE SAMPLES AND MAXIMUM
RESIDUE ( ) DETECTED IN PPB (^O/KO)
DDT
23 (156)
23 (205)
34 (172)
33 (90)
34 (90)
30 (65)
5 (16)
4 (35)
30 (96)
75
DlELDRIN
4 (13)
6 (19)
25 (25)
2 (10)
13
Each sample represents 15 or more mature mollusks.
TABLE C-2-—Distribution of organochlorine residues in the monitored species for each sampling station by date of
collection—Delaware
1 Blank - no sample collected; — = no residue detected above 5 ppb; T = >5 but <10 ppb]
YEAR
COMPOUND
RESIDUES IN PPB (^O/'KO)
JAN. FEB. MAR.
APR.
MAY JUNE JULY AUG. SEPT. OCT.
Nov. DEC.
STATION 1.—LEIPSIC RIVER—M. DEMISSVS—V SAMPLES'
1967
196S
1969
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
DDE
TDE
DDT
12 25
11 53
__ T
— 10
27 30
68 69
17 17
— - T 15
— — 14 18
— —
26 47
46 91
12 18
13 —
32 26
91 41
14 —
T —
T —
— —
33
77
23
10
33
45
22
17
33
19
21 17 22 18 32
51 47 29 47 77
51 47 — T 17
— 10 — — —
T 12 23 19
20 18 29 37
— — T 18
—
-
—
STATION 2.—SIMONS RIVER—M. DEMISSUS—25 SAMPLES'
1967
1968
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
13 17
43 37
_
-
28
65
18
—
31
65
15
15
23
78
39
—
31 43
150 89
24 19
19 12
39
100
16
10
37 25
79 47
28 38
T —
23
30
—
—
18
88
29
12
— 20
— 26
— 22
— —
29
75
18
-
11
16
-
—
29
66
16
—
264
PESTICIDES MONITORING JOURNAL
-------
TABLE C-2—Distribution of organochlorine residues in the monitored species for each sampling station by date of
collection—Delaware—Continued
RESIDUES IN
JAN. FEB.
MAR
STATION 2.— SIMONS
1969
DDE
TDE
DDT
— 13
— 22
- —
T
T
—
APR.
RIVER— M.
21
23
-
MAY
JUNE
DEMISSVS— 25
14
19
-
21
29
24
PPB OG/KG)
JULY AUG. SEPT. OCT. Nov. DEC.
SAMPLES !— Continued
13 21
26. 35
26 31
STATION 3.—BOWERS BEACH—C. VIRGIN 1C A—34 SAMPLES '
1966
1967
1968
1969
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
27 25 «> I2> 42 43
35 26 <2> «> 65 70
(21 (21 T 10
18 14 20 25 24 18
50 46 48 52 66 75
56 42 47 48 73 78
T - T — 10 T
12 13 14 15 12 16
52 48 42 49 41 79
37 47 36 39 39 70
— — — - — 20
14 15 11 — II _
41 40 40 42
66 64 56 98
24 68 17 25
11 13 16 16
82 49 41 52
53 34 32 44
25 T T 13
- — - —
60 39
57 29
29 11
— -
25
29
—
16
41
51
14
13
57
57
18
16
19
17
—
—
32
38
10
15
41
35
T
15
STATION 4.—MISPILLION RIVER—C. V1RGIN1CA—35 SAMPLES '
1966
1967
1968
1969
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
DDE
TDE
DDT
31 21 22
27 24 24
15 —
17 24 <=< 25 35 23 22 16 20 T 32 27
18 22 '=> 31 41 26 30 20 23 44 36 32
- - - T 11 T T _ T
— — 10 — 10 — ______
23 28 Lost 41 39 39 47 34 32 25 39 3g
40 31 40 36 34 33 23 25 18 32 27
T — 15 ________
40 50 36 29 36 26 35 28
29 31 28 25 32 26 22 20
— — - — ____
VOL. 6, No. 4, MARCH 1973
265
-------
TABLE C-2—Distribution of organochlorine residues in the monitored species for each sampling station by date of
collection—Delaware—Continued
YEAH
COMPOUND
RESIDUES IN PPB (/IO/KO)
JAN.
FEB. MAR. APR. MAY JUNE JULY Auo.
SEPT.
OCT.
Nov. DEC.
STATION J.—BROADK1LL RIVER—C. VIRG1NICA—34 SAMPLES »
1966
1967
1968
1969
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
28 18 23
23 11 17
- — -
25 29 23
21 22 16
— — -
40 38 43
24 28 26
— — —
17 23 24
13 18 21
— — T
32 39 43
21 45 32
- , — —
41 39 42
20 28 28
— — —
18
20
T
16 30 27 35
19 30 27 27
T 16 11 T
36 48 37 31
20 32 25 22
_ _ _ _
43 35
24 15
—
22
17
—
35
31
T
44
33
—
37
33
T
51
39
— t
STATION 6.—CAPE HENLOPEN—M. MERCENARIA—32 SAMPLES'
1966
1967
1968
1969
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
.DDT
12
11
-
12 13 — <" 14 12 20 12 T 14 16 16
16 12 — «> 14 14 24 14 T 13 15 15
___«>________
13 15 18 18 78 39 25 25 19 13 T 21
12 14 14 15 24 26 16 15 12 T T 11
____________
12 14 22 Lost 16 19 22 35
— — T 11 T 10 20
— — — — — — —
STATION 7.—THOMPSON ISLAND—M. MERCENAR1A—33 SAMPLES »
1966
1967
1968
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
— T
T 11
T —
T _ T T — ______
-T- -J-
-J-
____________
______ ______
— — — — — — — — — — — —
266
PESTICIDES MONITORING JOURNAL
-------
TABLE C-2—Distribution of organochlorine residues in the monitored species for each sampling station by date of
collection—Delaware—Continued
YEAR
RESIDUES IN PPB (/iG/Ko)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT. Nov. DEC.
STATION 7.— THOMPSON ISLAND— M. MERC EN ARIA— 33 SAMPLES '—Continued
1969
1966
1967
1968
1969
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
— ___ — — _
________
________
STATION 8.— ARROWHEAD POINT— M. MERCENARIA— 34 SAMPLES 1
— — T
24 — T
11 — —
(2) f
(21 1" .
(2) _
_. ___ _ ______
______ ______
— — — — — — ______
______ _
______ __
— — — — — — — —
STATION 9.—WEST GABLES—M. DEMISSUS UNLESS OTHERWISE INDICATED—33 SAMPLES l
1966
1967
1968
1969
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
11
25
32
19
29
21
10
14
—
18
37
19
24 18
35 28
30 22
16 13
16 17
— —
17
32
14
20
32
26
16
20
T
22
33
24
18
30
23
17
22
10
19
24
13
23
37
36
21
27
T
15 18
21 29
14 41
T T
11 T
•j-
13 »11
18 T
11 —
(3) • (3)
_ _
- -
13 19 19 21
13 29 30 33
21 21 22 26
— T 12 13
— T 16 18
- — T —
1 Each sample represents 15 or more mature mollusks.
3 Present but not quantified.
3 M. merceparia.
VOL. 6, No. 4, MARCH 1973
267
-------
SECTION D.—FLORIDA
Investigation of the effects of pesticide pollution on
estuarine fauna in Florida was initiated at the Gulf
Breeze Laboratory, Gulf Breeze, Fla., in 1959. During
the next 5 years, sufficient headway in the understand-
ing of uptake and flushing rates of persistent synthetic
compounds as well as the technology for handling
samples made a continuing monitoring program feasible.
Local oysters (Station 9, East Bay) were analyzed
monthly during 1964, and the concept of a national
monitoring program was developed and implemented
in 1965. The eastern oyster, C. virginica, was the only
species monitored in Florida; all samples were analyzed
at the Gulf Breeze Laboratory. The approximate loca-
tions of monitoring stations are shown in Fig. D-l. A
"summary of data on organochlorine residues in the
monitored species, C. virginica, is presented in Table
D-l, and the distribution of residues in this species for
each sampling station by date of collection in Table
D-2.
Oyster samples from Florida contained the highest levels
of DDT residues and the most persistent contamination
with PCB's observed in the entire monitoring program.
The polychlorinated biphenyl, Aroclor 1254®, was
identified in studies of estuarine fauna following a 1969
fish kill in Escambia Bay, Fla., (8). Station 9 is about
25 miles from the presumed source of this PCB pollution
and is in a contiguous but distinct drainage basin.
Monitoring samples from this station contained PCB
residues about one-third the magnitude of residues in
Escambia Bay oysters and continued to have residues of
similar magnitude for at least 3 years after the pre-
sumed primary source of PCB's had been eliminated.
The trend in DDT residues is most clearly shown in the
Station 9 data. Some DDT had been used in this
geographic area for agricultural purposes. However, its
primary use had been for the control of stable-fly
larvae, Stomoxys calcitrans, that develop in seaweed
windrows on estuarine beaches. In 1969, methoxychlor
was substituted for this purpose, and DDT residues
virtually disappeared from all succeeding monitoring
samples. Methoxychlor residues were not detected in the
monitored samples. There are not enough recent data to
determine DDT pollution trends in other estuaries along
the Florida Gulf coast.
The incidence of DDT in Florida samples (62%) is
about the average for all States monitored. The incidence
of dieldrin (7%) may be compared with the average
incidence of 15% for all States.
FIGURE D-l.—Diagram of coastal Florida showing
approximate location of monitoring stations
1. lona Point—Caloosahatchee River
2. Charlotte Harbor—Peace River
3. Coral Cove—Little Sarasota Bay
4. Manatee River
5. Crystal River
6. Suwanee River
7. St. Vincents Bar (North)—Apalachicola Bay
8. St. Vincents Bar (South)—Apalachicola Bay
9. East Bay—Blackwater River
TABLE D-l.—Summary of data on organochlorine residues in the monitored species (C. Virginica), 1965-72—Florida
STATION
NUMBER
,
2
3
4
5
6
LOCATION
lona Point
Charlotte Harbor
Coral Cove
Manatee River
Crystal River
Suwanee River
MONITORING
PERIOD
1967-69
1966-69
1966-69
1966-69
1966-71
1966-69
NUMBER OF
SAMPLES '
31
31
32
32
43
32
NUMBER OF POSITIVE SAMPLES AND MAXIMUM
RESIDUE ( ) DETECTED IN PPB (JIC/KO)
DDT
31 (5,390)
28 (338)
32 (129)
32 (159)
7 (27)
6 (22)
DIELDRIN
1 (11)
13 (271
PCB's '
268
PESTICIDES MONITORING JOURNAL
-------
TABLE D-l.—Summary of data on organochlorine residues in the monitored species (C. Virginica), 1965-72—
Florida—Continued
STATION
NUMBER
7
8
9
LOCATION
St. Vincents Bar (North)
St. Vincents Bar (South)
East Bay
Occasional stations (21)
MONITORING
PERIOD
1966-67
1966-67
1965-72
1966-71
Total number of samples
Percent of samples positive for indicated compound
NUMBER OF
SAMPLES '
17
16
84
56
374
NUMBER OF POSITIVE SAMPLES AND MAXIMUM
RESIDUE ( ) DETECTED IN PPB (/IG/KG)
DDT
12 (50)
10 (70)
46 (65)
26 (101)
62
DlELDRIN
3 (28)
3 (22)
7 (12)
7
PCB'S -
25 (390)
7
1 Each sample represents 15 or more mature mollusks.
' Calculated as Aroclor 1254®
TABLE D-2—Distribution of organochlorine residues in C. virginica for each sampling station by date of collection—Florida
[Blank = no sample collected; -r- = no residue detected above 5 ppb or no residue detected (PCB's); T = >5 but <10 ppb]
YEAR
COMPOUND
RESIDUES IN PPB (#G/KG)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT.
Nov. DEC.
STATION 1.—IONA POINT—31 SAMPLES1
1966
1967
1968
1969
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
91 320
94 170
190 630
760 1 ,200
44 560
2,800 3,600
- —
710
1,400
1,700
930 1,450 290 110
760 705 310 200
3,700 2,550 350 68
1,100 1,500 780 340
580 560 390 310
2,300 , 1,200 650 220
- — — -
940
400
1,100
30 13 24 35
39 20 48 79
— — — 28
53 60 87 72 140
110 160 160 150 220
57 32 97 110 68
180 — T 77 84
190 T 16 160 120
33 — — 69 140
— — 11 — —
T
T
—
240
310
520
82
120
60
—
STATION 2.—CHARLOTTE HARBOR—31 SAMPLES"
1966
1967
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
T
10
-
83 14 15 39 18 30 13 T
85 20 24 33 27 43 20 13
170 T 13 — T 13 T —
— — — — — — 14 11
T 17
16 23
— 15
T —
T —
— —
19 —
— 52
— 91
— 41
T 18
11 28
— 21
— 15
VOL. 6, No. 4, MARCH 1973
269
-------
TABLE D-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of collection—
Florida—Continued
YEAR
COMPOUND
RESIDUES IN PPB (/IG/KO)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT.
Nov. DEC.
STATION 2.—CHARLOTTE HARBOR—31 SAMPLES'—Continued
1968
1969
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
19 23
22 2&
14 13
— 11
14
1*
—
18 34 27 22
18 36 29 18
— 20 16 10
— 18 11 —
17
22
12
20 — T — T T
26 T 17 — 11 T
1*
27 — 16 — 13 19
STATION 3.—CORAL COVE—32 SAMPLES '
1966
1967
1968
1969
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
34
28
12
29
30
10
30
38
T
26
23
12
27
30
14
41
40
20
24 25 20 24
22 26 21 21
T 13 T 10
21 35 49 39
14 36 43 40
— 13 37 49
36
40
15
24 T
21 —
- —
25 23 12
24 20 16
17 T T
31 19 20
26 16 23
32 22 14
10 25
T 33
-j-
16 13
16 10
T T
28 21
28 23
28 11
17
16
T
10
10
T
23
29
T
STATION 4.—MANATEE RIVER—32 SAMPLES 1
1966
1967
1968
1969
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
37 39
30 45
19 19
26 24
24 29
26 13
22 32
33 46
— 17
22 31 18 T
24 41 23 T
10 13 T —
18 42 16 18
30 65 19 61
10 22 T 25
24
26
T
23 37 25
39 47 33
— 13 11
19 21 23 33
20 42 46 59
T 20 17 13
31 16 18 18
88 37 38 16
40 T 13 —
T 30
T 33
— 12
34 13
45 14
14 14
17 24
19 27
— 14
270
PESTICIDES MONITORING JOURNAL
-------
TABLE D-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of collection—
Florida—Continued
YEAR
COMPOUND
RESIDUES IN PPB (/IG/KG)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT.
Nov. DEC.
STATION 5.—CRYSTAL RIVER—43 SAMPLES1
1966
1967
1968
1969
1970
1971
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
_ 12 - — T —
_ _ _ — T —
— — — — — —
-T----T-- --
______ T — — — —
— — — — 13 — T — — — —
— — — T — 11 — ____
_ _ _ T - - - ____
_____16_ ____
_ _ _ _ _ — —
_ _ _ — — — —
— — — — — — —
_ _ _ _ _ _
______
______
— —
— —
— -
STATION 6.—SUWANEE RIVER—32 SAMPLES1
1966
1967
1968
1969
1966
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
— T — — T —
— — — — T —
— — — — — —
— 12 T — — — — _ — ___
T T
— TT — 11 11 — _____
— — — — ___ __
— — — — — — — — ___
— — — — — — — — ___
— — —
_ — _
— — —
STATION 7.— ST. VINCENTS BAR (NORTH)— 17 SAMPLES1
T 16 T 14 — _ n T
T 19 T 15 - _ ,o T
_T_ _____
— 10— — — — __
VOL. 6, No. 4, MARCH 1973
271
-------
TABLE D-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of collection—
Florida—Continued
YEAR
COMPOUND
RESIDUES IN PPB (#G/KG)
JAN. FEB.
STATION 7.— ST.
1967
DDE
TDE
DDT
Dieldrin
T —
— —
— —
11 —
MAR. APR.
VINCENTS BAR
T
T
—
—
MAY JUNE JULY AUG. SEPT. OCT. Nov. DEC.
(NORTH)— 17 SAMPLES J— Continued
22 T 13 — — T
23 T 10 — — —
T — T — — —
28 — — — — —
STATION 8.—ST. VINCENTS BAR (SOUTH)—16 SAMPLES »
1966 DDE
TDE
DDT
Dieldrin
1967 DDE
TDE
DDT
Dieldrin
18 25
21 30
— 15
— 13
T — 14 21 T
— — 13 22 T
— — — T 38
— — 15 22 —
17 — — — —
T — — — —
_ — — — —
— — — — —
13 T T —
12 T — —
T — — —
— — — —
STATION 9.—EAST BAY—84 SAMPLES '
1965
1966
1967
1968
1969
1970
1971
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
PCB's •
DDE
TDE
DDT
PCB's «
12 13 13 17 26 24
— 14 — 15 24 19
— — — 15 15 14
T 18 21 18 18 12
— 18 17 22 24 13
— 14 16 15 15 —
16 12 17 22 15 15
15 20 — — — —
10 12 — — — —
T 16 11 — 13 14
T 14 10 — 13 —
_ T — — — —
— T — T — —
______
______
— — — — 380 180
— — — _ — —
______
______
160 160 200 220 230 390
19 T T
18 T T
13 T T
15 14 T
11 — —
— — —
20 T T
23 — —
18 — 10
— T 20
— T —
— — —
— — 10
— — T
— — 14
_ _ _
_ _ _
_ _ _
170 73 92
— — —
_ _ _
_ _ _
190 230 100
— T T
f
-J-
— T T
— — T
— — —
T T T
_ _ _
_ _ _
y
— — —
— — —
T — —
— — —
_ _ _
_ _ _
_ _ _
— — —
SO 55 140
_ _ _
_ _ _
_ _ _
55 120 —
272
PESTICIDES MONITORING JOURNAL
-------
TABLE D-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of collection—
Florida—Continued
YEAR
COMPOUND
RESIDUES IN PPB UO/KG)
JAN. FEB. MAK.
APR. MAY JUNE JULY AUG. SEPT. OCT.
Nov. DEC.
STATION 9.—EAST BAY—84 SAMPLES »—Continued
1972
DDE
TDE
DDT
PCB'S "
__ — — — —
__ — — — —
— — — — — —
50 82 140 160 190 300
1 Each sample represents 15 or more mature mollusks.
' Calculated as Aroclor 1254®.
SECTION E.—GEORGIA
Monthly collections of the eastern oyster (C. virginica)
were made at 11 estuarine areas in Georgia during the
period Febiuary 1967 June 1972. Analyses were
done at the Gulf Breeze Laboratory until September
1969, and thereafter at the Marine Institute of the
University of Georgia. The approximate locations of
monitoring stations are shown in Fig. E-l. A summary
of data on organochlorine residues in the monitored
species, C. virginica, is presented in Table E-l, and the
distribution of residues in this species for each sampling
station by date of collection in Table E-2. The 15%
incidence of DDT residues in Georgia samples was next
to the lowest of all States monitored (Washington,
lowest at 11%). The maximum level of DDT observed
was also next to the lowest of any of the other States
monitored. By contrast, the largest dieldrin residue
detected in the nationwide program was in Georgia,
(230 ppb) and the incidence of dieldrin residues (21% )
was well above the average incidence (15%) for all
States.
The occurrence of substantial toxaphene residues in the
samples collected in St. Simons Sound was unexpected.
A special sampling program was initiated in the area
that included the placement of trays of oysters in creek
beds where oysters did not normally occur. Analyses of
these samples pinpointed the industrial source of the
toxaphene and precipitated a schedule for control of the
effluent discharge by the manufacturer. The magnitude
of toxaphene residues at Stations 8 1 1 illustrates well
the importance of dilution (distance) in the abatement
of pollution.
Polychlorinated biphenyl residues were analyzed for
beginning in 1969. A few samples collected in the
Ogeechee and Satilla River basins contained residues of
Aroclor 1254'®, but the amounts were not quantified.
DDT residue levels were generally low and there was an
approximate increase of 13% in the number of samples
VOL. 6, No. 4, MARCH 1973
with negligible residues in 1971 as compared to earlier
years. Stations 1 and 2 in the Savannah River basin,
however, showed a reversal of this trend in 1972 when
oysters contained substantially higher residue levels than
in 1971.
GEORGIA
ATLANTIC OCEAN
FIGURE E-l.—Diagram of coastal Georgia showing
approximate location of monitoring stations
I. Lazeretta Creek—Savannah River Basin
2. Wilmington River—Savannah River Basin
3. Ogeechee River—Ogeechee River Basin
4. St. Catherine Sound—Ogeechee River Basin
5. Sapelo Sound—Ogeechee River Basin
6. Doboy Sound—Ogeechee River Basin
7. Egg Island—Altamaha River Basin
8. St. Simons Sound—Satilla River Basin
9. Terry Creek—Satilla River Basin
10, Jekyll Island—Satilla River Basin
11, Satilla River—Satilla River Basin
273
-------
TABLE E-l—Summary of data on organochlorine residues in the monitored species (C. virginica), 1967-72—Georgia
STV.TION
NUMBER
2
3
4
5
6
7
8
9
10
11
LOCATION
Lazerelta Creek
Wilmington River
Ogeechee River
St. Catherine Sound
Sapelo Sound
Doboy Sound
Egg Island
St. Simons Sound
Tei -y Creek
Jekyll Island
Satilla River
Occasional stations (2)
MONITORING
PERIOD
1967-72
1967-72
1967-72
1967-72
1967-72
1967-72
1967-72
1967-72
1967-70
1967-72
1967-72
1968-69
Total number of samples
Percent of samples positive for indicated compound
NUMBER OF
SAMPLES *
64
65
65
65
65
64
65
65
16
62
64
4
664
NUMBER OF POSITIVE SAMPLES AND MAXIMUM
RESIDUE ( ) DETECTED IN PPB (AC/KG)
DDT
30 (96)
21 (86)
13 (50)
7 (15)
12 (50)
7 (27)
3 (52)
<3>
131
(3>
3 (15)
<3t
15
DFELDRIN
58 (230)
27 (90)
15 (26)
2 (T)
6 (12)
8 (14)
22 (23)
3 (T)
21
TOXAPHENE
64 (7,500)
16 (54,000)
37 (3,500)
8 (1,000)
3 (13,000)
19
PCB'S-'
1
1
2
1
2
8
1
2
NOTE: T = >5 but <10 ppb.
L Each sample represents 15 or more mature mollusks.
' Present but not quantified.
5 Presence of toxaphene prevented quantification of DDT and its metabolites.
TABLE E-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of collection—Georgia
YEAR
COMPOUND
JAN.
FEB.
M*R.
APR.
MAY
JUNE
JULY
AUG.
SEPT.
OCT.
Nov.
DEC.
RESIDUES IN PPB Ug/kg)
[Blank = no sample collected; — = no residue detected above 5 ppb or no residue detected (PCB's); T = >5 but <10 ppb]
STATION 1.—LAZERETTA CREEK—64 SAMPLES1
1967
1968
1969
1970
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
14
17
—
98
— 13
— 16
— —
22 42
— —
— —
— —
39 23
T 13
— —
— —
30 31
13
14
—
65
12
12
—
37
—
—
—
47
—
—
—
40
21
29
T
56
17
23
T
46
—
—
—
51
T
T
—
32
T T
13 11
— T
32 30
— —
— —
- —
— 20
— T
— 13
— —
16 35
T —
T —
— —
17 23
53 —
25 14
18 11
30 33
15 —
23 —
28 —
39 22
— —
— —
- -
28 23
— T
_ _
— —
80 T
— — — 12
— — — 16
— — — T
18 42 33 46
— — T
— — T
— — —
18 42 56
— — — T
— - — —
— — — —
20 180 230 20
— 23 — —
— — — —
- — — —
— - T T
274
PESTICIDES MONITORING JOURNAL
-------
TABLE E-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of collection-
Georgia—Continued
YEAR COMPOUND
JAN. FEB. MAS. APR.
MAY JUNE JULY AUG. SEPT. OCT. Nov. DEC.
RESIDUES IN PPB (/ig/kg)
[Blank =: no sample collected; — = no residue detected above 5 ppb or no residue detected (PCB's); T = >5 but <10 ppb]
STATION 1.— LAZERETTA CREEK— 64 SAMPLES '—Continued
1971 DDE
TDE
DDT
Dieldrin
1972 DDE
TDE
DD1
Dieldrin
— — 20 —
_ _ _ _
_ _ _ _
19 i9 T 17
7 1 23 !8
T 14 12
Y r
15 13 T T
STATION 2. WILMING
1967 DDE
TDE
DDT
Dieldrin
1968 DDE
TDE
DDT
Dieldrin
1969 DDE
TDE
DDT
Dieldrin
1970 DDE
TDE
DDT
Dieldrin
1971 DDE
TDE
DDT
Dieldrin
1972 : DDE
TDE
DDT
"M'eldrin
TIT
T T T
_ _ _
17 19 22
— — T T
_____
_ _ _ _
10 21 12 —
_ _ _ _
_ _ _ _
_ _ _ _
— — — 10
— 11 — T
T
— — — —
T T 10 25
— — 10 —
- - — —
_ _ _ _
T 12 12 T
T 12 15 12
— 13 T 11
— _ _ _
10 17 15 —
— — — — — T T —
y -j1
______ — —
13 10 -- T T — — 13
T T
T T
T
22 T
TON R.JYL-.R ..5 SAMPLES1
— — T 12 — -- — —
- — ,___ — —
_ _ ______
----- ______
— _ _ ._ _ — — _
__ _______
_ _ _____ — _ —
_________
— — — _ _ _ 90 T
— — — T 86 — — —
— — — — —
________
- T ______ T
— — - - T T T
— — — — — T T —
— — — —
T — — -.__ T T
T T
T T
T T
T T
VOL. 6, No. 4, MARCH 1973
275
-------
TABLE E-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of collection—
Georgia—Continued
YEAR
COMPOUND
JAN.
FEB. MAE. APR. MAY JUNE JULY Auo. SEPT. OCT. Nov. DEC.
RESIDUES IN PPB (0g/kg)
[Blank = no sample collected; — = no residue detected above 5 ppb or no residue detected (PCB's); T = >5 but <10 ppb]
STATION 3.—OGEECHEE RIVER—65 SAMPLES *
1967
1968
1969
1970
1971
1972
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
PCB's
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
T T T- — — T — — — T
T 10 — — — — ____T
— — _ — ______T
13 26 10 — — — — — — — —
— — 13 T — — — — — — — —
— — 13 11 — — — — — — — —
— — 24
— 18 16 — — — — — — — — —
— — — — — — — —
______ ______
______ ______
— — _T_ — — 16 — _ — T
_ (!)
— 10 — — — — — T 12 — — —
____________
____________
T T T — — — — — — — — —
____________
____________
______ ______
— T T T — — 11 — — — — —
- - T — T T
f
______
STATION 4.—ST. CATHERINE SOUND—65 SAMPLES »
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
PCB's
T
T
276
PESTICIDES MONITORING JOURNAL
-------
TABLE E-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of collection—
Georgia—Continued
YEAR
COMPOUND
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT. Nov. DEC.
RESIDUES IN PPB (/ig/kg)
[Blank = no sample collected; — = no residue detected above 5 ppb or no residue detected (PCB's); T = >5 but <10 ppb]
STATION 4.— ST. CATHERINE SOUND— 65 SAMPLES "—Continued
1970
1971
1972
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
______ ______
____________
— __ — — — — — — — — —
______ — ___ — —
____ — — — — — — — —
__________ _ _
_T_T — — — — — — — —
— — T — T T
1*
— — — — — —
STATION 5.—SAPELO SOUND—65 SAMPLES >
1967
1968
1969
1970
1971
1972
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
PCB's
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
T T T — T T — — — — T
T — T — T 22 — — — — T
T — — — T 23 — — — — —
,2 __________
— — — — —
— — — — — — — — — — — —
— — — — — — ______
— — T — — — — — ____
— — 13 — — — ______
— — 19 — — — — _____
— — — T — — — — — ___
— — — — — — — — — — (a) (a)
— 11 — T — _______
— y
------ ______
-p
--------____
— T T T — _______
- - T - T T
— — — — — T
_„ _ 'y
VOL. 6, No. 4, MARCH 1973
277
-------
TABLE E-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of collection—
Georgia—Continued
YEAR
COMPOUND
JAN.
FEB. MAX. APS. MAY JUNE JULY Auo. SEPT. OCT. Nov. DEC.
RESIDUES IN PPB
[Blank = no sample collected; — = no residue detected above 5 ppb or no residue detected (PCB's); T = >5 but <10 ppb]
STATION 6.—DOBOY SOUND—64 SAMPLES '
1967
1968
1969
1970
1971
1972
DDE
TDE
DDT
DDE
TDE
PDT
DDE
TDE
DDT
Dleldrin
PCB's
DDE
TDE
DDT
Dleldrin
DDE
TDE
DDT
Dleldrin
DDE
TDE
DDT
T T - - - T _ _ _ _ —
— T — — — || _ _ _ _ —
----- 11 - - - - -
— —
— — — — — — — — — — — —
_ _ __________
— — — — —
— — ____ ______
— — — — ________
— 14 14 13 — — — — — — — —
----------<»-
— 10 — — _______
— — __ _______
— — __ _______
T
— — — — — — — — — —
____________
____________
T T T — T — — — — — — —
— — T T T —
_ _ T — — —
— — — — — —
STATION 7.—EGG ISLAND—65 SAMPLES '
1967
1968
1969
DDE
TDE
DDT
Dleldrin
DDE
TDE
DDT
Dleldrin
DDE
TDE
DDT
Dieldrin
_____ ,5 _ - - - -
J9 __
_____ is _ — — — —
_____ _ _ _ _ _ 20
— _ — — _ — __ — — — —
— — — — — — ______
____________
13 15 23 14 — — — — — — — 21
— — _ — — — — — — — — —
— — — — — — _ — — — — —
— — — — — — — — — — — —
16 — 15 15 — — — — — — — T
278
PESTICIDES MONITORING JOURNAL
-------
TABLE E-2.—Distribution of organocMarine residues in C. virginica for each sampling station by date of collection—
Georgia—Continued
YEAR
COMPOUND
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT. Nov. DEC.
RESIDUES IN PPB (/ig/kg)
[Blank = no sample collected; — = no residue detected above 5 ppb or no residue detected (PCB's); T = >5 but <10 ppb]
STATION 7.— EGG ISLAND— 65 SAMPLES »— Continued
1970
1971
1972
DDE
TDE
DDT
Dleldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
-16----------
____ — — — — — — — —
_____ _ ____ — —
TTT-T----TTT
______ ___ — — —
_____-__ — — — — —
_______ ___ — — —
11TTT — — — — — — — —
|-
______
______
T
RESIDUES IN PPM (mg/kg)
[Blank = no sample collected; — = no residue detected above 0.1 ppm or no residue detected (PCB's);
T = >0.1 but <0.25 ppm]
STATION 8.—ST. SIMONS ISLAND—55 SAMPLES '
1967
1968
1969
1970
1971
1972
Toxaphene
Toxaphene
Toxaphene
Toxaphene
PCB's
Toxaphene
PCB's
Toxaphene
«> 2.5 1.5
0.8 5.0 6.0 4.3
2.0 1.2 2.5 7.5
3.8 3.8 7.2 3.3
- — — -
1.3 0.7 1.1 1.6
__..__
0.6 1.0 I.I 1.0
1.5
1.6
5.0
1.8
—
0.7
(2)
0.8
1.0 1.0
2.0 2.0
1.5 1.0
1.1 <1.0
(2)
0.1 0.6
- —
0.6
l.l 0.8 0.7 2.0 2.0
0.6 T — 5.4 2.8
1.0 1.5 1.6 1.6 1.8
0.8 T 0.6 0.7 1.6
- -----
T T 0.6 T 0.6
-----
RESIDUES IN PPM (mg/kg)
[Blank = no sample collected; — = no residues detected above 0.1 ppm; T ±= >0.1 but <0.25 ppm]
STATION 9.— TERRY CREEK— 16 SAMPLES '
1967
1968
1969
1970
Toxaphene
Toxaphene
Toxaphene
Toxaphene
12.0
23.0 6.0 54.0
9.0
6.2
"•7 18.0 13.0
5-0 6.3 12.0
12.0 17.0 8.0
8.2
VOL. 6, No. 4, MARCH 1973
279
-------
TABLE E-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of collection—
Georgia—Continued
YEAK COMPOUND JAN.
FEB.
MA*.
APR.
MAY
JUNE
JULY
AUG.
SEPT.
OCT.
Nov.
DEC.
RESIDUES IN PPM (mg/kg)
[Blank = no sample collected; — = no residue detected above 0.1 ppm; T = >0.1 but <0.26 ppm]
STATION 10.—JEKYLL ISLAND—62 SAMPLES ».«
1967
1968
1969
1970
1971
1972
Toxaphene
Toxaphene
PCB's
Toxaphene
PCB's
Toxaphtne
PCB's
Toxaplione
PCB's
Toxapheue
— T
0.7 2.1 0.7
— — —
1 .0 1 .0 1 .0
— —
0.8 - T
(2) (2)
05 T 0.6
(2)
0.3 T 1.0
0.5 0.4 0.4
0.7 0.5 T
— — —
1.0 —
— —
T — —
<2> (2) .
0.8 T T
(2)
0.6 T —
— — — 1.0
0.4 — — — — —
______
— -- 3.5 T — 0.7
C_M (SJ
— — — — — 0.6
______
— — — 0.5 T T
_______
1967
1968
1969
1970
1971
1972
RESIDUES IN PPM (mg/kg)
(Blank = no sample collected; — = no residue of DDT detected above 0.005 ppm or no residue detected above
0.1 ppm (toxaphene and PCB's); t = >0.1 but <0.25 ppm; T = >0.005 but <0.010 ppm]
STATION 11.—SATILLA RIVER—64 SAMPLES'
DDE
TDE
DDT
Toxaphene
DDE
TDE
DDT
Toxaphene
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
PCB's
DDE
TDE
DDT
i ( t t
1.0 0.5 0.7 1
1 Each sample represents 15 or more mature mollusks.
' Aroclor 1254® present but not quantified.
* Presence of toxaphene prevented quantification of DDT
and its metabolites in these samples.
280
' Toxaphene present but not quantified.
•• One sample each in April 1969, April 1970, and February 1972
contained a trace of dieldrin.
' DDT and its metabolites not detected in any samples.
PESTICIDES MONITORING JOURNAL
-------
SECTION R—MAINE
The monthly monitoring of Maine estuaries for per-
sistent synthetic residues was initiated in December 1965
and continued until November 1970. There were 10
principal stations; about 40 other sites were sampled
occasionally. Samples were analyzed at the Gulf Breeze
Laboratory until June 1969 and, thereafter, at the
Fisheries Research Station, Maine Department of Sea
and Shore Fisheries.
The soft clam (Mya arenarid) and the blue mussel
(Mytilus edulis) were the principal mollusks monitored
and, on occasion, both eastern oysters (Crassostrea
virginica) and horse mussels (Modiolus modiolus)
were collected at the same sites. In the laboratory,
the uptake of DDT was greater in the soft clam
than in other species tested as was the flushing rate,
and 90% of DDT residues was lost within 7 days
after the toxicant was removed. This may explain why
in simultaneous collections of two or more species of
mollusks, DDT residues in soft clams examined at 30-
day intervals were usually lower than those in the oyster
or horse mussel. A summary of data on organochlorine
residues in the monitored species, is presented in Table
F-l, and the distribution of residues in these species for
each sampling station by date of collection in Table F-2.
The Maine samples are characterized by the low in-
cidence (18%) of detectable DDT residues as compared
to most other monitored areas, despite the fact that
substantial amounts of DDT are reported to have been
used agriculturally in some watersheds in Maine. The
maximum magnitude of DDT residues detected was,
however, larger than that found in seven other States.
Analysis of occasional collections of fish and inverte-
brates other than mollusks revealed DDT residues larger
than those in mollusks. Presumably organochlorine
pollution in Maine estuaries was usually too low and too
transitory to be detected except in animals that retain
residues for a Ipng period of time.
Despite the generally low incidence of DDT residues at
most stations, there was sufficient continuity in detect-
able DDT residues at Station 10 on the Piscataqua River
to show a gradual decline from an average of about 28
ppb in 1966 to an undetectable level in 1970. A similar
trend is clearly shown in samples collected at Station 7,
Small Point.
FIGURE F-l.—'Diagram of coastal Maine showing
approximate location of monitoring stations
I. Mill Cove—St. Croix River
2. Machiasport—Machias River
3. Millbridge—Narraguagus River
4. Fort Point—Penobscot River
5. Thomaston—St. George River
6. Medomak—Medomak River
7. Small Point—Kennebec-Androscoggin River
8. Phippsburg—Kennebec-Androscoggin River
9. Biddeford Pool—Saco River
10. Eliot—Piscataqua River
TABLE F-l—Summary of data on organochlorine residues in the monitored species, 1965-70—Maine
STATION-
NUMBER
1
*
J
4
5
6
7
LOCATION
Mill Cove
Machiasport
Millbridge
Fort Point
Thomaston
Medomak
Small Point
MONITORING
PERIOD
1965-66
1965-70
1966-70
1965-70
1965-70
1967-70
1968-70
PRINCIPAL
MONITORED
SPECIES
M, arenaria
M. arenaria
M. arenaria
M. arenaria
M. arenaria
M. arenaria
M. edulis
NUMBER OF
SAMPLES '
12
52
37
42
42
23
18
NUMBER OF POSITIVE SAMPLES AND MAXIMUM
RESIDUE ( ) DETECTED IN PPB (#G/KG)
DDT
I (15)
1 (12)
1 (15)
1 (80)
2 (11)
12 (359)
DlELDRIN
1 (11)
VOL. 6, No. 4, MARCH 1973
281
-------
TABLE F-l.—Summary of data on organochlorine residues in the monitored species, 1965-70—Maine—Continued
STATION
NUMBER
8
9
10
11
LOCATION
Phippsburg
Biddeford Pool
Eliot
Occasional stations (40)
MONITORING
PERIOD
1965-69
1968-70
1966-70
1965-69
Total number of samples
Percent of samples positive for indicated compound
PRINCIPAL
MONITORED
SPECIES
M. arenaria
M. edulls
\t. arenaria
Mixed
NUMBER OF
SAMPLES 1
39
24
45
62
396
NUMBER OF POSITIVE SAMPLES AND MAXIMUM
RESIDUE ( ) DETECTED IN PPB (IM/KO)
DDT
7 (24)
7 (64)
22 (67)
16 (93)
18
DfELDRIN
9 (38)
4 (}8)
4
1 Each sample represents 15 or more mature mollusks
TABLE F-2.—Distribution of organochlorine residues in the monitored species for each sampling station by date of
collection—Maine
[Blank = no sample collected; — = no residue detected above 5 ppb; T = >5 but <10 ppb.]
YEAR
COMPOUND
RESIDUES IN PPB (iia/na)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT.
Nov. DEC.
STATION 1.—MILL COVE—M. ARENARIA—12 SAMPLES1
1965
1966
DDE
TDE
DDT
DDE
TDE
DDT
—
—
—
______ ______
______ ______
— — — ——— ______
STATION 2.— MACHIASPORT— M . ARENARIA— 52 SAMPLES 1
1965
1966
1967
1968
1969
DDE
TDE
DDT
DDE"
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
—
—
—
f
— — — — — — — — _ — __
— — — — — — — — — — — —
— - — — — — — — ____
— — — — — — — ____
— — — — — — — ____
-----_T-_
------T--
------T--
----- - — _ — __
— — — — — ______
282
PESTICIDES MONITORING JOURNAL
-------
TABLE F-2.—Distribution of organochlorine residues in the monitored species for each sampling station by date of
collection—Maine—Continued
YEAR
1970
COMPOUND
DDE
TDE
DDT
RESIDUES IN PPB (/IG/KO)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT. Nov. DEC.
STATION 2.— MACHIASPORT— M. ARENARIA— 52 SAMPLES >— Continued
— _____ _
— — — — — — . —
— — — — —
STATION 3.— MILLBRIDGE— M. ARENARIA— 31 SAMPLES »
1966
1967
1968
1969
1970
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
—
—
—
___ ______
___ ______
___ ______
_ _ _ _"_ _ _ — — —
____ _____ _
____ ______
__________ _
_____ ______
_____ ______
— — — 12 — —
______
— — — — — —
STATION 4.— FORT POINT— M. ARENARIA— 42 SAMPLES 1
1965
1966
1967
1968
1969
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
—
—
— — — — — — — — __
— — — — — — — — __
— - — — — — -— __
-------___
------____
— — — — — — ____
— __ _ __
----- -___
_
— — — 15
— — — — — — — — _
— — — — — —
— — — — — — _
VOL. 6, No. 4, MARCH 1973
283
-------
TABLE F-2,—Distribution of organochlorine residues in the monitored species for each sampling station by date of
collection—Maine—Continued
YEAR
COMPOUND
RESIDUES IN PPB (^O/KO)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT. Nov. DEC.
STATION 4.— FORT POINT— M. ARENARIA— 42 SAMPLES i— Continued
1970
DDE
TDE
DDT
— —
— —
— —
STATION 5.— THOMASTON— M. ARENARIA, UNLESS OTHERWISE INDICATED— 42 SAMPLES'
1965
1966
1967
1968
1969
1970
1967
1968
1969
1970
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
—
—
—
21
____ — _— 35 —
_______ 24 —
___,!___ --
_ ______.__ — —
_ ___ — _ — — — — —
_ _____ — — — — —
____ ____
____ ____
— — — — ____
»_ — - — - - —
_ _____ —
— - — — - — ~
— — — —
_ _ — - — —
— — — — — —
STATION 6— MEDOMAK— M. ARENARIA— 23 SAMPLES1
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
_ _____ ___
— — — — — ___
,, ._ T - - -
_ _ _ _ —
_ _ _ — —
— — — —
- - - - —
— — — — —
— — — — —
- _ ... _
— _ _ _
— _ — —
284
PESTICIDES MONITORING JOURNAL
-------
TABLE F-2. _ Distribution of organochlorine residues in the monitored species for each sampling
collection — Maine — Continued
station
by date of
YEAR
COMPOUND
RESIDUES IN PPB (HG/KC)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT. Nov^^^_
STATION 7.— SMALL POINT— M. EDULIS— 18 SAMPLES 1
1968
1969
1970
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
35 21 17 12 25 19
44 25 20 14 27 18
280 77 68 18 26 26
11 TTT12 — — — —
14 T T T 21 — — — —
18 15 13 T 13 49 — — —
— — —
_ _ _
— — —
STATION 8.— PH1PPSBURG— M. ARENARIA—39 SAMPLES*
1965
1966
1967
1968
1969
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
1968
1969
1970
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
—
—
-
________ _ —
— — — — — — — — __
________ __
___T---- ---
— — — — — — — — — — —
— — — 11 11 T — — ___
-j-
_____ ______
— _ — T19 — — — — Til
— — _ — — _
— — — — — —
— — — — — —
STATION 9— BIDDEFORD POOL— M. EDULIS— 24 SAMPLES >
- _T-TTTT
- — T - T T T T
~~ — 21 — 54 22 15 ]3
T — T — _ __
T
21 — 12 — _ _
— _ _ _ _ _
— — — — —
— — — — — — _
VOL. 6, No. 4, MARCH 1973
285
-------
TABLE F-2.—Distribution of organochlorine residues in the monitored species for each sampling station by date of
collection—Maine—Continued
YEAR
COMPOUND
RESIDUES IN PPB (JMJ/KO)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG.
SEPT. OCT.
Nov.
DEC.
STATION 10.—ELIOT—M. ARENAR1A, UNLESS OTHERWISE INDICATED—45 SAMPLES'
1966
1967
1968
1969
1970
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
DDE
TDE
DDT
T — 12
T - 16
21 T 32
32 — 38
_ _
— —
— -
— 16
—
T
18
10
' II
23
18
14
21
32
—
T
11
20
Lost
T
12
15
—
3 12
22
14
—
—
—
13 13
19 21
23 16
27 23
T —
14 T
18 30
10 T
—
—
—
—
T
T
T
— —
— —
— —
T - T T T —
11 — T 11 T —
T — T T T —
_____ T
— T T — T
— - 15 T — T
— — T 22 — 15
______
_ T — — —
— 12 — — —
_ T — — —
_____
_ _ _ _
_ _ _ _
_ _ _ _
_ _ _ _
_ _ _ _
— — _ —
1 Each sample represents IS or more mature mollusks.
i M. edulls.
•' M. demissus.
286
PESTICIDES MONITORING JOURNAL
-------
SECTION G.—MARYLAND
Eastern oysters, Crassostrea virginica, were collected in
upper Chesapeake Bay and its tributaries at irregular
intervals (usually twice yearly) from August 1966 to
November 1970. The sampling was made possible be-
cause of oyster surveys being conducted for other pro-
grams. All samples from the 10 locations in Maryland
were analyzed at the Gulf Breeze Laboratory. The ap-
proximate station locations are shown in Fig. G-l. A
summary of data on organochlorine residues in the
monitored species, C. virginica, is presented in Table
G-l, and the distribution of residues in this species for
each sampling station by date of collection in Table
G-2.
Maryland was fifth among all States, in the incidence of
DDT residues (81%), but the magnitude of residues
in oysters was surprisingly low in view of the size of the
Susquehanna River watershed and the extent of its
agricultural development. More selective monitoring
might show that the major pesticide burden of the river
is precipitated with silt in the headwaters of the Bay
and does not enter the trophic web of the estuarine
system extensively. DDT residues detected at monitoring
stations probably reflected pollution primarily in the
adjacent and usually small drainage basins.
Despite the small number of samples, the decline in
average DDT residues from 26 ppb in 1966 to 10 ppb
in 1970 together with a more than 150% increase in
samples containing less than 11 ppb suggests a real
change in average pollution levels.
FIGURE G-l.—Diagram of coastal Maryland showing
approximate location of monitoring stations
I. Franklin City—Chincoteague Bay
2. Pocomoke Sound
3. Tangier Sound
4. Honga River
5. Choptank River
6. Eastern Bay
7. Tollys Bar—Chesapeake Bay
8. Herring Bay—Chesapeake Baj
9. Cedar Point—Chesapeake Bay
10. St. Marys River
TABLE G-l.—Summary of data on organochlorine residues in the monitored species (C. virginica), 1966-70—Maryland
STATION
NUMBER
1
2
3
4
5
6
7
8
9
10
LOCATION
Franklin City
Pocomoke Sound
Tangier Sound
Honga River
Choptank River
Eastern Bay
Tollys Bar
Herring Bay
Cedar Point
St. Marys River
MONITORING
PERIOD
1966-70
1966-69
1966-70
1966-70
1966-70
1966-70
1967-70
1966-70
1966-70
1966-70
Total number of samples
Percent of samples positive for indicated compound
NUMBER OF
SAMPLES '
8
6
10
10
8
10
8
10
10
8
88
NUMBER OF POSITIVE SAMPLES AND MAXIMUM
RESIDUE ( ) DETECTED IN PPB (/io/no)
DDT
8 (43)
5 (47)
5 (48)
8 (43)
4 (30)
8 (70)
8 (44)
9 (46)
9 (70)
7 (33)
81
DlELDRIN
7 (22)
4 (18)
13
1 Each sample represents 15 or more mature mollusks.
VOL. 6, No. 4, MARCH 1973
287
-------
TABLE G-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—Maryland
[Blank = no sample collected; — = no residue detected above 5 ppb; T = >5 but <10 ppb]
YEAR
COMPOUND
RESIDUES IN PPB (iia/KO)
JAN. FEB. MAR. APR. MAY JUNE JULY
AUG. SEPT.
OCT. Nov.
DEC.
STATION 1.— FRANKLIN CITY— 8 SAMPLES'
1966
1967
1968
1969
1970
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
10 T
14 —
T —
ii in
— —
— 13
26 T T
17 T —
— 16 T
No Samples Collected
14
—
—
STATION 2.— POCOMOKE SOUND— 6 SAMPLES '
1966
1967
1968
1969
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
T
17 T
T —
T T
T T
— T
11
12
24
—
-
—
STATION 3.— TANGIER SOUND— 10 SAMPLES '
1966
1967
DDE
TDE
DDT
DDE
TDE
DDT
13 T
24 T
11 —
— T
- -
— 10
288
PESTICIDES MONITORING JOURNAL
-------
TABLE G-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—Maryland—Continued
YEAR
1968
1969
1970
COMPOUND
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
RESIDUES IN PPB (^O/KO)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT. Nov. DEC.
STATION 3.— TANGIER SOUND— 10 SAMPLES "—Continued
T 17 -
T 10 —
_ — —
—
—
—
— —
— —
STATION 4.— HONGA RIVER— 10 SAMPLES 1
1966
1967
1968
1969
1970
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
T 12
12 20
11 11
T T
12 T
— T
T T T
T
— 28 10"
—
—
—
— T
— 10
— —
STATION 5.— CHOPTANK RIVER— 8 SAMPLES '
1966
1967
1968
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
T T
11 13
— 12
T T
II T
- —
_
—
VOL. 6, No. 4, MARCH 1973
289
-------
TABLE G-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—Maryland—Continued
YEA*
COMPOUND
RESIDUES IN PPB (/IO/KO)
JAN.
FEB.
MAI.
An.
MAY JUNE JULY AUG.
SEPT.
OCT.
Nov.
DEC.
STATION 5.—CHOPTANK RIVER—8 SAMPLES >—Continued
1969
1970
DDE
TDE
DDT
DDE
TDE
DDT
STATION 6.—EASTERN BAY—10 SAMPLES1
1966
1967
1968
1969
1970
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDB
DDT
— 14
— 17
— —
11 T
IS 11
— T
— 11 11
— 11 —
— 48 16
10
T
—
11 10
T 10
— —
STATION 7.—TOLLYS BAR—« SAMPLES1
1967
1968
1969
DDE
TDE
DDT
Dfoldiin
DDE
TDE
DDT
Dfoldrin
DDE
TDE
DDT
Dleldrin
18 13
19 17
T 11
13 13
14 12
11 14
— T
15 —
15
14
—
16
15
13
16
11
290
PESTICIDES MONITORING JOURNAL
-------
TABLE G-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—Maryland—Continued
RESIDUES IN PPB (/WS/KO)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG.
SEPT. OCT. Nov. DEC.
STATION 7.— TOLLYS BAR— 8 SAMPLES > — Continued
1970 DDE
TDE
DDT
Dieldrin
16
17
—
22
T
10
—
15
STATION 8.—HERRING BAY—10 SAMPLES '
1966
1967
1968
1969
1970
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
10 T
15 —
T —
12 10
11 17
— T
— 13
T 12 12
T 14 11
— 20 11
10
11
—
13
14 _
16 _
—
18 12
STATION 9.—CEDAR POINT—10 SAMPLES 1
1966
1967
1968
1969
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
18
27
25
22 IS
20 16
T 13
T 21
— 13
— 16
T 11
T 12
— —
20
24
15
T
T
24
VOL. 6, No. 4, MARCH 1973
291
-------
TABLE G-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—Maryland—Continued
YEAH
COMPOUND
RESIDUES IN PPB (;UJ/KO)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG.
SEPT. OCT. Nov. DEC.
STATION 9.—CEDAR POINT—10 SAMPLES >—Continued
1970
DDE
TDE
DDT
—
—
—
STATION 10.—ST. MARYS RIVER—8 SAMPLES 1
1966
1967
1968
1QAQ
i yvy
1970
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
— 12
— 16
— T
15 T
17 T
-J*
T 11 T
— 11 —
— 11 12
15
11
—
Each sample represents 15 or more mature mollusks.
SECTION H.—MISSISSIPPI
Mississippi Sound and tributaries were monitored for
organochlorine residues in eastern oysters, C. virginica,
during the period August 1965 - June 1972. All samples
from the eight sampling stations were analyzed at the
Gulf Breeze Laboratory. Approximate station locations
are shown in Fig. H-l. A summary of data on organo-
chlorine residues in the monitored species, C. virginica,
is presented in Table H-l, and the distribution of resi-
dues in this species for each sampling station by date of
collection in Table H-2.
Only four States had a lower incidence of DDT residues
in oysters, and the maximum residue detected in
Mississippi (135 ppb) was lower than that in 12 of the
other 14 States. Maximum DDT residues appeared to be
more directly related to runoff from urban and in-
dustrialized centers rather than from agricultural areas.
In 1971, there was a more than 70% increase in the
number of DDT residues of less than 10 ppb as com-
292
pared to earlier years. This trend was reversed in the
first 6 months of 1972 when 44% of the residues were
more than 10 ppb as compared to only 25% in 1971.
MISSISSIPPI
GULF OF MEXICO
FIGURE H-l.—Diagram of coastal Mississippi showing
approximate location of monitoring stations
1. Pascagoula—Pascagoula River
2. Graveline—Graveline Bay
3. Deer Island—Biloxi Bay
4. Biloxi Bay—Biloxi Bay
5. Pass Christian (Inshore)—Mississippi Sound
6. Pass Christian (Offshore)—Mississippi Sound
7. Bay St. Louis—St. Louis Bay
8. St. Joseph Point—Mississippi Sound
PESTICIDES MONITORING JOURNAL
-------
TABLE H-l.—Summary of data on organochlorine residues in the monitored species (C. virginica), 1965-72—Mississippi
STATION
NUMBER
1
2
3
4
5
6
7
8
LOCATION
Pascagoula
Graveline
Deer Island
Biloxi Bay
Pass Christian (Inshore)
Pass Christian (Offshore)
Bay St. Louis
St. Joseph Point
MONITORING
PERIOD
1965-72
1965-72
1965-69
1965-72
1965-66
1965-72
1966-72
1969-72
Total number of samples
Percent of samples positive for indicated compound
NUMBER OF
SAMPLES »
78
79
49
78
13
78
66
29
470
NUMBER OF POSITIVE SAMPLES AND MAXIMUM
RESIDUE ( ) DETECTED IN PPB (JIG/KG)
DDT
47 (74)
56 (99)
33 (105)
71 (135)
7 (53)
29 (42)
31 (124)
11 (69)
61
DlELDRIN
8 (19)
3 (16)
7 (20)
1 (18)
4
Each sample represents 15 or more mature mollusks.
TABLE H-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—Mississippi
[Blank = no sample collected; — = no residue detected above 5 ppb; T = >5 but <10 ppb]
YEAS
COMPOUND
RESIDUES IN PPB (/WJ/KQ)
JAN.
FEB.
MAR.
APR.
MAY JUNE JULY AUG.
SEPT.
OCT.
Nov.
DEC.
STATION 1.—PASCAGOULA—78 SAMPLES1
1965 DDE
IDE
DDT
1966 DDE
TDE
DDT
1967 DDE
TDE
DDT
1968 DDE
TDE
DDT
1969 DDE
TDE
DDT
1970 DDE
TDE
DDT
T 17 13 19 —
T 41 10 19 —
— - — — —
13 12 II 14 T —
14 47 T 13 T —
T 10 — — — —
T T T T 10 —
— T T T 11 —
- T - - T —
T — 11 T 15 T
13 — 12 T 19 12
T
13 12 15 16 10 T
40 12 18 14 13 64
— — —
T T T 14 —
T — T 55 —
T — — T —
T T — — T 11
T - - _ _ 12
— — — — — —
— — T T T —
— — T
— — 17 — — —
--_-__
— — — — __
T — 11 12
T - 14 T
— — T —
- - - - T -
- - - - T -
— — — — — —
VOL. 6, No. 4, MARCH 1973
293
-------
TABLE H-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—Mississippi—Continued
YEA*
COMPOUND
RESIDUES IN PPB (JIG/KO)
JAN. FEB. MA*. API. MAY JUNE JULY AUG.
SEPT. OCT. Nov. DEC.
STATION 1.— PASCAGOULA— 78 SAMPLES *— Continued
1971
1972
DDE
TDE
DDT
DDE
TDE
DDT
_ _ _ _ T — —
— — — — 12 55 —
______ _
T T — 10 11 —
11 T — 10 — —
______
— — T
— — T
_ _ —
STATION 2.—GRAVELINE—79 SAMPLES »
1965
1966
1967
1968
1969
1970
1971
1972
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
10 T 12
27 T 10
T — —
14 12 23
11 10 66
13 — 10
15 16 13
22 23 19
T 12 —
— — 11
— — 14
— — —
— 14
— 16
— 10
15 — —
17 — —
_ _ _
T T —
T T —
— — —
29
10
T
24
36
T
11
18
T
T
T
—
14
17
T
—
—
—
15
—
—
21
60
T
T
T
—
16
23
T
15
15
T
15
14
—
12
23
—
T
T
—
22
68
—
T
18
—
22
25
T
T
12
—
10
11
—
—
—
—
16
—
—
T T T T T
T T T - 20
T — — — —
16 12 18 T 13 13
19 31 69 13 17 36
______
T — T 12 14 15
T — 12 23 18 23
— — 21 29 10 T
10 — — — — T
14 — T — — —
______
— — — 11 17
— — — 13 20
— — — — 10
T — — — — —
10 — — — — —
T — — — — —
— — — T
— — — T
— — — —
STATION 3.—DEER ISLAND—49 SAMPLES»
1965
DDE
TDE
DDT
10
21
T
T
T
—
— T
— 17
— —
T
17
—
294
PESTICIDES MONITORING JOURNAL
-------
TABLE H-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—Mississippi—Continued
YEAR
COMPOUND
RESIDUES IN PPB (AO/KO)
JAN. FEB. MAR. APR. MAY JUNE JULY Auo. SEPT. OCT.
Nov. DEC.
STATION 3.—DEER ISLAND—49 SAMPLES1—Continued
1966
1967
1968
1969
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
14
27
T
10
12
T
T
13
—
T
T
—
20
43
16
15
T
—
11
13
T
T
—
—
15
25
T
14
14
—
T
T
-
11
13
-
22
45
12
15
11
—
12
12
—
T
13
—
27 23
62 65
16 —
11 —
17 —
— —
12 —
12 —
— —
T —
•J-
— —
17 T — T — T
38 T — T — 11
— — — — — —
j1
_ _ _ _ — T
— — 12 — — —
____ — —
— — — — — —
____ — —
T T
27 34
— 22
STATION 4.—BILOXI BAY—78 SAMPLES'
1965
1966
1967
1968
1969
1970
1971
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
Dleldrin
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
14 16
43 30
14 T
16 19
31 40
15 23
- —
12 14
32 30
— T
T 18
28 46
— T
— —
T 24
26 65
— —
16
33
11
23
43
15
—
16
39
-
17
47
T
-
18
60
15
19
16
43
—
i
29
73
15
28
50
20
13
20
34
T
14
46
11
—
19
50
T
16
24
69
—
32 —
87 —
16 —
15 13
33 21
— T
— —
30 17
94 61
T —
20 17
67 54
T —
- -
28 —
78 —
14 —
15 —
T —
85 —
— —
14 T — T
23 11 — 23
T — — —
20 19 — T 15
47 48 — T 27
_ _ _ _ T
T T — '— T
43 25 17 17 19
— — T T —
— — - — —
T - — — T
52 25 24 T 43
— _ _ _ _
T — 14 14
22 25 42 28
— — is —
— — — 16
-
77 40 — 12 12
11 — 16 — —
22 — _
81 — 19
— — —
T
18
—
T
15
—
T
22
—
—
16
49
—
20
53
12
18
17
_
—
T
27
—
VOL. 6, No. 4, MARCH 1973
295
-------
TABLE H-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—Mississippi—Continued
YEA*
RESIDUES IN PPB (AO/KO)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT. Nov. DEC.
STATION 4.— BILOXI BAY— 78 SAMPLES ' — Continued
1972
DDE
TDE
DDT
T 10 12 18 17 T
20 28 35 58 63 30
— — — — — —
STATION 5.— PASS CHRISTIAN (INSHORE)— 13 SAMPLES '
1965
1966
DDE
TDE
DDT
DDE
TDE
DDT
T T — T —
T
_ _ _ _ _
T — — T 11 19 — —
T — — T 12 34 — —
JJ
STATION 6.—PASS CHRISTIAN (OFFSHORE)—78 SAMPLES '
1965 DDE
TDE
DDT
1966 DDE
TDE
DDT
1967 DDE
TDE
DDT
1968 DDE
TDE
DDT
1969 DDE
TDE
DDT
1970 DDE
TDE
DDT
Dieldrin
1971 DDE
TDE
DDT
1972 DDE
TDE
DDT
T — — 11 14
T — — 12 15
— — — T 13
T
_ _ T - -
— — — — —
T T T T T
— T T 11 11
1*
— — — T
— — — T
— — — T
12 13 15 11
T 22 22 16
— T — T
11 16 — —
-p
- - - 14
_____
T T 12 12 T
T T 10 16 T
-J-
T — — T —
T — — T —
T
— — — _ _ _ —
— ______
— ______
X
______ T
— — — — — • — —
_ ______
— — — — — — —
— — — — — — —
T - — — T T
13 — — — T T
— — — — — —
•T- _,_
y .
— — — — — — —
— — — 15 — — —
_ _ _ T
- - - ' - T
— — — — —
11
—
—
296
PESTICIDES MONITORING JOURNAL
-------
TABLE H-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—Mississippi—Continued
YEAI
COMPOUND
RESIDUES IN PPB UO/KO)
JAN.
FEB.
MAX.
APt.
MAY JUNE JULY Auo.
SEPT. OCT. Nov.
DEC.
STATION 7.—BAY ST. LOUIS—66 SAMPLES '
1966
1967
1968
1969
1970
1971
1972
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
Dfeldiin
DDE
TOE
DDT
Dkkbin
DDE
TDE
DDT
DDE
TDB
DDT
T T T
T T 14
_ _ _
T T T
T T T
— T —
— T T
T — 12
_ _ _
T 14 11
T 12
13 IS
_ _
17 20
_ _ _
_ _ _
_ _ _
T T —
T T —
_ _ _
T
T
—
T
T
—
—
—
—
T
17
21
T
—
—
—
—
11
11
T
T —
T —
— —
10 —
11 —
T —
T T
T 11
T —
— —
34 32
76 12
14 —
14 —
— —
13 —
— —
T —
— —
— —
_ _ _ —
_ _ — —
_ _ _ —
_ _ _ — 11 T
___ — — —
— — 10 — — —
___ — — —
______
____ — —
_ _ — T 10
— — — 11 12
— — — T 11
_ _ _ _ _
______
______
______
— — — — — —
f
— — _ _
— — — —
STATION 8.—ST. JOSEPH POINT—29 SAMPLES'
1969
1970
1971
1972
DDE
TDE
DDT
DDE
TDE
DDT
Dteldrin
DDE
TDE
DDT
DDE
TDE
DDT
— —
_ _
TTT 14 15 — _____
— — — 22 54 — — ____
— — — — — — — — — 24 —
18 — _________
— — — — — — __
— — — — — — — — — _
— — — — — — — — 24 —
T T — — T T
— 15 — — T —
— — — — — —
1 Each umple represents 15 or more mature molluaks.
VOL. 6, No. 4, MARCH 1973
297
-------
SECTION I.—NEW JERSEY
Samples of eastern oysters, Crassostrea virginica, were
collected at five principal stations in the New Jersey
waters of Delaware Bay during the period June 1966 -
June 1972. All samples were analyzed at th? Gulf
Breeze Laboratory. The approximate station locations
are shown in Fig. 1-1. A summary of data on organo-
chlorine residues in the monitored species, C. virginica,
is presented in Table 1-1, and the distribution of residues
in this species for each sampling station by date of
collection in Table 1-2.
Oyster samples collected in Delaware Bay were
characterized by a 100% incidence of DDT residues
and a relatively high incidence (24%) of dieldrin
residues as compared to other areas monitored.
The maximum DDT residue observed, 272 ppb, is low
compared to that in many other estuaries; most residues,
from New Jersey were less than half this amount. The
fact that DDE was the principal component of these
residues suggests that the pesticide had been metabolized
in other links of the trophic web before its acquisition
by the oyster.
DDT residues appear to have been somewhat higher in
the 1968-69 period than earlier, but the 1971 data show
a clear-cut trend towards decreased residue levels.
FIGURE 1-1.—Diagram of coastal New Jersey showing
approximate location of monitoring stations
1. Drum Beds—Delaware River
2. Maurice River—Delaware River
3. Dividing Creek—Delaware River
4. Lease 564/496D—Delaware River
5. Cohansey—Delaware River
TABLE 1-1.—Summary of data on organochlorine residues in the monitored species (C. virginica), 1966-72—New Jersey
STATION
NUMBER
1
2
3
4
5
LOCATION
Drum Beds
Maurice River
Dividing Creek
Lease 564/496D
Cohansey
Occasional Stations (7)
Total number of samples
Percent of samples positive for indicated compound
MONITORING
PERIOD
1966-72
1966-72
1966-71
1966-72
1966-72
1966-71
NUMBER OF
SAMPLES *
49
50
7
52
49
12
219
NUMBER OF POSITIVE SAMPLES AND MAXIMUM
RESIDUE ( ) DETECTED IN PPB (/JO/KG)
DDT
49 (213)
50 (143)
7 (125)
52 (278)
49 (245)
12 (166)
100
DIELDRIN
3 (12)
I (T)
1 (12)
28 (26)
16 (23)
3 (29)
24
PCB's »
2
1
2
1
3
NOTE: T = >5 but <10 ppb.
' Each sample represents 15 or more mature mollusks.
1 Present but not quantified,
298
PESTICIDES MONITORING JOURNAL
-------
TABLE 1-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—New Jersey
[Blank = no sample collected; — = no residue detected above 5 ppb or no residue detected (PCB's); T = >5 but <10 ppb]
YEA*
COMPOUND
RESIDUES IN PPB (AO/KO)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT.
Nov. DEC.
STATION 1.—DRUM BEDS—49 SAMPLES »
1966
1967
1968
1969
1970
1971
1972
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
PCB's
19
18
—
43 39 30 31 27
34 28 27 30 40
11 T — — 21
43 50 55 50 110 110
43 43 54 57 98 83
10 T T — T 13
49 19 48 51 73
35 18 15 45 44
_____
99 100 110
42 52 47
11 — —
— — 12
35 59 70 38
10 35 30 26
_ _ _ _
'10 > 52
20 29
52 —
T T
(Bt (8)
33
38
15
18
25
16
55
38
13
52
30
T
34
12
—
—
24
24
11
27
33
13
44
35
T
63
34
—
37
15
—
—
27
14
—
28 17
26 14
10 —
37 42
46 50
22 14
75 52
48 44
11 —
67 56 58
32 41 28
T T T
46 38
21 16
— —
— —
53
28
—
STATION 2.—MAURICE RIVER—50 SAMPLES i
1966
1967
1968
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
11
15
—
12 12 13 15
T T 19 28
17 — _ _
19 24 22 21 45
27 31 32 43 68
T — — — _
T
12
-
12 T
23 10
T —
37 17
38 - 21
16 T
14
15.
-
18
30
13
20
24
10
13
16
—
26 19
40 31
13 —
16 24
17 19
T T
T
•T
—
VOL. 6, No. 4, MARCH 1973
299
-------
TABLE 1-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—New Jersey—Continued
YEA»
COMPOUND
RESIDUES IN PPB (^O/KO)
JAN. FEB. MAX.
An.. MAY JUNE JULY Auo. SEPT. OCT.
Nov. DEC.
STATION 2.—MAURICE RIVER—50 SAMPLES»—Continued
1969 DDE
TDE
DDT
1970 DDE
TDE
DDT
1971 DDE
TDE
DDT
1972 DDE
TDE
DDT
Dieldrin
PCB's
21 16 77 75
21 15 26 68
— — — —
16 24 25
T 22 36
— — —
12 T 26
T — 29
— — —
14 » 17
— 17
— —
— T
— <»
13 22 12 T 19 13
14 23 18 11 14 T
_ T — — — —
32 17 13 18 13
34 16 15 19 10
— — — — —
23 14 T
29 13 —
— — —
STATION 3.—DIVIDING CREEK—7 SAMPLES1
1966
1967
1968
1969
IO7A
17 IV
1971
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
T
13
—
26 41 22
28 66 33
— 18 11
j2
49
64
T
60
35
—
• • " No S&iuples Collected ~
13
13
—
STATION 4.—LEASE 564/496D—52 SAMPLES '
1966
DDE
TDE
DDT
34
42
-
29
39
T
41
53
12
51
53
67
58
110
15
300
PESTICIDES MONITORING JOURNAL
-------
TABLE 1-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—New Jersey—Continued
YEA*
COMPOUND
RESIDUES IN PPB (^O/KO)
JAN. FEB. MAI. APR.
MAY JUNE JULY AUG. SEPT. OCT.
Nov. DEC.
STATION 4.—LEASE 564/496D—52 SAMPLES "—Continued
1967
1968
1969
1970
1971
1972
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
PCB's
47
48
T
10
35 38 37
50 54 60
T T T
11 14 15
100
87
13
13
130 140
61 67
15 16
14 16
150
45
—
13
•67
42
11
T
(•>
41 48 41
39 86 81
— — —
- 18 20
23 110
45 140
— 18
— —
66 95 27
51 74 34
_ _ _
12 18 21
180 75
98 92
— —
20 26
180 180
87 62
T —
19 19
•46
35
—
14
(8)
44
72
18
18
77
82
23
14
82
68
17
—
•35
18
—
—
190
78
—
T
29 38 26
47 56 41
17 19 10
— 12 —
51 69 67
46 57 57
16 T T
— — —
47 72 84
39 45 48
T 13 14
— 19 13
62 95 120
33 36 33
— — —
_ _ _
49
25
—
—
39 39
56 56
— —
— —
57
56
T
11
72
56
14
11
42 12
36 43
— —
— 12
56
18
—
—
STATION 5.—COHANSEY—49 SAMPLES «
1966
1967
1968
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
29
45
T
—
20 30 22
41 62 66
— — —
— 12 14
35 41
53 19
T 22
— 23
49
12
—
13
12
24
—
54
150
25
18
81
150
14
19
24
59
10
12
68
110
11
12
17
37
—
29
66
13
T
32
60
15
—
20
35
—
17 17
39 38
— T
— —
33 52
50 70
T T
— —
22
33
—
32
59
—
11
28
32
—
—
11
23
—
VOL. 6, No. 4, MARCH 1973
301
-------
TABLE 1-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—New Jersey—Continued
YEAI
COMPOUND
RESIDUES IN PPB (PG/KO)
JAK. FEB. MAI. API. MAY JUNE JULY Auo. SEPT. OCT.
Nov. DEC.
STATION 5.—COHANSEY—49 SAMPLES >—Continued
1969
1970
1971
1972
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
PCB's
33 59 57 36 37 29 24 42 38
46 43 76 49 46 45 44 48 42
— ___TT — — T
— _I6 — — — — — —
45 41 55 30 27 53 36 42
46 51 98 30 34 54 42 36
X — ^
— 11 21 _____
52 28 38 27 21 22
42 24 61 30 20 17
__ fp
13 _ 16 — — —
23 >40
114 49
— —
— T
— <«>
1 Each sample represents 15 or more mature mollusks.
' DDT values are approximate because of presence of unidentified PCB's.
3 Present but not quantified.
302
PESTICIDES MONITORING JOURNAL
-------
SECTION J.—NEW YORK
Several different species of moLIusks (Crassotrea vir-
ginica, Modiolus domissus, Mytilus edulis, Mercenavia
mercenaria, and My a arenaria) were collected at 16
principal sites in New York's coastal waters to monitor
organochlorine pollution during the period March 1966 -
June 1972. Samples were analyzed at the Gulf Breeze
Laboratory until February 1969 and thereafter by the
New York Conservation Department. Analyses of
aliquots of some of the samples collected during the
period October 1968 - July 1970 have been reported
by the cooperating agency (9) and do not differ sig-
nificantly from the data reported here.
Approximate station locations are shown in Fig. J-l. A
summary of data on organochlorine residues in the
monitored species is presented in Table J-l, and the
distribution of residues in these species for each sampling
station by date of collection in Table J-2.
The hard clam, M. mercenaria, was the principal species
collected because of its ubiquity and despite its recog-
nized inefficiency in storing organochlorine residues. This
lack of sensitivity to low levels of DDT pollution is
especially well documented in the analytical record of
samples collected in Conscience Bay, Station 6. Hard
clams were the only mollusk of four species collected
there in which DDT residues were undetected. DDT
pollution apparently disappeared at this station during
the period July 1968 - March 1969, but this was be-
cause of the substitution of hard clams for the blue
mussel as monitors.
These data emphasize the fact that in areas where hard
clams did show DDT residues, there were probably
significant levels of DDT in the water or food supply.
This parallels the situation in Delaware, where hard
clams collected in Delaware Bay (Cape Henlopen)
consistently had DDT residues while residues were
16 15
ATLANTIC OCEAN
FIGURE J-l.—Diagram of coastal New York showing
approximate location of monitoring stations
1. Mamaroneck
2.. Hempstead Harbor
3. Oyster Bay Harbor
4. Huntington Bay
5. Nissequogue River
6. Conscience Bay
7. Southold—Gardiners Bay
8. Flanders Bay
9. MecoxBay
10. Shinnecock Bay
11. Moriches Bay
12. Bellport—Great South Bay
13. Sayville—Great South Bay
14. Amityville—South Oyster Bay
15. East Bay
16. West Bay
usually not detected in hard clams collected in inner
bays. There was generally good agreement in the
magnitude of residues in two or more species, other
than the hard clam, collected at the same station on
the same day.
The New York samples ranked fifth among the States
in incidence and sixth in magnitude of DDT residues.
More samples (43%) contained dieldrin residues than
in any other area monitored. PCB's were present in
some samples in 1972, but they were not identified or
quantified.
Despite the large number of samples collected over a
period of 7 years, no clearly defined trends in pollution
levels can be identified. This may be the result of having
used a variety of species. The overall impression is one
of no significant change in DDT residue levels in
mollusks.
TABLE J-l.—Summary of data on organochlorine residues in the monitored species, 1966-72—New York
STATION
NUMBEI
1
2
3
4
5
6
7
8
9
LOCATION
Mamaroneck
Hempstead Harbor
Oyster Bay
Huntington Bay
Nissequogue River
Conscience Bay
Southold
Flanders Bay
Mecox Bay
MONITORING
PERIOD
1966-69
1966-72
1966-72
1966-72
1966-72
1966-72
1969-72
1966-72
1966-72
PRINCIPAL
MONITORED
SPECIES
M. mercenaria
M. mercenaria
M. mercenaria
M. edulis
M. edulis
M. edulis
C. virglnica
A/, mercenaria
C. virglnica
NUMBER OF
SAMPLES 1
36
74
73
74
74
73
34
69
67
NUMBER OF POSITIVE SAMPLES AND MAXIMUM
RESIDUE ( ) DETECTED IN PPB (/UJ/KG)
DDT
34 (96)
70 (201 )
54 (99)
72 (588)
70 (138)
61 (112)
32 (149)
63 (199)
65 (596)
DIELDRIN
27 (29)
61 (132)
48 (86)
52 (104)
58 (117)
52 (75)
26 (78)
15 (107)
14 (22)
VOL. 6, No. 4, MARCH 1973
303
-------
TABLE J-l.—Summary if data on organochlorine residues in the monitored species, 1966-72—New York—Continued
STATION
NUMBER
10
11
12
13
14
15
16
LOCATION
Sblnnecock Bay
Moriches Bay
Heliport Bay
Sayville
Amityville
East Bay
West Bay
MONITORING
PERIOD
1966-72
1966-72
1966-72
1966-72
1966-72
1966-72
1966-72
Occasional stations (8) 1967-72
PRINCIPAL
MONITORED
SPECIES
M, mercenarla
M. mercenaria
M. mercenarla
M. mercenarla
M. mercenarla
M. mercenarta
M. mercenarla
Mixed
Total number of samples
Percent of samples positive for indicated compound
NUMBER OP
SAMPLES '
13
71
71
74
73
57
57
9
1,059
NUMBER OF POSITIVE SAMPLES AND MAXIMUM
RESIDUE ( ) DETECTED IN PPB (^G/KG)
DDT
43 (188)
49 (83)
51 (132)
41 (107)
49 (64)
43 (98)
51 (111)
9 (159)
81
DffiLDRIN
19 (46)
13 (49)
10 (53)
16 (59)
13 (42)
13 (38)
19 (20)
3 (31)
43
1 Each sample represents 15 or more mature mollusks.
TABLE J-2.—Distribution of organochlorine residues in the monitored species for each sampling station by date of
collection—New York
[Blank = no sample collected; — = no residue detected above 5 ppb; T = >5 but <10 ppb]
YEAR
COMPOUND
RESIDUES IN PPB (#G/KG)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT. Nov.
DEC.
STATION 1.—MAMARONECK—M. MERCENARIA—36 SAMPLES1
1966
1967
1968
1969
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
— T — — — — T16T
— T — 13 T 12 18 49 29
14 13
___ ____2912
T 19 12 11 11 11 T T T 12 T T
35 50 31 27 27 28 20 15 26 28 32 30
24 27 11 11 11 15 T T — T 14 T
16 21 16 16 15 15 15 14 14 13 14 15
T T T1110T ___ — — T
27 25 27 30 27 26 22 20 20 18 23 19
T — — T 12 15 T — — — — —
,1 12 14 11 12 — 10 12 12 11 IS 14
— 10 —
18 24 13
_ _ _
11 11 —
304
PESTICIDES MONITORING JOURNAL
-------
TABLE J-2.—Distribution of organochlorine residues in the monitored species for each sampling station by date of
collection—New York—Continued
YEAR
COMPOUND
RESIDUES IN PPB US/KG)
JAN. FEB. MAP..
APR. MAY JUNE JULY Auo. SEPT. OCT.
Nov. DEC.
STATION 2.—HEMPSTEAD HARBOR—M. MERCENAR1A, UNLESS OTHERWISE INDICATED—74 SAMPLES"
1966 DDE
TDE
DDT
Dieldrio
1967 DDE
TDE
DDT
Dieldiin
1968 DDE
TDE
DDT
Dieldiin
1969 DDE
TDE
DDT
Dieldrin
1970 DDE
TDE
DDT
Dieldrin
1971 DDE
TDE
DDT
Dieldrin
1972 DDE
TDE
DDT
Dieldrin
T
17
—
—
11
29
11
12
—
12
—
47
"41
71
76
29
'10
22
18
10
' 19
41
22
19
14
36
13
15
13
31
—
16
10
29
T
70
11
33
15
30
'13
26
31
14
'17
—
24
14
_
—
—
—
13
35
13
16
13
31
10
14
"15
33
34
22
324
51
62
30
»13
33
34
16
323
48
44
19
—
—
—
12
30
11
17
13
34
10
15
"16
34
38
86
>13
28
29
25
"15
38
44
31
"34
67
53
21
—
—
—
12
33
15
15
10
26
10
20
18
18
12
85
"12
33
54
30
820
27
23
—
>9
15
9
—
T
29
13
—
14
39
28
19
T
27
—
16
15
19
T
13
"18
48
70
33
—
T
—
T
—
—
—
—
T
23
15
15
—
17
T
—
T
22
T
33
10
22
17
20
—
16
10
13
'24
48
26
—
T
25
16
17
—
18
—
—
—
10
—
38
10
35
18
18
"16
37
30
21
T
17
—
—
12
29
—
15
—
17
—
11
"34
93
74
132
321
71
28
26
11
29
—
—
13
30
T
16
T
24
—
50
'30
62
49
28
•18
40
35
22
»13
31
17
19
15 13
39 35
17 14
17 15
12 10
32 30
11 11
14 16
— T
22 23
— —
93 66
'23 "26
57 57
47 46
40 31
>18 '20
48 50
43 51
19 25
»22 »23
46 58
32 51
23 20
STATION 3.—OYSTER BAY HARBOR—M. MERCENARIA, UNLESS OTHERWISE INDICATED—73 SAMPLES »
1966
1967
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
— — —
— — —
— — 13
— _ _
13 T T T T
50 15 19 14 20
18 — — — —
10 — 14 13 14
—
—
—
—
T
22
13
14
— T — —
— — — —
— — — _
— T T T
26 17 13 T
T 12 — —
11 14 — 12
T
22
T
13
T
13
11
T
11
—
11
T
15
__
—
VOL. 6, No. 4, MARCH 1973
305
-------
TABLE J-2.—Distribution of organochlorine residues in the monitored species for each sampling station by date of
collection—New York—Continued
YEAR
COMPOUND
RESIDUES IN PPB (/ZG/KO)
JAN. FEB. MAR. APR. MAY JUNE JULY Auo.
SEPT. OCT.
Nov. DEC.
STATION 3.—OYSTER BAY HARBOR—M. MERCENARIA. UNLESS OTHERWISE INDICATED—73 SAMPLES "—Continued
1968
1969
1970
1971
1972
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDV
Dieldrin
ODE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
T
16
—
—
—
—
—
T
'19
38
16
18
' 18
36
24
12
T
T
—
—
T
T
—
T
•27
52
20
31
«19
38
T
25
M9
31
18
15
—
—
11
—
—
—
12
= 10
19
11
12
«22
45
15
27
—
T
—
—
T
T
—
—
"11
25
16
17
—
—
—
37
—
T
—
16
—
—
—
10
T — —
11 — T
— — —
_ _ _
»24 — —
28 11 15
32 — T
24 86 16
"21 T * 16
44 16 37
34 T 24
23 11 19
"13 —
32 T
18 —
18 10
'10 • 13
24 22
10 11
11 19
__ _
— — —
— — —
_ _ —
— — «27
— — 55
— — 17
— 17 —
T '22
18 48
T 20
16 29
— « 19 '23
— 30 41
— 13 10
— 21 20
___
—
—
—
T
13
—
15
•27
55
15
30
M9
34
17
14
T
—
10
—
T
—
18
* 24
50
16
26
'23
44
18
17
STATION 4.—HUNTINGTON BAY—M. EDUL1S, UNLESS OTHERWISE INDICATED—74 SAMPLES'
1966
19676
1968 «
1969
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
20 27
88 65
44 20
21 —
T —
27 40
- T
II 11
°— Ml
26 28
— —
1 T
5T
16
—
—
21
54
17
11
12
49
—
—
sT
32
—
T
6
—
—
—
19
57
12
11
16
48
T
12
18
35
21
11
= 32
75
81
—
19
54
24
—
15
46
14
—
6T
24
21
14
98
280
210
—
16
63
26
12
12
35
—
—
«30
104
46
104
"53
190
60
18
T
50
13
12
—
35
T
—
29
100
50
26
40
110
25
13
13
40
11
12
—
49
—
—
5 15
23
—
T
0 •>
— T
— —
— —
12 T
39 27
— —
11 —
— T
43 47
- —
— —
34 24
127 73
47 28
18 —
= 21
64
17
12
T
22
—
—
T
60
T
11
28
84
55
23
629
71
16
T
17
56
12
12
10
48
11
T
30
80
50
34
306
PESTICIDES MONITORING JOURNAL
-------
TABLE J-2.—Distribution of organochlorine residues in the monitored species for each sampling station by date of
collection—New York—Continued
YEAR
COMPOUND
RESIDUES IN PPB (/IO/KO)
JAN. FEB. MAR. APR.
MAY JUNE JULY Auo. SEPT. OCT.
Nov. DEC.
STATION 4.—HUNTINGTON BAY—M. EDVLIS, UNLESS OTHERWISE INDICATED—74 SAMPLES i—Continued
1970
1971
1972
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
38
90
61
18
21
70
35
17
—
T
—
—
15
58
40
16
18
57
30
18
14
33
20
13
23
60
49
17
15
50
24
22
17
40
15
15
_
22
12
38
11
37
22
14
15
36
18
12
20
50
40
22
21
51
22
—
10
20
—
—
13
59
40
21
14
41
15
60
21
146
55
26
15
63
22
21
20 19
70 40
29 44
21 26
26
74
27
14
20
66
28
22
33
14
T
13
24 21
89 73
28 29
17 17
21 19
56 60
23 28
— 14
STATION 5.—NISSEQUOGUE RIVER—M. EDVLIS, UNLESS OTHERWISE INDICATED—74 SAMPLES >
1966 DDE
TDE
DDT
Dieldrin
1967 DDE
TDE
DDT
Dieldrin
1968 DDE
TDE
DDT
Dieldrin
1969 DDE
TDE
DDT
Dieldrin
1970 DDE
TDE
DDT
Dieldrin
1971 DDE
TDE
DDT
Dieldrin
—
33
13
13
14
32
20
14
° —
—
—
T
16
37
25
27
14
30
24
14
23
53
27
17
16
33
18
18
-T
T
—
T
17
44
33
17
T
16
12
T
21
33
23
16
24
59
29
27
13
36
21
16
'T
14
T
T
= T
18
T
10
3T
17
T
17
21
47
37
20
21
50
33
27
17
40
23
18
12
30
22
16
—
16
T
16
3
15
T
20
24
45
50
23
23
18
45
27
17
44
42
—
»T
12
11
12
21
46
38
23
3
12
—
T
18
49
34
—
21
51
42
22
13
49
51
—
3
T
T
117
14
37
26
20
T
17
13
—
18
42
33
18
11
44
47
—
'15
22
14
—
18
48
34
24
10
38
20
27
19
43
30
—
14
38
32
19
—
31
25
—
• —
T
—
T
15
46
30
25
"17
T
—
—
17
38
29
—
20
49
33
15
E
—
—
10
34
17
24
3T
17
—
T
22
48
22
—
19
42
27
15
G
—
. —
—
14
35
30
21
14
38
22
24
"14
10
—
13
42 40
76 59
20 30
31 17
17 18
35 44
18 32
14 17
6 B
— T
— —
— li.
18 20
49 42
36 25
20 20
11 "T
29 14
17 —
12 T
11 » —
34 T
13 —
10 —
VOL. 6, No. 4, MARCH 1973
307
-------
TABLE J-2.—Distribution of organochlorine residues in the monitored species for each sampling station by date of
collection—New York—Continued
YEAR o»u»nTiun
RESIDUES IN PPB (AO/KQ)
JAN.
STATION 5.— NISSEQUOGUE
1972
DDE
TDE
DDT
Dieldrin
a —
12
—
10
FEB.
MAR. APR
RIVER— M. EDVLIS,
15
33
18
13
3T a —
12 T
T —
T —
MAY JUNE JULY Auo. SEPT. OCT. Nov. DEC.
UNLESS OTHERWISE INDICATED— 74 SAMPLES i— Continued
T T
20 14
10 T
T 12
STATION 6.—CONSCIENCE BAY—M. EDVLIS, UNLESS OTHERWISE INDICATED—73 SAMPLES »
1966
1967
1968
1969
1970
1971
1972
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
»T
18
IS
—
18 23 18
48 43 33
24 23 18
17 20 20
14 16 14
23 24 22
14 13 17
15 14 14
6 6 6
— — —
— — —
— T T
17 22
37 37
25 36
16 19
a T af • —
12 IS 13
T 10 —
T 14 22
•— —
10 18
— 14
— T
a —
—
12
12
18
34
21
30
15
27
19
14
16
23
M4
14
a
11
—
20
16
29
13
18
a
T
—
—
6
—
—
—
20
42
29
24
T
24
21
—
IS
24
32
15
25
49
36
26
MO
15
—
—
—
11
—
75
"18
26
15
—
22
44
41
22
12
24
21
—
•16
36
48
—
21
45
29
20
13
21
—
49
6
—
—
—
21
48
36
21
6 —
—
—
—
•18
22
20
T
24
59
28
23
• T
24
10
10
B_ JO
— 35
— 22
— —
21 20
44 36
35 24
16 13
6_ «_
— —
— —
— —
• 14 « 13
20 14
— T
— 15
26 » —
59 17
27 —
23 T
a a —
13 13
— T
10 17
21
34
22
—
18
31
21
15
6
—
—
—
17
29
15
26
24
46
24
16
T
16
T
T
24
46
30
25
17
29
18
14
•> —
-
—
—
23
39
26
18
23
37
15
T
12
23
11
T
•17
40
17
—
16
31
20
13
• —
—
—
T
21
36
17
13
18
26
14
10
•T
11
—
11
308
PESTICIDES MONITORING JOURNAL
-------
TABLE J-2.—Distribution of organochlorine residues in the monitored species for each sampling station by date of
collection—New York—Continued
YEAR
COMPOUND
RESIDUES IN PPB (/IO/KO)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT. Nov.
DEC.
STATION 7.—SOUTHOLD—C. V1RG1N1CA, UNLESS OTHERWISE INDICATED—34 SAMPLES»
1969
1970
1971
1972
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dleldrin
DDE
TDE
DDT
Dleldrin
« 16
32
21
—
ST 17
16 19
— 14
21 12
16 17
27 17
16 T
14 11
29
30
26
21
17
19
11
14
18
22
T
T
•22
48
28
—
27
32
23
20
18
17
12
16
16
21
11
10
•T
11
—
78
27
35
22
26
6
—
—
14
MO
18
—
T
6 r- -
T
—
T
• 10
13
—
T
21
28
31
39
« 22 5 T « T
38 14 T
89 — —
— — —
21 19 17
18 19 21
22 23 18
11 27 14
10 «— «_ • —
10 T T —
jq r ,
— — 12 —
"20
41
63
T
22 20
21 21
IS 12
IS 14
MS —
18 16
13 —
T T
STATION 8.—FLANDERS BAY—M. MERCENARIA, UNLESS OTHERWISE INDICATED—69 SAMPLES >
1966
1967
1968
1969
1970
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
Dleldrin
DDE
TDE
DDT
Dieldrin
56
54
89
15 18 25
20 27 42
— _ _
13 10 12
25 17 25
— — —
— T -12
12 32 25
— — -
- — —
14
26
—
11
28
25
29
23
32
—
17
41
—
»33
84
27
—
•T
22
12
—
22
21
15
'23
58
12
10
24
—
15
33
—
17
13
30
—
—
24
44
—
»20
53
12
19
59
—
14
28
—
107
3 14
29
—
—
—
—
—
17
46
—
10
44
—
10
19
—
11
' 14
29
T
T
11 T Ty 10 17
23 14 — 15 23
— — — — —
17 16 17 16 15
47 36 .42 37 33
T — — — —
T 10 — T 10
38 38 18 39 34
— — — — —
12 «20 — 13
23 49 T 28
— 75 — —
— 14 93 —
'T T »T
19 T 14
— ,
T — —
VOL. 6, No. 4, MARCH 1973
309
-------
TABLE J-2.—Distribution of organochlorine residues in the monitored species for each sampling station by date of
collection—New York—Continued
YEAR
COMPOUND
RESIDUES IN PPB (/«J/KG)
JAN. FEB.
STATION 8.— FLANDERS BAY— M.
1971
1972
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
— T
10 16
— —
10 T
T —
12 —
— —
T —
MAR. APR.
MERCENARIA.
— T
— 16
— —
12 13
— —
— 10
— —
— —
MAY JUNE JULY Auo. SEPT. OCT. Nov.
UNLESS OTHERWISE INDICATED— 69 SAMPLES >— Continued
"16 a— T 13 •- —
50 16 12 24 —
15 — — — —
T — — T —
— —
T —
— —
— —
DEC.
—
11
—
—
STATION 9.—MECOX BAY—C. VIRGINICA, UNLESS OTHERWISE INDICATED—67 SAMPLES 1
1966
1967
1968
1969
1970
1971
1972
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
300
240
56
63 67 77
66 60 62
— — —
100 53
81 48
— —
44 62 26
31 45 20
— — —
— — —
3 19 s 21
27 27
52 49
— —
30
24
—
—
45 37 26
52 39 26
13 T —
10 T 10
120
120
22
T
11
—
130
87
20
60
39
T
—
16
14
10
—
41
32
T
12
34
40
T
—
'22
41
20
a 37
67
29
150
120
38
'20
31
24
12
32
45
—
T
34
34
T
T
26
27
—
T
'T '27 '21 3— 46 42
13 45 29 — 46 37
— 19 — — 12 —
190 a 15 49 64 69 93
180 22 52 74 74 100
27 T 18 17 11 15
130 68 32 34 37 75
85 48 29 18 32 50
48 T — — — —
a21 a 19 ax > 10 3— a 12
29 21 10 10 — 23
10 — — — — —
. 22
* 16 21 22 36 ' T
36 25 38 48 10
32 T 13 13 —
10 — — 16 —
T 14 37
10 15 40
— — 15
— 10 19
35
38
12
—
83
83
—
110
73
T
30
18
—
'T
12
—
10
35
46
T
—
310
PESTICIDES MONITORING JOURNAL
-------
TABLE J-2.—Distribution of organochlorine residues in the monitored species for each sampling station by date of
collection—New York—Continued
YEAR
COMPOUND
RESIDUES IN PPB (AC/KG)
JAN. FEB. MAR. APR. MAY JUNE JULY Auo.
SEPT. OCT. Nov.
DEC.
STATION 10.—SHINNECOCK BAY—M. MERCENARIA, UNLESS OTHERWISE INDICATED—73 SAMPLES
1966
1967
1968
1969
1970
1971
1972
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
— — — »T
— — — T
— — — —
T T T 59 T t
12 12 12 50 T T
T
—
______
______
_ _ _ T "20 —
— T — T 38 T
— — — — 21 —
— — — — 14 T
'11 a_ a_ 3_ aio "18
18 13 T — 19 34
— — — — T 21
14 — 12 — — 12
'10 ' T = — 3 _
17 18 10 10
12 14 T —
10 10 — —
3 — 3_ 3_ an a_ an
T T — 22 11 165
— — — 12 T 12
— T —
— 14 —
— — —
— T —
— T —
— — —
_ — _
_ _ _
— — —
— '12 ' 14
T 21 20
- 12 T
— — T
'10 '12
18 22
24 20
T T
a _
— — T
— — —
— 44 18
— — T
— — 13
__ — — —
T — —
T — I--.
— — —
— — —
10 — T
— — —
MO • — M3
16 — 24
T — 22
46 — —
s_ s_ «T
— T 11
T _ _ —
12 — —
3_ „_ a_
— — T
— — —
T T —
STATION 11.—MORICHES BAY—M. MERCENARIA, UNLESS OTHERWISE INDICATED—71 SAMPLES *
1966
1967
1968
1969
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
__ — T ____M5 T
_ — — 10 — — — — 33 14
— — — — — — — — — —
TTTTTT TTTTT —
13 14 18 13 18 17 10 T 17 T 18 —
____________
T — T T — _______
10 — 14 T — — — T — 12 T T
— — — — — — — — — — — —
— — — — — — — — — — —
— 11 — — 13 — TT — — T
_______ __24 —
VOL. 6, No. 4, MARCH 1973
311
-------
TABLE J-2.—Distribution of organochlorine residues in the monitored species for each sampling station by date of
collection—New York—Continued
YEAR
COMPOUND
RESIDUES IN PPB (/IO/KO)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT.
Nov. DEC.
STATION 11.—MORICHES BAY—M. MERCENARJA, UNLESS OTHERWISE INDICATED—71 SAMPLES i—Continued
1970
1971
1972
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
—
T
—
—
T
10
—
T
3 17 =16
27 20
19 T
T —
T
13
T
—
"20
27
15
T
a 17
27
—
—
—
—
—
—
2 18
25
14
16
"20
26
15
T
T T 'T "20
14 20 21 40
— — 11 23
— T — —
— — —
11 — —
— _ _
— — 17
a 17 « 25
13 33
— 22
— 49
225
29
17
14
— "22
T 15
— 17
18 —
»25
19
17
T
' 10
26
T
T
'13
13
T
—
a 21
34
25
T
STATION 12.—BELLPORT BAY—M. MERCENARIA, UNLESS OTHERWISE INDICATED—71 SAMPLES'
1966 DDE
TDE
DDT
1967 DDE
TDE
DDT
1968 DDE
TDE
DDT
1969 DDE
TDE
DDT
1970 DDE
TDE
DDT
Dieldrin
1971 DDE
TDE
DDT
Dieldrin
1972 DDE
TDE
DDT
Dieldrin
57 — — 12
44 — 10 27
31 — — T
20 21 24 19 12 12
42 40 45 36 30 31
f
15 13 15 30 10 T
28 24 27 50 23 14
— — — — — —
— T — "25 T —
— 10 — 50 10 15
• — — — 33 — —
T — 11 10
13 — 26 17
-j-
-j-
— T T — —
10 12 11 13 10
_____
— — 12 T T
_____ T
— T T T — —
_____.—
— 16 — — — —
T T T 14 16 10
18 13 16 28 30 20
— — — — — —
11 10 15 T 13 14
25 19 26 20 22 27
«T-
T - T — — —
12 — T - — T
— — — — — —
*T- ,
18 — — — —
. _ _ — - _ —
T T — — —
21 17 — 11 —
_____
— — 37 T —
__ — — _ —
. . f
______
T — 10 — — 53
312
PESTICIDES MONITORING JOURNAL
-------
TABLE J-2.—Distribution of organochlorine residues in the monitored species for each sampling station by date of
collection—New York—Continued
YEAR
COMPOUND
RESIDUES IN PPB (/IG/KG)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT.
Nov.
DEC.
STATION 13.—SAYVILLE—M. MERCENARIA, UNLESS OTHERWISE INDICATED—74 SAMPLES'
966
1967
1968
1969
1970
1971
1972
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
_ _ _ T ____-T
__T10 — — — — — 1°
— — T T ___ — — —
TT11TTT T T T — T T
14 19 24 16 24 14 T 10 T — 16 19
T — — — T — — T — — — —
______ T____-
T --<-- __ __
______ ______
______ ___— »11 T
________ T T2413
______ ______
— — — — — — — — 59 — 19 12
— — — — — T — TT 10 — T
T 13 11 — T 16 T 14 16 12 T 20
— — — — — T 10 — TT — 10
— T — — — T — — — T10T
T — — — — — — ___
12 T 11 11 10 — — — — 16
— — — — — __ _ _ 12
T T — — — 13 _ 41 T 11
•22 '22 — 17 — —
56 38 — 16 — —
29 T T 10 — T
15 — _ 11 — _
STATION 14.—AMITYVILLE—M. MERCENARIA—73 SAMPLES'
1966
1967
1968
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
— — — — — ___T T
— — ,, — 18 — — — — 10 17
— — — — — — — — — —
TTTTTT — — _ _ T —
20 15 16 16 18 14 17 13 T 11 14 —
T---T — -T — ___
— — -T — — T — T — — _
— — — 13 — — 11 TT — 13 T
— — — — — — — — — — __
VOL. 6, No. 4, MARCH 1973
313
-------
TABLE J-2.—Distribution of organochlorine residues-in the monitored species for each sampling station by date of
collection—New York—Continued
YEAR
COMPOUND
RESIDUES IN PPB (JIG/KG)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG.
SEPT. OCT. Nov. DEC.
STATION 14.—AMITYVILLE—M. MERCENARIA—13 SAMPLES *—Continued
1969
1970
1971
1972
DDE
TDE
DDT
Dieldrln
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
— — — __•_____ 20 —
— T — T 14 — T — 11 T 11 —
— — — — — — — — — T33 —
— — — — T — — — T — T T
— — — 10 T T T — — — —
11 — T 20 19 15 15 10 T T 10
_ 1 -1 ___ __ __ -
— T — T — — — — 18 — T
— — — — — —
11 12 T 16 — T T 10 — 10
____ _ _ T — — —
— — 10 10 — — T — 42 —
T
16 T T T — —
T
11 — — — — —
STATION 15.—EAST BAY—M. MERCENAR1A, UNLESS OTHERWISE INDICATED—57 SAMPLES »
1966
1967
1968
1969
1970
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
T x
__T — — — — — 1819
__T — — — — — T —
T — TT — T — — — TTT
21 12 13 15 19 19 15 T T 13 T 14
T — — — T T TT — — — T
TTT — — — — — — —
16 10 12 — — T — 12 — T
____ _______
__________ T
16 10 — — 16 14 14 T T T 16
_________T
— — — — T 10 16 17 T 14 14
_ _ _ MO
T 12 T 21
— — — 18
— T — 16
314
PESTICIDES MONITORING JOURNAL
-------
TABLE J-2.—Distribution of organochlorine residues in the monitored species for each sampling station by date of
collection—New York—Continued
YEAR
COMPOUND
RESIDUES IN PPB (AC/KG)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT.
OCT. Nov. DEC.
STATION 15.—EAST BAY—M. MERCENARIA, UNLESS OTHERWISE INDICATED—57 SAMPLES'—Continued
STATION 16.—WEST BAY—M. MERCENARIA, UNLESS OTHERWISE INDICATED—57 SAMPLES 1
1971 DDE
TDE
DDT
Dieldrin
1972 DDE
TDE
DDT
= 12 »T
29 21
29 11
15 11
3 "J"
T 14
— T
= 18 " —
57 19
23 12
38 11
» _ 0
•p
— —
— T
15 26
10 23
— —
1966
1967
1968
1969
1970
1971
1972
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
1 Dieldrin
T T T
20 17 24
13 T 12
j j
20
—
_. T
19 20 T
— — -
— — -
— T
13 20
— 11
— 14
- 11
23
28
T
—
10 18
14
T
-T-----TT
- 15 — - 13 — — 26 22
— T — — — — — 14 13
— T 10 T T — T T T
24 27 32 — 17 T 20 14 19
18 17 14 — 13 — — — 13
12 10 — — — — — —
r T T —— — — — —
18 13 17 T 15 14 16 15 14
T__-____-
- 11 ----- 10
— 28 13 18 19 14 21 2i
33 T - - - 12 43
- T T 18 17 20 12 17
. 0 rj.
19 15
40 15
T x
"T '15 .._ - 10
22 47 15 14 25
12 44 _ T 20
10 16 - T 10
— "30 "13
T 59 28
— 22 T
— — —
Each sample represents 15 or more mature mollusks
M. edulis.
M. arenaria.
C. \irginica.
- M. mercenaria.
c M. demissus.
VOL. 6. No. 4, MARCH 1973
315
-------
SECTION K.—NORTH CAROLINA
The monthly collection of eastern oysters, Crassostrea
virginica, to monitor pollution was initiated in July 1966
and continued until July 1972. During the program, 17
stations were sampled routinely for periods ranging
from 3 to 6 years. All samples were analyzed by the
Gulf Breeze Laboratory.
Approximate station locations are shown in Fig. K-l. A
summary of data on organochlorine residues in the
monitored species, C. virginica, is presented in Table
K-l, and the distribution of residues in this species for
each sampling station by date of collection in Table
K-2.
North Carolina samples are noteworthy for the con-
tinuity of collections of a single species of mollusk at
short intervals over a relatively long period of time.
For this reason the data present a good picture of annual
and seasonal trends of a persistent synthetic pollutant
in this estuarine environment.
The incidence of DDT residues (75%) and maximum
magnitude (566 ppb) are about the median of the 15
Slates monitored. The 1 % incidence of dieldrin residues
was somewhat lower than most other states. PCB com-
pounds were not detected.
Although there are exceptions from one estuary to an-
other, the magnitude of DDT residues in oysters showed
little seasonal variation during the period 1967-69 when
maximum levels of DDT pollution were detected. The
overall decline in DDT residues (Part I. Table 7 and
Fig. 2) is notable and undoubtedly associated with the
decreased agricultural use of (his chemical in North
Carolina.
FIGURE K-l.—Diagram of coastal North Carolina showing
approximate location of monitoring stations
I. Wanchese—Croatan Sound
2. Salvo—Pamlico Sound
3. Wysocking Bay
4. Rose Bay
5. Bay River
6. Neuse River
7. Point of Marsh—Neuse River
8. West Bay
9. Back Bay—Core Sound
10. Jarrett Bay—Core Sound
11. North River
12. Newport River
13. Bogue Sound
14. White Oak River
15. New River
16. Wrightsville Beach—Wrightsville Sound
17. South port—Cape Fear River
18. Shallotte River
TABLE K-l.—Summary of data on organochlorine residues in the monitored species (C. virginica), 1966-72—North Carolina
SlA'IION
NUMBER
1 -
2 v*
.1
4 '
5 "
ft
7 *
8
9
10 •
II •
12 •
LOCATION
Wanchesc
Salvo
Wysocking Bay
Rose Bay
Bay River
Ncusc River
Point of Marsh
West Bay
Back Bay
.larrett Ba%
North River
Newport Rivei
MONI TORINO
PERIOD
1966-72
1966-72
1 966-70
1966-72
1966-72
1966-70
1966-72
1967-72
1966-67
1966-72
1966-72
1966-72
NUMBER or
SAMPLES l
72
71
43
71
71
4.1
7!
58
9
66
64
68
NrMBER OF POSITIVE SAMPLES AND MAXIMUM
RESIDUE ( ) DETECTED' IN PPB (^G/KO)
DDT
49 (264)
58 (566)
35 (64)
46 (121)
69 (310)
43 (176)
53 (139)
34 (74)
8 (103)
42 (106)
48 (172)
54 (121)
DIELDRIN
3 (14)
2 (12)
2 (19)
2 (10)
3 (13)
PESTICIDES MONITORING JOURNAL
-------
TABLE K-l.—Summary of data on organochlorine residues in the monitored species (C. virginica), 1966-72—
North Carolina—Continued
STATION
NUMBER
13
14
15 *
16
17 »
18
LOCATION
Bogue Sound
White Oak River
New River
Wrightsville Beach
Southport
Shallotte River
MONITORING
PERIOD
1967-72
1966-70
1966-72
1966-70
1966-72
1966-70
Total number of samples
Percent of samples positive for indicated compound
NUMBER OF
SAMPLES l
51
43
72
43
72
43
1,031
NUMBER OF POSITIVE SAMPLES AND MAXIMUM
RESIDUE ( ) DETECTED IN PPB (^G/KC)
DDT
33 (71)
30 (60)
61 (118)
35 (57)
32 (116)
38 (51)
75
DlELDRIN
1
* Data from these stations summarized in Part I. Table 7. and Fig. 2.
1 Each sample represents 15 or more mature mollusks.
TABLE K-2.—Distribution of organochlorine residues in C. virginica for each sampling station by dale of
collection—North Carolina
[Blank = no sample collected; — = no residue detected above 5 ppb; T = >5 but <10 ppb]
YEAR
COMPOUND
RESIDUES IN PPB (^G/KG)
JAN.
FEB.
MAR.
APR.
MAY JUNE JULY
AUG.
SEPT.
OCT.
Nov.
DEC.
STATION 1.—WANCHESE—72 SAMPLES'
1966
1967
1968
1969
1970
1971
1972
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
14 25 28 16
16 20 32 17
15 10 17 —
140 140 85 78
56 51 59 16
68 29 37 42
40 17 13 10
15 16 18 10
T T 11 —
T T 13 T
11 T 15 «•
— — T —
_ _ _
— _ _ _
— _ _ _
. . L -r
— — — _
— — — —
26 21
32 31
29 21
62 T
32 T
86 —
37 11
44 21
43 T
17 —
19 —
— —
— T
— —
— —
— T
— 10
— —
19 20
12 17
13 —
21 19
35 15
64 17
30 —
13 —
12 —
T 10
21 10
19 13
— —
— —
— —
—
— —
— —
12 22
T 17
T 22
20 24
15 11
13 17
43 35
22 27
60 49
— T
— T
— —
—
— 11
—
_
— —
20 20
18 32
— 11
43 28
57 10
64 53
29 21
16 15
57 —
T 12
13 15
•J1
__
_ „
— _
VOL. 6, No. 4, MARCH 1973
317
-------
TABLE K-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—North Carolina—Continued
YEAR
COMPOUND
RESIDUES IN PPB (/IO/KO)
JAN. FEB. MAR. APR. MAY JUNE JULY Auo. SEPT.
OCT.
Nov.
DEC.
STATION 2.—SALVO—71 SAMPLES '
1966
1967
1968
1969
1970
1971
1972
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
26
11
T
35
16
20
100
31
87
23
25
14
16
17
T
—
—
—
24
13
T
85
26
50
73
31
35
34
27
20
15
16
—
—
—
—
31
21
T
66
20
36
100
38
50
24
20
13
—
—
—
—
—
—
35
25
11
8S
24
21
59
27
22
19
16
10
17
18
—
T
—
—
24
21
19
65
37
28
120
51
67
27
33
19
21
21
11
13
T
—
21
30
21
34
29
19
39
38
16
14
20
—
—
—
—
13
22
—
12
—
—
17
21
29
58
38
87
27
37
28
—
—
—
12
14
—
14 14 14
17 — 17
— — —
24 120
19 56
44 390
28 95 45
21 40 33
36 240 76
— 15 13
— 21 15
— T T
12 11 —
17 — —
14 — —
_ _ _
— — —
_ _ _
32
28
28
74
45
190
58
37
120
10
19
—
10
—
—
—
—
—
23
17
15
87
29
66
66
35
80
15
13
11
19
16
11
—
—
—
STATION 3.—WYSOCKING BAY—43 SAMPLES »
1966
1967
1968
1969
1970
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
T 14 15 17 16 12
12 17 17 22 29 22
T 10 — — 14 13
T T T 18 13 15
T T — 20 20 13
.- — — — T 13
T T 12 T T
14 - — 16 T T
_ ._ _ T — —
T
T
T
17 —
17 —
— —
15 T
35 T
14 T
— T
— T
— —
T T
T
T 12
17 T T T
18 13 12 11
20 — — —
— — T 10
— — T 15
T T
T — T —
T — 13 —
21 — — —
T T 14
T 12 19
_ — — 12
.IIS
PESTICIDES MONITORING JOURNAL
-------
TABLE K-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—North Carolina—Continued
YEAR
COMPOUND
RESIDUES IN PPB (/K;/KG)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT.
Nov. DEC.
STATION 4—ROSE BAY—71 SAMPLES'
1966
1967
1968
1969
1970
1971
1972
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
12 T 23 15 16 15
18 14 32 26 32 30
T T T 11 16 40
T T 13 15 21 14
T T 12 15 27 25
— — — 22 13 T
11 16 11 T 12 T
15 18 12 — 17 T
20 — — — T —
17 13 14 T T
19 19 19 — 15
15 10 10 — —
— — 14 — —
_ _ _ _ T —
JJ
— — — — — —
— — 10 — 12 —
______
— — — — — —
— — — — — —
28
34
21
35
63
23
T
T
—
T
T
T
11
19
12
—
—
—
—
—
16 19 18 16 20
13 22 23 23 36
— 32 16 T 14
T T — 14 12
15 10 — 30 15
13 T — 19 T
_ T T T 16
— T 17 17 17
— T T T 29
T 12 T — 10
13 17 T — 14
69 — — — T
_____
— — — — —
— — — — —
— — — — —
_____
_____
_____
— — — — —
STATION 5.—BAY RIVER—71 SAMPLES 1
1966 DDE
TDE
DDT
1967 DDE
TDE
DDT
1968 DDE
TDE
DDT
1969 DDE
TDE
DDT
36
69
23
35
35
26
19
25
T
30
49
16
44
43
32
29
39
20
ei
100
35
37
24
28
36
36
37
39
56
20
52
49
47
43
56
37
32
65
27
75
71
55
54
59
32
29
56
19
22
33
T
45
80
17
36
16
29
22
46
27
18
24
13
37
71
26
55
78
25
15
48
27
23
28
18
18
16
18
52
73
34
T
T
—
13
15
12
T
T
14
23
46
19
28
43
51
T
—
—
13
T
—
30 26
61 60
11 20
25 24
34 46
— 16
24 16
36 21
29 T
T 20
20 35
— 21
VOL. 6, No. 4, MARCH 1973
319
-------
TABLE K-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—North Carolina—Continued
YEAR
COMPOUND
RESIDUES IN PPB (/U>/KO)
JAN. FEB. MAR.
APR.
MAY JUNE JULY Auo. SEPT. OCT.
Nov.
DEC.
STATION 5.—BAY RIVER—71 SAMPLES1—Continued
1970
1971
1972
DDE
TDE
DDT
DDE
TDE
DDT
Dleldrin
DDE
TDE
DDT
Dleldrin
24
33
17
12
15
—
—
16
11
—
—
40 36
43 29
33 18
16 —
13 —
10 —
.i. —
41 —
37 —
21 —
— —
42
31
14
22
21
—
-
71
48
37
—
55
94
22
110
170
30
10
85
130
87
12
10
27
—
16
19
—
—
43
91
43
—
15 11 13 — 23
23 12 22 16 39
18 — 14 — 13
52 49 18 17 13 T
97 96 34 26 11 —
27 22 — 18 12 —
______
STATION 6.—NEUSE RIVER-^t3 SAMPLES »
1966
1967
1968
1969
1970
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
17 16 24 29 21 16
29 28 41 50 42 30
— T T T T 16
19 20 15 30 49 32
24 25 15 44 110 68
11 T — 27 17 15
20 17 23 16 37 29
17 13 26 23 56 75
10 T 11 — 20 21
27
45
17
29
46
13
19
37
25
26
42
19
30
40
20
18
24
15
14
33
14
29
56
39
13
23
11
32
48
49
20
32
14
13
22
T
T
T
—
36
57
30
25
47
13
T
T
—
10
17
—
24
55
13
24
49
T
19
32
T
T
19
—
29
60
18
24
46
T
25
25
20
16
35
16
STATION 7.—POINT OF MARSH—71 SAMPLES»
1966
1967
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
33
37
38
T
15 17 22 20 20 33 13
29 24 28 32 39 82 26
15 10 — 11 45 24 27
26
26
28
—
22
24
33
15
20
25
—
11
19
10
29
31
19
—
12
19
27
16
33
16
—
11
18
15
20
40
16
—
18
25
10
320
PESTICIDES MONITORING JOURNAL
-------
TABLE K-2.—Distribution of organochlprine residues in C. virginica for each sampling station by date of
collection—North Carolina—Continued
YEAR
COMPOUND
RESIDUES IN PFB (AO/KO)
JAN. FEB. MAR.
APR.
MAY JUNE JULY AUG. SEPT. "OCT.
Nov.
DEC.
STATION 7.—POINT OF MARSH—71 SAMPLES >—Continued
1968
1969
1970
1971
1972
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
11
15
T
13
14
—
—
11
21
T
—
—
—
11
20
—
—
—
T
14
T
—
22
29
IS
T
T
—
—
—
—
12
18
—
12
17
T
—
20
21
—
T
T
—
—
—
—
18
23
21
T
13
—
—
T
12
—
T
32
—
T
—
—
11 15
18 20
10 16
T T
T 11
— —
— —
T
17
—
T —
— —
— —
T —
T —
— —
10 15 — T 14 11
13 19 — T 21 16
11 21 — — — —
10 37 — T T T
16 27 — T 11 10
18 48 — T 12 —
— 19 — — — —
T
_ _ _ _ T —
______
_____ T
_____ T
_____ T
STATION 8.—WEST BAY—58 SAMPLES '
1967 DDE
TDB
DDT
1968 DDE
TDE
DDT
1969 DDE
TDB
DDT
1970 DDE
TDE
DDT
1971 DDE
TDE
DDT
1972 DDE
TDE
DDT
18 25 22
25 39 22
T 11 13
T 11 25 T 16 11
— 11 30 T 13 T
— — 19 — T —
16 T 20 16 11 T
10 T 17 19 T 12
T — 12 12 10 T
— — —
— _ _
— — _
— T — — T —
— T — _ _ _
— — — — __
— 13 14 11 —
— 10 T T —
— — — — —
14 T — 16 T 15
19 T — 22 10 24
11 T — 17 12 15
— T — T — 10
— T — — — 16
— 15 — — — _
12 — — — T T
16 — — — 16 17
T - - _ _ T
— — — — T T
— — — — T 16
— — — ___
— — — T —
— — — T —
— — — T —
VOL. 6, No. 4, MARCH 1973
321
-------
TABLE K-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—North Carolina—Continued
YEAK
1966
1967
COMPOUND
RESIDUES IN PPB (/iG/KG)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT. Nov. DEC.
STATION 9.— BACK BAY— 9 SAMPLES '
DDE
TDE
DDT
DDE
TDE
DDT
10 — 15 17 24 10
12 — 11 32 74 14
14 — 12 15 T —
10 26 10
10 23 13
— 17 T
STATION 10.—JARRETT BAY—66 SAMPLES '
1966
1967
1968
1969
1970
1971
1972
DDE
^TJE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
22 — 13 14 T 12
22 — 11 16 T 18
15 — 19 16 — 14
11 17 14 29 19 17 10 12 12 14 10 21
T 12 19 24 22 18 12 T T 18 17 17
— T — 17 14 10 T 12 T 39 13 12
18 13 47 T 10 T — 13 T 18 — T
14 10 44 T — — — T T 13 — 15
TT15 — — — — TT66 — T
12 T 12 21 13 T 10 — — — 10 12
T 11 16 22 12 10 15 — — — 18 22
— T T — T — — — — — — 11
T
T
T
— T — — 11 — — — — T —
— T — — 10 — — — — T —
-p
— 12 T —
_ _ _ _
— — - —
STATION 11.—NORTH RIVER—64 SAMPLES'
1966
1967
DDT
TDE
DDT
Dieldrin
DDE
64
50
58
10
27 2
-------
TABLE K-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—North Carolina—Continued
YEAR
COMPOUND
RESIDUES IN PPB (IIIO/KO)
JAN. FEB. MAR.
APR. MAY JUNE JULY AUG. SEPT. OCT.
Nov. DEC.
STATION 11.—NORTH RIVER—64 SAMPLES'—Continued
1968
1969
1970
1971
1972
DDE
TDE
DDT
DDE
TDE
DDT
18 14 T 35
13 — — 25
T — — 43
18 13 16 32
T — T 15
— — 12 14
DDE T
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
T
T
— 12
— — —
- -
20 10
_
"
34 T 32 21 T 22 38 27
26 — 27 19 T 17 20 —
36 — 57 26 T 36 27 —
" 16 T — - 12 53 — T
12 - - - T 73 - T
— — — — 11 34 — T
11 — _____ T
11 — — — — — — —
— — ______
T T — — - —
_ _ — — — —
______
T
— —
- -
STATION 12.—NEWPORT RIVER—68 SAMPLES'
1966
1967
1968
1969
1970
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
i
1971
DDE
TDE
DDT
20 17
21 13
— T
T —
14 18 25 20 21 19 T T
21 24 85 27 29 30 12 15
T T II T — T 10 T
11 16 17 25 — T T T
14 16 23 31 — — T T
--T-____
21 21 20 27 17 12 T —
21 22 21 29 23 19 T —
— 10 T 13 T T — —
— - — — — _ _
16 12 12 18 T — T
14 T 10 23 T _ _
13 - T 10 _ _ _
-II - 13 _ _ ,7
-M - 18 - _ I6
- - — — — —
14
19
T
—
14
25
15
11
13
T
T
T
—
—
...
—
16
26
—
-
16
18
23
18
24
23
13
16
25
T
_
_
—
24
44
11
—
12
17
—
19
29
11
15
15
17
—
T
T
_
_
—
14
22
—
—
16
26
T
15
18
T
18
17
17
—
—
VOL. 6, No. 4, MARCH 1973
323
-------
TABLE K-2.—Distribution of organochlarine residues in C. virginica for each sampling station by date of
collection—North Carolina—Continued
1967
1968
1969
1970
1971
1972
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
31 20 15 T 10 10 13 15 14 15
26 23 17 T 12 12 14 — 13 T
12 T T — 11 16 24 T 12 T
12 T 16 15 T — T T 13 13 21 18
T — — T — — — T 13 13 17 11
— — — — — — — T 21 T 13 11
20 29 17 25 19 T 15 — — 12 13 12
15 23 11 20 19 11 16 — — 14 17 —
T 19 T 12 11 11 37 — — T 15 —
—
—
—
-p ,
____ ______
— — — — ______
-j-
______
— — — — — —
STATION 14.—WHITE OAK RIVER^»3 SAMPLES '
1966
1967
1968
1969
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
T 20 T
— 25 T
— T —
— — T — T -- T — T
— — — — T — 13 — T
______ 10 — T
TT13T — — — Til
TT13T — _ — Til
T — — _ — — _T T
T 12 14 T T — — — —
— — T — T — — — —
_________
29 T
31 T
— —
T T
— T
— -
T T
— T
— —
T T
T T
— —
T
T
—
T
T
—
14
T
T
—
—
—
324
PESTICIDES MONITORING JOURNAL
-------
TABLF K-2.—Distribution of organochlorinc residues in C. virginica for each sampling station by date of
collection—i\ortk Carolina—Continued
RESIDUES IN PPB (AC/KG)
.Us. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT. Nov. DEC.
STATION 14.— WHITE OAK RIVER— 43 SAMPLES '—Continued
1970
DDE
TDE
DDT
T
—
—
STATION 15.—NEW RIVER—72 SAMPLES'-
1966
1967
1968
1969
1970
1971
1972
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
39
49
16
42
38
31
25
19
-
21
19
-
11
—
—
19
18
—
16
18
T
16
14
T
18
19
T
28
24
T
T
T
—
17
21
—
30
30
T
35
31
T
21
27
T
29
22
T
15
12
—
24
21
—
45 20 16
59 23 13
14 — —
39 25 15
29 25 15
- - —
28 13 15
35 13 15
T — —
26 26 T
31 20 —
12 — —
T T —
- — —
— — —
11 T —
_ _ _
— — —
25 T 23 21 28 36
21 T 27 26 34 44
28 — T — — 14
11 19 16 28 21 28
13 28 20 34 26 37
— 11 — — — 21
T 10 14 15 15 14
— T 14 12 12 T
T T — — —
11 13 14 19 23 27
15 13 12 22 26 27
— T — 11 — 11
— — — — — 17
______
______
— — _ 12 _ 20
— — — 11 — 22
T -j-
STATION 16.—WRIGHTSVILLE BEACH^43 SAMPLES '
1966
1967
1968
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
11 14 15
12 18 12
_ _ _
II 16 19 14 T T T 10 —
14 18 15 14 T T 14 18 —
-TT-T- T 14 -
T T 10 13 T — _ I0 T
T — — 10 T — ___
T----__T_
15
16
13
T
12
22
T
—
—
19
24 1
14
—
T
12
—
VOL. 6. No. 4, MARCH 1973
325
-------
TABLE K-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—North Carolina—Continued
YEAR
COMPOUND
RESIDUES IN PPB (/IG/KC)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT. Nov. DEC.
STATION 16.— WRIGHTSVILLE BEACH— 43 SAMPLES '—Continued
1969
1970
DDE
TDE
DDT
DDE
TDE
DDT
TTTTT — — — — TT12
— — — T T — — — — T 11 16
,2
T
—
-
STATION 17.—SOUTHPORT—72 SAMPLES '
1966
1967
1968
1969
1970
1971
1972
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
11 21 11 29 T T
T 16 10 25 T T
12 — — — — —
T 13 11 T — T 10 14 11 T — T
— 12 T T — — 10 T — — — T
— T — — — — 17 30 15 — — —
18 13 T T 11 — — 10 11 T 17 17
I3TTTI4 — — — Til — T
T — — — — — — 13 T — 21 10
T — 12 T 11 — — — — __ _ _
__ 17 _ 12 _______
_ _ 87 — T — — — — — — —
_ T — — — — — T — — — —
______ ______
------ ______
______ __ — _ — —
______ ______
______ ______
._.____
______
— — — — — —
STATION 18.—SHALLOTTE RIVER-^13 SAMPLES'
1966
1967
DDE
TDE
DDT
DDE
TDE
DDT
- T T T
T —
-----
T 16 11 14 19 T T T 10 T
— 13 T 15 22 T 11 T T _
— T — — 10 — 10T T —
19
18
11
12
II
T
T
T
—
T
T
—
326
PKSTICIDES MONITORING JOURNAL
-------
TABLE K-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—North Carolina—Continued
YEAR
COMPOUND
RESIDUES IN PPB (AC/KG)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT.
Nov. DEC.
STATION 18.—SHALLOTTE RIVER—43 SAMPLES "—Continued
1968
1969
1970
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
13 14 16 15 - - - T T 13 - 17
11 10 11 12 — — — — — T — 10
TT_____--11-T
T T 12 11 15 T 14 T T 11 T T
— — 10 T 19 — 14 T — T 13 —
_ _ 19 — T — 16 16 12 T — —
T
-
—
Each sample represents 15 or more mature mollusks.
SECTION L.—SOUTH CAROLINA
Monthly collections of eastern oysters, Crassostrea
virginica, to identify estuarine pollution were made
from August 1965 through November 1969. The 17
stations (Fig. L-l) were monitored for periods ranging
from 1 to 5 years. All samples were analyzed at the
Gulf Breeze Laboratory. A summary of data on or-
ganochlorine residues in the monitpred species, C.
virginica, is presented in Table L-l, and the distribution
of residues in this species for each sampling station by
date of collection in Table L-2.
South Carolina samples are characterized by the uni-
formly low level of DDT residues and moderately low
incidence of positive samples. Samples from only three
other States indicated generally lower levels of DDT
contamination.
In those areas with adequate numbers of samples for
annual comparison, there was an obvious decline at most
stations in the magnitude and incidence of DDT resi-
dues in 1968-69 as compared to earlier years (Part
I. Table 6).
South Carolina was the only State in which mirex
residues were detected in mollusks. These residues were
observed only in the period March May 1969. They
were found at nine stations widely distributed along the
South Carolina coast. Largest residues were found in
samples collected in the Charleston area, i.e., Stations
8 and 9.
VOL. 6, No. 4, MARCH 1973
SOUTH CAROLINA
ATLANTIC OCEAN
FIGURE L-l.—Diagram of coastal South Carolina showing
approximate location of monitoring stations
1. North Santee Bay—Santee River
2. South Santee Bay—Santee River
3. Bull Creek
4. Price Creek
5. Inlet Creek
6. Hog Island Channel—Ashley, Cooper, and Wando Rivers
7. Wando River—Ashley, Cooper, and Wando Rivers
8. Ashley River—Ashley, Cooper, and Wando Rivers
9. Fort Johnson—Ashley, Cooper, and Wando Rivers
10. Steamboat Creek—North Edesto River
II. Toogoodoo Creek—North Edesto River
12. Big Bay Creek—South Edesto River
13. St. Pierre Creek—South Edeslo River
14. Whale Branch—Broad River
15. Skull Creek—Broad River
16. May Creek
17. New River
327
-------
TABLE L-l.—Summary of data on organochlorine residues in the monitored species (C. virginica"), 1965-69—South Carolina
STATION
NUMBER
1
2
3
4
5
6
7
g
9
10
11
12
13
14
15
16
17
LOCATION
North Santee Bay
South Santee Bay
Bull Creek
MONITORING
PERIOD
1965-68
1965-68
1969
Price Creek 1965-68
Inlet Creek 1965-68
Hog Island Channel
Wando River
Ashley River
Fort Johnson
Steamboat Creek
Toogoodoo Creek
Big Bay Creek
St. Pierre Creek
Whale Branch
Skull Creek
May Creek
New River
Occasional stations (6)
1965-68
1965-68
1965-69
1969
1965-69
1965-69
1965-69
1969
1965-68
1965-68
1969
1969
1965-68
Total number of samples
Percent positive for indicated compound
.NUMBER OF
SAMPLES '
41
40
12
42
42
41
42
54
12
54
53
54
12
41
39
12
12
7
610
NUMBER ot POSITIVE SAMPLES AND MAXIMUM
RF.SIDUE ( ) DETECTED IN PPB (/.G'KG)
DDT
10 (19)
14 (80)
2 (10)
25 (81)
21 (52)
33 (73)
31 (44)
45 (154)
4 (10)
26 (32)
40 (98)
32 (91)
7 (88)
21 (79)
12 (30)
3 (15)
1 (16)
5 (201)
54
Dieldrin
4 (19)
3 (19)
2 (13)
2 (12)
MlREX
2 (35)
8 (90) 1 (190)
1 (35)
1 (11)
1 (21)
2 (15)
4
1 (540)
1 (38)
1 (38)
1 (T)
1 (38)
3 (37)
1 (27)
2
Nt.TE: T = >5 but <10 ppb.
1 li.icr. -...nple represents 15 or more mature mollusks.
TABLK L-2.—Distribution of organochlorine residue!: in C. virginica for each sampling station by date of
collection—South Carolina
[Blank — no sample collected; — = no residue detected above 5 ppb; T — :-5 hut <10 ppb]
YEAR
COMPOUND
RESIDUES IN PPB (f-G KG)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT. Nov. DEC.
STATION 1.— NORTH SANTEE BAY-^U SAMPLES'
1965
1966
1967
DDE
T T T — — —
IDE T T T — — —
DDT - — — T — —
DDE _______ ______
TDE ______ _______
DDT — _____ ______
DDE ___ _ T T T — — — —
TDE
DDT
Dieldrin
.
_ _ _ ________
y -p __
— 15 ________
328
PESTICIDES MONITORING JOURNAL
-------
TABLE L-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—South Carolina—Continued
YEAR
1968
COMPOUND
DDE
TDE
DDT
Dieldrin
RESIDUES IN PPB (/iC/KC)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT. Nov. DEC.
STATION 1.— NORTH SANTEE BAY^tl SAMPLES '—Continued
T _ _ T — — — — — —
____ — — — — — 19 — —
15 — 12 19 — — — — — — — —
STATION 2.—SOUTH SANTEE BAY^»0 SAMPLES '
1965
1966
1967
1968
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
T T T — — —
T T — — — —
T — —
___._T---- - -
_________ — —
_________ — —
— — T __TTT — — —
•T-
— — — — — 13TT — — —
T46 — 10 T — — T — — — —
T — — T — — — — — — — —
f 34 j2
— 13 10 19 — — — — — — — —
STATION 3.—BULL CREEK—12 SAMPLES 1
1969
DDE
TDE
DDT
Dieldrin
Mirex
_ _ _ T T-______
J f
— — — 13 12 — — _ _____
- — 22 — 35 — — — — __-_
STATION 4.—PRICE CREEK—42 SAMPLES '
1965
1966
1967
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
T _ T — _ _
T ______
— _ — — __
T — T — — — - — I9T — T
-------- 36 -_T
— — — — — — — — 26 — _ _
— — 12 -- — — — — ____
T 'I I* T T T T 13 T _ 10 T
— T — — — 10 T12 — _TT
— T — — T 11 10 11 T — T —
VOL. 6, No. 4, MARCH 1973
329
-------
TABLE L-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—South Carolina—Continued
YEAR
RESIDUES IN PPB UG/KG)
JAN. FEB.
STATION
1968
DDE
TDE
DDT
Dieldrin
T T
10 T
T —
— 10
MAR. APR. MAY JUNE JULY AUG. SEPT.
4.— PRICE CREEK^2 SAMPLES1 — Continued
T 11 T — T — —
_ T _____
_______
_______
OCT. Nov. DEC,
T - -
— — —
_ _ _
— — —
STATION 5.—INLET CREEK—*2 SAMPLES 1
1965
1966
1967
1968
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
T T
T T
— T
T — — — U — _T
T — — — 14- --
T--_____
T T T 14 T T T 16
— — T 13 — — 15 21
— — — 12 T — 17 15
11 — 12 — — — — —
11 _ T — — — — —
________
— — T T
T
— — — —
— T T T
_ -p
— — — —
— — — T
_ _ _ —
— — — —
_ — _ —
_ _ _ _
— — — —
STATION 6.—HOG ISLAND CHANNEL—41 SAMPLES1
1965 DDE
TDE
DDT
1966 DDE
TDE
DDT
1967 DDE
TDE
DDT
1968 DDE
TDE
DDT
T
T
T
T 13 T 14 13 — 20
T T — — 15 T 15
T — — — — — -
15 20 20 16 14 11 10
14 13 — — 32 11 13
11 11 — — 12 20 14
T 13 12 20 — — T
— 10 — 16 — — 10
— — — 14 — — T
T T T T —
T — T — —
-p
— 14 10 — T
_____
----- —
19 T — T T
28 T — T T
26 11 — 16 —
— — T T
— - — -
— — — —
STATION 7.—WANDO RIVER—»2 SAMPLES '
1965
DDE
TDE
DDT
T
T
T
T
T
T
T
T
-
T
T
T
— T
— T
— —
330
PESTICIDES MONITORING JOURNAL
-------
TABLE L-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—South Carolina—Continued
YEAR
COMPOUND
RESIDUES IN PPB (^O/KO)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT.
Nov. DEC.
STATION 7.—WANDO RIVER—42 SAMPLES >—Continued
1966
1967
1968
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
10 — T
10 — —
T — —
10 12 16
17 13 17
— — —
14 13 11
18 T T
12 — —
11 10 —
— 12 —
— — —
T T T
— T T
— — T
14 13 —
T 14 —
— — —
—
—
—
—
12
10
T
10
T
— 10
— 13
— T
10 T
20 10
14 10
_ _
— —
— —
10 T T
12 11 14
TT
T T 11
T — 16
— — T
— — —
_ _ _
— — —
STATION 8.—ASHLEY RIVER—54 SAMPLES >
1965
1966
1967
1968
196?
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
Mirex
33
25
28
—
36
21
24
—
31
30
28
T
12
—
—
—
—
34 38
29 28
30 16
13 21
36 51
28 39
28 35
— 11
15 66
13 26
— 25
— 19
13 16
— —
— —
- —
— 190
35
33
22
20
26
21
18
—
35
31
23
—
—
—
—
—
—
25 16
23 39
14 —
90 —
42 11
69 —
43 19
— —
32 T
28 —
25 —
— —
15 —
— —
13 —
— —
— —
16 T
16 T
14 T
13 11
17 —
— —
— —
16 18
20 23
18 32
— —
13 15
16 15
14 11
— —
— —
— _
— —
— —
— —
T T
T 15
— 10
— T
— T
— 1!
— —
T 14
10 19
11 18
— —
T T
11 —
13 —
— —
— —
— —
— —
— —
— —
18
27
28
25
31
31
—
23
26
42
—
T
—
T
—
—
—
—
23
—
32
22
22
18
28
21
—
33
37
49
—
60
51
28
—
—
—
—
—
—
STATION 9.—FORT JOHNSON—12 SAMPLES 1
1969 DDE
TDE
DDT
Mirex
— — T T — —
______
______
— — 540 — — —
— — — T — T
— — — T — —
— — — — —
— — — — — —
VOL. 6, No. 4, MARCH 1973
331
-------
TABLE L-2.—Distribution of organochlorine residues in C. virginica for each sampling station by dale of
collection—South Carolina—Continued
YEAR
COMPOUND
RESIDUES IN PPB (^G/KO)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT.
Nov. DEC.
STATION 10.—STEAMBOAT CREEK—54 SAMPLES1
1965
1966
1967
1968
1969
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
Mire*
T T T — T —
T T — — — —
_ T — — — —
T T — 14 — 11 — T — T — —
— — — 11 — 11 — — T T — —
— — ____ — — T — — —
T 11 10 — 13 T — — T — — —
T T 10 — 14 T _ _ T — — T
— — T — T16 — — T — — T
TT — TT — T — — — — —
T
•p
-p ~p 'j1
— — — __ — ______
— — ______ — — _ —
3g
STATION 11.—TOOGOODOO CREEK—53 SAMPLES*
1965
1966
1967
1968
1969
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
Mirex
32 14 18 16 20
43 20 19 20 33
T T T — T
27 18 15 38 24 19 — 15 13 18 11
25 20 13 36 33 20 — 16 13 18 12
— T T 24 16 — — — 11 10 —
10 20 21 25 21 14 T — 12 T
T 17 18 23 22 15 — — — —
— — T — 14 T — — —
17 20 16 18 25 21 T 18 11 T T
14 16 — 16 20 16 T 19 10 — —
T — — — — — — 17 T — —
__________T
_ — — — — — — ____
— — — — — — — — — ——
— — 38 — — — — — — — —
16
16
—
22
26
T
30
26
12
13
T
—
—
—
—
—
STATION 12.—BIG BAY CREEK—54 SAMPLES 1
1965
DDE
TDE
DDT
T
T
—
T
T
—
T T
— T
— —
-j-
— T
— —
332
PESTICIDES MONITORING JOURNAL
-------
TABLE L-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—South Carolina—Continued
YEAR
COMPOUND
RESIDUES IN PPB (/JO/KG.)
JAN. FEB. MAR.
APR. MAY JUNE JULY AUG. SEPT. OCT.
Nov. DEC.
STATION 12—BIG BAY CREEK—54 SAMPLES1—Continued
1966
1967
1968
1969
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
Mlrex
11 —
T —
— —
11 14
T 12
T T
T T
11 —
T —
— —
— —
_ _
— —
j
— — T
— — —
14 12 12
14 11 15
11 12 13
T 13 12
T T —
— — —
— T T
_ _ _
— — 33
T — —
T
T
—
T
T
21
—
—
—
24
23
44
—
12 T — — — T
1" ___
— — — — — —
13 — T — — T
14 _ — — _ —
31 — — — — —
_ T — — — —
_ T — — — —
_ T — — — —
— — — 12 14 17
— — — 13 T 13
— — — 27 13 13
— — — — — —
STATION 13.—ST. PIERRE CREEK—12 SAMPLES'
1969
DDE
TDE
DDT
Mire*
— T — T T 22
— — — — — 21
— — — — 30 45
— — 38 — — —
15
— — — 15
— — — 29
— — — —
13
T
13
—
T
—
—
—
STATION 14.—WHALE BRANCH^tl SAMPLES 1
1965
1966
1967
1968
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
T T — T T T
T T — T — T
T T — — — T
— — — TT — — _ 1I33T
— — — T- — _— 11 20 —
— — — — — — — — _ 26 —
13 T — 11 T — T — 11 — — —
T — — T T — T — — ___
T--T--T-T___
11 T — 14 — — _ T T — — —
T T-14-__i2____
------- is -___
STATION 15.—SKULL CREEK—39 SAMPLES 1
1965
DDE
TDE
DDT
T
T
—
T
T
T
T
T
—
T —
_
— —
VOL. 6, No. 4, MARCH 1973
333
-------
TABLE L-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—South Carolina—Continued
YEAR
RESIDUES IN PPB (/IG/KG)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT. Nov. DEC.
STATION 15.— SKULL CREEK— 39 SAMPLES »— Continued
1966
1967
1968
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
— — — — 12 T — — — — T —
— — — — 12 T — — — — T —
— — _ _ T — — — — — — —
— — — 11 — T ___ —
— — — 14 — T — — — T
— — — T — 16 ___ T
X _
— — — — — — — — — — — —
— — — __ — ___ n — —
— — — — — — _____35
STATION 16.— MAY CREEK— 12 SAMPLES'
1969
DDE
TDE
DDT
Dieldrin
Mirex
— — — 1ST — — T — — — —
-___T_______
— — — __ — _ — _____
— — — 11 — — _ — _ — _ —
— — 23 37 27 — — — — — — —
STATION 17.— NEW RIVER— 12 SAMPLES'
1969
DDE
TDE
DDT
Dieldrin
Mirex
___T________
— — _ 11 ________
____________
___21 — — — — — — — —
----27 _______
1 Each sample represents IS or more mature mollusks.
334
PESTICIDES MONITORING JOURNAL
-------
SECTION M.—TEXAS
The eastern oyster, Crassostrea virginica, was used to
monitor pollution in Texas estuarine waters during the
period July 1965 June 1972. All samples were an-
alyzed at the Gulf Breeze Laboratory. Approximate
locations of the 13 sampling stations are shown in
Fig. M-l. A summary of data on organochlorine residues
in the monitored species, C. virginica, is presented in
Table M-l, and the distribution of residues in this species
for each sampling station by date of collection in Table
M-2. In some instances, more than one reef was sampled
at different times in a particular bay. In these instances,
the data have been integrated to reflect bay conditions
as a whole. At some times, floods resulting from tropical
storms decimated oyster reefs and inerrupted routine
monitoring. On at least one occasion, sample preparation
reagents were contaminated with chlordane leading to
spurious analytical results. Consequently, all findings of
chlordane have been omitted from the data tabulations.
In conjunction with oyster monitoring in Texas, many
samples of fish and other vertebrates were analyzed
throughout the monitoring program. These analyses
indicated, as might be expected, more kinds of pollutants
and of greater magnitude than those found in oysters.
PCB's, for example, were commonly found in fish
samples but were detected in only five collections of
oysters. In the Arroyo Colorado, Station 12, findings of
consistently large DDT residues in oysters were par-
alleled by DDT residues about 10 times larger in fish.
A causal relationship between DDT residues in the
eggs and reproductive failure of the spotted sea trout,
Cynoscion nebulosus, there in 1969, has been postu-
lated (5).
Although the incidence of DDT residues was higher in
eight other States, samples from monitoring stations in
Texas bays that receive runoff from the agricultural areas
were consistently contaminated with DDT. The maxi-
mum DDT residue detected, 1,249 ppb, was in an isolated
sample; more typically the residues in contaminated
areas were in the range of 100 500 ppb of DDT.
Toxaphene of presumaaw
tected in only one sample.
There is a clearly defined trend of declining DDT resi-
dues in oysters. In 1971, there was a more than 50%
increase in the number of samples containing negligible
DDT residues (i.e., <11 ppb) over previous years and
a 75% decrease in the number of samples in the 100 -
1,000 ppb range.
TEXAS
GULF OF MEXICO
FIGURE M-l.—Diagram of coastal Texas showing
approximate location of monitoring stations
1. Trinity Ray—Trinity-San Jacinto River basins
2. Galveston Bay—Trinity-San Jacinto River basins
3. Tres Palacios Bay—Lavaca River Basin
4. Lavaca Bay—Lavaca River Basin
5. San Antonio Bay, North—Guadalupe-San Antonio River Basin
6. San Antonio Bay, South—Guadalupe-San Antonio River Basin
7. St. Charles Bay—San Antonio-Nueces Coastal Area
8. Aransas Bay—San Antonio-Nueces Coastal Area
9. Copano Bay—San Antonio-Nueces Coastal Area
10. Red Fish Bay—San Antonio-Nueces Coastal Area
11. Nueces Bay—Nueces River Basin
12. Arroyo Colorado—Rio Grande Coastal Area
13. Lower Laguna Madre—Rio Grande Coastal Area
TABLE M-l.—Summary of data on organochlorine residues in the monitored species (C. virginica), 1965-72—Texas
STATION
NUMBER
1
2
3
4
5
6
LOCATION
Trinity Bay
Galveston Bay
Tres Palacios Bay
Lavaca Bay
San Antonio Bay, North
San Antonio Bay, South
MONITORING
PERIOD
1965-69
1965-72
1965-72
1965-72
1965-72
1965-72
NUMBER OF
SAMPLES '
47
71
74
66
59
75
NUMBER OF POSITIVE SAMPLES AND MAXIMUM
RESIDUE ( ) DETECTED IN PPB (/IO/KO)
DDT
28 (51)
60 (88)
71 (974)
59 (400)
38 (78)
40 (488)
DlELDRIN
I (20)
31 (87)
6 (18)
4 (24)
8 (27)
3 (56)
ENDRIN
1 (10)
TOXA-
PHENE a
PCB's '
2
VOL. 6, No. 4, MARCH 1973
335
-------
TABLE M-l.—Summary of data on organochlorine residues in the monitored species (C. virginica), 1965-72—
Texas—Continued
STATION
NUMBER
7
8
9
10
11
12
13
LOCATION
St Charles Bay
Aransas Bay
Copano Bay
Red Fish Bay
Nueces Bay
Arroyo Colorado
Lower Laguna Madre
Occasional stations (16)
Total number of samples
MONITORING
PERIOD
1966-72
1965-67
1967-71
1966-72
1965-68
1965-71
1965-67
1965-72
Percent of samples positive for indicated compound
NUMBER OF
SAMPLES 1
66
19
51
67
20
48
24
41
728
NUMBER op POSITIVE SAMPLES AND MAXIMUM
RESIDUE ( ) DETECTED IN PPB (AQ/KG)
DDT
33 (93)
18 (83)
24 (96)
52 (82)
20 (450)
48 (710)
15 (57)
24 (1,249)
73
DlELDRIN
11 (80)
2 (48)
4 (33)
45 (46)
1 (46)
16 (64)
18
ENDRIN
3 (18)
18 (32)
3
TOXA-
PHENE*
1
PCB's »
2
1
<1
1 Each sample represents 15 or more mature mollusks.
' Present but not quantified.
TABLE M-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date
of collection—Texas
[Blank = no sample collected; — = no residue detected above 5 ppb or no residue detected (PCB s); T = >5 but <10 ppb.]
YEAR
COMPOUND
E RESIDUES IN
JAN. FEB. MAR. APR. MAY JUNE
PPB (0G/KG)
JULY AUG. SEPT. OCT.
Nov.
DEC.
STATION 1.—TRINITY BAY—47 SAMPLES 1
1965
1966
1967
1968
1969
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
T — T T T 11
T — — T 10 16
— — — — — —
12 18 15 18 — — — — — T T
17 27 28 33 — — — — — T 12
___ ________
T T 12 — — T — — — T T T
T 12 18 — — T — — — T T —
_____ T __ — T — —
TTTTT — — T — 10 T
11 T T 11 T — — — — T —
______ _____
_ T - - - - T
— 13 — — — — —
_ _____ —
_ 20 — — — — —
336
PESTICIDES MONITORING JOURNAL
-------
TABLE M-2.—Distribution of organochlorine residues in C. virginica for each sampling station hy date of
collection—Texas—Continued
YEAR
COMPOUND
RESIDUES IN PPB (/IG/KG)
JAN. FEB.
MAR. APR. MAY JUNE JULY AUG. SEPT. OCT.
Nov. DEC.
STATION 2.—GALVBSTON BAY—71 SAMPLES'
1965 DDE
TDE
DDT
1966 DDE
TDE
DDT
Dieldrin
1967 DDE
TDE
DDT
Dieldrin
1968 DDE
TDE
DDT
Dieldrin
1969 DDE
TDE
DDT
Dieldrin
1970 DDE
TDE
DDT
Dieldrin
1971 DDE
TDE
DDT
Dieldrin
1972 DDE
TDE
DDT
Dieldrin
15 18 13 11 —
36 43 32 33 —
•T-
- — — — —
T 11 15 13 — T
26 29 44 34 15 18
•j-
12 14 15 — — —
10 16 T 14 20 T
24 43 36 37 46 41
_ T — — — —
13 25 20 — — —
T 24 —
.44 64 23
_ _ _
30 19 —
13 15 T 12 T
35 34 34 32 18
— 10 T — —
18 19 11 14 —
T 11 —
39 38 48
_ _ _
16 23 30
T T T T —
18 T 26 15 T
— — _ _ _
42 26 87 24 36
— T T T T
_ - - 11 17
— — — — —
_ _ — T T T
_ _ — T 24 24
___ — — —
_ _ _ — 12 —
_ T T 11 11 13
_ 10 T 21 23 32
_ — — 11 — —
__ — — 14 19
T T T 14 T —
30 13 15 46 34 49
_ _ _ 13 — —
— — — — 19 14
T 10 — 19
13 11 T 19
_ _ _ _
— — — —
____U_
20 — — 17 31 32
______
— — 13 — 24 18
— — — T T
— — — 17 17
— — — T 11
65 46 — — 26
STATION 3.—TRES PALACIOS BAY—74 SAMPLES 1
1965
DDE
TDE
DDT
11
T
—
11
T
—
T T 21
— — T
— — T
93
29
65
VOL. 6, No. 4, MARCH 1973
337
-------
TABLE M-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—Texas—Continued
YEAR
COMPOUND
RESIDUES IN PPB (/IO/KQ)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT.
Nov. DEC.
STATION 3.—TRES PALACIOS BAY—74 SAMPLES '—Continued
1966
1967
1968
1969
1970
1971
1972
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
Li-T
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
78
36
43
42
14
—
270
66
83
—
25
19
—
—
70
48
31
—
—
—
—
100
25
15
—
78
36
53
42
17
T
120
57
21
18
24
18
T
—
36
29
—
13
43
30
—
59
20
T
T
250
44
80
67
31
11
230
23
81
—
83
35
27
—
no
51
19
17
14
12
—
190 300
53 89
59 130
51 34
19 14
T 15
320 300
590 77
64 22
— —
91
44
21
—
230
44
56
13
13
12
—
47 210
22 97
12 23
58 18
24 —
11 —
91 62
62 95
T 17
— —
55 95
62 15
15 —
— —
44 33
38 55
31 —
— —
10 13
23 27
— —
97 21 11 23 26
29 T — T T
— — — — —
12 18 72 150 240
— — 41 52 57
— — T 71 38
43 20 19 22 24
10 42 13 19 15
_ T — — —
— — — — —
58 43 25
56 T 40
— — 12
— 10 —
41 — 50 16
72 — — 12
— — — —
_ _ — —
15 T — 14 T
44 — — 15 T
— — — — —
STATION 4.—LAVACA BAY—66 SAMPLES 1
1965
1966
1967
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
33 40 43 56
12 17 T 27
11 18 T 16
22 16 20 25
13 T 12 14
— — — T
51 140
25 30
21 23
14 T
T —
14 —
T — T 13 22
— — — T T
— — — T 10
39 26 17 T T 11
16 11 — — — —
— — — — — —
25 14 14 T 19 26
13 - - - - 12
34 — - — - T
338
PESTICIDES MONITORING JOURNAL
-------
TABLE M-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—Texas—Continued
YEAR
COMPOUND
RESIDUES IN PPB (/K)/KO)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT.
Nov. DEC.
STATION 4.—LAVACA BAY—66 SAMPLES'—Continued
1968 DDE
TDE
DDT
Dieldrin
1969 DDE
TDE
DDT
1970 DDE
TDE
DDT
Dieldrin
PCB's
1971 DDE
TDE
DDT
Dieldrin
1972 DDE
TDE
DDT
Dieldrin
39 38 39 69 46 140
16 17 22 62 39 120
26 40 18 49 34 140
24
20 19 33 41 33
T — 11 19 22
— — — 14 16
— 120 30 «>
— 42 16 «'
— 26 11 «>
— — 10 —
(3)
48 43 43 22 T
29 15 — — —
T — — — —
— — — — —
18
T
T
21
24
13
—
~~
— 12 — 26 40
_ _ - 18 33
_ — — 15 48
37 on — — 16
53 a, — — T
(» — — —
_ — — — —
(3)
T — 12 T 13
— — 25 — —
_ — — — —
_ — — — 14
STATION 5.—SAN ANTONIO BAY (NORTH)—59 SAMPLES 1
1965
1966
1967
1968
1969
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
T T T T 11 30
T T T T T 25
— T — T T 16
— — — — — 11
31 30 32 29 33 29 16 — T 13 15 19
24 27 30 i 23 27 22 10 — — — T 14
14 16 15 — 18 13 — — — — — —
— — 17 — — — — — _ — __
17 20 22 29 1-3- 12 T — 11 14
13 18 18 30 T — — — T 13
---T----I4-
— 10 — — — _ _ _ _ _
- No Samples Collected* •- --
— — — T 18
— — — T 12
- - - - T
VOL. 6, No. 4, MARCH 1973
339
-------
TABLE M-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—Texas—-Continued
YEAR
1970
1971
1972
COMPOUND
RESIDUES m PPB (#O/KG)
JAN. FEB. MAR. APR. MAY JUNB JULY AUG. SEPT. OCT. Nov. DEC.
STATION 5.— SAN ANTONIO BAY (NORTH)— 59 SAMPLES i— Continued
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
— — 18 14 17 — — — — — — —
__ — — 33 — ______
— __ — __ __ — — — —
— ___ — _ 27 — — — 17 —
T — — _ _ _ _ 10 _ T 13
12 — — ______ 11 _
— __ ____i9___
— — — ______ 12 17
12 12 T
— — —
_ _ _
Y
STATION 6.—SAN ANTONIO BAY (SOUTH)—75 SAMPLES t
1965
1966
1967
1968
1969
1970
1971
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
Endrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
DDE
TDE
DDT
— T T T 12
— — T T 10
— — T T T
13 13 14 19 14 T — — — T 15
T — — 14 10 — — — — — T
T _ — _ 10 _ _ _ _ _ T
17 20 20 14 T — — — T
11 10 11 — — — — — T
10 — 11 — ' — — — — T
21 T — — 110 — — —
19 — — — 310 — — —
13 — — — 68 — — —
56 — — — 14 — — —
10 — — — — — — —
T 16 20 16 T — — — T T
— — — 14 T — — — T T
Y .
14 — — — — — ____
__ — — 11 — — — — — 13
— — — — 25 — — — — — 41
— — — — — — — — — — —
___ — T — — — — — T
— — — — — — — — — — —
— — — — — — — — — — —
17
T
T
10
T
—
T
T
—
—
—
12
IS
T
—
—
—
—
13
10
T
340
PESTICIDES MONITORING JOURNAL
-------
TABLE M-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—Texas—Continued
RESIDUES IN PPB (/to/so)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT. Nov. DEC.
STATION 6.— SAN ANTONIO BAY (SOUTH)— 75 SAMPLES »— Continued
1972
DDE
TDE
DDT
13 16 T
_ _ _
— — —
STATION 7.—ST. CHARLES BAY—66 SAMPLES>
1966
1967
1968'
1969
1970
1971 *
1972'
DDE
TDE
DDT
Dieldrln
DDE
TDE
DDT
Dieldrln
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
11 T 11 11 — — — — T T 15
___ _____ — — T
___ T_____-T
_^-_ _____ — — 11
15 20 17 23 16 — — — 12 15 16 17
T T T 52 47 — — — — — T 12
T T — — 23 — — — — — — 10
13 12 — 28 39 78 80 — — — — 15
22 30 — 24 19 — T — — — — —
— 20 — 19 16 — ______
— 43 — 11 23 — ______
— — 14 12 T T _____T
— — T 21 17 15 ______
— — — _ T — ______
49 — — — — — 27 — T — — —
—
— — — — —
— — — — — — — TT 13
_______ TT T
— — — — — — — 21 10 15
14 23 27 16
T f 10 —
14 15 — —
STATION 8.—ARANSAS BAY—19 SAMPLES'
1965
1966
DDE
TDE
DDT
DDE
TDE
DDT
T
T
—
12 16 20 16 16 10 T T
T 45 57 _ 43 35 30 33
T — T_14___
21
T
—
T 27
20 26
— —
15 15
42 43
— T
VOL. 6, No. 4, MARCH 1973
341
-------
TABLE M-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—Texas—Continued
YEAR
RESIDUES IN PPB (na/Ka)
JAN. FEB.
STATION
1967
DDE
TDE
DDT
Dieldrin
23 24
54 49
T T
— —
MAR. APR.
8.— ARANSAS
27 —
56 —
— _
— 28
MAY JUNE JULY AUG. SEPT. OCT. Nov. DEC.
BAY— 19 SAMPLES1 — Continued
T
—
—
48
STATION 9.—COPANO BAY—51 SAMPLES L
1967 DDE
TDE
DDT
1968 DDE
TDE
DDT
1969 DDE
TDE
DDT
1970 DDE
TDE
DDT
1971 DDE
TDE
DDT
—
—
14 18 — 21 T 12
23 28 — 24 — —
T
— T — 50 15 17
T — — 21 27 23
— — — T 18 T
17 15 25 14 — —
11 T T 14 — —
T T — T — —
10 13 15 17 10 —
10 11 10 39 10 —
— — — — — —
— — — T — 21
— 96 — 20 — 30
«p
T — — — — —
______
______
— — — — — 15
_____ T
_____ T
______
______
______
— —
— —
— —
STATION 10.—RED FISH BAY—67 SAMPLES '• •
1966
1967
1968
1969
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
29
25
12
T 15 17 — T 14
T 21 32 — 14 39
— T T — — 13
23 25 18 12
14 26 43 18
— 12 21 —
15 T 10 15 — T
27 T 19 27 18 T
13 — T 19 — —
24 12
21 18
12 —
T —
19 —
14 —
10 10
21 18
— T
— —
— —
— —
T T T T
T 11 T T
— — — —
11 — — 10
15 21 — 17
23 T — T
12 — 11 17
— — 15 22
— — T 13
342
PESTICIDES MONITORING JOURNAL
-------
TABLE M-2.—Distribution of organochlorine residues in C. virginica for each sampling .station by date of
collection—Texas—Continued
YEAR
COMPOUND
RESIDUES IN PPB (^O/KO)
JAN. FEB. MAS. APR. MAY JUNE JULY AUG. SEPT. OCT.
Nov. DEC.
STATION 10.—RED FISH BAY—67 SAMPLES i—Continued
1970
1971
1972
DDE
TDE
DDT
PCB's
DDE
TDE
DDE
DDE
TDE
DDT
PCB's
16
20
T
-
—
—
—
14
11
22
—
14
19
10
—
—
—
—
20
15
30
—
18
29
13
—
17
14
—
16
16
45
—
17 10 12
25 23 38
12 — —
_ _ —
— T —
_ — —
— — —
18 — T
12 — 14
42 — 34
— — °"
14 12 — — — T
25 18 — — — 29
_ 10 — — — 15
(3)
_ 13 T T T 12
13 — T T 11
— 17 26 17 22 26
'
STATION 11.—NUECES BAY—20 SAMPLES1
1965
1966
1967
1968
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
Endrin
DDE
TDE
DDT
T
—
—
34 22 32 120 18 18 T T
30 17 26 200 20 16 — 12
12 — 14 130 — — — —
33
31 30 43 46 34 29 20 32 57 51
36 61 110 110 48 52 20 20 25 22
T 22 20 26 22 49 17 37 35 15
— 11 13 19 — — _____
— 18 12 11 — — _ — — _
45
28
15
STATION 12.—ARROYO COLORADO—48 SAMPLES1
1965
1966
DDE
TDE
DDT
Dieldrin
Endrin
DDE
TDE
DDT
Dieldrin
Endrin
170
520
20
19
—
80 120 120 74 96 230 300 270 98
110 140 130 70 69 140 230 93 57
21 '9 17 — 26 31 53 24 —
32 23 ™ 18 16 30 45 27 14
18 n H - 22 23 28 13 _
24 55
33 80
T 17
T 29
— 32
12 180
— 50
— 19
— 18
— 14
64
80
16
34
19
63
58
12
20
12
VOL. 6, No. 4, MARCH 1973
343
-------
TABLE M-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—Texas—Continued
YEAR
COMPOUND
RESIDUES IN PPB (/IG/KO)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT.
Nov. DEC.
STATION 12.—ARROYO COLORADO-^18 SAMPLES '—Continued
1967
1968
1969
1970
1971
DDE
TDE
DDT
Dieldrin
Endrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
Endrin
DDE
TDE
DDT
Dieldrin
Toxaphene
120
73
27
23
11
* 260
110
15
14
23
35
32
21
T
140 210
110 180
— 28
16 42
29 19
48
150
—
—
330 220
100 48
57 35
16 17
120 140
20 25
19 22
18 23
y
170
75
19
46
—
320
100
110
18
110
29
T
25
—
65
14
—
11
—
110
49
26
19
—
180
35
77
14
130
25
T
13
. —
280
61
—
27
—
160 160 79
63 92 49
24 23 16
33 30 16
— 12 —
160
68
49
33
260 280 86 100 110 210 54
63 55 28 33 30 T 21
48 22 — — T — 24
25 18. 17 12 T 16 —
96
54
60
25
12
380 220
46 78
— —
24 38
(3)
STATION 13.—LOWER LAGUNA MADRE—24 SAMPLES'
1965
1966
1967
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
T T — —
— — — —
— — — —
_ _ — — 27 T T 13 — 12 — T
19 _
II
_____ 46 — — — — — —
II 15 13 13 10 12 T —
*J-
— — — — 14 T — —
* Each sample represents 15 or more mature mollusks.
1 DDT present but not quantified due to presence of PCB's in sample.
9 Present but not quantified.
* Dieldrin data omitted because of possible sample contamination.
344
PESTICIDES MONITORING JOURNAL
-------
SECTION N.—VIRGINIA
The eastern oyster, Crassostrea virginica, was monitored
at 10 principal stations in estuarine areas of Virginia
during the period July 1965 February 1972. Samples
were analyzed at the Gulf Breeze Laboratory until June
1968, and thereafter at the Virginia Institute of Marine
Science. The approximate station locations are shown
in Fig. N-l. A summary of data on organochlorine res-
idues in the monitored species, C. virginica, is presented
in Table N-l, and the distribution of residues in this
species for each sampling station by data of collection
in Table N-2.
The 87% incidence of DDT residues in Virginia samples
and the maximum residue of 678 ppb were fourth highest
of the States monitored. The higher residues were clearly
associated with intensive truck farming (Station 2) and
a combination of urban and industrial development
(Station 9).
The presence of PCB's was noted in 1970 samples, but
not until 1971 was equipment acquired to identify and
quantify these compounds. The residue of 2,800 ppb of
Aroclor 1254® detected in oysters in the Elizabeth
River, a highly industrialized area, has prompted a
special study to pinpoint the source of this pollution.
Trends in DDT residues in Virginia oysters differ some-
what from other areas in that, while the larger residues
(those above 100 ppb) decreased by 66% in 1971,
100% of the 1971 samples contained residues in excess
of 11 ppb as compared to 82% in earlier years. It ap-
pears that DDT residues are more widely dispersed but
at relatively lower levels, presumably through the proc-
esses of recycling.
FIGURE N-l.—Diagram of coastal Virginia showing
approximate location of monitoring stations
1. Machipongo River
2. Cherrystone Inlet—Chesapeake Bay
3, Bowlers Rock—Rappahannock River
4. Urbanna—Rappahannock River
5. Bell Rock—York River
6. Pages Rock—York River
7. Deep Water Shoals—James River
8. Nansemond Ridge—James River
9. Hospital Point—Elizabeth River
10. Lynnhaven Bay
TABLE
N-l.—Summary of data on organochlorine residues in the monitored species (C. virginica), 1965-72—Virginia
STATION
NUMBER
1
LOCATION
Machipongo River
2 Cherrystone Inlet
3 ! Bowlers Rock
4
5
6
7
Urbana
Bell Rock
Pages Rock
Deep Water Shoals
MONITORING
PERIOD
1965-72
1965-72
1965-72
1965-72
1965-72
1965-72
1965-72
NUMBER OF
SAMPLES 1
67
68
70
69
69
68
69
NUMBER OF POSITIVE SAMPLES AND MAXIMUM
RESIDUE ( ) DETECTED IN PPB (JIG/KG)
DDT
56 (127)
67 (678)
62 (60)
59 (45)
35 (54)
50 (100)
69 (144)
DlELDRIN
2 (11)
1 (T)
38 (40)
PCB's 2
1 (390)
2 (510)
2 (400)
2 (270)
2 (450)
2 (400)
3 (1,000)
VOL. 6. No. 4. MARCH 1973
345
-------
TABLE N-l.—Summary of data on organochlorine residues in the monitored species (C. virginica), 1965-72—
Virginia—Continued
STATION
NUMBER
8
9
10
LOCATION
Nansemond Ridge
Hospital Point
Lynnhaven Bay
Occasional stations (4)
MONITORING
PERIOD
1965-72
1966-72
1965-70
1965-67
Total number of samples
Percent positive for indicated compound
NUMBER OF
SAMPLES '
64
58
62
5
669
NUMBER op POSITIVE SAMPLES AND MAXIMUM
RESIDUE ( ) DETECTED IN PPB (/IO/KG)
DDT
63 (128)
58 (300)
61 (113)
5 (241)
87
DffiLDRIN
29 (22)
38 (24)
4 (16)
17
PCB's *
2 (1,000)
3 (2,800)
3
NOTE: T = >5 but < 10 ppb.
1 Each sample represents 15 or more mature mollusks.
TABLE N-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of collection—Virginia
[Blank = no sample collected; — = no residue detected above 5 ppb or no residue detected (PCB's); T = >5 but <10 ppb]
YEAR
COMPOUND
RESIDUES IN PPB (^O/KO)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT.
Nov. DEC.
STATION 1.—MACHIPONGO RIVER—67 SAMPLES t
1965
1966
1967
1968
1969
1970
1971
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
DDE
TDE
DDT
PCB's
12 T 11 14 19 17
— — — T 14 18
— — — — — —
— 34 T T 17 14
— 59 — — 12 13
— 10 — — — T
T — T — T 18
— — — — — 10
— — — — — —
T T T T T 13
— T — — T 13
- T - - - T
______
15 10 — — 11 24
14 T — — T 35
— 11 — — — 17
10
—
—
—
34 15
20 13
73 T
11 —
19 —
17 —
— —
T T
— T
— T
15 T
20 T
10 —
— 11
— 16
— —
— —
T
13
T
18
T
—
—
18 T
28 T
24 —
— —
T T
— —
— —
— —
— —
— —
— T
— T
— —
T
T
—
T
T T
T T
— —
17
T
—
'390
13 15
T T
— —
— —
Y
— —
— —
T 11
— —
— —
11 T
T —
— —
20 T
27 T
16 —
— —
11
T
—
346
PESTICIDES MONITORING JOURNAL
-------
TABLE N-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—Virginia—Continued
YEAR
RESIDUES IN PPB (#O/KO)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT. Nov. DEC.
STATION 1.— MACHIPONGO RIVER— 67 SAMPLES1 — Continued
1972
DDE
TDE
DDT
12
—
—
STATION 2.—CHERRYSTONE INLET—68 SAMPLES 1
1965
1966
1967
1968
1969
1970
1971
1972
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
PCB'j
DDE
TDE
DDT
PCB's
45 42
49 46
23 26
49 31
74 52
17 14
19 27
18 20
T —
24 11
16 14
— —
32 33
30 42
T 16
43
18
—
'510
60 35
89 60
230 71
33 43 36 32 90 41
45 40 36 37 110 86
25 15 11 16 130 59
32 37 45 37 34 44
36 53 75 68 61 63
— 12 10 21 110 110
35 59 42 40 146 63
46 58 55 52 210 172
T 21 12 17 322 42
21 17 12 T 15 35
22 16 13 14 10 31
— — — — — 20
— 20 28 24
— 21 29 26
— — — 23
30 22
30 14
— 14
— —
16
14
T
14
35
T
55
81
120
20
76
12
17
22
11
T
T
T
24
42
35
25
66
20
26
42
22
33
31
T
24
34
13
22
34
T
29
23
T
= 350
25
32
20
19
62
11
24
29
13
33
31
T
16
10
—
18
19
—
45
55
35
36
73
14
20
18
T
T
15
T
35
39
17
19
22
T
STATION 3.—BOWLERS ROCK—70 SAMPLES
1965
1966
DDE
TDE
DDT
DDE
TDE
DDT
16
23
21
11 10 — 13 11 13 ,3
T T — T 11 15 15
— T —
T
T
T
T '
T
—
T T T
T T T
T T —
— — T
— — T
- — —
11
12
—
_
-
VOL. 6, No. 4, MARCH 1973
347
-------
TABLE N-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—Virginia—Continued
YEAR
COMPOUND
RESIDUES IN PPB (^O/KO)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG.
SEPT. OCT. Nov.
DEC.
STATION 3.—BOWLERS ROCK—70 SAMPLES '—Continued
1967
1968
1969
1970
1971
1972
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
• PCB's
DDE
TDE
DDT
— T T
— — T
— — —
— T T
— — T
_ _ _
T T 11
10 T 10
_ _ _
16 10 T
14 T —
T 32 —
17
12
—
10 T T
11 T 11
_ _ _
T T 13
— — 14
— — —
12 10 T
14 10 T
— T —
T 10 10
11 10 17
— — T
11
T
—
T
10 — — T
14 15 — T
11 18 — T
10 10 — 17
12 15 — 17
— T — T
T T T 11
T T 10 18
— T T 14
T T — T
12 T T 12
— — T T
T
—
—
—
T T
T T
T —
T 12
T T
— —
T 11
14 13
T T
— T
T T
— —
14
12
—
400
STATION 4.—URBANA—69 SAMPLES'
1965
1966
1967
1968
1969
1970
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
10 T
T —
— —
12 10
— T
_ _
T T
T —
— —
T T
T 11
T T
T
10
16
— — 10 T
— — — —
— — — —
11 T T T
T T T T
_ — — —
T 11 T 11
T T — —
— — — —
— T T T
— — T T
— — — —
— T 10 15
— T T 17
, "J"
T
13
14
15
—
—
T
T
—
14
13
T
T
T
—
T
T
T
10
16
19
—
—
—
T
T
T
T
T
—
T
13
—
—
T
—
T
T
T
T
—
—
—
—
—
—
—
—
T
T
—
10
17
14
T
—
—
—
—
—
T
11
12
T
T
—
11
18
16
—
—
—
T
—
—
—
—
—
11
T
—
T
12
T
T
10
—
T
T
—
13
10
—
T
T
T
11
12
T
T
T
—
T
10
—
T
T
T
348
PESTICIDES MONITORING JOURNAL
-------
TABLE N-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—Virginia—Continued
YEAR
COMPOUND
RESIDUES IN PPB (AO/KO)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT. Nov. DEC.
STATION 4.— URBANA— 69 SAMPLES '—Continued
1971
1972
DDE
TDE
DDT
• PCB's
DDE
TDE
DDT
10 T 10
T — T
— , — —
T — 270
10
T
—
STATION 5—BELL ROCK—69 SAMPLES *
1965
1966
1967
1968
1969
1970
1971
1972
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
PCB's
DDE
TDE
DDT
PCB's
11 T T T 11 16
24 T 13 10 13 21
19 T 10 T — 13
-p
— — — — — — ______
— — — — — — — — — — — —
_T — T — — — — — — — T
— T — — — — — — — 12 — T
— — — — — — ______
— — — — — — T T — T Til
— — — — — — 10 T — T T T
— — _ — __ _____n
— — — — — T — TT 11 17
— — — — — T — T 14 20 T
— — — — — — — — — 10 —
T T — T T 10 10 — — — — T
T 12 11 11 T 18 13 T T — — T
------ T _ _ _ _ _
T _ T
— T T
_ _ _
— — 3390
T
T
—
M50
STATION 6.—PAGES ROCK—68 SAMPLES 1
1965
DDE
TDE
DDT
10
15
17
T
12
14
11
17
14
11
14
12
17
20
24
19
16
13
VOL. 6, No. 4, MARCH 1973
349
-------
TABLE N-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—Virginia—Continued
COMPOUND
RESIDUES IN PPB (AG/KO)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT.
Nov. DEC.
STATION 6.—PAGES ROCK—68 SAMPLES '—Continued
1966
1967
1968
1969
1970
1971
1972
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
Dicldrin
DDE
TDE
DDT
DDE
TDE
DDT
PCB's
DDE
TDE
DDT
14 — — T 10 11 — T — T
13 — — — Til — T — T
T — — — — — _ ii _ _
— — T 10 — T T — T T
— — — T — T T — 1614
— — — — — — — TUT
TT — T — 1213T — T
— T — — — — 14 10 — 10
— — — — — — — ___
— — T — T 13 — 14 18
— — T — T T — 12 16
T T T
L — — 1 1
. •y
— T — T T T TT — 10
T 20 — T T 29 12 T — T
— — — — — 11 T — — —
X
— —
— —
3 f
T
T
—
— T
— T
— —
T 13
11 14
— —
T 10
T 11
— T
T
T
—
—
90 T
10 T
— —
T
T
—
MOO
STATION 7.—DEEP WATER SHOALS—69 SAMPLES 1
1965
1966
1967
1968
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
37
43
15
—
21
29
12'
—
15
15
—
—
11
15
—
-
26
30
13
14
14
12
—
—
24 26
24 32
— —
— 23
19 31
21 41
— 19
— 40
17 15
15 15
— —
12 12
30
45
14
34
19
25
13
28
15
19
11
16
40
63
20
38
20
32
12
21
23
29
14
16
21
52
63
14
37
57
30
16
20
37
18
22
18
26
15
T
18
31
35
T
23
41
22
17
T
25
24
11
14
25
18
T
10
17
T
—
11
21
T
—
17
33
28
12
T
T
—
13
22
13
—
18
29
16
12
13
20
10
—
15
20
T
30 40
41 56
17 23
11 13
19 17
28 32
15 10
— —
21 19
23 20
11 24
— —
12 19
T 18
T T
T —
350
PESTICIDES MONITORING JOURNAL
-------
TABLE N-2.—-Distribution of organochlorine residues in C. virginka for each sampling nation hy date of
collection—Virginia—Continued
YEAR
COMPOUND
RESIDUES IN PPB (^O/KG)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT.
Nov. DEC.
STATION 7.—DEEP WATER SHOALS—69 SAMPLES '—Continued
1969 DDE
TDE
DDT
Dieldrin
1970 DDE
TDE
DDT
Dieldrin
1971 DDE
TDE
DDT
Dieldrin
3 PCB's
1972 DDE
TDE
DDT
Dieldrin
PCB's
T T 10 11 T 10
10 T 12 T T 16
_ — — — T T
- Lost - — T 11
41 19 10 27 10 12
43 22 11 12 T 21
60 — T — — 17
T — 14 — — 12
40
35
T
31
1,000
15
15
—
10
» 760
18 14 17 20 28
12 16 23 28 40
— !6 21 20 29
13 T — T 14
T 40 20 25 12 17
22 7) 40 43 17 28
T 10 14 25 T 12
16 -- — — —
T 16
13 21
— —
T 17
— 560
STATION 8.—NANSEMOND RIDGE—64 SAMPLES '
1965
1966
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
' Dieldrin
1967
1968
1969
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
28 14 16 30
35 14 17 34
15 - — 13
— — — 17
14 17 22 18
17 16 20 18
T T 11 T
— - — 15
12 15 14 12
14 16 15 12
— T T —
— 11 — 10
— T 10 T
T T 11 T
- - - -
— — —
30
29
13
14
15
17
T
12
.12
47
45
21
r
T
-
—
36
52
29
22
14
20
12
T
24
32
27
15
11
23
10
10
16
59
53
17
14
24
11
12
23
38
23
T
T
r
-
-
17
43
39
T
34
55
29
14
18
35
20
14
14
29
15
-
50
37
23
-
17
29
25
-
13
18
_._
-
T
16
T
—
-
—
_-
—
11
14
T
-
T
14
T
-
10
15
10
-
13
24
10
—
T
12
—
—
16
28
13
11
27
37
24
T
11
16
16
—
17
24
13
10
16
13
T
—
36
49
31
11
11
19
10
—
20
25
10
11
16
22
T
—
16
26
12
T
VOL. 6, No. 4, MARCH 1973
351
-------
TABLE N-2.—Distribution of organochlorine residues in C. virginica for each sampling station by date of
collection—Virginia—Continued
YEAR
COMPOUND
RESIDUES IN PPB (/IG/KO)
JAN. FEB. MAR.
APR.
MAY JUNE JULY AUG. SEPT. OCT.
Nov. DEC.
STATION 8.—NANSEMOND RIDGE—64 SAMPLES'—Continued
1970
1971
1972
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
PCB's
DDE
TDE
DDT
Dieldrin
12 15 — 10 16 10 T T
18 40 13 27 23 14 12 14
22 11 35 — 13 11 10 T
T — T T — — — —
22 11 16
18 13 15
T — —
15 T 20
3 1,000 — "440
16
14
—
—
STATION 9.—HOSPITAL POINT—58 SAMPLES1
1966
1967
1968
1969
1970
1971
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
'PCB's
42 52
48 53
31 29
T 16
68 92
79 67
52 55
13 19
12 17
T 11
— —
— T
15 32
21 54
T 29
T 14
140
120
40
13
26
17
T
—
60
48
18
15
31
32
11
10
15
35
—
—
82
73
32
18
34
25
—
—
57
49
20
12
22
21
19
10
63
60
31
15
33
42
20
11
48
48
34
12
15
28
11
T
40
40
20
T
2,800
83
130
89
20
37
76
58
16
50
93
83
13
15
28
27
T
66
96
39
—
29
67
36
—
30
67
35
10
15
33
15
10
T
22
10
-
37
63
43
—
11
55
31
-
20
62
19
T
28
70
41
T
18
37
19
—
20
36
22
—
20
59
62
10
14
30
12
—
33
84
46
19
24
57
28
—
27
49
—
12
—
24
53
35
—
39
83
63
15
13
24
10
10
32
92
71
10
13
30
14
—
26 27
42 40
27 24
— T
43* 54
78 64
100 37
16 19
11 26
17 21
T T
11 —
29
56
36
T
T 15
13 23
— 11
— —
27
32
—
24
960
352
PESTICIDES MONITORING JOURNAL
-------
TABLE N-2.—Distribution of organochlorine residues in C. virginica for each sampling station b\ date of
collection—Virginia—Continued
YEAR
RESIDUES IN PPB (AC/KG)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT. Nov. DEC.
STATION 9.— HOSPITAL POINT— 58 SAMPLES '—Continued
1972
DDE
TDE
DDT
Dieldrin
a PCB's
34
32
—
Lost
1,440
STATION 10.—LYNNHAVEN BAY—62 SAMPLES 1
1965
1966
1967
1968
1969
1970
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
19 20 17 32 16 25
25 29 20 39 20 41
— — — T — 11
16 22 19 29 15 24
19 27 21 36 18 35
T T — T T 20
— — — 16 — —
19 T 29 18 27 30
22 — 43 18 28 45
T — 12 — 10 27
— — 13 — — _
18 16 12 11 16 12
16 22 21 11 21 16
— T T — T —
— — — _ — _
— 20 — T 11 28
— 26 — — — 29
T - - - - T
26
49
17
10
36
59
15
34
57
22
—
15
T
12
—
18
33
12
—
16
21
—
17
33
17
—
14
21
T
—
27
28
17
—
13
10
T
—
16
24
—
21
33
16
—
11
13
—
—
14
18
T
—
18
20
10
14
24
—
—
14
22
T
20
32
17
—
16
20
T
—
11
20
10
—
13
14
T
—
17
27
—
20
27
11
—
15
12
—
—
20
28
—
10
18
12
T
31
40.
T
—
14
26
T
18
25
10
—
19
20
T
—
18
23
—
—
14
23
T
1 Each sample represents 15 or more mature mollusks.
•J Calculated as Aroclor 1242®.
•• Calculated as Aroclor 1254®.
VOL. 6, No. 4, MARCH 1973
VS53
-------
SECTION O.—WASHINGTON
The Pacific oyster, Crassostrea gigas, was used to moni-
tor 19 estuarine sites at monthly intervals in the period
October 1965 December 1968. All samples were
analyzed at the Gulf Breeze Laboratory. The approxi-
mate station locations are shown in Fig. O-l. A summary
of data on organochlorine residues in the monitored
species, C. gigas, is presented in Table O-l, and the
distribution of residues in this species for each sampling
station by date of collection in Table O-2.
The monitoring program was terminated in Washington
after 3 years because of the absence of detectable DDT
residues in mcst samples. This was due to the absence of
DDT pollution and not because of any lack of sensitivity
on the part of the monitored species. Analyses of
samples of *he Pacific oyster in California waters had
demonstrated its ability to store organochlorine residues
at levels comparable to other molluscan species in the
same estuary.
The overall incidence of DDT residues in Washington
samples was only 11%. The maximum residue detected,
176 ppb, was the obvious result of a sfnglc pollution
incident. Station 18 was the only one demonstrating a
continuing, but low-level pollution problem. The fact
that residues at this station were primarily DDT rather
than one of its metabolites suggests a direct application
of the pesticide to coastal waters. Analytical data are too
few, even at Station 18, to indicate any trend in DDT
pollution. The overall picture is that of an estuarine area
of the United States that was remarkably free from DDT
pollution in the period 1965-68.
FIGURE O-l.—Diagram of coastal Washington showing
approximate location of monitoring stations
11.
1. Stackpole Harbor—Willapa Bay
2. Olson Slough—Willapa Bay
3. Bear River—Willapa Bay 12.
4. Naselle River—Willapa Bay
5. Nemah River—Willapa Bay 13.
6. Stony Point—Willapa Bay 14.
7. South Bend—WiMapa Bay 15.
8. Bearclslee Slough— 16.
Grays Harbor 17.
9. Oyehut—Grays Harbor 18.
10. Scquim Bay 19.
North Bay Reserve—
Puget Sound
Oakland Bay Reserve—
Puget Sound
Mud Bay—Puget Sound
Padilla Bay—Padilla Bay
Swinomish—Padilla Bay
Scott Point—Samish Bay
Rock Point—Samish Bay
Lummi—Lummi Bay
Blaine—Drayton Harbor
TABLE O-l.—Summary of data on organochlorine residues in the monitored species (C. gigas), 1965-68—Washington
STATION
NUMBER
1
2
3
4
5
6
7
8
9
10
11
12
LOCATION
Stackpole Harbor
Olson Slough
Bear River
Naselle River
Nemah River
Stony Point
South Bend
Beardslee Slough
Oyehut
*•• •uim Bay
North Bay Reserve
Oakland Bay Reserve
MoNMORING
PERIOD
1965-68
1966-68
1965-68
1965-68
1965-68
1965-68
1965-68
1965-68
1966-68
1966-68
1965-68
1965-68
NUMBER OI
SAMPLES '
38
30
38
38
39
39
39
NUMBER OF POSITIVE SAMPLES AND MAXIMUM
RESIDUE ( ) DETECTED IN PPB (^G/KG)
DDT
9 (25)
7 (55)
3 (17)
! (11)
4 (21)
10 (176)
6 (23)
37 2 (27)
36
31
33
33
DlELDRIN
1 (120)
354
PESTICIDES MONITORING JOURNAL
-------
TABLE O-l.—Summary of data on organochlorine residues in the monitored species (C. gigas), 1965-68—
Washington—Continued
STATION
NUMBER
13
14
15
16
17
18
19
LOCATION
Mud Bay
Padilla Bay
Swinomish
Scott Point
Rock Point
Lummi
Blaine
Occasional stations (2)
MONITORING
PERIOD
1965-68
1965-68
1965-68
NUMBER OF
SAMPLES '
32
39
38
1965-68 ; 39
1965-68 , 37
1965-68
1965-68
1966
Total number of samples
Percent of samples positive for indicated compound
38
38
1
695
NUMBER OF POSITIVE SAMPLES AND MAXIMUM
RESIDUE ( ) DETECTED IN PPB (MO/KG)
DDT
8 (17)
1 (T)
4 (10)
23 (99)
11
DrELDRIN
<1
NOTE: T = >5 but <10 ppb.
' Each sample represents 15 or more mature mollusks.
TABLE O-2.—Distribution of organochlorine residues in C. gigas for each sampling station by date of collection—Washington
[Blank = no sample collected; — = no residue detected above 5 ppb; T = >5 but <10 ppb]
YEAR
COMPOUND
RESIDUES IN PPB (/IG/KG)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT. Nov. DEC.
STATION 1.— STACKPOLE HARBOR— 38 SAMPLES'
1965
1966
1967
1968
1966
1967
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
T T —
T — —
T — —
— — TTT13 11 _ T — — —
— — T — — 12 — — — — — —
- — -- — — — — — — — —
----T-__ ___
-J-
— — — — — — — — ___
-------_____
.
— - — - — — - — — ___
STATION 2.— OLSON SLOUGH— 30 SAMPLES '
— 20 T — — — —
19 10 — — — —
— 16 — — — — —
-T-T — T-— ___
— 11 — T — 12 — _ ___
— 14 — 14 — 14 — — — — _
VOL. 6, No. 4, MARCH 1973
355
-------
TABLE O-2.—Distribution of organochlorine -residues in C. gigas for each sampling station by date of
collection—Washington—Continued
YEAR
1968
1965
1966
1967
1968
1965
1966
1967
1968
1965
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
Dieldrin
DDE
TDE
DDT
RESIDUES IN PPB (/US/KO)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT. Nov. DEC.
STATION 2.— OLSON SLOUGH— 30 SAMPLES ' — Continued
T — __________
15 — 14 — — — — — — — — —
STATION 3.— BEAR RIVER— 38 SAMPLES '
— — T
_ _ _
— — —
_____T______
— — ___ 12 ______
____________
-J*
_____T_- ___
_____T-_ ___
— — — — — — — — — — — —
____________
— — — — — — — — — — — —
STATION 4.— NASELLE RIVER— 38 SAMPLES >
— — —
_ _ _
— — —
— — — — — _ _ — ___ —
____________
— — — — — — — — — — — —
— _ — ____ — _ — —
— — — — — — — — ___
________ ___
____________
________ — — — —
H_____ — — — — — —
______ 120 — — — — —
STATION 5.— NEMAH RIVER— 39 SAMPLES >
— T —
— — —
— — —
356
PESTICIDES MONITORING JOURNAL
-------
TABLE O-2.—Distribution of organochlorine residues in C, gigas for.each sampling station by date of
collection—Washington—Continued
RESIDUES IN PPB (^G/KG)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT. Nov. DEC.
STATION 5.— NEMAH RIVER— 39 SAMPLES '—Continued
1966
1967
1968
1965
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
_ _ _ — — 11 T — — — — —
___ — — 10 ______
______ ______
______ ______
______ ______
___ — _ — ______
_______ — — — — —
_____ — _ — _ — — —
T — — — — — — — — — — —
STATION 6.— STONY POINT— 39 SAMPLES 1
DDE
TDE
DDT
T T —
14 T —
T — —
1965
1966
1967
1968
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
T T —
14 T —
T — —
T — — T T T 13 — — — — —
T — — T — 11 11 — — — — —
— — — — — — ______
— — — — — — — — — — — —
— — _ — __ _ 26 — — — —
— — — — — — — 150 — _ _ _
— — — —
— — — — — — — — — — — —
T — T — — — ______
STATION 7.—SOUTH BEND—39 SAMPLES 1
1965
1966
1967
1968
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
— _ _
T — T T T — 10 — — — __
T — 16 T — — 13 — ____
— — — T — _______
— T
— T — _______
— T — — __ _____
— —
— — ____ ___
~ ~ ~ — — — — — — — __
VOL. 6, No. 4, MARCH 1973
357
-------
TABLE O-2.—Distribution of organochlorine residues in C. gigas for each sampling station by date of
collection—Washington—Continued
YEAR
COMPOUND
RESIDUES IN PPB (/WJ/KO)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT. Nov. DEC.
STATION 8.— BEARDSLEE SLOUGH— 37 SAMPLES i
1965
1966
1967
1968
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
—
—
—
— — — — — —
— — — — — — — — — — — —
— — — — — — ______
— — __T — j — — — — —
____!!_ !!_____
— — _ _ 11 _ io_ — __ —
— — — — — — — _ — — — —
____________
____________
STATION 9.— OYEHUT— 36 SAMPLES 1
1966
1967
1968
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
______ ______
— — — — — — ______
______ ______
______ ______
— — — — — — — — — — — —
— — — — — — — — — — — —
______ ______
____________
— — — — — — — — — — — —
STATION 10.— SEQUIM BAY— 31 SAMPLES '
1966
1967
1968
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
_ _______
— _______
— _______
— — — — — — — — — — —
— — — — — — — — — — —
— — — — — — — — — — —
____________
____________
— — — — — — — — — — — —
358
PESTICIDES MONITORING JOURNAL
-------
TABLE O-2.—Distribution of organochlorine residues in C. gigas for each sampling station by date of
collection—Washington—Continued
VTTID
TEAR
COMPOUND
RESIDUES IN PPB (AG/KC)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT. Nov. DEC.
STATION 11.— NORTH BAY RESERVE— 33 SAMPLES 1
1%5
1966
1967
1968
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
—
—
—
___ _____ — — —
___ ____ — — — —
___ ________
______ _____ _
______ __ — — — —
______ ______
___ — _ — _ — —
_________
— — — — — — — — —
STATION 12.— OAKLAND BAY RESERVE— 33 SAMPLES '
1965
1966
1967
1968
1965
1966
1967
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
—
—
—
— — — — — — — — — — —
___ ________
___ ________
— — — — —
______ ______
______ ______
— — — —
— — — — — — — — —
— — — — — — — — —
STATION 13.— MUD BAY— 32 SAMPLES 1
_
—
--- ---_____
— — — — — — — — — — —
___
— — - — — — — — — ___
VOL. 6, No. 4, MARCH 1973
359
-------
TABLE O-2.—Distribution of organochlorinc residues in C. gigas fur each sampling station by date of
collection—Washington—Continued
YEAR
COMPOUND
RESIDUES IN PPB ; m, KG)
JAN. FED. MAS. APR. MAY JUNE JLH AUG. SEPT. OCT. Nov. DEC.
STATION 13.— MUD BAY— 32 SAMPLES '— Contimwd
1968
DDE
TDE
DDT
________
STATION 14.— PADILLA BAY— 39 SAMPLES '
1965
1966
1967
1968
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
T T —
y ___ f
_________ ___T T T —
12 T T
T_-________ —
STATION 15.— SWINOMISH— 38 SAMPLES '
1965
1966
1967
1968
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
_ _ _
— — —
— — -
_________ __
______ ______
______ ______
_T__________
_______ ______
------ ____ _ _
______ ______
STATION 16.— SCOTT POINT— 39 SAMPLES '
1965
DDE
TDE
DDT
— — —
— — —
_ _ _
360
PESTICIDES MONITORING JOURNAL
-------
TABLE O-2.—Distribution of organochlorine residues in C. gigas for each sampling station by date of
collection—Washington—Continued
YEAR
COMPOUND
RESIDUES IN PPB (^O/KO)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT.
Nov. DEC.
STATION 16.—SCOTT POINT—39 SAMPLES '—Continued
1966
1967
1968
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
STATION 17.—ROCK POINT—37 SAMPLES »
1965
1966
1967
1968
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
STATION 18.—LUMMI—38 SAMPLES »
1965
1966
1967
1968
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
_ _ _
_ _ _
T T
------ -___15_
-----T -____,„
T — TTTUTT — T — T
10 -TUT 14 T T T T — T
25 19 29 44 42 74 34 14 15 21 21 19
T - — _ 10 T T T — — _
— — — — T T — — _-__
17 19 22 33 25 24 14 17 - - -
VOL. 6, No. 4, MARCH 1973
361
-------
TABLE O-2.—Distribution of organochlorine residues in C. gigas for each sampling station by date of
collection—Washington—Continued
YEAR
COMPOUND
RESIDUES IN PPB (/KJ/KO)
JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT. Nov. DEC.
STATION 19.— ELAINE— 38 SAMPLES'
1965
1966
1967
1968
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
DDE
TDE
DDT
— — —
_ _ _
— — — — —
— — — — — — — — — — — —
— — — — — — ______
— — — — —
— — — — — — — — — — —
— — — — — — — — — — — —
— — — — „_
— — — — — — — — — — —
— — — — — — — — — — —
1 Each sample represents IS or more mature mollusks.
362
PESTICIDES MONITORING JOURN.
-------
CONTRIBUTION NO. 156
-------
RESIDUES IN FISH, WILDLIFE,
AND ESTUARIES
Accumulation and Movement of Mirex in Selected Estuaries
of South Carolina, 1969-71 '
P. W. Borthwick2, T. W. Duke3, A. J. Wilson, Jr.3, J. I. Lowe2,
J. M. Patrick, Jr.2, and J. C. Oberheu3
ABSTRACT
In conjunction with a fire ant eradication program during
which mirex was aerially applied to coastal areas near
Charleston, S. C., field studies were conducted to monitor
the movement and accumulation of mirex in the estuarinc
system.
Collections of background and periodic posttreatmenl
samples of water, bottom sediments, shrimp, crabs, fish, and
estuary-dependent birds and mammals were analyzed for
mirex using electron-capture gas chromatography.
The data revealed that (I) mirex was translocated from
treated lands and high marsh to estuarinc biota—all animal
classes sampled contained mirex: and (2) biological concen-
tration of mirex occurred—especially in predators such as
racoons and birds.
Mirex residue ranges for respective sample categories were:
water «10.01 ppb); sediment (0-0.07 ppm); crabs (0-0.60
ppm); fishes (0-0.82 ppm); shrimps (0-1.3 ppm); mammals
(0-4.4 ppm); and birds (0-17.0 ppm). No mass mortalities
were observed during the study.
Introduction
Mirex, a chlorinated hydrocarbon, is the insecticide
component of a bait used in the Southeastern United
States to control the imported fire ant (Solenopsis saevis-
sima richteri Forel). This bait was developed after
various pesticides and pesticide-bait formulations applied
to control the ants proved to be toxic to nontarget or-
1Contribution No. 156 from the Gulf Breeze Environmental Research
Laboratroy, U.S. Environmental Protection Agency, Gulf Breeze.
Fla. 32561, an Associate Laboratory of the National Environmental
Research Center, Corvallis, Oreg.
"Gulf Breeze Environmental Research Laboratory, U.S. Environmental
Protection Agency, Gulf Breeze, Fla. 32561.
a Bureau of Sport Fisheries and Wildlife, U.S. Department of the In-
terior, Atlanta, Ga. 30323.
ganisms. Large-scale applications of dieldrin and hep-
tachlor were especially destructive to fish and wildlife
(2). Mirex was developed specifically to control fire ants
and, until recently, was not considered to be toxic to
nontarget organisms.
Independent experiments conducted under controlled
conditions in the laboratory at Gulf Breeze, Fla., and at
Bears Bluff, S. C., showed this chemical to be toxic to
decapod crustaceans, including juvenile blue crabs and
penaeid shrimp (1, 5, 6). Because of these and other
results and concern of commercial fishermen that ap-
plication of mirex to marsh areas could adversely affect
fishery resources, application of mirex to the coastal en-
vironment was suspended and this cooperative study was
undertaken.
The Gulf Breeze Laboratory (formerly a biological
laboratory of the Bureau of Commercial Fisheries, Fish
and Wildlife Service, U.S. Department of the Interior)
entered an agreement with the U.S. Department of
Agriculture in September 1969, to study the accumula-
tion and movement of mirex in the estuarine environ-
ment near Charleston. S. C. The Bureau of Sport
Fisheries and Wildlife of the Fish and Wildlife Service
also agreed to participate in the study which terminated
July 1, 1971.
The Gulf Breeze Laboratory was responsible for de-
signing the study; collecting samples of bottom sediment,
water, shrimp, crabs, and fish; and for analyzing all
samples. The Bureau of Sport Fisheries and Wildlife was
responsible for collecting birds and mammals.
The purposes of this investigation were (1) to observe
the possible movement of mirex from treated areas near
PESTICIDES MONITORING JOURNAL
-------
Charleston, S. C., to the estuarine environment and (2)
to determine levels of mirex in organisms, particularly
crabs and shrimp, before, during, and after treatment of
the area. The investigation began approximately 1 week
before the first treatment was applied. Pretreatment
samples were collected to establish "background" levels
of mirex in the environment. The short time period be-
tween the start of the investigation and the application
of mirex, however, precluded studies necessary to de-
termine the ecological impact of this chemical on the
study area.
Methods
STUDY AREA
The estuaries in which these studies were conducted
border on a fire ant treatment area that extended 30
miles on either side of a line from Columbia to Charles-
ton, S. C. The boundary of the treatment area ended
approximately 12 miles from the coast (Fig. 1). Mirex,
therefore, was not applied directly to the salt marsh,
except in an experimental plot near Toogoodoo Creek.
The topography and biota of the estuarine environment
arc unique and often present special problems to environ-
mental studies. Estuaries along this portion of the East
Coast of the United States are protected by barrier islands
and are supplied silt-laden fresh water by creeks and
rivers on the mainland. These typical "Spartina"
marshes support transient populations of crabs, shrimp,
and fish that develop to maturity in the estuary, then re-
turn to the sea. In addition, resident populations of shell-
fish and some fin-fish inhabit the estuary throughout their
lives. Many predatory birds and mammals depend upon
the estuarine organisms for food.
MONITORING STATIONS
FIGURE 1.—Sampling sites in xelecivd eituuricx of South Carolina, 1969-71
VOL. 7, No. 1, JUNE 1973
-------
Descriptions of sampling sites with the dates of mirex
applications are given in Table 1.
Perodically, levels of mirex in water, sediment, and
biota were monitored at (1) four stations near the main
inland mirex-treated area and within and near a 2-square
mile plot of salt marsh that was treated experimentally,
(2) six stations located on the major rivers that drain the
main inland mirex-treated areas, and (3) a station in a
semi-enclosed tidal pond 4 miles from the main inland
mirex-treated area; the banks of the pond (2.5 acres)
were treated with mirex by hand spreader.
APPLICATION OF MIREX
The mirex 4X Bait formulation contained 84.7% corn-
cob grits, 15.0% soybean oil, and 0.39f mirex. The bait
was applied at a rate of 1.25 Ib per acre or 1.7 g of
technical mirex. This produced approximately 16 par-
ticles of bait per square foot.
TABLF I.—Description i>f sampling sites mid dales of mirex application,
South Carolina. 1969-71
MAP
LOCATION
NUMBER
(Fio. 1)
A
H
C
1)
1
-
.1
4
S
r,
7
NAME AND LOCATION
OF SAMPLING
SITU
Toogoodoo Creek
Lat. 32" 41' N
Long. 80° 18' W
do.
do.
do.
Riverland Terrace Pond
Lat. 32° 46' N
Long. 79° 59' W
Stono River ( Intracoastal
Waterway) at Lop
Bridge Creek
Lat. 32° 45' N
Long. 80° 08' W
Ashley River at
Runnymcadc Plantation
Lat. 32" 5.V N
Long. 80° 05' W
Cooper River at U.S.
Naval Ammunition
Depot
Lat. 32° 57' N
Long. 79° 56' W
Ashley River at
Oldtown Creek
Lat. 32° 48' N
Long. 79° 58' W
Wando River at
Bcresford Creek
Lat. 32° 53' N
Long. 79° 53' W
South Santee River
(Intracoastal Waterway)
at Alligator Creek
Lat. 32° 08' N
Long. 79° 19' W
REMARKS
Within the main inland mirex-treated area on the upper
reaches of the left branch of the Creek. Tidal marshlands
predominate along the north bank; pine woodlands lie
lo the south. Several homes are in the area. Also treated
experimentally by helicopter.
Begins at the mouth of Swinton Creek and continues east-
ward on lower part of the Creek; partially within the
mainland treated zone; marsh and woodland areas are
on each bank. A few homes arc along the south bank.
Also treated experimentally by helicopter.
Extends southward from the fork of Toogoodoo Creek,
jusl south of the experimental area treated by helicopter.
Extensive farmlands arc on the west bank, tidal marsh-
lands on the cast.
Begins 2 miles south of the helicopter-treated area and
continues to the mouth of Toogoodoo Creek on the
Inlracoastal Waterway. Uninhabited low marsh areas on
both sides of the creek arc riddled with many small
lidal creeks.
Seven miles from main inland treatment area, includes
one of two adjoining 1-acrc ponds in Riverland Terrace
(a residential area). The cast pond floods and drains
with ihe tides into Wappoo Creek (Intracoastal Water-
way), via two large culverts. The banks of the cast pond
were treated above the high tide mark by hand spreader.
homes along the cast side of the creek; the west side is
bordered by lidal marshes.
Old plantations and rural homes are located on the east
hank, and lidal marshes lie along the west side. This
fresh water station is inside the main inland mirex-trealcd
area.
Bordered on the west bank by high wooded ground. On
[he cast side, tidal marshes predominate. This fresh water
station is inside the main inland mirex-trcated area.
Six miles from Ihe main inland treated area. The Citadel
Military College is on the cast bank, Charles Townc
Landing on the west. Several industrial plants and homes,
in addition lo tidal marshes, are on this portion of the
river.
Six miles from the main inland treated area. This unin-
habited area is bordered by expansive marshlands and
marsh islands.
Six miles from the main inland treated area. This location
is uninhabited and near the Cape Remain Migratory Bird
Refuge.
APPLICATION DATES
1ST
10/14-15/69
do.
do.
do.
12/3/69
10/23/69
10/22/69
.
10/17/69
10/22/69
10/17/69
9/18/69
2o
6/3-4/70
do.
do.
do.
7/24/70
6/18/70
6/11/70
6/10/70
6/10/70
6/8/70
5/20/70
3D
10/27-28/70
do.
do.
do.
12/1/70
_
—
—
—
PESTICIDES MONITORING JOURNAL
-------
Mirex 4X Bait was applied by fixed wing multi-engine
aircraft (PV-2) to the inland treatment area, by heli-
copter to the Toogoodoo experimental plot, and by hand
spreaders to the banks of a tidal pond. All applications
were supervised by USD A.
The inland treatment area was divided into blocks that
varied from 350,000 to 450,000 acres. An electronic
guidance system (Decca Survey System) was set up in
each block. This system consisted of three transmitting
and receiving stations (one master station and two slave
stations). The master and one of the slave stations pro-
duced an electronic tracking signal. Equipment mounted
in the application aircraft received this signal which was
fed through a computer into a dacometer. By use of the
dacometer, the aircraft pilot could follow the signal
from one point to another along the tracking path.
Uniform application was made across each block by
flying along a series of parallel signals. The master sta-
tion and the second slave station produced a ranging
signal that designated the points along the tracking path
where the spraying was cut off. These signals, tracking
and ranging, were converted to a numbering system and
the system oriented to a map of the area being treated.
To allow for better control, helicopter applications of
the mirex bait at the recommended rate were made to
800 acres of the 1,200-acrc Toogoodoo Experimental
Area. The Bell G-2-A helicopter used for spraying was
equipped with two side-mounted hoppers (300 pounds
capacity each) with electrically operated hopper gates.
agitators, spinner-vane wheels, and spreaders for "posi-
tive control" at the ''cut-off point." Flying at an air-
speed of 40 miles per hour and an altitude of 80 feet
produced a 75-foot wide swath: at 40 feet, a 60-foot
wide swath. Helium-filled kytoons were used to mark
each swath path. Bait distribution was even at both
altitudes, but wind caused considerable aerial drift.
Approximately 2.5 acres surrounding the tidal pond
were treated three times at 6-month intervals by hand-
operated seed spreaders.
SAMPLING
Materials Sampled
Pesticides entering the estuarine environment become
part of the biogeochemical cycles continually in opera-
tion within that environment. Therefore, the water, sedi-
ment, and various biota were sampled to determine
routes of movement and reservoirs of the chemical in
the study area. Common and scientific names of aquatic
animals, birds, and mammals collected for mirex an-
alysis are listed in Table 2. Crabs and shrimp were of
special interest because of their sensitivity to mirex.
Bottom and filter-feeding fish were also collected since
they could accumulate mirex from food web organisms
VOL. 7, No. 1, JUNE 1973
that were often difficult to obtain because of their
transitory nature and movement to deeper water during
the winter. In general, representatives of all species of
aquatic organisms caught in the trawl were analyzed for
mirex.
In selecting the birds and mammals for residue analysis,
an effort was made to pick species that were (a) directly
dependent on the estuarine environment for food: (b)
most likely to live and feed in the area where they were
collected; and (c) plentiful enough to allow periodic
collections with a minimum of difficulty.
The raccoon was selected as the mammal for sampling
since it is the most abundant carnivore in the salt marsh,
where it preys heavily upon crustaceans and shellfish.
It was more difficult to decide upon a bird species.
Numerous gulls, shore birds, wading birds, and other
water birds are found in the estuary, but most of them
are migratory by nature, moving up and down the coast
in search of food or as a result of weather changes. Few
water birds are year-round residents. The clapper rail
was finally selected as the most sedentary and widely
distributed bird in the estuaries; when rails were not
available at a particular sampling station, wading birds
were collected. All animals collected during this study
are listed in Table 2.
Co/lection of samples
Aquatic animals were collected with a 12-foot, %-inch
bar mesh, otter trawl towed at 3 to 6 knots for 20
minutes at each station. Pond collections were made
with a 15-foot, 14-inch mesh haul seine. Occasionally,
animals near the surface were taken with dip nets; fidder
crabs and oysters were collected by hand. Raccoons
were captured with wire live-traps; birds were hunted
on foot or from a boat.
Several methods for collecting water (including carbon
filtration) were considered, but because of the high silt
content of the well-mixed water, water was collected
just below the surface in a 1-gallon glass jug and sealed
with a teflon-lined cap. A modified grab sampler was
used to collect bottom sediment at each station. A
sampler especially designed to collect the upper few
centimeters of bottom sediment was used in specified
instances.
Frequency of sampling
A pretreatment (background) and numerous posttreat-
ment samples were taken at all stations. Quarterly collec-
tions of birds and mammals were scheduled around
the first week in September, December, March, and
June. Periodically, this schedule was altered by 2 or 3
weeks to select the week of highest tides for the best
-------
TABLE 2.—Common and scientific names of aquatic animals, birds, and mammals collected for mirex residue analysis
CRABS
Blue crab
Common mud crab
Mud crab
Portunid crab
Sand fiddler
Brown shrimp
Brown-spotted shrimp
Grass shrimp
River shrimp
White shrimp
American eel
Allan tic.croaker
Atlantic menhaden
Atlantic silversidc
Atlantic thread herring
Bay anchovy
Blackcheek tonguetish
Black drum
Black sea bass
Bluefish
Fourspot flounder
Hogchoker
Mummichog
Pinfish
Sailfin molly
Sea catfish
Searobin
Sheepshead
Silver perch
Snook
Southern kingfish
Spot
Spotted hake
Spotted seatrout
Callinecies sapidus -Rathbun
Panopeus herbstii H. Milne Edwards
Rithropanopcus harrlsii Gould
Callinectes ornaius Ordway
II ca pugilaior (Hose)
SHRIMPS
Penaeus aztecus Ives
Penaeus duorarum Burkenroad
Palaemonetes pugio Holthuis
Macrobraclrium ohione (Smith)
Penaeus setiferus (Linnaeus)
FISHES
Anguilla rostrala (Lesueur)
Micropogon undulatus (Linnaeus)
Brevoortia tyrannus (Latrobe)
Menidia mcnidia (Linnaeus)
Opisthonema oglinum (Lesueur)
Anclwa miichilli (Valenciennes)
Symphurus plagiusa (Linnaeus)
Pogonias cromis (Linnaeus)
Ccntropristis striata (Linnaeus)
Pomatomus saltatrix (Linnaeus)
Paralichthys oblongus (Mitchill)
Trincctes maculatus (Bloch and
Schneider)
Fundulus heteroclitus (Linnaeus)
Lagodon rhomhoides (Linnaeus)
Poccilia latipinna (Lesueur)
Arius telis (Linnaeus)
Prionotus sp.
A rchosargus probatocephalus
(Walbaum)
Bairdiella chrysura Lacepede
Centroponius undccimalis (Bloch)
Menticirrhus americaniis (Linnaeus)
Lciottonius xanrhurus Lacepede
Urophycis regius (Walbaum)
Cynoscion nchulosus (Cuvier and
Valenciennes)
FISHES— Continued
Star drum
Striped killifish
Striped mullet
Weakfish
White catfish
White mullet
Winter flounder
Stellifer lanceolatus (Holbrook)
Fundulus majalis (Walbaum)
Mugil cephalus Linnaeus
Cynoscion regalis (Bloch and Schneider)
tctalurm catus (Linnaeus)
Mugil curema Valenciennes
Pseudopleuronectes arnericanus
(Walbaum)
MISCELLANEOUS AQUATIC ANIMALS
American oyster
Brief squid
Nudibranch
Southern periwinkjc
Crassostrea virginlca (Gmelin)
Lolligunciila bre\'is (Blainville)
Doris sp.
Littorina irrorata (Say)
BIRDS
American bittern
American egret
Anhinga
Belted kingfisher
Clapper rail
Common snipe
Green heron
Least bittern
Little blue heron
Louisiana heron
Marsh hawk
Pied-billed grebe
Plover
Snowy egrel
Sora rail
Virginia rail
White ibis
Willet
Yellow-crowned night heron
Botaurus lentiginosus (Rackett)
Casmerodius albus (Linnaeus)
Anhinga anhinga (Linnaeus)
Mcgaceryle alcyon (Linnaeus)
Rallus longirostris Boddgert
Capella galllnago (Linnaeus)
Butorides \-irescens (Linnaeus)
Ixobrychus cxilis (Gmelin)
Florida caendea (Linnaeus)
Hydranassa tricolor (Muller)
Circus cyancus (Linnaeus)
Podilymbus podiceps (Linnaeus)
Cliarndriux sp.
Leitcophoyx thula (Molina)
Porzana Carolina (Linnaeus)
Rallus limicola Vieillot
Eudocimus albus (Linnaeus)
Catoptrophorus semipaltnatus (Gmelin)
Nyctanassa riolacea (Linnaeus)
MAMMALS
Opossum
Raccoon
Didelpliis rirginiana (Linnaeus)
Procyon lotor (Linnaeus)
rail hunting. Samples were taken at the six river stations
24 hours and 3 months after each of two applications
of mirex to the inland treatment area by fixed wing
aircraft. Biweekly collections were made at the four
stations in the Toogoodoo Creek Plot during the entire
18 months of the study; mirex was applied to the ex-
perimental plot by helicopter three times at 6-month
intervals to high marsh only, i.e., marsh not normally
covered by tidal waters. Samples were taken from the
tidal pond 24 hours after each of three hand-spread
treatments and at irregular intervals between applica-
tions.
ANALYTICAL PROCEDURES
Preparation of samples
Crabs, shrimp, and fish were prepared separately by
pooling whole individuals, but birds and mammals were
10
prepared individually. Muscle tissue from breast and
upper wing in birds and thigh in raccoons and oil glands
and eggs from birds were analyzed. All samples were
ground and mixed thoroughly in a blender. A 30-g
subsample was blended with a desiccant mix composed
of 10% QUSO (a microfine precipitated silica) and
90% anhydrous sodium sulfate. This mixture was al-
ternately frozen and blended until a free-flowing powder
was obtained.
Sediment samples were spread on sheets of aluminum
foil and dried at room temperature. The dry sediment
was pulversized to a fine powder in a blender.
At this stage of preparation, the samples were wrapped
in aluminum foil, packaged in plastic bags, and mailed
to the Gulf Breeze Laboratory for extracting and
pesticide residue analysis.
PESTICIDES MONITORING JOURNAL
-------
Water samples were refrigerated for up to 2 weeks
until they were mailed to Gulf Breeze for analysis.
Analysis of samples
Tissues of shrimp, crabs, and fish mixed with the
desiccant were extracted for 4 hours with petroleum
ether in a Soxhlet apparatus. Extracts were concentrated
to approximately 10 ml and transferred in 3- to 4-ml
portions to a 400-mm by 20-m.m chromatographic
column that contained 76 mm of unactivated Florisil.
After each portion settled in the column, vacuum was
applied to evaporate the solvent. This was repeated after
each addition and after three 5-mt petroleum ether
rinses of the extraction flask. The vacuum pump was
disconnected after all solvent had evaporated, and the
residue was eluted from the column with 70 ml of 9:1
mixture of acetonitrile and distilled water. The eluate
was evaporated to dryness and the residue transferred
to a Florisil column (7) with petroleum ether.
Sediment samples were dried at room temperature and
extracted for 4 hours with 10% acetone in petroleum
ether in a Soxhlet apparatus. Extracts were concentrated
to approximately 10 ml and transferred to a Florisil
column (7).
Water samples were not filtered before being extracted
with petroleum ether. The extracts were dried with
anhydrous sodium sulfate and reduced to an appropriate
volume.
The extracts of all substrates were identified and meas-
ured by electron capture gas chromatography. Extract
volumes were adjusted to obtain a sensitivity of 0.01
ppm (mg/kg) for tissue and sediment samples and 0.01
ppb (^g/liter) for water samples. Operating conditions
of the two 152.4-cm by 3.2-mm glass columns used
were:
Liquid phase:
Solid Support:
Temperatures:
Oven:
Injector and detector
N2 flow rate:
Laboratory tests indicated recovery rates for mirex were
greater than 85%. Data in this report do not include a
correction factor for percent recovery. All residues re-
ported are on a wet-weight basis, except those of sedi-
ments, which are reported on a dry-weight basis. Thin
layer chromatography. "p" values, and mass spectro-
metry were used to confirm the presence of mirex.
STATISTICAL ANALYSIS OF DATA
All statistical comparisons were made with the x-'-test
for independent samples dr), and differences were consid-
VOL. 7, No. 1, JUNE 1973
2Tr OV-101
10(1 120 Ga.s
Chrom Q
188" C
210' C
25 ml min
1 :1 2Tc OV-ini
100/120 Gas
Chrom Q
180° C
210° C
25 ml 'min
ered real at the 0.01 level of significance. The move-
ment of mirex into the aquatic environment and its
consequent accumulation in populations of estuarrne
animals were presumed to he greatest in areas where a
significantly greater proportion of samples was positive
for mirex (>0.01 ppm).
Average residues reported were computed by assuming
that samples where mirex was not detected «0.01 ppm)
actually had no residue.
Bird and mammal residue data were not analyzed statis-
tically: however, average mirex residues in muscle
tissues are tabulated for herons and egrets (Table 10),
clapper rails (Table 11), and raccoons (Table 13).
Results
PRETREATMENT SAMPLES
Mirex was not detected in any pretreatment samples of
crabs, shrimp, fish, sediment, or water taken from the
six monitoring stations. Toogoodoo stations, or the
Riverland Terrace pond. Mirex residues were found in
about one-third of the pretreatment samples of migra-
tory birds (Table 9). Only one raccoon was collected
before treatment, and this sample was free of mirex
residues. Background residues in birds collected in the
Charleston area are discussed in the section on "Signifi-
cance of Data."
WATER AND SEDIMENT SAMPLES
Mirex was not detected in water samples during the
study. No attempt was made, however, to concentrate
water samples (such as carbon filtration of large volumes
(if water).
Sediment samples were negative, except in six instances
(three samples from Riverland Terrace pond and three
from the Ashley River within the treated zone). Ac-
cordingly, mirex residues in water and sediment are
not reported in tabular form.
ACCUMULATION OF MIREX IN BIOTA
Biological concentration of mirex occurred in
cstuarine food web as shown below:
the
SAMPLL
Water
Sediment
Crabs
Fishes
Shrimps
Mammals
Birds
Ri'.smui: RANGT
( ppm )
< 0.01 pph
n^-o.n?
0-0. 60
0-0. 82
0-1.3
0-4.4
0-17,0
PF.RCF.NT OF POSTTREAI MENT
SAMPLES WITH MIREX RESIDUES
0
3
31
15
10
54
78
-0= < 0.01 ppm
Differences in the amounts of mirex accumulated by
the animals were probably caused by variables such as
proximity to the treated area, duration of exposure to a
11
-------
mirex-contaminated habitat, seasonal habits, avoidance
ability, and position in the food web.
Additional variation may depend upon parameters such
as method of application, amount and frequency of rain-
fall, surface runoff, variations in sea level, and degrada-
tion.
Levels of mirex found in the biota are listed in Tables
5 to 14.
Discussion
ANIMAL MORTALITIES
Procedures used in this study were neither capable of,
nor intended to comprehensively detect mortality of
aquatic organisms. The sampling areas were, however,
inspected for dead or affected animals during each
sampling period, and mass mortalities would probably
have been detected visually or in trawl catches. No mass
mortalities of organisms were observed during the study.
Our laboratory experiments (5) suggest that mortalities
in a population of marine crustaceans due to mirex
would not all occur at the same time. Symptoms of
mirex poisoning exhibited by shrimp and crabs prior to
death are irritability, uncoordinated movement, loss of
equilibrium, and paralysis. An affected crab may live
several days or even weeks after the initial exposure to
mirex. Animals in advanced stages of poisoning would
be highly susceptible to prcdation by larger carnivores
and could be swept out of estuaries hy tidal action.
Thus, affected animals could be removed from the
system without leaving visible evidence of their condi-
tion. Further, any dead animals would generally enter
the detritus pool soon after death.
TOOGOODOO CREEK EXPERIMENTAL AREA—
/,200-acre plot (Fit>. /) treated hy helicopter on Oct.
14-15. 1969; June 3-4. 1970: and Oct. 27-2K. 1970
Movement of mirex from trcaied lands above the high-
tide mark undoubtedly occurred after each of the three
treatments, especially the first. The mechanisms of
transporting mirex from treated land areas to the
Toogoodoo Creek estuary arc poorly understood. Sur-
face runoff, into the drainage system (especially after
heavv rain) is one suspected cause.
All species sampled contained mirex. Residues first
appeared in a shrimp sample 2 weeks after the first
treatment. From then on. the relative frequency of
mirex-positivc samples and the "average" levels of
mirex residues fluctuated greatly. Mirex was present,
however, in at least'one sample in 33 of 44 collections.
Some of the statistically significant relationships occur-
ring within these fluctuations are discussed in the follow-
ing paragraphs.
Application effects
The relative number of samples that contained mirex
appeared to increase during the first 10 weeks after each
treatment and then to decrease in the 10- to 20-week
period (Table 3). After the first and third treatments,
the number of positive samples appeared to decrease
even further in the third interval, 20 to 32 weeks.
These decreases for each individual treatment were not
statistically significant; however, when data from all
treatments were evaluated together, the relationship be-
tween time elapsed since spraying and the decreasing
number of positive samples proved to be real.
Decreases in the percent occurrence of mirex residues
from the first to the third treatment were significant.
This could be due to: (1) mirex being translocated
rapidly from the estuarine biota to reservoirs in fatty
tissues of predacious birds and mammals, (2) transient
estuarine animals (e.g., crabs, shrimp) translocating
mirex during emigration from nursery areas of Toogoo-
doo Creek, (3) possible differences in the manner in
which mirex bait was applied by the two helicopter
pilots, or (4) degradation of mirex by physical, chemical,
or biological processes.
Location effects
The relative number of samples positive for mirex also
fluctuated with station location. Stations A and B were
located within the treated area, and Stations C and D
were downstream from the treated area (Fig. 1). Mirex
levels at these stations gave some indication of the move-
ment of mirex from treated land areas into untreated
areas downstream.
As expected, more animals from the treated area con-
tained mirex than did those from downstream stations
(Table 4). This relationship was statistically significant
after the first and third applications, but was only.ap-
parent after the second treatment. The frequencies of
positive samples at stations located within the treated
zone (A and B) were not significantly different. As
shown in Table 4, there was an apparent decrease in
frequency of positive samples with increased distance
from the treated zone.
Species effects
The percent occurrence of mirex was higher in crab
samples than in fish or shrimp samples. Although this
difference was apparent at Stations A through D, it was
statistically significant only at Station B. Overall, how-
ever, the higher residues in crabs were significant after
the first two helicopter treatments, but was not statisti-
cally significant after the third treatment. The frequency
of positive samples appeared.unrelated to size of crabs
or to species of fish.
PESTICIDES MONITORING JOURNAL
-------
TABLE 3.—Percent occurrence of mirex in crab, shrimp,
and fish samples by time of sample collection in respect to
mirex application at Toogoodoo Creek Experimental Area
APPLICATION
First (Oct. 14-15,
Second (June 3-4,
Third (Oct. 27-28,
1969)
1970)
1970)
Overall Percent Occurrence
PERCENT OCCURRENCE OF MIREX BY
SAMPLING TIME IN WEEKS
SINCE APPLICATION
0-10
37
22
28
28
10-20
36
9
12
17
20-32
18
2
8
TOTAL WEEKS
30 (0-32 weeks)
16 (0-20 weeks)
12 (0-32 weeks)
19
TABLE 4.—Percent occurrence of mirex in aquatic animals
by sampling site with respect to treated area
SAMPLING
SITE
A
B
C
D
3
4
2
5
6
7
LOCATION WITH
RESPECT TO
TREATED AREA
Inside treated area
Inside treated area
Just outside treated area
2 miles downstream
from treated area
Upstream stations located inside
main inland treated area
Downstream stations located out-
side main inland treated area
PERCENT OCCURRENCE
MKEX IN ALL POST-
TREATMENT SAMPLES
OF CRABS, SHRIMP,
AND FISH
29%
24%
17%
8%
70%
70%
30%
0%
25%
0%
MONITORING STATIONS— mirex was applied to inland
treatment areas (Fig. 1) by fixed-wing aircraft during
September-Ocotber 1969 and May-June 1970
Trends in the data were similar to those observed in the
Toogoodoo Creek Experimental Area. The greatest
number of positive samples occurred shortly after treat-
ment and diminished with time. Samples positive for
mirex were significantly more frequent within the
treated area, upstream Stations 3 and 4, than at sites
located outside the treated area, downstream Stations
2, 5, 6, and 7 (Table 4).
Although significant differences in percent occurrence
were noted between these two groups, individual stations
showed no significant differences because too few
samples were taken and too few positive samples oc-
curred for x2-analysis. Also, for the individual monitor-
ing stations, the relative number of samples containing
mirex did not vary depending on the type of animal
sampled.
A more frequent occurrence of mirex residues was
apparent after the second application than after the
first, but this increase was not significant.
VOL. 7, No. 1, JUNE 1973
RIVERLAND TERRACE POND—a 2.5-acre zone around
the pond treated by hand-operated seed spreader on
Dec. 3, 1969; July 24, 1970; and Dec. 1, 1970
Although mirex was applied three times to the banks of
the pond, it was obviously not accumulated by sampled
biota. During the study, only two crab samples con-
tained mirex. In addition, three sediment samples were
positive. The method of treatment (mirex applied by
hand to a narrow bank around the pond above the high-
tide mark on hard mud banks) might have caused the
occurrence of the mirex in the sediment samples. "Crab
samples" of sediment often consisted of as many as 20 to
25 "grabs" taken near the edge of the pond and one or
more particles of bait could have been picked up with the
sample.
Special attention was given Riverland Terrace Pond to
observe any individual- or .mass-mortalities in the pond
biota. Migration of animals was controlled by means of
retaining screens (V^-inch mesh) placed over two cul-
verts that flood and drain the treated pond. Daily screen
checks were made for a 3-week "period to reveal any
distressed, moribund, or dead animals. Crabs, shrimp,
and fish observed in the pond or on the screens never
appeared to be affected.
During several pond collections after treatment, seine-
hauls revealed large populations of grass shrimp (mostly
Palaemonetes pugio) and many of the females were
gravid. On one occasion, grass shrimp were held for
several weeks in an aquarium, where the shrimp re-
mained healthy and their eggs seemed to develop and
hatch normally. At no time was mirex detected in the
grass shrimp population.
SIGNIFICANCE OF DATA
Surprisingly, mirex appeared in one-third of the birds
in the pretreatment sample. Since the study area had
not been previously treated with a large-scale applica-
tion of mirex. the birds must have accumulated the
residues from some other area. A large acreage around
Savannah, Ga., had been treated with three successive
applications of mirex in a pilot study of the feasibility
of eradication. This area is the likely source of the mirex
residues. Thus, migration is an important factor in in-
terpreting the study data. Stewart's (9) report that
northern clapper rails banded at Chincoteague, Va.
migrated southward to winter in the coastal marshes of
the South Atlantic States, including South Carolina, sup-
ports this view.
Measurable levels of mirex appeared at all stations,
demonstrating that tidal flushing, biological transport, or
some other mechanism can distribute the chemical
throughout the estuary, regardless of precautions taken
13
-------
to avoid treatment in the tidal zone. This finding is
evidence that mirex can become widespread in animal
food webs.
The occurrence and amount of mirex in birds and mam-
mals varied considerably at all stations. This is to be
expected since all of these animals are more or less
migratory, and food sources of individuals vary. Simi-
larly, Keith (4) found that levels of insecticide residues
in fish-eating birds vary considerably within local popula-
tions of most species. Even so, average residues that
appeared at the different stations correlated well with
station location in respect to treated area, with the
highest residues occurring at stations within a treated
area and the lowest at stations farthest from the source
of contamination.
Approximately 78% of the 179 birds collected after
treatment began contained measurable residues of
mirex, whereas residues were present in only 54% of
the raccoons. The greater mobility of birds is doubtless
the reason for this difference. In any case, occurrence of
mirex residues as well as the quantity of residue in the
animal appear directly related to drainage and distance
from a treated area.
Residues in animals collected from the 2-square-mile
treatment area on the Toogoodoo creek marshes (Sta-
tions A, B, and C) were not as great as those from
animals collected within the inland treatment zone. This
is not surprising because water as well as food-chain
organisms in the Toogoodoo marshes were flushed
twice daily by 4- to 6- foot tides.
Local and seasonal migrations of the sampled species
would tend to mask any evidence of residue buildup
during the course of the study. Even so, the absence of
any individuals with greatly elevated residue levels in-
dicates that average levels did not continue to rise
beyond levels reached in the first few months following
treatment. Raccoons were the most sedentary animals
sampled. Data in Table 12 indicate that there was no
gradual buildup of mirex residues in raccoons, although
some seasonal variations are apparent.
The highest mirex residue (17.0 ppm) found in any
animal analyzed occurred in a kingfisher. The highest
level found in a raccoon was 4.40 ppm. All birds and
mammals that contained residues in excess of 1.00 ppm
are listed in Table 14. All animals on this list, except the
kingfisher from Station D and two raccoons from Sta-
tion 2, came from stations classified as having a "high"
mirex exposure potential.
See Appendix for chemical name of mirex
Acknowledgment
Special thanks are due Dr. Nelson Cooley for editorial
help, Dave Hansen for assistance with statistical an-
alysis, Jerry Forester and Johnnie Knight for analysis of
samples, and Madeleine Brown and Steve Foss for
assistance in manuscript preparation—all of the Gulf
Breeze Environmental Research Laboratory, U.S. En-
vironmental Protection Agency.
We also thank the following persons who supervised the
application of mirex or approved or conducted various
aspects of the collection and preparation of samples:
Julian Mikell, Plant Protection Division U.S. Depart-
ment of Agriculture; Charles Bearden and Michael
McKenzie, Division of Marine Resources, S.C. Wildlife
Resources Department; Alston Badger, Bears Bluff Field
Station, U.S. Environmental Protection Agency; Frank
McKinney, Grice Marine Biological Laboratory, College
of Charleston.
LITERATURE CITED
(1) Bookhout, C. G., A. J. Wilson, Jr., T. W. Duke, and
J. I. Lowe. 1972. Effects of mirex on the larval develop-
ment of two crabs. Water, Air, Soil Pollut. 1(2):165-180.
(2) Carson, R. 1962. Silent Spring, p. 161-169. Houghton
Mifflin Co., Boston, Mass.
(3) Frear, D. E. H. 1969. Pesticide Index, 4th ed. 399 p.
College Science Publishers, State College, Pa. 16801.
(4) Keith, J. O. 1968. Insecticide residues in fish-eating birds
and their environment. Aves 5(1):28-41.
(5) Lowe, J. I., P. R. Parrish, A. J. Wilson, Jr., P. D.
Wilson, and T. W. Duke. 1971. Effects of mirex on
selected estuarine organisms. In Transactions of the 36th
North Am. Wildl. Natl. Resour. Conf. p. 171-186.
(6) McKenzie, M. D. 1970. Fluctuations in abundance of the
blue crab and factors affecting mortalities. S. C. Wildl.
Resour. Dep., Marine Resour. Div., Tech. Rep. No. 1,
45 p., Charleston, S. C.
(7) Mills, P. A., J. H. Onley, and R. A. Gaither. 1963. Rapid
method for chlorinated pesticide residues in nonfatty
foods. J. Assoc. Off. Anal. Chem. 46(2):186-191.
(8) Siegel, S. 1956. Nonparametric statistics for the be-
havioral sciences. McGraw-Hill Book Co., Inc., New
York, N. Y.
(9) Stewart, R. E. 19$4. Migratory movements of the north-
ern clapper rail. Bird Banding 25(1): 1-5.
14
PESTICIDES MONITORING JOURNAL
-------
TABLE 5.—Mirex residues in shrimps by sampling site and sampling time, South Carolina, 1969-71
[— = not detected]
INTERVAL NUMBER IN
SPECIES FROMMIREX CoMPOSITE
APPLICATION SAMPLE
TO SAMPLING
SIZE OF
INDIVIDUALS
IN COMPOSITE
SAMPLES
(INCHES)
MlREX
RESIDUE
(PPM —
WHOLE
BODY)
SAMPLING SITE A— TOOGOODOO CREEK
White shrimp Background 3
First application
(Oct. 14-15, 1969)
White shrimp 24 hrs 4
2 wks 4
4 wks 10
Brown shrimp 30 wks 13
Brown-spotted shrimp 32 wks 10
Second application
(June 3-4, 1970)
Brown-spotted shrimp 24 hrs 12
2 wks 12
4 wks 12
6 wks 12
8 wks 12
White shrimp 10 wks 12
12 wks 12
14 wks 12
16 wks 12
18 wks 11
20 wks 12
Third application
(Oct. 27-28, 1970)
White shrimp 24 hrs 12
2 wks 4
Brown-spotted shrimp 20 wks 5
24 wks 5
28 wks 4
30 wks 12
32 wks 12
2.5
2.5
2.5
4-5
2.5-3.5
3.5-4
3.5-5.5
5-5.5
5.5-6
5-6
3.5-5
3.5-4.5
2.5-3.5
3-4.5
4-4.5
5.5-6
5-6
5-6
5-6
2.25-2.5
2.5-3.5
3.5-4.5
2.5-5
2-4
—
—
—
.014
—
—
.014
—
—
.024
—
—
—
—
—
—
—
—
—
—
—
—
—
—
SAMPLING SITE B— TOOGOODOO CREEK
White shrimp Background 4
First application
(Oct. 14-15, 1969)
White shrimp 24 hrs 4
2 wks 4
4 wks 10
Brown-spotted shrimp 30 wks 2
32 wks 3
Second application
(June 3-4, 1970)
Brown-spotted shrimp 24 hrs 12
2 wks 12
4 wks 2
6 wks 5
8 wks 12
White shrimp 10 wks 12
12 wks 12
14 wks 12
16 wks 12
18 wks 12
20 wks 12
Third application
(Oct. 27-28, 1970)
White shrimp 24 hrs 12
2 wks 3
Brown-spotted shrimp 18 wks 3
22 wks 5
24 wks 10
26 wks 4
28 wks 12
30 wks 11
32 wks 12
3
3
3-4
4-5
3-5
4
5-6
5-5.5
4-6
4-7
4-5
3-4
2-3
3.5-4.5
4-4.5
5-6
5
5-6
4.5-6
3-4.5
2-4
3-4
3-4.5
3-5.5
2.75-5
3-4.5
—
—
.040
.052
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
INTERVAL
SPECIES FROM MlRE>
NUMBER IN
' COMPOSITE
APPLICATION SAMPLE
TO SAMPLING
SAMPLING SITE
SIZE OF
INDIVIDUALS
IN COMPOSITE
SAMPLES
(INCHES)
RESIDUE
(PPM —
WHOLE
BODY)
C— TOOGOODOO CREEK
White shrimp Background 3
First application
(Oct. 14-15, 1969)
White shrimp 24 hrs
2 wks
4 wks
6 wks
Brown-spotted shrimp 30 wks
32 wks
Second application
(June 3-4, 1970)
Brown-spotted shrimp 24 hrs.
2 wks
4 wks
6 wks
8 wks
White shrimp 10 wks
12 wks
14 wks
16 wks
18 wks
20 wks
Third application
(Oct. 27-28, 1970)
White shrimp 24 hrs
2 wks
Brown-spotted shrimp 20 wks
Grass shrimp 24 wks
Brown-spotted shrimp 26 wks
28 wks
30 wks
32 wks
SAMPLING SITE
6
4
10
10
1
3
9
12
12
12
4
1
12
12
12
12
10
12
10
4
132
9
12
12
12
5
4-5
3
4-5.5
3.5-4
5.5
6-7
4-5
3-5
4-6
5-6
4
4.5
2.5-4
4.5-5.5
4-4.5
5.5-6
5-6
5-6
5-7
3.5-4.5
.75-1.25
3.5-5.5
3.5-5
4-5
3.5-5
—
—
-_
.020
.014
—
—
.015
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
D— TOOGOODOO CREEK
White shrimp Background 2
First application
(Oct. 14-15, 1969)
White shrimp 24 hrs
2 wks
4 wks
6 wks
8 wks
Brown-spotted shrimp 24 wks
30 wks
Second application
(June 3-4, 1970)
Brown-spotted shrimp 24 hrs
2 wks
4 wks
6 wks
White shrimp 10 wks
Brown-spotted shrimp 12 wks
White shrimp 14 wks
16 wks
18 wks
20 wks
Third application
(Oct. 27-28, 1970)
White shrimp 24 hrs
2 wks
Brown-spotted shrimp 20 wks
24 wks
2
2
7
10
15
2
2
12
12
2
12
4
12
12
12
12
10
12
1
2
6
6
5
4.5
5-6
4-5
3.5-5
4.5-5
3-3.5
4-5
5-6
5
5-6
3.5-5.5
3-4.5
4.5-5.5
4-5
5.5-6
5-6
5-6
4.5
5
3.5-4.5
—
.027
—
_,
_
_
_
—
VOL. 7, No. 1, JUNE 1973
15
-------
TABLE 5.—Mirex residues in shrimps by sampling site and sampling time, South Carolina, 1969-71—Continued
[— = not detected]
INTERVAL .
SPECIES FROM MIREX
APPLICATION
TO SAMPLING
NUMBER IN
COMPOSITE
SAMPLE
SIZE OF
INDIVIDUALS
IN COMPOSITE
SAMPLES
(INCHES)
MIREX
RESIDUE
(PPM —
WHOLE
BODY)
SAMPLING SITE D—TOOGOODOO CREEK—Continued
Grass shrimp
24wks
26wks
28wks
30wks
32wks
100
12
12
1
6
.75-1.25
2.5-4
3-4
4.5
3.5-5.5
STATION 1—RIVERLAND TERRACE POND (EAST)
Grass shrimp
First application
(Dec. 3, 1969)
Grass shrimp
Grass shrimp
Background 178
48hrs
5 mos
144
77
Background 100
Second application
(July 24, 1970)
White shrimp 72hrs
Grass shrimp 3 mos
Brown-spotted shrimp 3 mos
Grass shrimp
4 mos.
5
146
5
176
.75-1
.75-1
1
.75-1.25
2-3
1-1.25
2.25-3
.75-1.25
Third application
(Dec. 1, 1970)
Grass shrimp
48 hrs
2wks
6wks
3 mos
159
167
193
126
STATION 2— STONO RIVER, LOG
White shrimp
Brown shrimp
First application
(Oct. 23, 1969)
White shrimp
Background
Background
24 hrs
13
9
4
.75-1.25
.75-1.25
.75-1.25
.75-1.25
BRIDGE CREEK
Medium
Small
3-4
—
_
Second application
(June 18, 1970)
Brown-spotted shrimp 24 hrs
White shrimp 3 mos
12
12
4.5-5.5
3.5-4.5
STATION 3—UPPER ASHLEY RIVER,
RUNNYMEADE PLANTATION
River shrimp Background 9 1.5-2.5 —
First application
(Oct. 22, 1969)
Second application
.(June 11. 1970)
Brown-spotted shrimp 24 hrs 3 3-4 .11
White shrimp 3 mos 2 4.5 —
. SIZE OP MntEx
iJy NUMBER IN INDIVIDUALS RESIDUE
SPECIES 4 MIREX CoMPOSITE ,N rnxi.osiTE (PPM—
APPLICATION SAMPLE SAMPLES V(moiE
TO SAMPLING ([NCHES) BQDY)
STATION 4 — COOPER RIVER, U.S. NAVAL
AMMUNITION DEPOT
First application
(Oct. 17, 1969)
River shrimp 24 hrs 1 3 1
White shrimp 24 hrs 5 2.5-3
Second application
(June 10, 1970)
White shrimp 3 mos 10 3-4.5
STATION 5— LOWER ASHLEY RIVER,
OLD TOWN CREEK
White shrimp Background 6 4
First application
(Oct. 22, 1969)
White shrimp 24 hrs 4 3-4
Second application
(June 10, 1970)
Brown-spotted shrimp 24-hrs 3 2.5-3
White shrimp 3 mos 7 3-6
.3
.26
—
—
—
—
—
STATION 6— WANDO RIVER, BERESFORD CREEK
White shrimp Background 4 4.5-5.5
First application
(Oct. 17, 1969)
White shrimp 24 hrs 3 4-6
Second application
(June 8, 1970)
Brown-spotted shrimp 24 hrs 12 3.5-4.5
White shrimp 3 mos 12 3.5-4
8 mos 12 3.5-5
—
—
.015
—
—
STATION 7— SOUTH SANTEE RIVER, ALLIGATOR CREEK
White shrimp Background 8 2.5-4
First application
(Sept. 18, 1969)
White shrimp 24 hrs 9 2-2.5
Second application
(May 20, 1970)
Brown-spotted shrimp 3 mos 12 3.5-4
White 'shrimp 8 mos 12 4.5-5
—
—
—
—
16
PESTICIDES MONITORING JOURNAL
-------
TABLE 6.—Mirex residues in crabs by sampling site and sampling time, South Carolina. 1069-77
[— = not detected]
SIZE OF MIREX
NT^r'AL NUMBER IN INDIVIDUALS RESIDUE
SPECIES ARp°PMrA™N CoMPOSITE IN COMPOSITE (PPM-
APPUCATION SAMPLE SAMPLES WHOLE
TO SAMPLING (INCHES) BODy)
SAMPLING SITE A— TOOGOODOO CREEK
Blue crab
First application
(Oct. 14-15, 1969)
Blue crab
Second application
(June 3-4, 1970)
Blue crab
Third application
(Oct. 27-28, 1970)
Blue crab
Background
24hrs
4 wks
12 wks
14 wks
16 wks
22 wks
26 wks
28 wks
30 wks
24hrs
4 wks
6 wks
8 wks
10 wks
12 wks
14 wks
16 wks
18 wks
20 wks
24hrs
2 wks
4 wks
6 wks
8 wks
10 wks
14 wks
16 wks
18 wks
20 wks
22 wks
24 wks
26 wks
28 wks
30 wks
32 wks
1
2
5
2
1
1
3
4
2
4
3
5
3
5
8
12
12
6
1
s
3
3
3
11
12
7
12
10
6
8
8
5
5
1
2
5
5
3
1-2.5
4.5-5
4.5
2.5
1-3
3-4
3-4
3-6
'2-3
2.5-5
3-6
2-5
5-6
1.5-2.5
2-2.5
2.5-5.5
5.5
1-5
6
1-5
2-5
1-2
1-2.5
1-3
1-1.25
1-1.75
1.5-2
1.25-1.5
1-3
3.5-5
1.5-3
2.5
3.25-4.5
2-5.5
—
.12
.19
.12
.19
.040
.015
.026
.016
.19
—
.052
.053
—
—
—
.024
—
—
—
—
.013
—
—
—
.022
—
—
—
.016
—
—
—
—
SAMPLING SITE B— TOOGOODOO CREEK
Blue crab
First application
(Oct. 14-15, 1969)
Blue crab
Second application
(June 3-4, 1970)
Blue crab
Fiddler crab
Background
24hrs
4 wks
8 wks
14 wks
18 wks
26 wks
32 wks
4 wks
8 wks
10 wks
12 wks
14 wks
16 wks
18 wks
21 wks
1
1
1
1
1
2
2
3
6
3
4
12
6
1
1
28
6
6
6
3
3
4.5
3-4
4-6
2.5-5
2-6
4-5
1.25-2.25
3-5.5
5
5.5
33-.15
—
—
.24
.089
.20
—
.025
.035
—
.017
—
—
—
—
—
INTERVAL
TO SAMPLINO
SIZE OF MIREX
N'UMHIRIN INDIVIDUALS RESIDUE
COMPOSITE IN COMPOSITE (PPM—
SAMPLE SAMPLES WHOLE
(ISCnta) BODY)
SAMPLING SITE B— TOOGOODOO CREEK— Continued
Third application
(Oct. 27-28, 1970)
Blue crab
Fiddler crab
Blue crab
Fiddler crab
Blue crab
24hrs
24hrs
4 wks
6 wks
6 wks
8 wks
10 wks
12 wks
14 wks
16 wks
18 wks
20 wks
22 wks
24 wks
26 wks
28 wks
32 wks
6
30
2
11
29
5
4
3
8
9
7
9
4
12
4
5
1
1-2.5
.33-.7S
3-5
1-2.5
.33-. 75
1-2
1.5-5
.75-3
.75-2
2-5
1 .25-2
1-2
2-4
1.5-2.5
1-4.5
3-6
3.5
.049
—
.012
.12
—
.041
.027
.038
—
.020
—
—
—
—
—
—
—
SAMPLING SITE C— TOOGOODOO CREEK
Blue crab
First application
(Oct. 14-15, 1969)
Blue crab
Fiddler crab
Second application
(June 3-4, 1970)
Blue crab
Fiddler crab
Third application
(Oct. 27-28, 1970)
Blue crab
Fiddler crab
Blue crab
Fiddler crab
Blue crab
Background
24hrs
2 wks
4 wks
6 wkx
8 wks
12 wks
14 wks
18 wks
20 wks
22 wks
24 wks
26 wks
28 wks
30 wks
32 wks
24hrs
2 wks
6 wks
8 wks
12 wks
14 wks
16 wks
20 wks
21 wks
24hrs
24hrs
2 wks
4 wks
6 wks
6 wks
8 wks
14 wks
16 wks
20 wks
22 wks
24 wks
26 wks
28 wks
30 wks
!
1
1
9
4
1
2
1
2
2
8
2
1
2
1
25
1
3
6
4
4
1
2
2
26
3
27
1
3
4
31
4
3
7
8
2
7
8
8
I
5
6
6
1-2
2-3
6
5.5
2
4.5
4.5
1-2
1-2
3
4-5
4
.5-1
4.5
6-6.5
3-4.5
4-5
2-4
5
4.5
6
.5-.7S
3-5
.S-.75
6
4-5
4.5
.5-1
1.5-2.5
1-1.75
1-2.5
1-2
1.5-2.5
1.5-3
1-2.5
1.25-3.25
2
—
—
—
.015
.032
.090
.050
.056
.025
.038
—
—
—
—
—
.013
—
.027
—
—
—
—
—
—
.010
—
—
VOL. 7, No. 1, JUNE 1973
17
-------
TABLE 6.—Mirex residues
in crabs by sampling site and sampling lime, South Carolina, 1969-71—Continued
[— = not detected]
SPECIES
SIZE OF MIREX
v7AL NUMBER IN INDIVIDUALS RESIDUE
FROM MIREX COMPOSITE IN COMPOSITE (PPM —
APPLICATION SAMPLE SAMPLES WHOLE
TO SAMPLING (INCHES) BODY)
SAMPLING SITE D— TOOGOODOO CREEK
Blue crab
First application
(Oct. 14-15, 1969)
Blue crab
Second application
(June 3-4, 1970)
Blue crab
Third application
(Oct. 27-28, 1970)
Blue crab
Mud crab
Blue crab
STATION
Blue crab
First application
(Dec. 3, 1969)
Blue crab
Background
24hrs
2 wks
4 wks
6 wks
8 wks
12 wks
14 wks
18 wks
20 wks
22 wks
24 wks
26 wks
28 wks
30 wks
32 wks
24hrs
4 wks
6 wks
10 wks
12 wks
14 wks
18 wks
20 wks
24 hrs.
2 wks
6 wks
8 wks
10 wks
12 wks
14 wks
1 6 wks
20 wks
24 wks
26 wks
28 wks
30 wks
32 wks
1
6
3
12
3
12
2
2
3
3
2
6
1
2
1
T
5
4
6
1
12
2
2
3
8
4
5
12
4
5
3
12
10
12
2
12
2
1
1— RIVERLAND TERRACE
Background
5 mos
3
8
7
1-1.5
1-2
1-1.25
1.5-3
1-1.75
5.5
5.5
1.5-5
3
3
2-3
4
4-5
5
3-6
3-6
3-4
4-6
4.5
1-2.5
4-5
5-6
2-5
1.5-2.5
2-5
1.5-4.5
1-2
1.5-4.5
1-5
1-2
1-2
1.5-2.5
.5-1.25
2.5-3.5
1 .75-3.5
2-3
2.5
POND
1-2.5
.5-1.25
—
—
—
.065
.030
.051
—
—
—
_
—
—
—
—
—
—
.098
—
.015
—
—
—
—
—
—
—
—
—
—
—
—
—
—
\ —
—
—
—
(EAST)
—
—
Second application
(July 24, 1970)
Blue crab 72 hrs
3 mos
Third application
(Dec. 1, 1970)
Blue crab
Fiddler crab
Blue crab
48 hrs
48 hrs
2 wks
3 mos
15
13
12
4
3
12
.5-2
.75-1.5
.5-2
.5-.7S
1-3
1.5-4.5
.024
.026
STATION 2—STONO RIVER, LOG BRIDGE CREEK
Dlue crab
First application
(Oct. 23, 1969)
Background
1.5-3
SPECIES
INTERVAL
FROM MIREX
APPLICATION
TO SAMPLING
NUMBER IN
COMPOSITE
SAMPLE
SIZE OF
INDIVIDUALS
IN COMPOSITE
SAMPLES
(INCHES)
MIREX
RESIDUE
(PPM
WHOLE
BODY)
STATION 2—STONO RIVER—Continued
Second application
(June 18, 1970)
Blue crab
24 hrs
3 mos
8 mos
8
2
12
2.5-6
5-5.5
1.25-4
.010
.031
—
STATION 3—UPPER ASHLEY RIVER,
RUNNYMEADE PLANTATION
Blue crab
First application
(Oct. 22, 1969)
Blue crab
Second application
(June 11, 1970)
Blue crab
Background 2
24 hrs 3
24 hrs 3
1.5
2-3
5
—
.60
.27
STATION 4—COOPER RIVER, U.S. NAVAL
AMMUNITION DEPOT
Blue crab
First application
(Oct. 17, 1969)
Second application
(June 10, 1970)
Blue crab
Mud crab
Background 1
24 hrs 4
3 mos 1
8 mos 3 1
4
6-8
4.5
.2S-.5
—
.042
STATION 5—LOWER ASHLEY RIVER, OLD TOWN CREEK
Blue crab
First application
(Oct. 22, 1969)
Blue crab
Second application
(June 10, 1970)
Blue crab
Background 1
24 hrs 9
3 mos 2
24 hrs 5
3 mos 4
8 mos 8
5.5
1
1-2.5
1.5-2.5
3-5
1-4
—
—
—
STATION 6—WANDO RIVER, BERESFORD CREEK
Blue crab
First application
(Oct. 17, 1969)
Blue crab
Second application
(June 8, 1970)
Blue crab
Background
3 mos
24 hrs
3 mos
8 mos
2
2
I
8
12
1.5-3
5
2-5
5-6
1.25-3.25
—
—
.025
STATION 7—SOUTH SANTEE RIVER, ALLIGATOR CREEK
Blue crab
Background
5.5
First application
(Sept. 18, 1969)
Blue crab
Second application
(May 20, 1970)
Blue crab
24 hrs
3 mos
24 hrs
3 mos
8 mos
1
2
2
4
12
4.5
1.5-3
2-3
4-5
1-4
.012
18
PESTICIDES MONITORING JOURNAL
-------
TABLE 7.—Mirex residues in fishes by sampling site and sampling time, South Carolina, 1969-71
[•— = not detected]
INTERVAL NuMBER ,N
SPECIES FROM MIREX CoMPOSITE
APPLICATION SAMPLE
TO SAMPLING
SIZE OP
INDIVIDUALS
IN COMPOSITE
SAMPLES
(INCHES)
MIREX
RESIDUE
(PPM —
WHOLE
BODY)
SAMPLING SITE A— TOOGOODOO CREEK
Blackcheek tonguefish Background 2
Southern kingnsh Background 1
First application
(Oct. 14-15, 1969)
Silver perch 24 hrs 2
2 wks 2
4 wks 2
8 wks 11
Atlantic menhaden 16 wks 10
18 wks 10
Spot 30 wks 13
Second application
(June 3-4, 1970)
Spot 24 hrs 12
2 wks 7
4 wks 12
6 wks 5
8 wks 5
12 wks 6
14 wks 12
16 wks 6
Third application
(Oct. 27-28, 1970)
Weakfish 24 hrs 3
Silver perch 2 wks 3
Striped mullet 6 wks 1
10 wks 7
Silver perch 20 wks 2
Atlantic menhaden 20 wks 2
Bay anchovy 22 wks 12
Silver perch 26 wks 4
Spot 28 wks 12
30 wks 12
32 wks 12
3
7
3.5
3.5
4.5
3.5-4.5
3.5-4.5
4-5
3-4
3-4
3.5-5
4.5-5.5
3.5-4
3-4
2-3
4-5
3-5
4.5-7
4.5-5.5
7
7-9
4.5
4.25
2-3
5.5-7
1.5-3
2.25-3.25
2-3
—
—
—
—
.073
.028
—
—
.015
—
—
—
—
—
.060
.043
—
—
.017
—
—
—
—
—
—
—
—
—
SAMPLING SITE B— TOOGOODOO CREEK
Silver perch Background 1
First application
(Oct. 14-15, 1969)
Silver perch 24 hrs 2
2 wks 1
8 wks 1
Spotted seatroul 8 wks 1
Atlantic menhaden 14 wks 12
16 wks 10
18 wks 4
Spot 28 wks 3
Blueflsh 30 wks 1
Spot 32 wks 2
Second application
(June 3-4, 1970)
Spot 24 hrs 12
2 wks 6
4 wks 12
6 wks 12
8 wks 1
Striped mullet 8 wks 2
Silver perch 10 wks 6
Spot 12 wks 12
Silver perch 14 wks 7
Spot 16 wks 4
Silver perch 18 wks 4
20 wks 1
Third application
(Oct. 27-28, 1970)
Silver perch 24 hrs 8
Spot 2 wks 1
3.5
3.5
4.5
4
7
2-4
3.5-4.5
4-5
2-2.5
7
3-4
3.5-4.5
3.5-5.5
3-4.5
3-4
3.5
4-5
4.5-6
3.5-4
4-5
3-4.5
6-6.5
5
4-5
6
—
—
—
—
.039
—
—
—
—
—
—
—
—
—
—
—
—
.027
.016
—
—
—
—
.034
—
SIZE OF MIREX
INTERVAL NuMBER IN JNDIVIDUALS RESIDUE
SPECIES APPLICATION COMPOSE IN COMPOSITE (PPM-
APPLICATION SAMPLE SAMPLES WHOLE
TO SAMPLING (INCHES) BODY)
SAMPLING SITE B— TOOGOODOO CREEK— Continued
Striped mullet 6 wks
Spot 20 wks
Fourspot flounder 22 wks
24 wks
Spot 28 wks
30 wks
32 wks
3
4
3
2
12
5
11
5.5-6.5
6.5-7.5
2.5
3-4
1.5-2.75
3-4.5
2.5-4.25
.018
—
—
—
—
—
—
SAMPLING SITE C— TOOGOODOO CREEK
Blackcheek tonguefish Background
First application
(Oct. 14-15, 1969)
Hogchoker 24 hrs
Silver perch 2 wks
Blackcheek tonguefish 4 wks
Silver perch 6 wks
Atlantic menhaden 14 wks
20 wks
Mixed fish 22 wks
24 wks
Searobin 26 wks
Spotted hake 26 wks
Spot 30 wks
32 wks
Second application
(June 3-4, 1970)
Spot 24 hrs
2 wks
4 wks
6 wks
Weakfish 8 wks
Spot 10 wks
Silver perch 12 wks
14 wks
16 wks
Spot 18 wks
Third application
(Oct. 27-28, '1970)
Silver perch ' 24 hrs
2 wks
Bay anchovy 26 wks
Spot 28 wks
30 wks
32 wks
3
1
2
7
5
12
4
2
3
1
1
3
6
10
2
2
12
3
5
12
12
3
3
1
2
12
12
5
12
4
4
4
4-5
3-4
3.5-4.5
4
2-4
2.5-4
7
4
2-3
4-6
3.5-4.5
4-6
4.5
3-4
4-5
3.5-4.5
2.5-3.5
3-4
4
5-6.5
4.5
4
2-3
2.25-3.5
3-4.5
2.5-4.5
—
—
—
—
.017
—
—
—
—
—
—
—
—
—
— .
—
—
—
—
—
—
—
—
.028
—
—
—
—
SAMPLING SITE D— TOOGOODOO CREEK
Blackcheek tonguefish Background
First application
(Oct. 14-15, 1972)
Blackcheek tonguefish 24 hrs
2 wks
4 wks
Silver perch 6 wks
8 wks
Atlantic menhaden 12 wks
14 wks
16 wks
18 wks
Mixed fish 20 wks
Silver perch 24 wks
Spotted hake 24 wks
Weakfish 26 wks
Spotted hake 26 wks
Mixed fish 26 wks
Spotted hake 28 wks
Bluefish 30 wks
Spot 32 wks
2
2
2
3
10
12
2
10
10
3
2
1
2
1
1
3
3
2
7
3
3.5
4
3-4.
3.5-4
3-4
4
3-5
3.5-4.5
3.5-4
3-4.5
7
4
10.5
8.5
2-4
6
3-6
3-6
—
—
—
.
—
—
_
—
VOL. 7, No. 1, JUNE 1973
19
-------
TABLE 7.—Mirex residues in fishes by sampling site and sampling time, South Carolina, 1969-71—Continued
[— = not detected]
INTERVAL NUMBER IN
SPECIES FROMMIREX COMPOSITE
APPLICATION SAMPLE
TO SAMPLING
SAMPLING SITE D— TOOGOODOO
Second application
(June 3-4, 1970)
Spot 24 hrs
2wks
4wks
lOwks
Silver perch 12 wks
14wks
16 wks
Blackcheek tonguefish 18 wks
Third application
(Oct. 27-28, 1970)
Silver perch 2 wks
Winter flounder 8 wks
Spotted hake 18 wks
Spot 20 wks
Spotted hake 24 wks
Silver perch 26 wks
Spot 28 wks
30 wks
32 wks
12
8
2
2
8
7
2
4
10
1
i
1
7
2
12
2
12
SIZE op MIREX
INDIVIDUALS RESIDUE
IN COMPOSITE (PPM —
SAMPLES WHOLE
(INCHES) BODY)
CREEK — Continued
4.5-5.5
3-5
4
3.5-5.5
2.5-3.5
4
3.5-5
4-5
4-4.5
10.5
3-4.5
4.5
3.5-6
3-5
2-3
3-3.5
3-4.5
—
—
—
—
.046
—
—
—
—
—
—
—
—
—
—
—
—
STATION 1— RIVERLAND TERRACE POND (EAST)
Mummichog Background
White mullet Background
Atlantic silverside Background
First application
(Dec. 3, 1969)
Atlantic silverside 48 hrs
Mummichog 12 days
Atlantic silverside 12 days
White mullet 5 mos
Second application
(July 24, 1970)
Silver perch 72 hrs
SaiLfin molly 3 mos
Atlantic silverside 4 mos.
Third application
(Dec. 1, 1970)
Sailfin molly 48 hrs
White mullet 2 wks
Mummichog 2 wks
Atlantic silverside 2 wks
6 wks
White mullet 6 wks
3 mos
13
2
30
55
13
49
23
12
17
27
18
25
20
11
25
12
42
STATION 2— STONO RIVER, LOG
Blackcheek tonguefish Background
Black drum Background
Spotted seatrout Background
First application
(Oct. 23, 1969)
Silver perch 24 his
Second application
(June 18, 1970)
Spot 24 hrs
Silver perch 3 mos
Spot 8 mos
4
1
1
2
12
12
21
1-2
4-5
1.5-2.5
1.5-2
1-2.5
1.5-2
1.25-1.5
2-2.5
.5-1
2-3
1-1.5
.75-1
1.25-2.25
1.75-2
1.5-2.5
2.5-3.75
1
BRIDGE CREEK
3
5
7
3-4
2.5-3.5
4-5
1.5-2.5
—
—
—
—
—
—
—
—
—
—
—
— .
—
—
—
—
—
—
—
—
—
—
.054
INTERVAL NUMBER IN INDIVIDUALS
SPECIES APPLICATION CoMPOSITE IN COMPOSITE
APPLICATION SAMPLE SAMPLES
TO SAMPLING (INCHEs)
MIREX
RESIDUE
(PPM —
WHOLE
BODY)
STATION 3— UPPER ASHLEY RIVER,
RUNNYMEADE PLANTATION
Atlantic croaker
Hog choker
First application
(Oct. 22, 1969)
Hog choker
White catfish
Striped mullet
Second application
(June 11, 1970)
Spot
Silver perch
Hog choker
White catfish
Background
Background
24 hrs
2 mos
2mos
3 mos
3 mos
24 hrs
3 mos
3 mos
8 mos
1
3
4
8
6
1
I
8
3
8
7
STATION 4— COOPER RIVER, U.
AMMUNITION DEPOT
Spot
First application
(Oct. 17, 1969)
Silver perch
Mixed fish
White catfish
Second application
(June 10, 1970)
Spot
Hogchoker
Silver perch
White catfish
Spot
Bay anchovy
Background
24 hrs
2 mos
3 mos
24 'hrs
24 hrs
3 mos
8 mos
8 mos
8 mos
STATION 5— LOWER ASHLEY
Spot
Winter flounder
First application
(Oct. 22, 1969)
Background
Background
Blackcheek tonguefish 24 hrs
Atlantic menhaden
Second application
(June 10, 1970)
Spot
Silver perch
Atlantic menhaden
Striped mullet
Star drum
3 mos
24 hrs
3 mos
8 mos
8 mos
8 mos
3
2
2
1
9
6
5
1
2
14
5
3
1-2.5
2-3
2-3
12.5
8
3-5
4
2-2.5
3.5-7
S. NAVAL
2
3.5
4-6
13
2-3.5
1.5-2
5-5.5
5.5
4.5-5.5
1.5-2.25
RIVER, OLD TOWN
3
1
3
12
8
9
5
2
43
3.5
8
3
2-4
2-3
4-4.5
4.5
5.5
1-1.75
—
—
.053
.35
.086
—
.82
.20
.096
.19
—
.21
—
.036
.12
—
.14
,045
—
—
CREEK
—
—
—
—
—
—
—
—
20
PESTICIDES MONITORING JOURNAL
-------
TABLE 7.—Mirex residues in fishes by sampling site and sampling time, South Carolina, 1969-71—Continued
[— = not detected]
SIZE OF MIREX
INTERVAL NUMBER IN INDIVIDUALS RESIDUE
SPECIES FROM MIREX cOMPOSrrE IN COMPOSITE (PPM —
APPLICATION SAMPLE SAMPLES WHOLE
TO SAMPLING (INCHES) BODY)
STATION 6— WANDO RIVER, BERESFORD CREEK
Silver perch Background 2 4.5 —
First application
(Oct. 17, 1969)
Pinfish 24hrs 1 7 —
Second application
(June 8, 1970)
Spot 24 hrs 12 2.5-3.5 .016
3 mos 12 3.5-5 —
Silver perch 8 mos 22 1.5-2 —
TABLE 8. — Mirex residues in miscellaneous organisms by
[— = not
SIZE op
MAL NUMBER IN INDIVIDUALS MIREX
SPECIES FROM MIREX COMPOSITE IN COMPOSITE RESIDUE
APPLICATION SAMPLE SAMPLES (PPM) i
TO SAMPLING (,NCHES)
SAMPLING SITE A— TOOGOODOO CREEK
Second application
(June 3-4, 1970)
American oyster 12wks 12 2-4 —
SAMPLING SITE B— TOOGOODOO CREEK
First application
(Oct. 14-15, 1969)
Brief squid 32 wks 10 4-6 —
Second application
(June 3-4, 1970)
American oyster 12 wks 12 2-4 —
Third application
(Oct. 27-28, 1970)
American oyster 6 wks 28 2-4 —
10 wks 18 2-4 —
12 wks 10 2-4 —
SAMPLING SITE C— TOOGOODOO CREEK
First application
(Oct. 14-15, 1969)
American oyster 32 wks 25 2-4 —
Southern periwinkle 32 wks 40 ,5-,75 —
Phytoplankton
(dry weight) 32 wks —
Second application
(June 3-4, 1970)
American oyster 12 wks 12 2-4 —
Third application
(Oct. 27-28, 1970)
American oyster 6 wks 22 2-4 —
10 wks 13 2-4 —
12 wks 12 2-4 —
Nudibranch 16 wks 18 .5-1
SIZE OF MIREX
INTERVAL NuMBER IN INDIVIDUALS RESIDUE
SPECIES FROM MIREX COMPOSITE IN COMPOSITE (PPM-
APPLICATION s SAMPLES WHOLE
TO SAMPLING 3AMPLE (INCHEs) BODY)
STATION 7— SOUTH SANTEE RIVER, ALLIGATOR CREEK
Atlantic croaker Background 24 —
Hog choker Background 4 2-2.5 —
Spot Background 3 2-3 —
First application
(Sept. 18, 1969)
Spot 24hrs 3 2-3 -
Sheepshead 3 mos 1 8.5 —
Second application
(May 20, 1970)
Spot 24hrs 26 —
Silver perch 24 hrs 1 5 —
3 mos 6 3.5-6.5 —
Spot 8 mos 12 3.75-4.5 .011
sampling site and sampling lime, South Carolina, 1969-71
detected)
SIZE OF
iw'AL NUMBER IN INDIVIDUALS MIREX
SPFCIM FROM MIREX COMPOSITE IN COMPOSITE RESIDUE
APPLICATION SAMPLE SAMPLES (PPM)'
TO SAMPUNG (INCHES)
SAMPLING SITE D— TOOGOODOO CREEK
Second application
(June 3-4, 1970)
STATION 1— RIVERLAND TERRACE POND (EAST)
First application
(Dec. 3, 1969)
Egg masses from 186
gravid grass
shrimp 5 mos —
Third application
(Dec. 1, 1970)
Dead white mullet 2 wks 1 5 —
STATION 5— LOWER ASHLEY RIVER, OLD TOWN CREEK
Second application
(June 10, 1970)
Dead striped mullet 24 hrs 1 11.5 —
STATION 7— SOUTH SANTEE RIVER, ALLIGATOR CREEK
First application
(Oct. 18, 1969)
Pied-billed grebe
(breast muscle)
(drowned in net) 3 mos
1 Residues are whole-body basis unless otherwise indicated.
VOL. 7, No. 1, JUNE 1973
21
-------
TABLE 9.—Mirex residues in birds by sampling site and sampling lime. South Carolina. 1969-7]
[— = not detected]
QUARTERLY
COLLECTION
MIREX
SPECIES RESIDUE
(PPM) I
SAMPLING SITES A-B-C— TOOGOODOO CREEK
December 1969
March 1970
May 1970
September 1970
December 1970
February 1971
May 1971
SAMPLING
December 1969
March 1970
Green Heron
Kingfisher
Clapper Rail
do.
do.
do.
do.
do.
do.
Oil glands from 5 Clapper Rails
Clapper Rail
do.
do.
do.
do.
do!
Oil glands from 6 Clapper Rails
Green Heron
do.
do.
j_
QO.
Yellow Crowned Night Heron
Little Blue Heron
Clapper Rail
Clapper Rail
do.
do.
do.
do.
do.
do.
do.
do.
do.
do.
do.
do.
Clapper Rail
do.
10 Rails (Muscle)
10 Rails (Fat)
Willet
Kingfisher
Clapper Rail
do.
do.
d-
o.
do.
do.
do.
do.
do.
do.
do.
Oil glands from 6 Clapper Rails
Louisiana Heron
Clapper Rail
Louisiana Heron
do.
Little Blue Heron
do.
American Egret
do.
SITE D— TOOGOODOO CREEK
.99
1.30
.29
1.40
.05
.10
.15
1.90
.29
.52
_
.06
.05
.08
.10
.06
.29
.09
.17
m
.u /
.11
—
.u
—
.17
.02
—
.02
.05
.63
.02
.19
.04
—
.15
.07
1.20
—
.15
.04
.13
.10
03
.19
.04
.07
.05
.75
—
.58
.11
.01
.94
.14
.01
.22
5.40
.05
Attempts to collect birds unsuccessful
Clapper Rail
do.
—
.11
QUARTERLY
COLLECTION
SAMPLING
May 1970
September 1970
December 1970
February 1971
May 1971
STATION
September 1969
December 1969
March 1970
May 1970
September 1970
December 1970
February 1971
May 1971
MIREX
SPECIES RESIDUE
(PPM) 1
SITE D— TOOGOODOO CREEK— Continued
Plover
Clapper Rail
do.
Kingfisher
do. 1
do.
Clapper Rail
do.
do.
Clapper Rail
do.
do.
Clapper Rail
Kingfisher
Snowy Egret
Green Heron
Louisiana Heron
do.
2— STONO RIVER, LOG BRIDGE CREEK
American Egret
Little Blue Heron
Clapper Rail
Clapper Rail
do.
Clapper Rail
do.
do.
Sora Rail
Clapper Rail
do.
Clapper Rail
do.
do.
Clapper Rail
do.
Clapper Rail
do.
Clapper Rail
Little Blue Heron
—
.61
—
.50
.04
—
—
—
.06
.03
.03
.06
—
.17
.04
.03
.12
.19
.55
.90
—
.18
.11
—
.08
.05
.10
.21
.16
.11
.06
.09
.13
.09
.70
STATION 3— UPPER ASHLEY RIVER,
September 1969
December 1969
March 1970
May 1970
September 1970
December 1970
RUNNYMEADE PLANTATION
Green Heron
do.
do.
Snowy Egret
American Bittern
Snipe
do.
Anhinga (Juvenile)
Anhinga
Kingfisher
(internal organs)
Pied-Billed Grebe
Anhinga
Snipe
do.
do.
do.
.25
.15
.13
.69
1.80
.11
1.10
_
1.70
17.00
8.30
•jo
.f.O
.35
.05
.34
."'PJ
22
PESTICIDES MONITORING JOURNAL
-------
TABLE 9.-—Mirex residues in birds by sampling site and sampling time, South Carolina, 1969-71—Continued
[— — not detected]
QUARTERLY
COLLECTION
STATION
February 1971
May 1971
SPECIES
MIREX
RESIDUE
(PPM) l
3_ UPPER ASHLEY RIVER— Continued
Snowy Egret
Marsh Hawk
Least Bittern
Louisiana Heron
.60
1.50
2.20
1 m
1 . 1U
COLLECTION
QUARTERLY
SPECIES
MIREX
RESIDUE
MIREX
STATION 5— LOWER ASHLEY RIVER— Continued
May 1971
STATION
Clapper Rail
do.
do.
Eggs
6— WANDO RIVER, BERESFORD
STATION 4— COOLER KlVhR. U.S. NAVAL
September 1969
December 1969
March 1970
May 1970
September 1970
December 1970
February 1971
May 1971
C-T-ATT/-1 >J ^ T
MA11UN 5 L
September 1969
December 1969
March 1970
May 1970
September 1970
December 1970
February 1971
AMMUNITION DEPOT
Louisiana Heron
do.
Attempts to collect
Snipe
do.
Grebe
Louisiana Heron
Green Heron
Clapper Rail
do.
Clapper Rail
American Bittern
Grebe
Least Bittern
Clapper Rail
/-jvirpD A QtTT PV T?T\/T^T) O'
UWx^lv Aorim 1 KlVCtv, >*Ji
Louisiana Heron
American Egret
Clapper Rail
do.
Clapper Rail
do.
Sora Rail
Virginia Rail
Clapper Rail
do.
Eggs
Clapper Rail
do.
Clapper Rail
do.
Clapper Rail
Sora Rail
—
birds unsuccessful
.14
.89
—
.38
1.00
.60
.94
.19
.04
1.10
2.80
.18
r T"l TOWM PRFFTf
September 1969
December 1969
March 1970
May 1970
September 1970
December 1970
February 1971
May 1971
Snowy Egret
American Egret
Attempts to collect birds
Clapper Rail
Snipe
Clapper Rail
White Ibis
Kingfisher
do.
Sora Rail
Clapper Rail
do.
Clapper Rail
do.
Clapper Rail
do.
Green Heron
Little Blue Heron
.10
—
.04
.05
CREEK
.89
unsuccessful
.05
—
.47
.29
—
—
—
.04
—
.82
.25
.71
.07
.14
.17
STATTnMT SnilTtr SAMTCT3 DT\7T31> A T T T« ATnB <~TJCP V
.11
.35
.09
.07
.09
.06
—
—
.08
.04
.07
.06
.09
.01
'
September 1969
December 1969
March 1970
May 1970
September 1970
December 1970
February 1971
May 1971
Snowy Egret
Little Blue Heron
Clapper Rail
do.
Grebe
Snowy Egret
do.
Kingfisher
do.
Snowy Egret
American Egret
Snowy Egret
Louisiana Heron
Clapper Rail
Snowy Egret
—
—
—
—
—
.
.20
.06
.51
.45
.13
.17
.10
.11
1 Residues are breast and upper wing muscle unless otherwise indicated.
VOL. 7, No. 1, JUNE 1973
23
-------
TABLE 10.—Average mirex residues in muscle tissue of
herons and egrets at each station,
South Carolina, 1969-71
TABLE 11.—Average mirex residues in muscle tissue of
clapper rails at each station, South Carolina, 1969-71
STATION
A-B-C
D
2
3
4
5
6
7
Overall
average
Total
birds
AVERAGE RESIDUES
Sept.
1969
.06
(2)
.18
(3)
.00
(2)
.00
(2)
.45
(2)
.00
(2)
.12
(13)
Dec. Mar.
1969 1970
.99
(D
.69
(1)
.84
(2)
IN PPM AND NUMBER OF BIRDS
May Sept. Dec. Feb.
1970 1970 1970 1971
.07 .11
(6) (1)
.60
(D
.69
(2)
.10 .48 .15
(2) (2) (2)
.08 .69 .48 .25
(8) (2) (2) (4)
( )
May
1971
1.13
(6)
.06
(4)
.70
(D
1.10
(1)
.16
(2)
.11
(1)
.61
(15)
AVERAGE RESIDUES IN PPM
STATION ^ t
1969
A-B-C
D
2 .19
(D
3
4
5
6
7 .00
(2)
Dec.
1969
.60
(7)
.73
(2)
.23
(2)
Mar.
1970
.07
(6)
.06
(2)
.10
(3)
.08
(2)
.05
(D
May
1970
.11
(D
.31
(2)
.07
(2)
.00
(2)
.47
(D
AND NUMBER op BIRDS ( )
Sept.
1970
.09
(13)
.00
(3)
.16
(3)
.02
(2)
.02
(2)
Dec.
1970
.07
(12)
.04
(3)
.08
(2)
.77
(2)
.06
(2)
.54
(2)
Feb.
1971
.14
(11)
.06
(1)
.11
(2)
.19
(1)
.09
(1)
.39
(2)
May
1971
.01
(D
.09
(D
.18
(1)
.05
(3)
.10
(D
Overall
average .19 .47 .07 ,16 .07 .23 .15 .07
Total
birds (1) (13) (14) (8) (23) (23) (18) (7)
TABLE 12.—Mirex residues in mammals by sampling site and sampling lime, South Carolina, 1969-71
[— — not detected]
QUARTERLY
COLLECTION
MIREX
SPECIES RESIDUE
(PPM) i
SAMPLING SITE A— TOOGOODOO CREEK
December 1969
March 1970
May 1970
September 1970
December 1970
February 1971
May 1971
SAMPLING
December 1969
March 1970
May 1970
Raccoon
Fat
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
SITE B— TOOGOODOO CREEK
Raccoon
Fat
Raccoon
Fat
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
.14
.97
.05
1.30
.20
.02
.26
.02
.16
.88
.16
.07
.43
.03
.66
.12
QUARTERLY
COLLECTION
SPECIES
MIREX
RESIDUE
(PPM)'
SAMPLING SITE B—TOOGOODOO CREEK—Continued
September 1970
December 1970
February 1971
May 1971
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
.21
.04
.12
.16
.04
SAMPLING SITE C—TOOGOODOO CREEK
December 1960 Raccoon —
Raccoon —
March 1970 Attempts to collect raccoons unsuccessful
May 1970 Attempts to collect raccoons unsuccessful
September 1970
December 1970
February 1971
May 1971
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
.15
.24
.02
.08
.04
.09
.09
.02
24
PESTICIDES MONITORING JOURNAL
-------
TABLE 12.—Mirex residues in mammals by sampling site and sampling time, South Carolina, 1969-71—Continued
[— = not detected]
QUARTERLY
COLLECTION
SPECIES
MIREX
RESIDUE
(PPM) '
SAMPLING SITE D— TOOGOODOO CREEK
December 1969
March 1970
May 1970
September 1970
r- .. .nber 1970
February 1971
May 1971
Raccoon
Raccoon
Fat
Attempts to collect raccoons
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
STATION 2— STONO RIVER, LOG BRIDGE
December 1969
March 1970
May 1970
September 1970
December 1970
February 1971
May 1971
Raccoon
Fat
Raccoon
Fat
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
unsuccessful
.01
.04
.01
.04
CREEK
1.40
.07
.07
1.90
.20
.04
STATION 3— UPPER ASHLEY RIVER,
RUNNYMEADE PLANTATION
December 1969
March 1970
May 1970
September 1970
December 1970
February 1971
May 1971
Attempts to collect raccoons
Attempts to collect raccoons
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Opossum
unsuccessful
unsuccessful
.56
.12
.88
.10
.19
1.90
.09
3.30
STATION 4— COOPER RIVER, U.S. NAVAL
AMMUNITION DEPOT
December 1969
March 1970
May 1970
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
.80
.39
.60
4.40
.28
QUARTERLY
COLLECTION
SPECIES
MIREX
RESIDUE
(PPM) i
STATION 4— COOPER RIVER— Continued
September 1970
December 1970
February 1971
May 1971
Raccoon
Raccoon
Attempts to collect
Attempts to collect
Attempts to collect
STATION 5— LOWER ASHLEY RIVER,
December 1969
March 1970
May 1970
September 1970
December 1970
February 1971
May 1971
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
.90
1.30
raccoons unsuccessful
raccoons unsuccessful
raccoons unsuccessful
OLD TOWN CREEK
.06
.03
.02
.05
STATION 6— WANDO RIVER, BERESFORD CREEK
December 1969
March 1970
May 1970
September 1970
December 1970
February 1971
May 1971
STATION 7— SOUTH
September 1969
December 1969
March 1970
May 1970
September 1970
December 1970
February 1971
May 1971
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Opossum
Opossum
.04
.16
.02
.02
.04
.80
2.20
SANTEE RIVER, ALLIGATOR CREEK
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
Attempts to collect
Attempts to collect
Raccoon
Raccoon
Raccoon
Raccoon
Raccoon
raccoons unsuccessful
raccoons unsuccessful
.06
.03
1 Residues for thigh muscles unless otherwise indicated.
VOL. 7, No. 1, JUNE 1973
25
-------
TABLE 13.—Average mirex residues in muscle tissue of
raccoons at cadi station, South Carolina, 1969-71
TABLE 14.—Summary of birds and mammals containing
mirex residues in excess of 1.0 ppm, South Carolina, 1969-71
AVERAGE RESIDUES IN
STATION g_ _,
1969
B
C
D
2
3
4
5
6
7 .00
(D
Overall
average .00
Total
raccoons (1)
Dec.
1969
(1)
.00
(2)
.00
(2)
.00
(2)
.00
(2)
.60
(2)
.00
(2)
.02
(2)
.00
(2)
.08
(17)
Mar.
1970
f\c
.UJ
(D
.25
(2)
.00
(1)
2.50
(2)
.00
ti \
(2;
.00
(2)
.00
(2)
.46
(12)
PPM AND NUMBER OF RACCOONS ( )
May
1970
•je
, IJ
(2)
.20
(4)
.03
(2)
.74
(2)
.00
(D
.28
(D
.00
(2)
.08
(2)
.27
(16)
Sept.
1970
(2)
.11
(2)
.20
(2)
.01
(2)
.99
(2)
.52
(3)
1.10
(2)
.03
(2)
.00
(I)
.37
(18)
Dec.
1970
nt
.Ul
(2)
.00
(2)
.05
(2)
.00
(2)
.00
(2)
.05
(2)
.03
(2)
.02
(2)
.00
(2)
.02
(18)
Feb.
1971
no
.Uo
(2)
.08
(2)
.07
(2)
.02
(2)
.12
(2)
.19
(1)
.00
(2)
.02
(2)
.03
(2)
.06
(17)
May
1971
.44
(2)
.10
(2)
.06
(2)
.00
(2)
.00
(2)
1.00
(2)
.03
(2)
.20
(1)
.03
(D
.22
(15)
STATION
A-B-C
A-B-C
A-B-C
A-B-C
D
3
3
3
3
3
3
3
4
4
4
ANIMAL
SPECIES
B:
Kingfisher
Clapper Rail
Clapper Rail
American Egret
Kingfisher
American Bittern
Snipe
Anhinga
Kingfisher
Marsh Hawk
Least Bittern
Louisiana Heron
Louisiana Heron
Grebe
Least Bittern
MIREX
RESIDUES
IN PPM
IRDS
1.30
1.40
1.90
5.40
1.50
1.80
1.10
1.70
17.00
1.50
2.20
1.10
1.00
1.10
2.80
MONTH OF
SAMPLE COLLECTION
December 1969
December 1969
December 1969
May 1971
September 1970
December 1969
March 1970
May 1970
May 1970
February 1971
May 1971
May 1971
September 1970
February 1971
May 1971
MAMMALS
A
2
2
3
3
4
4
6
Raccoon
Raccoon
Raccoon
Raccoon
Opossum
Raccoon
Raccoon
Opossum
1.30
1.40
1.90
1.90
3.30
4.40
1.30
2.20
May 1970
May 1970
September 1970
May 1971
May 1971
March 1970
September 1970
May 1971
26
-------
CONTRIBUTION NO. 157
-------
998 JOURNAL FISHERIES RESEARCH BOARD OF CANADA, VOL. 30, NO. 7, 1973
Relation Between Simple Dynamic Pool and Surplus Production
Models for Yield from a Fishery1
A. L. JENSEN
Environmental Protection Agency
Gulf Breeze Environmental Research Laboratory? Sabine Island, Gulf Breeze, Fla. 32561, USA
JENSEN, A. L. 1973. Relation between simple dynamic pool and surplus production models
for yield from a fishery. J. Fish. Res. Board Can. 30: 998-1002.
Dynamic pool models without self-regenerating properties are continuous age models,
and surplus production models are continuous time models. Self-regenerating dynamic pool
models are continuous age-discrete generation models and, also, discrete time-discrete age mod-
els. In a steady state specification of the regulatory function and direct estimation of biomass
results in the surplus production model. Estimation of biomass by specifying the functions with
respect to age for size of a cohort and individual weight and application of the coefficient of
fishing mortality result in the dynamic pool model. A third approach, not applied in fisheries,
is to specify the regulatory function and functions with respect to age of cohort size and in-
dividual growth in weight. In a steady state all methods for calculating yield give the same
results if the functions specified are realistic. Specification of the functions requires that many
assumptions be made. The dynamic pool model may be more accurate than the surplus
production model because the regulatory function may be more difficult to determine than
the functions with respect to age of cohort size and growth in individual weight.
JENSEN, A. L. 1973. Relation between simple dynamic pool and surplus production models
for yield from a fishery. J. Fish. Res. Board Can. 30: 998-1002.
Des modeles pools dynamiques depourvus d'autor6g&i6ration sont des modeles 4 age
continu, alors que les modeles surplus-production sont des modeles a temps continu. Les mo-
deles pools dynamiques a autoreg6neration sont des modeles a age continu-gen^ration dis-
continue; ils sont egalement des modeles a temps discontinu-age discontinu. Dans un 6tat
d'equilibre, la specification de la fonction regulatrice et I'estimation directe de la biomasse
aboutissent au modele surplus-production. Par ailleurs, I'estimation de la biomasse par speci-
fication des fonctions qui ont trait £ 1'age correspondent a la taille d'une cohorte et au poids
individuel d'une part, et 1'application du coefficient de mortalite due a la peche d'autre part,
aboutissent au modele pool dynamique. Une troisieme m6thode, non appliqu6e aux peches,
consiste a specifier la fonction regulatrice et les fonctions qui ont trait a 1'age correspondant
4 la taille d'une cohorte et a la croissance ponderale individuelle. Dans un 6tat d'equilibre,
toutes les methodes de calcul de rendement donnent les memes r&ultats, pourvu que les fonc-
tions specifiers soient realistes. La specification des fonctions requiert 1'elaboration de plusieurs
hypotheses. Le modele pool dynamique peut etre plus prfcis que le modele surplus-production,
parce que la fonction regulatrice est parfois plus difficile a etablir que les fonctions qui ont
trait a 1'age correspondant 4 la taille de la cohorte et 4 la croissance ponderale individuelle.
Received January 9, 1973
./'THEMATICAL models applied to fish populations of models have been compared by Schaefer and
have been grouped into two basic categories: dy- Beverton (1963) and by Silliman (1971).
namic pool models such as those of Baranov (1918), These models differ in a fundamental way that
Thompson and Bell (1934), Ricker (1944), and does not appear to have been explicitly discussed
Beverton and Holt (1957); and surplus production in the literature. In dynamic pool models without
models such as those of Hjort et al. (1933), Graham self-regenerating properties the variables are con-
(1935), and Schaefer (1954, 1957). These two types tinuous functions of age, whereas in surplus
production models the variables are continuous
'Contribution No. 157, Gulf Breeze Laboratory, functions of time. In this note the dynamic pool
2Associate Laboratory of the National Environmental model without self-regeneration properties and the
Research Center, Corvallis, Oreg., USA. surplus production model will be reviewed with
attention drawn to their time and age properties,
Printed in Canada (J2790) and then the models will be compared.
-------
NOTES
999
Mathematical symbols used are:
B(0 = population size in biomass at time /;
BO, = environmental carrying capacity in terms of
biomass;
B = annual biomass accumulation;
/(B(/)) = regulatory function that gives instantaneous
rate of change in biomass;
F = fishing mortality coefficient;
FI = coefficient of effect of fishing on population
density;
F2 = coefficient of effect of fishing on average
individual weight;
g = coefficient of growth in weight per individual
averaged over the entire population;
K = coefficient of individual fish growth in weight
or length;
k = coefficient of population growth in number
of individuals and weight;
N(jc,0 = number of individuals of age x at time t;
N(x) = number of individuals of a cohort alive at
age x;
N^ = environmental carrying capacity in terms of
the number of individuals;
N(/) = total number of individuals in population at
time t;
P(0 = population size at time t in biomass or
numbers;
R = recruitment;
r = coefficient of growth in number of individuals;
t = time measured in years;
v = greatest age attainable by an individual fish;
W(x,t) — average weight of an individual of age x
at time t;
W(x) = average weight of an individual of age x;
W,,, = maximum attainable weight of an individual
fish;
W(/) = average weight of all individuals in the
population at time /;
x = age measured in years;
XQ = theoretical age at which length equals zero;
Y = yield in weight;
YE = equilibrium yield in weight;
Z = total instantaneous mortality coefficient.
In the above list, symbols of the form B(0 indicate that
B is a function of / and should not be confused with
multiplication.
Review of typical dynamic pool model—The dynamic
pool model for change in yield with respect to age is
given by
dY
dx
dx
(1)
where F is a constant and N(.x) and W(x) are un-
specified functions of age. Change in the number of
individuals in a cohort with respect to age is given by
which after integration becomes
N(x) = R e-Z*.
(3)
A number of different models have been applied to
describe weight as a function of age; Richards (1959)
has shown that these models are related. Von Berta-
lanffy's equation
(4)
has frequently been applied for this purpose and appears
to adequately describe the growth of many species of
fish. Substitution of equations (3) and (4) into equation
(1) gives the simple Beverton and Holt yield equation
dY
dx
= F R e-z* Woo (1 - e-KC*-*,))3.
(5)
dN
d*
= -Z N(x)
(2)
This model has recently been discussed in considerable
detail by Gulland (1969). In equations (3), (4), and (5)
population density, weight, and yield are all clearly
functions of age. Under steady state conditions it has
been shown that yield per generation equals annual
yield from an entire population (Thompson and Bell
1934; Beverton and Holt 1957). In this situation annual
yield can be calculated using equation (5), but it is con-
stant with respect to time. In practice, to compensate
for variation in recruitment, equation (5) is usually
divided by recruitment to obtain an equation for yield
per recruit (Gulland 1972). In most fisheries variation in
recruitment is important and has led to investigation of
stock-recruitment relations (Ricker 1958).
Self-regeneration properties of dynamic pool models,
such as those proposed by Ricker (1954) and Beverton
and Holt (1957), produce a continuous age-discrete
generation model. These self-regenerating dynamic
pool models usually have been applied to obtain yield
per generation (Ricker 1958; Larkin and Ricker 1964;
Tautz et al. 1969; Waller et al. 1971). A simple book-
keeping procedure can easily be established to obtain
yield per year from a population consisting of several
age-groups (Walters 1969). Continuous age models such
as the Von Bertalanffy growth equation may be applied
to calculate the constants in self-regenerating dynamic
pool models, but these continuous age-discrete genera-
tion models are also discrete time-discrete age models
as is clearly revealed by the nature of Walters' (1969)
computer program. Self-regenerating dynamic pool
models may offer the best approach for obtaining realistic
models. However, these models are complex and are
difficult to compare with the simpler models; their
inclusion in this study would result in an undesirable
degree of complexity.
Review of typical surplus production model- — The
general form of this model for an exploited population
under average environmental conditions was formulated
by Schaefer (1954) as
(6)
-------
1000
JOURNAL FISHERIES RESEARCH BOARD OF CANADA, VOL. 30, NO. 7, 1973
If P(f) is taken as biomass, then
P(0 = B(0 = N(/) W(0
(7)
where N(0 is the total number of individuals of all ages
at any point in time
= f
Jo
N(0 = I N(x,0 Ax (8)
Jo
and W(r) fhe average weight of an individual at time t
W(0 =
f
Jo
Ax
f
Jo
(9)
Ax
It can be shown (Jensen 1972), by applying the equation
-•>
ao,
which is obtained by differentiating equation (7), that, if
change in biomass with respect to time is given by the
linear surplus production model
f- = *«"(BT^)-
. . .
dN
then suitable equations for —— and dW/d/ are given by
At
dN
At
dW —
rN(Q
(12)
where k •= r + g. Yield with respect to time is given by
the equation
•- = F N(0 W(0-
(14)
Substitution of equations (12) and (13) into equation
(14) gives
Comparison of simple dynamic pool and surplus pro-
duction models under steady state conditions — Virtually
no exploited fish population is in a steady state but, as
a first approximation in modeling a fish population, a
steady state is usually assumed (Gulland 1972). In
the steady state the difference between dynamic pool
and surplus production models results from specifying
different functions. In a steady state, where dB/df = 0,
dN/d? = 0, and dW/d/ = 0, annual yield from a fishery
is given by
YE = FB = B/(B)
(16)
which is obtained from equations (6) and (7). Biomass
accumulation of a cohort during its lifetime in a steady
state is given by
f'
Jo
Ax
(17)
where N(x) and W(x) are unspecified functions of age.
Biomass accumulation from an entire population during
1 year of life under steady state conditions is given by
r
Jo
N(0 W(/) At
(18)
but N(0 and W(0 are constant with respect to time.
Substituting equations (8) and (9) into expression (18)
gives
(19)
and, after cancellation of similar terms and integration
with respect to time from 0 to 1, expression (19) becomes
the same as expression (17). Under steady state conditions
the biomass accumulation of a cohort during its lifetime
equals the biomass accumulation of an entire population
during 1 year.
Substitution of expression (17) into equation (16) for
annual biomass accumulation gives
YE = F I N(JC]
Jo
' fN
dx.
(20)
From equations (16) and (20) it is clear that yield in a
steady state can be calculated by four different ap-
proaches :
'. (15)
(1) Specify the form of no functions and apply the
Clearly, equations (12), (13), and (15) give population equation
size, average individual weight, and yield as functions
of time. YE = F B. (21)
-------
NOTES
1001
This method was applied by Ricker (1945, 1958), who
separated the life of the fish into segments and applied
equation (21) to each segment and then summed over the
segments to obtain yield from the entire population.
(2) Specify the form of the regulatory function, /(B),
and apply the equation
YE=/(B)B.
(22)
This is the surplus production approach. Examples of the
application of these models to specific fisheries are given
by Ricker (1958), Schaefer (1954), and Gulland (1972).
Ricker reviews the assumptions of this approach.
(3) Specify the form of W(x) and N(x) and apply the
equation
YE
d*.
(23)
This is the dynamic pool approach. Examples of the
application of these models are given by Gulland (1972),
Beverton and Holt (1957), and Gulland (1969). Ricker
(1958) and Beverton and Holt (1957) review the assump-
tions of this approach.
(4) Specify the form of/(B), N(x), and W(x) and apply
the equation
f
YE = /(B) W(x) N(x) Ax. (24)
Equation (24) has not been applied. Application
of equation (24) is equivalent to applying both the
dynamic pool and surplus production models to
the same fish population. Gulland (1972) has ap-
plied both the linear surplus production model
and the Beverton and Holt yield equation to the
eastern Pacific yellowfin tuna fishery, but he did
not combine the two models into a single equation.
Equation (24) combines the surplus production
concept of the environmental carrying capacity
with the age-specific events of the dynamic pool
model. Application of equation (24) requires many
of the assumptions of both equations (22) and (23).
Equation (24) may not be of practical value, but
it is of interest for it shows that in a steady state
the constant for fishing mortality, F, of the dynamic
pool model equals the regulatory function, /(B),
of the surplus production model.
All four expressions for yield are equal and theo-
retically give the same result. However, in fitting
specific models to data different results may be
obtained with the four yield equations if the func-
tions selected for /(B), N(JC), and W(*) are not rea-
listic. For example, if biomass is calculated by
functions N(;c) and W(x) that poorly approximate
mortality and growth, the annual biomass accumula-
tion estimated directly from the weight of the catch
may not equal the annual biomass accumulation
calculated by expression (17). Therefore, yield
calculated by the surplus production approach
(equation 22) may not equal yield calculated by the
dynamic pool approach (equation 23).
The dynamic pool yield equation may be more
accurate than the surplus production yield equation
because the form of the functions N(x) and W(x)
can be determined more easily than the form of
the function /(B). To obtain data on the form
of W(*) and N(*) in a steady state, either observa-
tions can be made throughout the lifetime of a
cohort or observations can be made on the entire
population at a single point in time. To obtain data
on the form of/(B), estimates of yield and biomass
are required for different steady states. The dynamic
pool model must be used if the parameters it con-
tains, such as age of entry into the fishery, are of
interest. Gulland (1972) has discussed in consider-
able detail the practical aspects of applying both
the dynamic pool and surplus production models.
Schaefer and Beverton (1963, equation 1) give a
general equation that describes the dynamics with
respect to time of an exploited fish stock. The terms
in this equation for changes with respect to time
were confused with the terms for changes with
respect to age that are contained in the expressions
for biomass in both the dynamic pool and the sur-
plus production models. This led to the incorrect
conclusion that the dynamic pool model describes
a population in terms of recruitment, growth, and
natural and fishing mortality, whereas the surplus
production model combines these effects into a
common function of the mean population size.
Equation (23) shows that yield calculated by the
dynamic pool model is the product of the fishing
mortality coefficient and annual biomass accu-
mulation. Annual biomass accumulation is estimated
analytically using functions N(JC) and W(jt). In
the dynamic pool model the terms for recruitment,
growth, and natural and fishing mortality are
contained in the expression for annual biomass
accumulation. Equation (22) shows that yield cal-
culated by the surplus production model is the
product of the regulatory function and annual
biomass accumulation. In practice annual biomass
accumulation is estimated directly from the biomass
of the catch without aid of the functions \V(x)
and N(x). In the surplus production model the
terms for recruitment, growth, and natural and
fishing mortality are all combined in the expression
for annual biomass accumulation. The regulatory
function is a function of biomass; therefore, the
regulatory function is also a function of recruitment,
-------
1002
JOURNAL FISHERIES RESEARCH BOARD OF CANADA, VOL. 30, NO. 7, 1973
growth, and natural and fishing mortality. In ad-
dition, however, the regulatory function contains
terms that have no counterpart in the dynamic
pool model. These terms are the coefficient for
growth in number of individuals with respect to
time, r; the coefficient of growth of average indivi-
dual weight, g; and the environmental carrying
capacity in terms of biomass, Bm. The regulatory
function is a complex function.
In summary, the surplus production model is a
function of time and the simple dynamic pool
model is a function of age. In the steady state the
differences between the dynamic pool and surplus
production models result from specifying different
functions: (1) In the dynamic pool model analytical
functions are applied to determine biomass. In
surplus production models biomass is estimated
directly from the weight of the catch; (2) In dynamic
pool models the proportion of the annual biomass
accumulation that is captured by fishing is estimated
directly by the coefficient of fishing mortality.
In surplus production models the proportion of the
annual biomass accumulation that is captured by
fishing is equated to the regulatory function. The
regulatory function gives the rate of growth of
biomass per unit of biomass that would occur in
the absence of fishing. If fishing were stopped,
biomass would increase to the environmental
carrying capacity, and the rate of biomass increase
at the moment fishing stopped would equal the
coefficient of fishing mortality. The dynamic pool
and surplus production models are not based on
mutually exclusive theories, but when specific
functions are defined the results calculated by these
two methods may not agree. The dynamic pool and
surplus production approaches are only two of
four possible approaches for calculating yield under
steady state conditions. Some of the four approaches
may be more accurate than others.
Acknowledgments — I thank R. P. Silliman for valuable
suggestions that he made on a draft of this note.
BARANOV, F. I. 1918. On the question of the biological
basis of fisheries [in Russian]. Nauch.-Issled. Ikhtiol.
Inst. Izv. 1: 81-128.
BEVERTON, R. J. H., AND S. J. HOLT. 1957. On the
dynamics of exploited fish populations. H.M.S.O.,
London. 533 p.
GRAHAM, M. 1935. Modern theory of exploiting a
fishery, and application to North Sea trawling. J.
Cons. Cons. Perma. Int. Explor. Mer 10: 264-274.
GULLAND, J. A. 1969. ' Manual of methods for fish
stock assessment. Part 1. Fish population analysis.
FAO Man. Fish. Sci. No. 4, Rome. 154 p.
1972. Population dynamics of world fisheries.
University of Washington Press, Seattle, Wash. 336 p.
HJORT, J., G. JAHN, AND P. OTTESTAD. 1933. The
optimum catch. Hvalradets Skr. 7: 92-127.
JENSEN, A. L. 1972. Population biomass, number of
individuals, average individual weight, and the linear
surplus-production model. J. Fish. Res. Board Can.
29: 1651-1655.
LARKIN, P. A., AND W. E. RICKER. 1964. Further
information on sustained yields from fluctuating en-
vironments. J. Fish. Res. Board Can. 20: 647-678.
RICHARDS, F. J. 1959. A flexible growth function for
empirical use. J. Exp. Bot. 10: 290-300.
RICKER, W. E. 1944. Further notes on fishing mor-
tality and effort. Copeia 1944: 23-44.
1945. A method of estimating minimum size
limits for obtaining maximum yield. Copeia 1945:
84-94.
1954. Stock and recruitment. J. Fish. Res.
Board Can. 11: 559-623.
1958. Maximum sustained yields from fluctuating
environments and mixed stocks. J. Fish. Res. Board
Can. 15: 991-1006.
SCHAEFER, M. B. 1954. Some aspects of the dynamics
of populations important to the management of com-
mercial marine fisheries. Bull. Inter-Amer. Trop.
Tuna Comm. 1: 27-56.
1957. A study of the dynamics of the fishery for
yellowfin tuna in the eastern tropical Pacific Ocean.
Bull. Inter-Amer. Trop. Tuna Comm. 2: 247-268.
SCHAEFER, M. B., AND R. J. H. BEVERTON. 1963.
Fishery dynamics — their analysis and interpretation,
p. 464-483. In M. N. Hill [ed.] The sea. John Wiley
& Sons, New York, N.Y.
SILLIMAN, R. P. 1971. Advantages and limitations of
"simple" fishery models in light of laboratory experi-
ments. J. Fish. Res. Board Can. 28: 1211-1214.
TAUTZ, A., P. A. LARKIN, AND W. E. RICKER. 1969.
Some effects of simulated long-term environmental
fluctuations on maximum sustained yield. J. Fish.
Res. Board Can. 26: 2715-2726.
THOMPSON, W. F., AND F. H. BELL. 1934. Biological
statistics of the Pacific halibut fishery. (2) Effects
of changes in intensity upon total yield and yield per
unit of gear. Rep. Int. Fish. Comm., No. 8. Seattle,
Wash. 49 p.
WALLER, W. T., M. L. DAHLBERG, R. E. SPARKS, AND
J. CAIRNS, JR. 1971. A computer simulation of the
effects of superimposed mortality due to pollutants
on populations of fathead minnows (Pimephales
promelas). J. Fish. Res. Board Can. 28: 1107-1112.
WALTERS, C. J. 1969. A generalized computer simu-
lation model for fish population studies. Trans.
Amer. Fish. Soc, 98: 505-512.
-------
CONTRIBUTION NO. 158
-------
Copyright 1973 by the Society of Proto/oologists
Reprinted from THE JOLRNAI. OF PROTOZOOLOGY.
J. PROTOZOOI.., 20(3), 443-4-45 (1973).
Made in United Sidles of America
The Polychlorinated Biphenyls, Aroclors® 1248 and 1260: Effect on and
Accumulation by Tetrahymena pyriformis*
NELSON R. COOLEY, JAMES M. KELTNER, JR. and JERROLD FORESTER!
Environmental Protection Agency, Gulf Breeze Environmental Research Laboratory,
Sabine Island, Gulf Breeze, Florida 32561; Associate Laboratory of the
National Environmental Research Center, Corvallis, Oregon
SYNOPSIS. Effects of 2 polychlorinated biphenyls, Aroclor 1248 and 1260, on axenic Tetrahymena pyriformis strain W
were investigated and compared with published data on Aroclor 1254. Aroclors 1248 and 1260 at 1 ing/liter in the presence
of 0.1% (v/v) polyethylene glycol 200 reduced significantly (P < 0.005) growth rates and 96-hr populations of T.
pyriformis grown at 26 C. Both toxicants were ~ 0.001 as toxic as Aroclor 1254. Ciliates were exposed for 7 days to
concentrated Aroclors 1248 40X, 1254 60X, and 1260 79 X over initial concentrations in the media. Accumulation of
Aroclors increased with increased chlorination. It is suggested that if levels in the environment reached those used in these
studies, the chief ecologic effect of Aroclor 1254 would be reduction of availability of the ciliates as food and as nutrient
regenerators, but with Aroclors 1248 and 1260, this effect would be secondary to accumulation of the toxicants by the
ciliates. Accumulation of polychlorinated biphenyls by ciliates would permit the toxicants to enter aquatic food chains. Thus
the compounds could exert toxic effects at higher trophic levels.
Index Key Words: Tetrahymena pyriformis strain W; axenic cultivation; Aroclor 1248; Aroclor 1254; Aroclor 1260; toxic
effect on, and accumulation by ciliates.
THE Aroclors are mixtures of polychlorinated biphenyls
(PCBs) or terphenyls. The PCBs are related structurally
to DDT and some are toxic to and accumulated by animals and
man (17). Aroclor 1254, reported from water, sediment, and
biota of Escambia Bay, Florida (5), in laboratory experiments
is toxic to and accumulated by the ciliate, Tetrahymena pyri-
formis, and by shrimp, fiddler crabs, oysters, and fishes (4, 5,
9, 10, 13, 14). The ubiquity and significance of these and re-
lated PCBs in the environment have been reviewed (6, 5, 17).
Individual Aroclors are identified by 4-digit numbers, the
first 2 indicating the type of molecule and the last 2, the weight
percentage of chlorine in the molecule. For example, Aroclor
1248 is a polychlorinated biphenyl that contains 48% chlorine.
The Aroclors have been used in lubricants; as plasticizers in
paints, plastics, and chlorinated rubbers; as heat-exchange fluids
in industrial heating systems; and as dielectric compounds in
large electrical transformers and capacitors (17). Recently, use
of these compounds has been restricted to their dielectric ap-
plication (17).
We report here the effects of Aroclors 1248 and 1260 on
growth of populations of T. pyriformis and the accumulation
of these compounds by this ciliate. The data are compared with
our previous observations on response of T. pyriformis to Aroclor
1254 (4).
MATERIALS AND METHODS
Tetrahymena pyriformis strain W was grown in optically
matched culture tubes containing 10 ml of proteosc peptone
[2% (w/v)] medium, supplemented with 0.1% (w/v) de-
hydrated yeast extract and 0.5% (w/v) glucose, at 26 C. Cul-
ture tubes were slanted at 60° to enhance aeration.
©Registered Trademark, Monsanto Co., 800 N. Lindbergh
Blvd., St. Louis, Mo. Mention of commercial products does not
constitute endorsement by the Environmental Protection Agency.
* Contribution No. 158, Gulf Breeze Environmental Research
Laboratory.
t We thank Dr. John O. Corliss for the cultures of T. pyriformis
strain W from which our stock was derived, the Monsanto Com-
pany for the samples of Aroclors 1248 and 1260 used in this
investigation, and David J. Hansen and Dr. Alvin L. Jensen for
y«^7}^ ^j,,.:-^ ..p *k«f -f~».'-ti_-.ii syv'"":/! of our data.
Stock solutions of toxicants in polyethylene glycol 200 were
prepared, and chemical residue analyses in media and cells were
performed by methods already described (4), with one excep-
tion. Previously, petroleum ether was used in extraction before
gas-chromatographic analysis. In this study, a mixture of equal
parts of ethyl and petroleum ethers was used.
Aroclors were tested in the presence of 0.1% (v/v) poly-
ethylene glycol 200 for effect on Tetrahymena growth and for
accumulation by the ciliates. This concentration of polyethylene
glycol had no toxic effect on the ciliates (4) and we had no
reason to suspect that effects of polyethylene glycol and Aroclors
were additive. Aroclor 1248 was tested at 0.01, 0.1, and 1 mg/
liter. Aroclor 1260 was tested for effect on population growth
at 0.001, 0.01, 0.1, 1, and 10 mg/litcr and for accumulation at
0.001, 0.01, 0.1, and 1 mg/liter. Each concentration was tested
for effect on population growth in 6 replicate 96-hr experiments
and for accumulation in 3 replicate 7-day experiments. Initial
concentrations of each Aroclor in the media were confirmed by
electron-capture gas chromatography, and were within 17% of
the desired concentrations.
Growth was measured as absorbance at 540 nm. Observations
were made at 0, 4, 8, 16, 24, 36, 48, 60, 72, 84, and 96 hr and
the data graphed. Exponential growth rate of each population
was estimated as the quantity b of the least squares estimate
of the line )• = a + bx of the exponential part of the growth
curve. The calculated regression lines for this segment of the
growth curves closely fitted the experimental data—for Aroclor
1248, r > 0.89; for Aroclor 1260, r > 0.87. In addition, popu-
lations were compa-cd at 96 hr, when densities of control popu-
lations were maximal. Values for all treatments in 6 replicates
were subjected to analysis of variance with 2-way classification
and individual treatments were compared by Scheffe's test (16).
Differences were considered significant at P < 0.05.
RESULTS
Aroclor 1248
Effect mi population growth.—Exponential growth rate of the
ciliaie (Table 1) was reduced significantly by Aroclor 1248
[variance ratio, Ft-.. ,.-.. = 6.77; P < 0.005], growth rates of popu-
lations exposed to 1 mg/lilrr of [he compound being 18.9%
-------
444
EFFECTS OF AROCLORS ON T. pyriformis
TABLE 1. Comparison of the effect of Aroclors 1248 and 1260 on growth of T. pyriformis strain W at 26 C.
Concentration
Toxicant (mg/liter)
AROCLOR 1248 0
0.01
0.1
1.0
AROGLOR 1260 Series 1
0
0.001
0.01
0.1
1.0
Series 2
0
1.0
10.0
Mean- growth
rate*
(6)
0.0164
0.0168
0.0161
0.0133
0.0168
0.0176
0.0179
0.0171
0.0126
0.0194
0.0157
0.0113
Difference
(%)
+ 2.2
- 1.8
-18.9t
+ 4.8
+ 6.5
+ 1.8
-25.0§
-19.1**
-41.1**
Mean population
density at
96 hr*
(absorbance)
0.7732
0.7883
0.7915
0.6992
0.8472
0.8487
0.8533
0.8317
0.6577
1.0175
0.8788
0.7075
Difference
(%}
+ 1.9
+ 2.4
- 9.6*
+ 1.8
+ 7.2
-18.3
-22.411
-13.6tt
-30.5tt
* Means of 6 replicate experiments.
tFo. IB = 6.77, P < 0.005; t Fa ls, = 9.35, P < 0.005; § F«
< 0.005; ttF0> 10) = 217.90, P < 0.005.
= 5.85, P < 0.005; II F«. » — 6.06, P < 0.005; ** Fa. *» = 485.00, P
less than those of control populations. There was no statistically
significant difference between growth rates of control and ex-
perimental populations exposed to lower concentrations or among
growth rates of experimental populations exposed to lower con-
centrations.
Population density at 96 hr (Table 1) was reduced (9.6%)
significantly by 1 mg/liter [Fa, m = 9.35; P < 0.005], but
not by lower concentrations of the toxicant.
Accumulation.—Tetrahymena pyriformis accumulated Aroclor
1248 from media containing 0.01, 0.1, or 1 mg/liter during 7-
days exposure. Uptake of the toxicant from the medium was
linear with increasing concentration. When initial concentration
in the medium was plotted against mean concentration in the
cells, the data agreed closely (r = 0.998) with the calculated
regression line Yx — 0.9322^ - 1.4746. Concentration factors
ranged from 14.8 to 40.6. Ciliates exposed to 0.01 mg/liter ab-
sorbed 15.1-25.5% (x = 21.6%) of the toxicant in the medium,
those exposed to 0.1 mg/liter absorbed 13.4-19.2% (x = 15.7%),
and those exposed to 1 mg/liter absorbed 14.9-19.2% (x =
16.8%).
We observed no indication of change in isomeric composition
of Aroclor 1248 as was reported for Aroclor 1254 in tissues of
fish (9) and shrimp (14).
Aroclor 1260
Effect on population growth.—Exponential growth rate of the
ciliate (Table 1) was reduced significantly by Aroclor 1260
[F «. »> = 5.85, P < 0.005, in the 1st series; F a. «> = 485.00,
P < 0.005, in a 2nd series], growth rates of organisms exposed
to the highest concentrations being 19.1-25.0% less at 1 mg/liter
and 41.1% less at 10 mg/liter than those of control populations.
There was no statistically significant difference between growth
rates of control populations and populations exposed to lower
concentrations of the toxicant.
At 96 hr, population density was reduced significantly [F u, 20)
= 6.06, P < 0.005, in the 1st; and F e, ,„> = 217.9, P < 0.005
in the 2nd experimental series]. The reduction ranged 13.6-
22.4% in ciliate populations exposed to 1 mg/liter and 30.5%
in those exposed to 10 mg/liter. No significant reductions in
population density were observed in the presence of a lower
concentration of the toxicant.
Accumulation.—Tetrahymena pyriformis accumulated Aroclor
1260 from medium that contained 0.001, 0.01, 0.1, or 1 mg/liter
during 7-days exposure. Uptake of the toxicant from the me-
dium was linear with increasing concentration. When initial
concentration in the medium was plotted against mean concen-
tration in the cells, the data agreed closely (r = 0.917) with
the calculated regression line ¥„ = 0.06967^-0.2191. Concen-
tration factors ranged 21-79. Ciliates exposed to 0.001 mg/liter
absorbed amounts that ranged from detected-but-unquantifiable
to 61.7% (x = 38.2%) of the toxicant in the medium; those
exposed to 0.01 mg/liter, 16.4-49.3% (x = 36.8%), those ex-
posed to 0.1 mg/liter, 15.1-68.1% (x = 41.7%), and those ex-
posed to 1 mg/liter, 26.4-81.0% (x — 53.3%).
We observed no indication of change in isomeric composition
of Aroclor 1260 as was reported for Aroclor 1254 in tissues of
fish (9) and shrimp (14).
Comparison of accumulation of Aroclor 1248 and Aroclor
1260 by T. pyriformis by the i-test (differences considered sig-
nificant at P < 0.05) (16) revealed that (a) when concentration
of each compound in the medium increased, concentrations of
Aroclor 1248 in the cells increased at the same rate, as did
Aroclor 1260 (P > 0.05) and (b) the mean amounts of Aroclor
1248 and Aroclor 1260 accumulated were not significantly dif-
ferent (P > 0.05).
DISCUSSION
Protozoa, algae, and bacteria form the broad base of aquatic
food chains. Ciliates are among the most numerous organisms
of the estuarine benthos (1, 7) and may be more important as
nutrient regenerators, particularly of nitrogen and phosphorus,
than are bacteria (11, 12). Also, some ciliates, including T.
pyriformis, can concentrate certain persistent pesticides and
PCBs (4, 8), and thus help translocate them up the trophic
pyramid. It has been shown that PCBs build up in food chains
(6). It is possible, therefore, that PCBs acting-on or through
these ciliates could be toxic at higher tronhir 1w»i<> «:+v~_
-------
EFFECTS OF AROCLORS ON T. pyriformis
445
through disruption of nutrient cycles or through translocation
and bioaccumulation in the food chain (2, 3, 17).
Tests of Aroclor 1254 included concentrations found in nat-
ural waters and sediments (5). Testing similar concentrations
of Aroclor 1248 and 1260 permitted comparison of effects and
accumulation of the 3 compounds. All were toxic to and ac-
cumulated by T. pyriformis, although in different degree. Aro-
clor 1254 was the most toxic, as judged by significant reduction
of both growth rate and 96-hr population density at 0.001
mg/liter concentration (4), whereas Aroclors 1248 and 1254
did not reduce significantly either population growth or 96-hr
population density at concentrations below 1 mg/liter. Concen-
tration of these PCBs by T. pyriformis increased with increas-
ing chlorination of the toxicants: Aroclor 1248, 40 X; Aroclor
1254, 60 X; and Aroclor 1260, 79 X. This trend appears to be
in agreement with the suggestion that PCBs with fewer chlorines
tend to be metabolized or excreted faster than those more chlo-
rinated, so that the latter compounds tend to increase in food
chains (10, 17).
At the concentrations tested, the chief effect of Aroclor 1254
on natural populations of T. pyriformis and ciliates that respond
similarly might be reduction of population density. This would
reduce their availability as food organisms and nutrient re-
generators. Conversely, with Aroclors 1248 and 1260, reduction
of population density is secondary in importance to accumula-
tion of the toxicants by the ciliates. The ability of these ciliates
to concentrate them would enable these PCBs to enter aquatic
food chains, thereby permitting the toxicants to pass to, and
possibly exert their effect at higher trophic levels.
REFERENCES
1. Borror, A. C. 1963. Morphology and ecology of the benthic
ciliated protozoa of Alligator Harbor, Florida. Arch. Protistenk.
106, 465-534.
2. Burdick, G. E., Harris, E. J., Dean, H. J., Walker, T. M.,
Skea, J. & Colby, D. 1964. The accumulation of DDT in lake
trout and the effect on reproduction. Trans. Am. Fish. Soc. 93,
127-36.
3. Butler, P. A. 1969. Monitoring pesticide pollution. BioScience
19, 889-91.
4. Cooley, N. R., Keltner, J. M., Jr. & Forester, J. 1972. Mirex
and Aroclor® 1254: Effect on and accumulation by Tetrahymena
pyriformis strain W. /. Protozool. 19, 636-8.
5. Duke, T. W., Lowe, J. I. & Wilson, A. J., Jr. 1970. A
polychlorinated biphenyl (Aroclor® 1254), in water, sediment,
and biota of Escambia Bay, Florida. Bull. Environ. Contam.
Toxicol. 5, 171-80.
6. Dustman, E. H., Stickel, L. F., Blus, L. J., Reichel, W. L. &
Wiemeyer, S. N. 1971. The occurrence and significance of poly-
chlorinated biphenyls in the environment. Trans. 36th N. Am.
Wildl. Nat. Resour. Con/. 118-33.
7. Fenchel, T. 1967. The ecology of marine microbenthos.
I. The quantitative importance of ciliates as compared with
metazoans in various types of sediments. Ophelia 4, 121-37.
8. Gregory, W. W., Jr., Reed, J. K. & Priester, L. E., Jr. 1969.
Accumulation of parathion and DDT by some algae and protozoa.
/. Protozool. 16, 69-71.
9. Hansen, D. J., Parrish, P. R., Lowe, J. I., Wilson, A. J., Jr.
& Wilson, P. D. 1971. Chronic toxicity, uptake, and retention of
Aroclor® 1254 in two estuarine fishes. Bull. Environ. Contam.
Toxicol. 6, 113-9.
10. Jensen, S., Johnels, A. G., Olsson, M. & Otterlind, G. 1969.
DDT and PCB in marine animals from Swedish waters. Nature
(London) 224, 247-50.
11. Johannes, R. E. 1965. Influence of marine protozoa on
nutrient regeneration. Limnol. Oceanogr. 10, 434-42.
12. 1968. Nutrient regeneration in lakes and oceans,
in Droop, M. R. & Ferguson Wood, E. J., eds., Advances in
Microbiology of the Sea, Academic Press, New York 1, 203-13.
13. Nimmo, D. R., Wilson, P. D., Blackman, R. R. & Wilson,
A. J., Jr. 1971. Polychlorinated biphenyl absorbed from sediments
by fiddler crabs and pink shrimp. Nature 231, 50-2.
14. , Blackman, R. R., Wilson, A. J., Jr. & Forester, J.
1971. Toxicity and distribution of Aroclor® 1254 in pink
shrimp Penaeus duorarum. Mar. Biol. (Berlin) 11, 191-7.
15. Peakall, D. B. & Lincer, J. L. 1970. Polychlorinated
biphenyls: another long-life widespread chemical in the environ-
ment. BioScience 2O, 958-64.
16. Snedecor, G. W. & Cochran, W. G. 1971. Statistical
Methods. 6th ed. Iowa State Univ. Press, Ames.
17. U. S. Interdepartmental Task Force on PCBs. 1972.
Polychlorinated Biphenyls and the Environment. Final Report.
COM-72-10419. 192 pp. (National Technical Information Ser-
vice, U. S. Dept. of Commerce, Springfield, Va. 22151.)
-------
CONTRIBUTION NO. 159
-------
Differential Responses of Marine Phytoplankton
to Herbicides: Oxygen Evolution
by TERRENCE A. HOLLISTER
Environmental Protection Agency
Gulf Breeze Environmental Research Laboratory, Sabine Island
Gulf Breeze, Fla. 32561
and
GERALD E. WALSH
Associate Laboratory of the National Environmental Research Center
Corvallis, Ore.
INTRODUCTION
Marine unicellular algae vary in their responses to a variety
of toxicants, including chlorinated hydrocarbon insecticides (UKELES
1962, MENZEL et al. 1970), organophosphate insecticides (DERBY and
RUBER 1970), and fungicides (UKELES 1962). Little is known, however,
about toxicities of herbicides to marine unicellular algae. Responses
of four algal species to 30 herbicidal formulations have been reported
(WALSH 1972) and the urea and triazine herbicides were the most
toxic. Four urea herbicides also caused depression of the carbo-
hydrate contents of six species of algae, and effect was directly
proportional to salinity of the growth medium (WALSH and GROW 1971).
The work reported here was done to learn if marine unicellular
algae differ in their responses to herbicides. We tested 18 species
against the substituted ureas, neburon and diuron, and the triazines,
atrazine and ametryne.
MATERIALS AND METHODS
The algae were obtained from the culture collections of the
Woods Hole Oceanographic Institution, Scripps Institution of
Oceanography, and Indiana University. All were maintained and
tested in a growth medium composed of artificial seawater —'
supplemented with trace elements and vitamins. The supplements,
per liter of medium, were: 30 mg N32EDTA, 14 mg FeCl2'6H20,
34 mg H3B03, 4 mg MnCl2 • 4H20, 2 mg ZnS04'7H20, 6 mg K-jPO^,
100 mg NaNO.,, 40 mg Na SiO -9H 0, 5ug CuSO,, 12 pg CoCl ,
50 ug thiamine hydrochloride, I pg vitamin B^2, and 0.01 ug biotin.
Salinity was 30 parts per thousand and the pH ranged between 7.9
and 8.1. The medium was sterilized by autoclaving for 15 minutes
at 121°C.
Five ml of stock algae were inoculated into 100 ml of growth
medium and incubated at 20°C under 6,000 lux illumination from
fluorescent tubes with alternating 12-hour periods of light and
~~ Contribution No. 159 from the Gulf Breeze Laboratory.
2 /
— From Rila Products, Teaneck, New Jersey. Mention of commercial
products does not constitute endorsement by the Environmental
Protection Agency.
291
Bulletin of Environmental Contamination ft Toxicology,
Vol. 9. No. 5 © 1973 by Springer-Verlag New York Inc.
-------
darkness for three days. Then, the cultures were centrlfuged gently
and resuspended in growth medium to an optical density of 0.100 at
525 m]i on a Fisher electrophotometer. The algal cultures were not
axenic.
Effects of herbicides were easured as inhibition of oxygen
evolution. Both ureas and tri ,^j.nes inhibit photosynthesis and
move quickly into the cells (ZWEIG 1969) and the concentrations
required for inhibition of both growth and photosynthesis are the
same (WALSH 1972).
Concentrations of herbicides in the suspending media ranged
from zero to those which inhibi sd evolution of oxygen by approxi-
mately 25, 50, and 75%. Concer Cations were calculated as parts
per billion (ppb) of the technical preparation. From each cell
suspension, 5.0 ml were placed in reaction vessels of a Gilson
photosynthesis model differential respirometer. After equilibrating
at 20°C for 30 minutes, oxygen evolution was measured for 60 minutes
with a C02 buffer in the well of the reaction vessel (UMBREIT et al.
1964). Duplicate flasks were analyzed and each test was performed
three times.
All data were subjected to statistical analysis. Mean
percentage inhibition after 60 minutes was calculated and EC50
values (effective concentrations at which evolution of oxygen was
507» that of untreated cell suspensions) were calculated by the
least squares method. Concentrations were converted to logarithms
and responses to probits and the standard error obtained for each
series of tests.
RESULTS AND DISCUSSION
The EC50 values for the four herbicides and 18 algal species
tested are shown in Table 1 and summarized by family in Table 2.
Atrazine was the least toxic; ametryne, neburon, and diuron were
approximately equal in toxicity. Species of the family Bacillari-
ophyceae were generally the least sensitive, requiring as much as
5.8 times more ametryne to reduce oxygen evolution by 507» than did
species of the other families.
Wide variations occured in response to the toxicants among
the individual species of the families Chlorophyceae, Bacillari-
ophyceae, and Chrysophyceae. A measure of the range of responses
among species was calculated by derivation of the ratio of the
highest EC50 to the lowest EC50. The ratio, here called the
"Difference Factor", is given for each family and herbicide in
Table 2. Difference Factors were greatest in the Bacillariophyceae,
being as high as 11.9 for neburon-treated algae. In that case,
the EC50 for Cyclotella nana was 11 ppb, whereas for Nitzschia
(Indiana strain 684) it was 131 ppb.
292
-------
TABLE 1. EC50 (ppb) of neburon, diuron, atrazine, and ametryne on oxygen evolution by marine
unicellular algae. Standard errors (SE) were derived by unweighted probit analysis.
Fami ly
Species
Chlorophyceae
Ch1amydomonas sp.
Dunaliella tertiolecta
Platymonas sp.
Chlorella sp.
Neochloris sp.
Chlorococcum sp.
Bacillariophyceae
Thalassiosira fluviatilis
Navicula inserta
Amphora exigua
Achnanthes brevipes
Stauroneis amphoroides
Cyclotella nana
Nitzschia closterium
Nitzschia (Ind. 684)
Ch ry s ophy ce a e
Monochrysis lutheri
Isochrysis galbana
Phaeodactylum tricornutum
Rhodophyceae
Porphyridium cruentum
Neburon
EC 50
37
10
12
22
39
20
108
124
82
23
17
11
120
131
12
20
40
SE
5
3
5
3
6
3
9
11
5
4
3
4
13
9
4
5
7
Diuron
EC 50
37
10
17
19
28
20
95
93
31
24
31
39
50
169
18
10
10
SE
3
3
3
2
5
4
10
12
4
1
2
7
6
17
3
3
3
Atrazine
EC50
60
159
102
143
82
80
110
460
300
93
348
84
287
434
77
100
100
SE
8
18
8
8
7
7
19
15
21
11
67
19
68
84
23
17
19
Ametryne
EC50
41
40
24
32
36
10
58
97
26
19
65
55
62
135
14
10
10
SE
5
6
4
3
7
3
7
9
4
I
11
8
6
11
4
4
5
24
24
79
35
-------
TABLE 2. Average EC50 values (ppb) for four herbicides and four families of marine unicellular algae.
The Difference Factor (DF) is the ratio of the highest to the lowest EC50 among the algal species.
Family
Chlorophyceae
Bacillariophyceae
Chrysophyceae
Rhodophyceae
Number of
Species Tested
6
8
3
1
Neburon
EC50 DF
23 3.9
77 11.9
24 3.3
24
Diuron
EC50 DF
22 3.7
67 7.0
13 1.8
24
Atrazine
EC 50
104
265
92
79
DF
2.6
5.5
1.3
_
Ametryne
EC 50
31
65
11
35
DF
4.1
7.1
1.4
_
-------
These data show that when bioassay analyses are conducted for
effects of herbicides on marine unicellular algae, two factors are
particularly important: (1) the response in relation to familial
taxonomic position, and (2) the wide range of responses by individual
species within a given family. It is necessary, therefore, to use
several species from each of several families in algal bioassay
studies to obtain realistic data concerning effects of herbicides
on algae.
ACKNOWLEDGEMENT
We thank E. I. DuPont de Nemours and Co. for providing the
urea herbicides, and Geigy Agricultural Chemicals for providing
the triazine herbicides used in these experiments.
REFERENCES
DERBY, S. B. (SLEEPER) and E. RUBER. Bull. Environ. Contam.
Toxicol. 5:553-558, (1970).
MENZEL, D. W., J. ANDERSON, and A. RANDTKE. Science 167:
1724-1726, (1970).
UKELES, R. Appl. Microbiol. 10:532-537, (1962).
UMBREIT, W. W., R. H. BURRIS, and J. F. STAUFFER. Manometric
Techniques, 4th ed., Burgess Pub. Co., Minneapolis, Minn.,
305 pp. (1964).
WALSH, G. E. Hyacinth Control J. _10:45-48, (1972).
WALSH, G. E. and T. E. GROW. Weed Sci. JL9:568-570, (1971).
ZWEIG, G. Residue Rev. 2_5:69-79, (1969).
295
-------
CONTRIBUTION NO. 163
-------
Comp. Biochem. PhysioL, 1974, Vol. 49B, pp. 375 to 379. Pergamon Press. Printed in Great Britain
SOME ASPECTS OF MYOSIN ADENOSINE
TRIPHOSPHATASE OF PINK SHRIMP (PENAEUS
DUORARUM}*
W. P. SCHOOR
U.S. Environmental Protection Agency, Gulf Breeze Environmental Research Laboratory,
Sabine Island, Gulf Breeze, Florida 32561, U.S.A. (Associate Laboratory of the National
Environmental Research Center, Corvallis, Oregon)
(Received 22 October 1973)
Abstract—1. Myosin ATP'ases of shrimp and rabbit muscle behave similarily
except at temperatures above 30°C where the activity in shrimp started to
decline rapidly.
2. There is a correlation between the effects of temperature on myosin
ATP'ase and behavior of shrimp in nature; this compares well with similar
observations in crayfish (Maruyama, 1958).
3. Data obtained emphasize that under certain conditions caution must
be used in determining initial enzymatic activities.
INTRODUCTION
PURIFICATION of myosin adenosine triphosphatase (ATP'ase, E.G. 3.6.1.3) is
usually carried out by modification of the method of Szent-Gyorgyi (1951) in
combination with one of a variety of buffers. The characteristics of proteins in
solution are such, however, that changes in ions, as well as changes in pH and
temperature, can affect their conformation (Flory, 1956). If the proteins possess
catalytic function, altered conformation may cause changes in their specific
activity. In the research reported here, some aspects of shrimp myosin ATP'ase
in solution were investigated, using the method of Szent-Gyorgyi for purification
and a minimal amount of histidine buffer.
MATERIALS AND METHODS
Pink shrimp (Penaeus duorarum) from the Gulf of Mexico near Tampa, Florida, were
held in large tanks with flowing sea water and fed fish muscle for 2 weeks prior to use.
Sea water (February-May) averaged 10°C and 23 %„ salinity. Heads of shrimp were removed
and tails diced after removal of exoskeleton, nerve cord and digestive tract. Ten g of diced
muscle were homogenized in a blender for 30 sec with 50 ml of 0-5 M KC1 that contained
0-002 M histidine at pH 7-0 (KCl-histidine buffer), and centrifuged in a Beckman Model
L 3-50 ultracentrifuge at 70,000 g (max.) for 45 min, using a SW-25.2 rotor. The super-
nate, usually 35-40 ml, was decanted and diluted 1 : 19 with 0-5 mM histidine at pH 7-0.
The resulting flocculate was allowed to settle for 30 min before centrifugation at 17,000 g
(max.) for 10 min, using a SW-50.1 rotor. The supernate was decanted and sufficient
* Contribution No. 163, Gulf Breeze Environmental Research Laboratory.
375
-------
376 W. P. SCHOOR
KCl-histidine buffer added to bring the volume to 25 ml. The resulting solution was ad-
justed to 0-5 M in KC1 by the addition of powdered KC1. This procedure was repeated
twice more. The final enzyme solution was centrifuged at 100,000 g (max.) for 1 hr to
remove aggregates and diluted to the desired enzyme concentration with KCl-histidine
buffer.
The specific activity of the myosin ATP'ase is expressed here as the number of micro-
equivalents of inorganic phosphate (Pj) liberated per gram of protein per second (/n-equiv.
P,/g per sec). The ATP'ase activity was determined by the pH-stat technique (Kay &
Brahms, 1963) with a Radiometer pH Meter 26, Titrator II, Autoburette ABU 12, Titi-
graph SBR-2-c, and G-222-B and K 401 electrodes. The reaction mixture was kept at
constant temperature in a jacketed vessel connected to a Lauda K-2/R Circulator. The
reaction volume was 5 ml and no adjustment was made for the small error in concentration
caused by dilution with base. The order of addition of reagents was inconsequential, but
it was convenient to add the enzyme last. Unless otherwise indicated, only initial activities
are given. Reagent blanks were determined in the absence of myosin. The Biuret method
with bovine serum albumin as the standard was used for protein assay.
RESULTS
The enzymatic activities for shrimp myosin ranged from 3 to 5 /u-equiv.
Pj/g per sec and were linear up to 2 mg of protein per assay, after which the
activity declined.
Table 1 shows the change in the specific activity as a function of pH and time.
Specific activity between pH 6-5 and 8-0 increased with increasing pH and remained
constant for at least 4 min. After 1 min at pH 8-5 and 9-0, the activity of the
ATP'ase decreased. True initial activity at those pH values may have been under-
estimated because of rapid inactivation of the enzyme.
TABLE 1—EFFECT OF pH ON SPECIFIC ACTIVITY OF MYOSIN ATP'ase OF SHRIMP
(Penaeus duorarum)*
Specific activity (min)
pH 1 2 3
5-5
6-5
7-0
7-5
8-0
•8-5
9-0
0
0-70
0-92
1-58
3-32
5-22
3-00
0
0-70
0-92
1-58
3-32
1-38
1-38
0
0-70
0-92
1-58
3-32
0-94
0-94
0
0-70
0-92
1-58
3-32
0-34
0-34
* Assay conditions: l-44mg protein/assay, 0-5 M KC1, 0-1 M CaCl2, 2 mM
ATP, 25°C. Values represent the average of three determinations; error was
±10 per cent maximum.
Table 2 shows the same type of enzymatic response as a function of temperature.
There was no change in the specific activity of myosin ATP'ase during the 3-min
reaction interval between 20 and 25°C. The specific activities rose during the
first minute up to 30°C but declined at 35°C, at which temperature the true initial
-------
MYOSIN ADENOSINE TRIPHOSPHATASE OF PINK SHRIMP 377
TABLE 2—EFFECT OF TEMPERATURE ON THE SPECIFIC ACTIVITY OF MYOSIN ATP'ase
OF SHRIMP (Penaeus duorarum) *
Specific activity (min)
Temperature =
(°C) 1 3
20 2-29 2-29
25 2-92 2-92
30 3-48 1-53
35 3-36 0-10
* Assay conditions: 1-44 mg protein/assay, 0-5 M KC1, 0-1 M CaCl2, 2 mM
ATP, pH 8-0. Values represent the average of three determinations; error
was ± 10 per cent maximum.
activity may have been underestimated because of rapid inactivation of the enzyme.
Table 3 shows the effects of storage at 0°C and CaCl2 on the shrimp ATP'ase
system. Activation by the addition of CaCl2 continued as the specific activity
decreased with increasing length of storage at 0°C. Concentrations of MgCl2
above 10~* M completely inhibited the enzymatic activity.
TABLE 3—EFFECTS OF STORAGE AT 0°C AND CaCl2 ON THE SPECIFIC ACTIVITY OF
MYOSIN ATP'ase OF SHRIMP (Penaeus duorarum) *
Storage tim<
(hr)
1
24
48
Specific activity (CaCl2, moles/1.)
0
0-41
<0-10
<0-10
0-04
2-93
2-11
0-71
0-08
4-28
2-28
1-00
0-10
4-36
1-79
0-94
0-12
3-00
0-87
0-15
* Assay conditions: 1-50 mg protein/assay, 0-5 M KC1, 2 mM ATP, pH 8-0,
25°C. Values represent the average of three determinations; error was ±10
per cent maximum.
DISCUSSION
A reduction in specific activity at concentrations above 2 mg protein per assay
suggests that protein aggregation is taking place in the manner described for rabbit
myosin (Lowey & Holtzer, 1959; Johnson & Rowe, 1961) anu for cod myosin
(Mackie, 196'5). Other physical properties of the structural proteins of cod and
carp, and rabbit myosin also appear to be closely related (Hamrar, 1955). The
CaQ2 activation effect still remained, even though me isolated enzyme system
was unstable when stored at 0°C.
In these studies, shrimp myosin ATP'ase beha'- td similarly to rabbit myosin
ATP'ase (Schoor & Mandelkern, unpublished results) under id'Trica! assay con~
ditions. The range of CaCla activation arid complete loss of activity ic the presence
-------
378 W. P. SCHOOR
of 10~4 M MgCl2 was identical in both enzyme systems. However, differences in
the maximum activities did exist, specific activities ranging from 8 to 10 /x-equiv.
P4/g per sec in rabbit myosin and 3-5 /^-equiv. Pj/g per sec in shrimp myosin.
Initial activities of the rabbit myosin remained linear for at least 5 min at
temperatures up to 40°C; that of shrimp myosin decreased rapidly at 35°C.
"Total" muscle ATP'ases show approximately the same activity in fishes, frog,
mouse, bird and turtle at 30°C, but wide differences occur at 0°C (Steinbach,
1949; Davidson & Richards, 1954).
The cold-blooded animals showed higher activities at the lower temperatures
than did warm-blooded ones. Although these data agree well with my findings,
it must be remembered that "total" ATP'ase activity includes actomyosin ATP'ase,
which was not measured in this study. Field studies have shown that pink shrimp
tolerated water temperatures of 30-31°C, appeared hyperactive around 32°C and
at 35°C had a low survival rate (Heald, personal communication). The same is
true for the protozoeal stage of the pink shrimp (Thorhaug et al., 1971). The
actomyosin ATP'ase of crayfish (Cambarus clarkii) had an optimal activity at
pH 7-0 at 30-35°C, the activity quickly declining at 37°C (Maruyama, 1958).
This temperature is somewhat higher than the maximum found for the pink shrimp
myosin ATP'ase, but parallels the higher temperatures to which the crayfish is
exposed in its shallow water habitat.
Even so, until the temperature dependence of other shrimp enzyme systems has
been established, it is speculative to suggest that inactivation of myosin ATP'ase
is the reason for the reduced survival rate of shrimp at temperatures above 32°C.
Nevertheless, observed differences in the effects of temperature on enzyme systems,
having similar functions, seem to be a good example of adaptability of such
systems in warm- and cold-blooded animal species.
REFERENCES
DAVIDSON J. A. & RICHARDS A. G. (1954) Muscle apyrase activity as a function of temperature
in the cockroach, crayfish and minnow. Archs Biochem. Biophys. 48, 484—486.
FLORY P. J. (1956) Role of crystallization in polymers and proteins. Science, Wash. 124,53-60.
HAMOIR G. (1955) Fish proteins. Adv. Protein Chem. 10, 227-288.
JOHNSON P. & ROWE A. J. (1961) The spontaneous transformation reactions of myosin.
Biochim. biophys. Acta S3, 343-360.
KAY C. M. & BRAHMS J. (1963) The influence of ethylene glycol on the enzymatic adenosine
triphosphatase activity and molecular conformation of fibrous muscle proteins. J,
biol. Chem. 238, 2945-2949.
LOWEY S. & HOLTZER A. (1959) The aggregation of myosin. J. Am. Chem. Soc. 81,1378-
1383.
MACKIE I. M. (1965) The effect of adenosine triphosphate, inorganic pyrophosphate and
inorganic tripolyphosphate on the stability of cod myosin. Biochim. biophys. Acta 115,
160-172.
STEINBACH H. B. (1949) Temperature coefficients in muscle apyrase systems J cell comb
Physiol. 33, 123-131.
SZENT-GYORGYI A. (1951) Chemistry of Muscular Contraction, 2nd End., p. 146. Academic
Press, New York.
-------
MYOSIN ADENOSINE TRIPHOSPHATASE OF PINK SHRIMP 379
THORAUG At, DEVANY T. & MURPHY B. (1971) Refining shrimp culture methods: the effect
of temperature on early stages of commercial pink shrimp. Proc. 23rd Ann. Session,
Gulf and Carib. Fish Inst., pp. 125-132.
Key Word Index—Pink shrimp (Penaeus duoraruni); myosin ATP'ase; rabbit; tempera-
ture effects; purification; comparison.
-------
COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY
Volume 49 Number 2A 1 °Ct°ber
PAGE
D. J. WEIDLER, A. M. EARLE, G. G. MYERS and P. J. GARDNER : Effect of metabolic inhibition
on sodium ion exchange in the ventral nerve cord of Melanoplus differentials 207
R. A. BALOGUN: Studies on the amino acids of the tsetse fly, Glossina morsitans, maintained
on in vitro and in vivo feeding systems 215
JAMES R. SNAPPER, S. M. TENNEY and F. V. McCANN: Observations on the amphibian
"diaphragm" ^23
H. M. SHAW and T. J. HEATH: The gall bladder of the guinea pig: its concentrating and
contractile abilities 231
A. M. CHERET, A. SOUMARMON, M. LEWIN, A. AMAR-COSTESEC and S. BONFILS: Comparative
study of pyloric and fundic acid proteases in the guinea pig gastric mucosa: evidence for
a pyloric pepsinogen 241
THOMAS H. DIETZ: Active chloride transport across the skin of the earthworm, Lumbricus
terrestris L. 251
D. W. ENGEL, E. M. DAVIS, D. E. SMITH and J. W. ANGELOVIC: The effect of salinity and
temperature on the ion levels in the hemolymph of the blue crab, Callinectes sapidus,
Rathbun ' 259
W. KENNETH DERICKSON : Lipid deposition and utilization in the sagebrush lizard, Sceloporus
graciosus: its significance for reproduction and maintenance 267
J. E. MclNERNEY: Renal sodium reabsorption in the hagfish, Eptatretus stouti 273
M. DEBORAH SMITH and PETER C. BAKER: The maturation of indoleamine metabolism in the
lateral eye of the mouse 281
ROBERT L. WINDERS, MARK O. PARSER, KENNETH F. ATKINSON and FELICE MANFREDI:
Parameters of oxygen delivery in the species Marmota flaviventris at sea level and 12 000
feet 287
ANDREW E. DIZON, E. DON STEVENS, WILLIAM H. NEILL and JOHN J. MAGNUSON: Sensitivity
of restrained skipjack tuna (Katsuzoonus pelamis) to abrupt increases in temperature 291
JOHN H. CROWE and KAREN A. MAGNUS: Studies on acarine cuticles—II. Plastron respiration
and levitation in a water mite 301
BRIAN G. D'AousT and LYNWOOD S. SMITH: B»nds in fish 311
JOE R. LINTON and M. S. PHOCTOR: Isolation and characterization of melanocyte stimulating
factors from the pituitary gland of the grey mullet, Mugil cephalus 323
KARIN D. RODLAND and F. REED HAINSWORTH: Evaporative water loss and tissue dehydration
of hamsters in the heat 331
PAUL J. HIGGINS and CHARLES S. RAND: A comparative immunochemical study of the serum
proteins of several Galapagos iguanids 347
JOHN PATRICK STONE and WALTER CHAVIN : Response of dermal melanophores to epinephrine
after removal of the epidermal barrier 357
D. L. DAHLMAN: Hemolymph characteristics of developing adult tobacco hornworms reared
as larvae on tobacco leaf or synthetic diet 369
TERENCE T. YEN, RAY W. FULLER and DONA VAN V. PEARSON : The response of "obese" (ob/ob)
and "diabetic" (db/db) mice to treatments that influence body temperature - 377
JOHN S. TUCKER and FLORENCE L. HARRISON: The incorporation of tritium in the body water
and organic matter of selected marine invertebrates 387
IMRE Zs.-NAOY: Some quantitative aspects of oxygen consumption and anaerobic metabolism
of molluscan tissues—A review 399
J. V. BANNISTER: The respiration in air and in water of the limpets Patella caerula (L.) and
Patella lusitanica (Gmelin) 407
-------
CONTRIBUTION NO. 164
-------
AROCLOR© 1254: EFFECT ON COMPOSITION OF
DEVELOPING ESTUARINE ANIMAL
COMMUNITIES IN THE
LABORATORY*
David J. Hansert
U.S. Environmental Protection Agency, Gulf Breeze Environmental Research Laboratory,
Gulf Breeze. Florida. U.S.A. 32561
(Associate Laboratory of the National Environmental Research Center,
Corvallis, Oregon)
ABSTRACT
Aroclor® 1254, a polychlorinated biphenyl (PCB), affected the composition
of communities of estuarine animals that developed from planktonic larvae in
salt water that flowed through 10 control aquaria and 10 aquaria contaminated
with 0.1. 1 or 10/tg/l of this PCB. Communities that developed in control aquaria
and aquaria that received 0.1 /xg/1 of PCB in water for four months were
dominated O75%) by arthropods, primarily the amphipod Corophium volu-
lalor. In aquaria receiving 1 and 10 pg/l, the number of arthropods decreased
and the number of chordates, primarily the tunicate Mogula manhallensis,
increased; over 75% of the animals in 10 /ig/1 aquaria were tunicates. Numbers
of phyla, species, and individuals (particularly amphipods, bryozoans, crabs,
and mollusks) were decreased in this PCB, but there was no apparent effect
on the abundance of annelids, brachiopods, coelenterates, echinoderms or nemer-
teans. The Shannon-Weaver index of species diversity was not altered by
Aroclor 1254.
INTRODUCTION
Polychlorinated biphenyls (PCBs) have been manufactured for various uses
(Broadhurst 1972) for over 40 years, but their occurrence in aquatic ecosystems
was not confirmed until 1966 (Anonymous 1966). Since then, PCBs have been
detected in estuarine organisms from 6 of 15 of the coastal United Slates (Butler
1973). One PCB, Aroclor 1254, was discovered in the water, sediment and biota
of Escambia Bay, Florida (D-ukeetal. 1970).
Chronic and acute toxicity experiments conducted at the Gulf Breeze Labora-
tory have established that Aroclor 1254 is toxic to some estuarine organisms. A
concentration of 100 ju.g/1 of Aroclor was acutely toxic (48 to 96 hours) to pink
shrimp, Penaeus duorarum, and oysters, Crassostrea virginica. but not to pinfish,
Lagodon rhomboides (Duke et al. 1970). Chronic toxicity was up to 100 limes
* Contribution No. 164, Gulf Breeze Environmental Research Laboratory. ©Registered trade-
mark, Monsanto Company, St. Louis, Missouri, U.S.A. Mention of commercial products or trade
names does not constitute endorsement by the Environmental Protective Agency.
CJiiitribuliuii!. in Mnrine Science, Vol. IS. 1974.
-------
20 David J. Hansen
greater than acute toxicity. In exposures lasting more than two weeks, 1 /ig/1 of
Aroclor 1254 killed pink shrimp (Nimmo etal. 1971 a), whereas 5 ,/xg/l killed pin-
fish and spot, Leiostomus xanthurus (Hansen et al. 1971) and significantly re-
duced oyster growth rate (Fairish et al. 1972), but it was not lethal to blue crabs,
Callinectes sapidus (Duke et al. 1970). Aroclor 1254 is, therefore, toxic to certain
estuarine species exposed separately. However, its effect on communities of estu-
arine animals is not known. This study reports experiments that determine the
effect of this chemical on development of estuarine animal communities in the
laboratory.
MATERIALS AND METHODS
I investigated the effect of Aroclor 1254 on development of estuarine communities by com-
paring the number, species, and diversity of animals that grew from planktonic larvae in appar-
atuses continuously contaminated with 0.1, 1 or 10 /tg/1 of PCB for four months, 18 May to 25
September 1970, with animals from an identical apparatus that was not contaminated.
SYRINGE PUMP
PRIMARY CONSTANT HEAD BOX
'SIPHON
/(2,300 ml/min)
SECONDARY CONSTANT HEAD BOX
DRAIN
STAND PIPES
(200 ml/min)
FIG. 1.—Apparatus used to test the effect of Aroclor 1254 on composition of estuarine animal
communities.
-------
PCB Affects Estuarine Communities 21
The apparatus used in this investigation is illustrated in Fig. 1 (only one of four identical
apparatuses is shown). Sea water with its natural component of plankton was pumped from the
estuary adjacent to the laboratory into the primary constant head box. Salinity of the water
ranged from 10 to 34%0 (average, 29.7%0) and temperature ranged from 22 to 33 C (average, 28.5
C). In contaminated apparatuses Aroclor 1254 was added to water after it was siphoned, at the
rate of 2,300 ml/min, from the primary into the secondary constant head box; the control appar-
atus received the same flow of water. Water then flowed from the secondary constant head box to
each of 10 adjacent aquaria-10 replicates for each treatment. (Treatment includes control and con-
taminated apparatuses.) Flow rate through each aquarium was maintained at 200 ml/min each
by adjusting the height of a 2.7 mm diameter hole in each of the 10 standpipes in the secondary
constant head box. Each aquarium was 44 cm long, 9 cm wide and 14 cm high. Water depth in
each aquarium was maintained at 8 cm. PCB-free sand was placed in each aquarium to a depth
of 6 cm. Planktonic larvae colonized this sand and the walls of the aquaria. The siphon and
constant head boxes were cleaned weekly so that all aquaria received planktonic larvae from a
common source-the incoming water. Water leaving each aquarium flowed through a V-shaped
opening and into a common drain for the apparatus.
Aroclor 1254, dissolved in polyethylene glycol 200, was metered by a syringe pump into the
water as it entered the secondary constant head box of each experimental apparatus. The same
amount of polyethylene glycol (2 ml/day, 0.68 mg/1) was metered into the control apparatus.
Solvent-induced effect was not expected because: (1) polyethylene glycol 200 did not affect
development of two species of crabs at this concentration (Epifanio 1971); (2) concentrations up
to 1% (v/v) were not lethal to grass shrimp, Palaemonetes pugio, or sheepshead minnows,
Cyprinodon variegatus, in 96 hours in static tests (Hansen unpublished data); and (3) the toxicity
of 5 jitg/1 of Aroclor 1254 to brown shrimp, Penaeus aztecus, and pinfish was not increased by
increasing the concentration of solvent up to 100 times (0.1 to 10.0 mg/1) (Hansen unpublished
data).
Concentrations of Aroclor in test water and sediment were determined by gas chromatography.
Methods of analysis for water are described by Nimmo et al. (1971 a) and for sediment by
Nimmo et al. (1971b), except that an OV-101 column was used. Recovery rates were above 70%,
but data in this report do not include a correction factor for recovery. Water from the secondary
constant head box of each apparatus was analyzed twice a month (Table 1). Concentrations
throughout this box were uniform; water from each standpipe, analyzed once during the 10 /ig/1
exposure, averaged 7.9 ^g/1 (range 6.8-8.7 /ig/1). Sediment cores from 4 of 10 aquaria from each
apparatus were analyzed at the end of the exposure (Table 1).
At the end of the four-month exposure, animals were scraped from the side of the aquaria and
the contents of the aquaria siphoned into a 1 mm mesh sieve. During the last three months of the
experiment, animals that left or were lost from the aquaria or were cleaned from the secondary
constant head box and the drainage area were also collected in a 1 mm mesh sieve. Animals
TABI.E 1
Range and average concentration of Aroclor® 1254 in water and sediment from experiment and
control apparatuses. Water was analyzed twice monthly and sediment was analyzed
at the end of the four month experimenta. Limit of quantification was 0.1 /tg/1
in water and 0.015 mg/kg (dry weight) in sediment. Correction
for recovery (>70%) is not included.
Concentration in water, jug/1 Concentration in sediment, /ig/g
Measured Measured
Nominal Average Range Average Range
Control
0.1
1.0
10.0
None
<0.1
0.6
6.7
<0.1 -0.1
0.48-0.72
5.2 -7.8
None
0.1
0.36
2.0
0.05-0.18
0.25-0.42
1.5 -2.5
-------
to
to
TABLE 2
Animals collected from control aquaria and from aquaria contaminated for four months with
Aroclor® 1254. Ten aquaria were used for each treatment. Number of animals
o
a
Sj
and number o
f aquana from
which they
were coil
ected are .
Listed
Aroclor 1254
Ta\on
Annelida
Armandia agilis
Capilella capilala
Cirratulus sp.
Dasybranchus sp.
Eupjrnalus dianthus
E. protulicola
Heteromastus filiformis
Laeonereis culveri
Lumbrinereis parvapedata
Marphysa sanguinea
Media mast us californ iensis
Neanthes succinea
Noiomastus hemipodus
Podarke, near guanica
Polydora websteri
Polydora sp.
Prinospio sp.
Spiophanes bombyx
Strcblospio benedicti
Capitellidae sp.
Spionidae sp.
Unidentified sp. # 1
Unidentified sp. #2
Anima
8
34
1
0
6
8
0
1
1
1
0
8
0
1
28
2
1
1
6
0
6
2
1
Control
Is Aquaria
5
10
1
0
5
4
0
1
1
1
0
6
0
1
10
2
1
1
4
0
1
2
1
0.1
Animals
11
30
0
0
8
13
0
0
0
0
0
11
0
1
35
1
2
0
7
0
0
0
0
Aquaria
4
8
0
0
6
9
0
0
0
0
0
7
0
1
10
1
1
0
4
0
0
0
0
1 f
Animals
3
34
1
1
11
5
2
0
0
0
0
12
1
0
51
1
0
0
3
0
0
2
1
Aquaria
2
9
1
1
6
4
1
0
0
0
0
6
1
0
8
1
0
0
3
0
0
1
1
10;
Animals
0
23
0
0
9
17
0
0
0
0
1
9
0
0
22
0
0
0
0
3
0
1
0
IB/I
Aquaria
0
8
0
0
6
9
0
0
0
0
1
7
0
0
4
0
0
0
0
1
0
1
0
tc
S
Total ^
animals ^
22
121
2
1
34
43
2
1
1
1
1
40
1
2
136
4
3
1
16
3
6
5
2
-------
TABLE 2—Continued
Aroclor 1254
Taxon
Arthropoda
Balanus sp.
Caprella sp.
Corophium volutalor
Neopanope texana
Upogebia affinis
Decapod larvae, unident. sp.
Pycnogonidae, unident. sp:
Brachiopoda
Gloltidia pyramidata
Chordata
Bostrichobranchus pilularis
Branchiostoma caribaeum
Molgula manhaltensis
Coelenterata
Leptomedusae', unident. sp.
Zoantharia, unident. sp.
Echinodermata
Hemipholis elongata
?Amphioplus sp.
Ectoprocta
Membranipora tenuis*
Mollusca
Abra aequalis
Amygdalum papyria
Anachis translirata
Andara iransversa
Barnea costata
Bittium varium
A:iima
6
0
1338
2
0
0
0
1
100
2
91
5
0
4
1
6
15
10
2
6
5
16
Cnnlrnl
Is Aquaria
4
0
10
2
0
0
0
1
1
1
7
5
0
2
1
6
8
6
2
4
4
7
0.1
Animals
2
11
1693
0
0
0
3
0
2
1
141
4
0
17
0
6
11
4
0
4
1
4
Aquaria
2
1
10
0
0
0
1
0
2
1
10
4
0
4
0
6
6
3
0
4
1
4
1 *
Animals
9
3
736
0
0
4
28
0
3
0
434
8
5
15
0
4
5
0
0
3
1
7
'B/l
Aquaria
6
3
10
0
0
3
6
0
1
0
9
8
3
6
0
4
5
0
0
3
1
4
10 pg/l
Animals Aquaria
2
0
3
0
1
0
1
0
29
0
499
7
0
0
0
0
0
6
0
6
1
1
1
0
3
0
1
0
1
0
5
0
10
7
0
0
0
0
0
6
0
5
1
1
Total
animals
19
14
3770
2
1
4
32
1
134
3
1164
24
5
1
36
16
31
20
2
19
8
28
to
S'
o
S
3
K
to
OJ
-------
to
J.ABLE z — txxnunuea
Aroclor 1254
Taxon
Caecum pulchellum
Crassostrea virginica
Crepidula fornicata
Epitonium humphreysi
Laevicardium mortoni
Lyonsia hyalina
Mactra fragilis
Mitrella lunata
Modiolus americanus
M. demissus
Mulinia lateralis
Musculus lateralis
Nassarius albus
N. vibex
Ostrea equestris
Retusa canaliculata
Rissoina catesbyana
Tagelus divisus
Tellina alternata
Eolidacea, unident sp.
Gastropoda, unident. sp.
Nemertea
Oerstedia dorsalis
Unidentified sp.
Control
Animals Aquaria
0
4
0
0
9
2
0
2
1
1
5
7
1
0
1
1
1
0
9
1
2
2
1
0
3
0
0
5
1
0
2
1
1
3
4
1
0
1
1
1
0
7
1
1
2
1
0.1
Animals
2
5
0
0
3
1
1
0
0
1
0
3
0
0
0
0
0
1
3
0
0
0
0
Ag/1
Aquana
2
4
0
0
3
1
1
0
0
1
0
2
0
0
0
0
0
1
3
0
0
0
0
Animals
0
4
0
1
2
1
1
1
1
2
1
2
2
1
1
0
0
1
7
0
0
0
0
Aquaria
0
3
0
1
2
1
1
1
1
1
1
1
2
1
1
0
0
1
7
0
0
0
0
10 ii
Animals
0
4
3
0
4
0
0
0
1
0
0
3
0
0
0
0
0
0
1
0
0
0
0
Aquaria
0
4
3
0
3
0
0
0
1
0
0
2
0
0
0
0
0
0
1
0
0
0
0
Total
animals
2
17
3
1
18
4
2
3
3
4
6
15
3
1
2
1
1
2
20
1
2
2
1
R
S:
B
§
§
Colonies: Counted as one animal.
-------
PCB Affects Estuarine Communities 25
retained by the sieve were placed in fingerbowls of seawater, relaxed with HgCl2, preserved in
50% isopropanol and identified.
To determined effect of Aroclor 1254 in each treatment, an index of species diversity as well
as the number and percent occurrence of various species in each treatment (contaminated and
control aquaria) were compared. A species diversity index provides a numerical means of
assessing community structure that is independent of sample size, expresses the relative impor-
tance of each species and is dimensionless. Modifications of the Shannon-Weaver (1963 formula,
B
H' = — S Pjlogp,, where PJ is the proportion of the ith species in the collection and s = the
number of species, have been used in freshwater (Wilhm and Dorris 1968) and saltwater
(Bechtel and Copeland 1970) to assess effects of pollution on natural communities. In unpolluted
areas, many species of animals are abundant and diversity is high, but pollution can decrease
diversity by making a few species very abundant and all others rare. In this study, the Shannon-
Weaver formula (Iog2) was used to determine the usefulness of the species diversity index in
assessing the effect of Aroclor 1254 on community structure in laboratory experiments.
Pooled data from each Aroclor concentration and control were compared statistically using the
X2 test for independent samples. Data from each of the 10 aquaria receiving one treatment were
compared with data from 10 aquaria receiving a different treatment using the Mann-Whitney
"U" test (Siegal 1956). Differences were considered real at a = 0.01.
RESULTS
A large number and variety of animals were found in all aquaria (Tables 2 &
3). Of the 67 species from nine phyla, 27 were mollusks (15 pelecypods and 12
gastropods), 23 annelids, 6 arthropods, 3 chordates, 2 coelenterates, 2 enchino-
derms, 2 nemerteans, 1 brachiopod and 1 bryozoan. Anthropods were most abun-
dant (3,842) of the 5,897 animals followed by chordates (1,302), annelids (448),
mollusks (219) and animals from other phyla (86). The two most abundant
animals were the amphipod, Corophium volutator (3,770), and the tunicate,
Molgula manhattensis (1,164).
Species composition and abundance of individual species varied among the 40
aquaria. The number of species in each aquarium ranged from 5 to 23 (average
13). Annelids and mollusks were present in all aquaria. Animals from other
phyla were present in from 1 to 36 aquaria (Table 3). Seventeen species were
found in all treatments and 26 were in only one.
Aroclor 1254 prevented animals of certain phyla from colonizing (Table 3).
Although nine phyla were found, the number of phyla represented in any
aquarium ranged from three to seven. The number of phyla in the control, 0.1
and 1 /ig/1 aquaria averaged 5.7, 5.4 and 5.7, respectively. Fewer phyla, average
4.1, were in aquaria contaminated with 10 jug/1 of Aroclor because fewer aquaria
contained arthropods, and none contained bryozoans.
The total number of species found in each apparatus ranged from 2'". to 52 but
the percentage of species in each phylum was similar in all four treatments
(Table 4). In each apparatus, more species of mollusks were found than species
from any other phylum. The relative numbers of molluscan species ware similar
for all treatments (40—44 percent). The number of annelid species was only
slightly less (29-35 percent). Although fewer arthropods and cbordstes were
found, the relative numbers of each were similar in all treatments.
-------
26 David J. Hansen
TABLE 3
Number of control and experimental aquaria that contained animals, by phylum. Ten aquaria
were used for each control and contaminated apparatus. Experimental aquaria
were contaminated continuously with Aroclor 1245 for four months.
Phylum
Annelida
Arthropoda
Branchiopoda
Chordata
Coelenterata
Echinodermata
Ectoprocta
Mollusca
Nemertea
Total phyla in apparatus
Average number of phyla per aquarium
Control
aquaria
10
10
1
7
5
3
6
10
3
9
5.7
Aroclor 1254 aquaria
0.1 W5/1
10
10
0
10
4
4
6
10
0
7
5.4
l.O/ig/1
10
10
0
9
9
6
4
10
0
7
5.7
10.0/ig/l
10
4
0
10
7
0
0
10
0
6
4.1
TABLE 4
Number of species, by phylum, that developed from planktanic larvae in control apparatus and in
apparatuses contaminated continuously for four months with 0.1, 1 or 10/tg/l
of Aroclor 1254. Each apparatus consisted of 10 aquaria.
Aroclor 1254
Taxon
Annelida
Arthropoda
Chordata
Mollusca
Other phyla
Total
Control
Number Percent
18
3
3
21
7
52
34.6
5.8
5.8
40.4
13.4
100.0
0.1 jug/1
Number Percent
10
4
3
14
3
34
29.4
11.8
8.8
41.2
8.8
100.0
i pg/l
Number Percent
14
4
2
19
4
43
32.6
9.3
4.6
44.2
9.3
100.0
10 /.g/l
Number Percent
8
4
2
10
1
25
32.0
16.0
8.0
40.0
4.0
100.0
TABLE 5
Number of species, by phylum, that developed from planktonic larvae in 10 control aquaria and
10 aquaria in each apparatus contaminated continuously for four months
with 0.1, 1 or 10 /ig/1 of Aroclor 1254.
Aroclor 1254
Control
Average
Taxon
Annelida
Arthropoda
Chordata
Mollusca
Other phyla
Total
number per
aquarium
5.7
1.6
0.9
6.3
1.8
16.3
Range
2-10
1-2
0-3
2-10
0-3
7-23
0.1
Average
number per
aquarium
5.1
1.4
1.3
3.6
1.4
12.8
Ag/l 1 .0 /ig/1
Average
Range
3-7
1-4
1-2
1-7
0-3
9-18
number per
aquarium
4.5
2.8
1.0
3.9
2.1
14.3
Range
2-6
2-4
0-2
1-6
1-4
7-18
10.0 Mg/1
Average
number per
aquarium
3.9
0.6
1.5
2.7
0.7
9.4
Range
3-5
0-3
1-2
1-5
0-1
5-15
-------
PCB Affects Estuarine Communities 27
The number of species in each aquarium of an.apparatus was altered by Aro-
clor 1254 (Tables 2 & 5). The total number of species and the number of species
from each phylum in the ten control, 0.1 and 1 /Ag/1 contaminated aquaria were
similar. However, there were significantly fewer species and the species compo-
sition differed in the ten aquaria contaminated by 10 /ig/1 of the PCB. The great-
est shifts in species composition were found in arthropods, bryozoans, and mol-
lusks. Although there were significant reductions in the number of molluscan
species in the 10 /Ag/1 aquaria, there was no difference in the gastropod—pelecy-
pod ratio.
The total number of animals in each aquarium did not differ significantly
among the four treatments; whereas the number and percentage occurrence of
species was markedly different (Tables 2 & 6). Arthropods (primarily the tube-
dwelling amphipod, Corophium volutator) were the dominant animals in the
control (76 percent) and 0.1 /j.g/1 PCB (84 percent) aquaria. In these aquaria,
chordates (primarily Molgula manhattensis) were secondarily abundant. Arth-
ropods were also abundant (55 percent) in aquaria that received 1 /ig/1 but a
significant decrease in their abundance and an increase in abundance (31 per-
cent) of chordates occurred. Dominance was different in aquaria contaminated by
10 |itg/l; 80 percent of the animals were chordates. This difference from commun-
ities dominated by arthropods in control aquaria and in aquaria contaminated
by 0.1 ju.g/1 of Aroclor 1254 to communities dominated by chordates in aquaria
receiving the highest concentration of this PCB was the most striking PCB-
induced effect in this experiment.
The abundance of animals of other phyla, although less striking, was also
altered by PCB. There were more mollusks in the control aquaria than in treated
aquaria, but the percentage of their occurrence was not different in the PCB
environments. Colonies of the encrusting bryozoan, Membranipora tenuis, were
not counted, and therefore their numbers are not adequately represented in Table
6. However, their exclusion from the ten aquaria contaminated with 10 /j.g/1
was significant. Abundance of polychaetes was not altered by any of the three
concentrations of PCB.
The Shannon-Weaver (1963) index of species diversity calculated for each
aquarium did not differ among the control and three contaminated apparatuses
(Table 7). Species diversity is a function of two components, richness (number
of species) (Table 5) and equitability or relative number of each species (Table
7) (Lloyd and Ghelardi 1964). In my study, species diversity is not correlated
(r = 0.094) with richness of species but is correlated (r — 0.882) with relative
abundance of each species: J- = calculated diversity-^ maximum diversity (Pielou
1966). (Maximum diversity is defined as species diversity where all species are
equally abundant.) Equitability did not differ between treatments because com-
munities in this study were usually dominated by one species and were not rich
in species. Therefore, the effect of Aroclor was not on species diversity but on
species composition.
-------
to
00
O
a
TABLE 6
Average number per aquarium and average percent frequency per aquarium, of animals, by phylum (range in parentheses), that developed from
planktonic larvae in 10 control aquaria and 10 aquaria that for four months received 0.1, 1 or 10 /tg/1 of Aroclor 1254.
Phylum
Aroclor 1254
Control
Number Pez-centage
o.i neA
Number Percentage
Number Percentage
10 /tg/1
Number Percentage
a
3
Annelida 11.6(3-23) 6.5(1.8-21.4)
Arthropoda 134.6(6-406) 75.8(12.7-94.0)
Oiordata 19.3(0-112) 10.9(0-64.7)
Mollusca 10.1(3-18) 5.7(0.7-28.6)
Other phyla 2.0(0-4) 1.1(0-7.1)
Total 177.6(14-432) 100.0
11.9(4-21) 5.8(1.3-42.8) 12.8(7-22) 9.0(3.9-38.5)
170.9(14-528) 83.6(28.6-96.2) 78.0(8-199) 54.9(20.5-86.9)
14.4(1-52) 7.0(0.6-19.4) 43.7(0-130) 30.8(0-59.1)
4.4(1-10) 2.2(0.8-12.2) 4.4(1-9) 3.1(1.3-33.3)
2.7(0-11) 1.4(0-6.5) 3.2(1-7) 2.2(0.9-7.7)
204.3(49-589) 100.0 142.1(24-239) 100.0
8.5(5-16) 12.9(5.3-66.7)
0.7(0-4) 1.1(0-2.8)
52.8(3-160) 80.4(25.0-88.6)
3.0(1-6) 4.6(1.9-9.0)
0.7(0-1) 1.0(0-3.6)
65.7(12-186) 100.0
-------
TABLE 7
Shannon-Weaver index of species diversity and index of species richness in the ten control aquaria
and ten aquaria contaminated with 0.1,1 or 10 /ig/1 or Aroclor 1254.
Species diversity
Equitability (J)
Mean
1.80
0.47
Control
Range Std. error
0.57-2.83 0.24
0.14-0.89 0.07
Mean
1.42
0.38
0.1 yltg/1
Range
0.36-3.17
0.10-0.81
Std. error
0.26
0.07
Mean
2.07
0.55
Aroclor 1254
l.O^g/1
Range
0.94-3.26
0.25-0.86
Std. error
0.20
0.06
Mean
1.62
0.53
10.0 jug/1
Range
1.03-2.19
0.26-0.94
Std. error
0.13
0.06
to
o
1
3
§
to
to
-------
30 David J. Hansen
TABLE 8
Species and total number of animals collected from the effluents of 10' control aquaria and
10 aquaria contaminated for four months with 0.1, 1 or 10 /ig/1 Aroclor 1254.
Taxon
Annelida
Eupomatus dianthus
E. protulicola
Neanthes succinea
Total
Arthropoda
Balanus sp.
Caprella sp.
Clibanaris tricolor
Ccrophium volutator
Eurypanopeus depresus
Neopanope texana
Pagurus longicarpus
Pinnixa chaetopterana
Upogebia affinis
Decapod zoea, unident. sp.
Portunidae, unident. sp.
Pycnogonidae, unident. sp.
Total
Chordata
Bostrichobranchus pilularis
Branchiostoma caribaeum
Molgula manhattensis
Total
Coelenterata
Leptomedusae*, unident. sp.
Echinodermata
Hemipholis elongata
Ectoprocta
Membranipora tenuis*
Mollusca
Anadara ovalis
A. transversa
Bittium alternata
B. varium
Crassostrea virginica
DoridelLx obscura
Laevicardium mortoni
Mitrella lunata
Musculus lateralis
Nassarius albus
Tagelus divisus
Eolidacea, unident. sp.
Total
Totals: Animals
Species
Control
1
0
1
2
10
0
0
7
5
10
1
1
1
0
2
0
37
3
1
72
76
1
1
1
1
12
0
3
2
12
1
2
1
2
1
1
38
156
27
0.1 us/I
1
1
2
4
2
8
1
15
2
6
0
0
1
0
1
0
36
0
0
103
103
1
0
1
0
0
1
,0
0
1
0
0
0
0
2
1
5
150
18
1 Mg/l
0
1
2
3
2
4
0
32
5
3
0
0
0
1
0
3
50
0
1
304
305
1
3
1
0
4
0
1
1
2
0
2
1
0
0
1
12
375
20
10/ig/l
0
0
0
0
1
0
0
1
0
0
0
0
0
0
0
0
2
0
0
35
35
1
0
0
0
1
0,
0
1
1
0
2
0
0
0
3
8
47
10
Colonies: Counted as one animal.
-------
PCB Affects Estuarine Communities 31
Conclusions based on abundance and diversity of animals collected at the end of
the four-month exposure were corroborated by the abundance and diversit)- of
animals that migrated from and were washed from each apparatus during the
exposure (Table 8). The total number of animals and species from aquaria with
10 jU.g/1 was markedly lower than those from the other aquaria. Arthropods
were abundant in collections from the effluents of control aquaria and aquaria
with 0.1 and 1 /j.g/l but rare from the aquaria with 10 ju.g/1. The effect of Aroclor
on crabs collected from the aquaria could not be assessed at the end of the expos-
ure because only two were found in all aquaria. However, presence of exoskele-
tons in seven of 10 control, six of 10 0.1 /j.g/1 and eight of 10 1 jj.g/1 aquaria and
absence of exoskeletons in the 10 /u.g/1 aquaria strongly suggest that crabs were
sensitive to the highest concentration of Aroclor 1254. This sensitivity was sub-
stantiated by collections of crabs from the effluents of the aquaria. Eurypanopeus,
Neopanope, Pinnixa and portunids were abundant in effluents of control, 0.1 and
1 ju.g/1 aquaria but absent from the effluent of the 10 ^g/1 aquaria. The bryozoan,
M. tenuis, was absent in the effluent from the 10 jug/1 aquaria, but present in the
effluent from the other aquaria. Mollusks were most abundant in effluent from
the 10 control aquaria.
DISCUSSION AND CONCLUSIONS
The polychlorinated biphenyl, Aroclor 1254. influenced the composition of
animal communities that developed from planktonic larvae in sea water which
entered the test aquaria. The primary influence was that the dominant species in
the control aquaria was the amphipod, C. volutator, and the dominant species in
aquaria receiving 10 /ug/1 PCB was the tunicate, M. manhattensis. Also, there
were fewer species and the number of animals was markedly but not significantly
fewer in the 10 ju.g/1 aquaria. The abundance of arthropods, chordates, bryozoans
and mollusks differed significantly but abundance of annelids, brachiopods, coel-
entrates, echinoderms or nemerteans apparently did not differ.
Differences in community structure that were apparent at the lowest concen-
tration (0.1 /tig/1) became more pronounced as the concentration of Aroclor 1254
increased to 10 fj.g/1- Control aquaria were dominated by arthropods (76 percent).
with lesser numbers of animals from eight other phyla. Aquaria contaminated
by 0.1, 1 or 10 /xg/1 contained fewer mollusks than did control aquaria; however,
the percentage occurrence of mollusks was not altered by the PCB. Aquaria
receiving 1 /xg/1 or 10 /ig/1 had more tunicates and fewer arthropods than did con-
trol aauaria. Aquaria receiving 10 ju.g/1 were dominated by tunicates (80 psrcent)
with lesser numbers of five other phyla; only 1 % of the animals were arthropods.
Animals most reduced in numbers at the highest PCB concentration included
the amphipod, C. volutator; the xanthid crabs, Eurypanopeus dcpressus and Neo-
panope texana; the bryozoan, M. tenuis; the gastropod. Bittium varium. and the
pelecypods, Abra aequalis and Tellina alternata.
Few of these changes could have been predicted from information from current
literature, because only one species in this study had been challenged previously
-------
32 David J. Hansen
•with PCB and only a few are phylogenetically related to previously challenged
species. Arthropods, particularly amphipods and crabs were sensitive to Aroclor
1254 in this experiment and in the experiments by Nimmo et al. (1971 a) in
which pink shrimp were killed by 1 /xg/1. Juvenile blue crabs appeared resistant
to 5 jug/1 (Duke et al. 1970). In my experiment, the numbers of xanthid crabs
was reduced by 10 ju.g/1 indicating that larval stages may be particularly sensitive
to PCBs. Sensitivny of crab larvae has been shown with the insecticides dieldrin
(Epifanio 1971) and mirex (Bookhout et al. 1972). Aroclor 1254 was lethal to
two estuarine fishes at 5 /ug/1 (Hansen et al. 1971) but in this study lower chord-
ates (tunicates) seemed unaffected and were most abundant in the PCB-stressed
communities. Lethal effects of PCBs on mollusks are not known. However, growth
of oysters was reduced significantly without mortality by 5 ^g/1 of Aroclor 1254
(Parrish et al. 1972). In my experiment fewer mollusks occurred in all exposure
concentrations possibly because the larval stages are sensitive or because of fac-
tors other than the presence of Aroclor. Studies on protozoans (Cooley, Keltner
and Forester 1972) provided the only other data I am aware of on the sensitivity
of estuarine animals of other phyla to PCBs.
The Shannon-Weaver species diversity index has been used as an indicator of
the effects of some types of pollution on animal communities in estuaries, but in
this experiment, the index was not decreased even though composition of the com-
munities were greatly altered. This species diversity index did not decrease as
the concentration of PCB increased because the index was proportional to the
relative number of each species present in the aquaria, and communities that
developed at each treatment concentration were dominated by one species of
animal. If Aroclor 1254 affected the composition of established communities in
an estuary as it did the developing communities in this experiment, this species
diversity index could not be used to estimate effects of this pollutant in the
environment.
One purpose of this experiment was to determine whether the effect of a tox-
icant on developing estuarine animal communities can be investigated in the lab-
oratory. Preliminary experiments at this laboratory indicated that the structure
of communities of organisms setting in aquaria was altered by presence of the
insecticide Dursban® (J. I. Lowe, personal communication2). My analysis of his
data indicated that replicate aquaria were necessary to separate the effects of a
toxicant from the effects of an efficient predator or from the effects of an animal
with great reproductive capacity. The use of ten replicate aquaria for each treat-
ment in my experiment readily separated the effect of Aroclor 1254 from other
factors that might influence community structure. My experiment also showed
that small aquaria can be used, provided that larger animals can emigrate before
they drastically affect community structure and thus mask effects of the toxicant.
Emigrating animals must be caught and enumerated so the effects on them can
be assessed.
- Mr. Jack I. Lowe, Environmental Protection Agency, Gulf Breeze, Fla. 32561.
® Registered trademark: Dow Chemical Co.
-------
PCB Affects Estuarine Communities 33
ACKNOWLEDGMENTS
I am grateful to Mr. Edward Matthews, who helped during the four-month experiment, and
to Mr. Johnnie Knight, who prepared water and sediment samples for chemical analysis. I am
particularly indebted to Dr. Nelson R. Cooley, because identification of the numerous species of
animals would have been most difficult without his help.
LITERATURE CITED
ANONYMOUS. 1966. Report of a new chemical hazard. New Scient. 32: 612.
BECHTEL, T. J. and B. J. COPELAND. 1970. Fish species diversity indices as indicators of
pollution in Galveston Bay, Texas. Conir. mar. Sci. Univ. Tex. 15: 103-133.
BOOKHOUT, C. G., A. J. WILSON, JR., T. W. DUKE and J. I. LOWE. 1972. Effects of Mirex
on the larval development of two crabs. Water, Air and Soil Pollut. 1: 165—180.
BROADHURST, M. G. 1972. Use and replaceability of PCBs. Environ. Health Perspectives.
2: 81-102.
BUTLER, P. A. 1973. Organochlorine residues in estuarine mollusks—1965-1972. Pestic.
Monit. J. 6 (4).
COOLEY, N. R., J. M. KELTNER, JR. and J. FORESTER. 1972. Mirex and Aroclor® 1254:
Effect on and accumulation by Tetrahymena pyriformis strain W. /. Protozool. 19: 636-638.
DUKE, T. W., J. I. LOWE and A. J. WILSON, JR. 1970. A polychlorinated biphenyl
(Aroclor 1254®) in the water, sediment and biota of Escambia Bay, Florida. Bull. Environ.
Contam. Toxicol. 5: 171-180.
EPIFANIO, C. E. 1971. Effects of dieldrin in seawater on the development of two species of
crab larvae, Leptodius floridanus and Panopeus herbstii. Mar. Biol. 11(4): 356-362.
HANSEN, D. J., P. R. PARRISH, J. I. LOWE, A. J. WILSON, JR. and P. D. WILSON. 1971.
Chronic toxicity, uptake and retention of Aroclor® 1254 in two estuarine fishes. Bull.
Environ. Contam. Toxicol. 6: 113-119.
LLOYD, M. and R. J. GHELARDI. 1964. A table for calculating the equitability component
of species diversity. /. Anim. Ecol. 33: 217-225.
NIMMO, D. R, R. R. BLACKMAN, A. J. WILSON, JR. and J. FORESTER. 1971 a. Toxicity
and distribution of Aroclor® 1254 in the pink shrimp Penaeus duorarum. Mar. Biol. 11(3):
191-197.
NIMMO, D. R., P. D. WILSON, R. R. BLACKMAN and A. J. WILSON, JR. 1971b. Poly-
chlorinated biphenyl absorbed from sediments by fiddler crabs and pink shrimp. Nature,
Land. 231: 50-52.
PARRISH, P. R., J. I. LOWE, A. J. WILSON, JR. and J. M. PATRICK, JR. 1972. Effects of
Aroclor® 1254, a PCB, on oysters, Crassostrea virginica (Bivalvia: Protobranchia: Ostreidae).
ASBBull. 19(2): 90.
PIELOU, E. C. 1966. The measure of diversity in different types of biological collections.
J. Theor. Biol. 13: 131-144.
SHANNON, C. E. and W. WEAVER. 1963. The mathematical theory of communication.
University of Illinois Press, Urbana.
SIEGAL, S. 1956. Non Parametric Statistics for the Behavioral Sciences. McGraw-Hill Book
Co., Inc., N.Y. 312 p.
WILHM, J. L. and T. D. DORRIS. 1968. Biological parameters for water quality criteria.
B ioScience \ 8 (6): 47 7-481.
-------
CONTRIBUTION NO. 165
-------
THE MICROBIAL DEGRADATION OF OIL POLLUTANTS
ESTUARINE MICROBES AND ORGANOCHLORINE PESTICIDES
(A BRIEF REVIEW)1
A. W. BOURQUIN
Gulf Breeze Environmental Research Laboratory2
U.S. Environmental Protection Agency
Gulf Breeze, Florida 32561
Little is known about microbiological degradation of organochlorine
pesticides in the estuarine and oceanic environments. Since mircoorganisms
are probably the main instruments of pesticide breakdown, and possibly offer
an array of mechanisms by which pollution may be reduced, research is needed
to learn the pathways of microbial degradation in the marine environment.
Table 1 lists a number of microorganisms, chiefly soil and aquatic,
with demonstrated ability to partially degrade organochlorine pesticides in
various environments. An excellent review of the interaction between haloge-
nated pesticides and microorganisms has been given by Pfister and Matsumura
(28). Lichtenstein and Schulz (18) found that soil bacteria converted aldrin
to its more stable epoxide, dieldrin; the peak of dieldrin formation occurred
56 days after treatment. A bacterium, Proteus vulgaris, isolated from the
gut of a mouse, converted DDT to ODD (k) and some soil actinomycetes degrade
polychloro-nitrobenzene (PCNB) and dechlorinate DDT (8). Most of the reports
listed in Table 1 were concerned with pure cultures and few involved more
than one or two step transformations. However, other investigators have re-
ported extensive degradation by soil microorganisms leading to speculation
that biodegradation could result in mineralization of organochlorine compounds
in the presence of certain microbial assemblages and environmental parameters.
Bixby et al. (6) reported a soil fungus, Tr-ichoderma koningi, which degraded
dieldrin to carbon dioxide with cleavage of the chlorinated ring structure.
Focht (10) reported that an aquatic fungus, isolated from sewage effluent,
metabolized chlorinated bacterial degradation products to water, carbon di-
oxide, and hydrochloric acid.
Many soil microorganisms also occur in water, thus environmental re-
lationships and microbial associations similar to those in soil may exist.
The essential differences between terrestrial and aquatic environments rela-
tive to microbial activity appear to be: (a) usually fewer nutrients per
unit mass and less biochemical activity are found in water than in soil, and
(b) usually fewer adsorptive surfaces for microbial growth in water than in
soil. Reports have indicated that fresh surface waters do not have a charac-
teristic bacterial flora (12). However, certain microorganisms, such as
Beneckea and Caulobacter, have been designated as typical marine or estuarine
genera. Differences between estuarine, freshwater, and soil ecosystems make
1 Gulf Breeze Contribution No. 165.
2 Associate Laboratory of the National Environmental Research Center,
Corvallis, Oregon.
237
-------
TABLE 1
Microorganisms Known to Metabolize Organochlorine Pesticides
Genera
Pesticides
Environment
Reference
(see Literature Cited)
Bacter ia
Artfoobaater
Bacillus
Clostridium
Esaherichia
Hydrogenomonas
Klebsiella
Miarooooaus
Proteus
Pseudomonas spp.
Ps eudomonas spp.
Pseudomonas spp.
Un identif ied
Unidentified
Actinomycetes
Nooardia
Streptomyaes
Fung i
Aspergillus
Fusarium
Muoor
Triehoderma
Yeast
Saaaharomyces
Algae
Ch lamydomonas
Chlorella and
Dunaliella
Endrin, DDT
Endrin, DDT
Lindane
DDT
DDT
DDT
Endrin, Aldrin, DDT
DDT
Endrin, Aldrin, DDT
Heptachlor
Dieldr in
Dieldr in, Aldrin,
Endrin, DDT
Lindane, Aldrin
DDT, PCNB
PCNB
PCNB
DDT
Dieldr in
Dieldrin
DDT
Lindane
Aldrin
Soi1-aerobic
Soi1-aerobic
Aquatic-anaerobic
Aquatic-anaerobic
Aqua t i c-a na erob i c
Aquatic-anaerobic
So i1-aerobic
Aquatic-aerobic
Soi1-aerobic
Aquatic-aerobic
Soi1-aerobic
Marine-aerobic
Soi1-aerobic
Soi1-aerobic
Soi1-aerobic
Soi1-aerobic
Aquatic-aerobic
Soi1-aerobic
Soi1-aerobic
Patil et al., 1970 (25)
Patil et al., 1970 (25)
McRae et al., 1969 (19)
Mendel and Walton, 1966 (22)
Focht, 1972 (10)
Wedemeyer, 1966 (32)
Patil et al., 1970 (25)
Barker et al., 1965 (*»)
Patil et al., 1970 (25)
Bourquin et al., 1971 (7)
Matsumura et al., 1968 (21)
Patil et al., 1972 (27)
Lichtenstein and Schulz, 1959 (18)
Chacko et al ., 1966 (8)
Chacko et al., 1966 (8)
Chacko et al., 1966 (8)
Focht, 1972 (10)
Anderson et al., 1970 (3)
Bixby et al., 1971 (6)
to
TO
to
o
o
TO
03
v.
Aquatic-anaerobic Kallerman and Andrews, 1968 (16)
Aquatic-aerobic
Mar ine-aerobic
Sweeney, 1968 (31)
Patil et al., 1972 (27)
-------
THE MICROBIAL DEGRADATION OF OIL POLLUTANTS
the estuarine area a unique environment for study of microbial degradation.
Because of this uniqueness, data from soil and freshwater ecosystems cannot
necessarily be extrapolated to estuarine systems. Therefore, research is
needed to learn more about degradation pathways in the marine environment.
Several investigators have reported that degradation of pesticides by
aquatic microorganisms is similar to degradation by soil microorganisms.
Miles et al. (2k) reported that soil microorganisms metabolized heptachlor to
l-hydroxy-2,3-epoxychlordene. Bourquin et al. (7) reported similar results,
and proposed a pathway for microbial transformation of heptachlor in the
aquatic environment. Metabolism of DDT occurs in soil, freshwater, and lake
sediments (26). The similarity of transformations of these compounds may be
due to similarity of microflora in the different environments. However, mi-
crobial differences as well as environmental factors exist between aquatic
and terrestrial ecosystems. For example, most cultivated soils to which in-
secticides are applied are more aerobic than aquatic sediments. Although DDT
is converted to ODD and other products in anaerobic systems, it is stable in
aerobic systems (2,13).
Estuarine sediment is a reservoir for pesticides transported by
rivers. Because organochlorine pesticides are strongly sorbed on soil and
other particulate material (30), including microorganisms (17), they are
found on suspended particulates in rivers and are incorporated into estuarine
sediment (2). These sediments are often enriched by decomposing organic mat-
ter and are anaerobic beneath the surface. Although the rate of carbon turn-
over due to microbial activity in the sea may not be substantially different
from that in fresh water, estuaries are areas of rapid microbial transforma-
tions (35). The latter play an important role in estuarine nutrition.
Biodegradation of organochlorine pesticides in estuarine or oceanic
environments has been little studied despite the known persistence of the
pesticides. Patil et al. (27) studied microbial metabolic transformations of
DDT, dieldrin, aldrin, and endrin in samples of marine water, bottom sedi-
ments, and surface films. Transformations of DDT and cyclodiene insecticides
occurred in samples with biological materials such as surface films, plankton,
and algae, but not in waters from the open ocean. Pure cultures of marine
microorganisms also metabolized the pesticides. l-n general, patterns of deg-
radation that have been observed in terrestrial and aquatic ecosystems closely
resemble those found for the marine environment (27). For example, production
of 6,7-t-dihydroxy-dihydro-aldrin was the major metabolite found in soil
fungi (20), in an aquatic bacterium (33), in algal cultures (5), and in pure
cultures of marine algae, bacteria, and surface films (27). Similar results
were obtained from aldrin, endrin, and DDT, except that algal cultures ap-
peared to convert DDT to a "DDOH-like compound" (2,2-bis (p-chlorophenyl)).
The strong degradation activity associated with surface films is significant.
Surface films are areas of high biological activity (9) and concentrators of
dissolved organics (3*0 and pesticides (29). Such films provide the environ-
ment necessary for selection of hydrocarbon-degrading microorganisms and a
relatively high nutrient concentration for proliferation of cells. Presence
of pollutants, crude oil or pesticides, in an already enriched area of micro-
bial activity may select for hydrocarbon-degrading microorganisms.
The organically-enriched estuarine environment provides an opportunity
for study of co-metabolism of pesticides by microorganisms. As noted by Focht
239
-------
Center for Wetland Resources, LSU-SG-?3-013 1973
and Alexander (11), "Co-metabol ism is the adventitious biological transforma-
tion of organic compounds which provides neither energy nor structural com-
ponents to the organism." Many bacteria break down certain compounds while
metabolizing other substrates but do not utilize the co-substrate as a source
of energy or carbon for growth. Relevance of this phenomenon to natural soil
ecosystems was noted by Horvath and Alexander (15). Focht and Alexander (11)
demonstrated degradation of DDT by sewage bacteria that grew on diphenyl-
methane, an analogue of DDT. Co-metabolism has not been reported in the es-
tuarine environment. Natural conditions, however, predispose estuaries to
such metabolism because of the large microbial communities that exhibit a
wide variety of physiological activities. These include degradation of rela-
tively recalcitrant large molecular weight compounds such as complex polysac-
charides and some petroleum products (1,23)-
Synergism within the microbial ecosystem, including bacteria, yeasts
and fungi, in the estuarine environment is another factor to consider when
studying breakdown of pesticides. In the soil, microorganisms act synergis-
tically to degrade molecules considered resistant to attack by single species
(1A). However, in estuarine and oceanic environments, such complex interre-
lationships among microorganisms have not been investigated adequately.
Studies of interactions of estuarine microbial assemblages can provide impor-
tant data for understanding microbial degradation in estuarine systems.
Estuaries, periodically flushed by tides, provide the near shore poi —
tions of the open ocean with many organic nutrients in solution or in the
form of partially degraded organic detritus. Organic matter from estuaries
and other biologically productive waters often forms a slick, or calm streak,
on a rippled sea. As noted, surface slicks are areas of high biological ac-
tivity and could provide nutrients necessary for co-metabolic transformation
of pesticides in off-shore marine locales. Microbiology of naturally occur-
ring surface slicks and oil slicks caused by spills or seepage is an impor-
tant but neglected area of estuarine research.
Pathways of microbial attack upon chlorinated hydrocarbons in the es-
tuarine environment need to be investigated since breakdown by microorganisms
is probably the main natural process of pesticide degradation. Although mi-
crobiological processes might reduce environmental pollution attributed to
use of persistent pesticides, detailed studies of degradative pathways are
required to assess the degree of hazard caused by breakdown products. Re-
quired information on microbial degradation of organochlor ine pesticides in
estuaries will be supplied when we answer the following questions:
What types of microorganisms are involved in transformation of organo-
chlorine pesticides? Are they the same types that predominate in organic
detritus formation or are they species selected by exposure to pesticide
pol lut ion?
What is the degree of degradation of specific compounds?
Does co-metabolism occur in the estuary, and is it a means of de-
grading pesticides?
What effects do additional hydrocarbons, such as oil, have on micro-
bial degradation of pesticides in the estuary?
m
Is synergistic activity within the estuarine microflora a factor in
icrobial degradation of pesticides?
-------
THE MICROBIAL DEGRADATION OF OIL POLLUTANTS
What is the role of microbial intracel 1 ular accumulation and adsorp-
tion in biodegradation and/or biological magnification of pesticides?
What environmental factors in the estuary prevent or inhibit or ac-
celerate microbial breakdown of pesticides?
What types of pesticides are easily degraded?
What are the effects of degradation products on estuarine macro- and
microflora and fauna?
Considering all the above questions, are similar reactions, selections
and effects occurring in the water column and in the sediments?
Such studies will provide data for an accurate picture of the role of
estuarine microorganisms on the fate of organic pollutants. Such data are
needed to formulate water quality criteria for pesticide regulation in the
estuarine environment.
LITERATURE CITED
1. Ahearn, D. G., and S. P. Meyers. 1972. The role of fungi in the decompo-
sition of hydrocarbons in the marine environment. In H. A. Walters
and D. G. Hueck van der Plas (eds.), Biodeterioration of Materials,
2:12-18. Applied Science Publishers, Ltd., London.
2. Albone, E. S., G. Eglinton, N. C. Evans, J. M. Hunter, and M. M. Rhead.
1972. Fate of DDT in Severn Estuary sediments. Environ. Sci.
Technol. 6:914-919.
3. Anderson, J. P. E., E. P. Lichtenstein, and W. F. Whittingham. 1970.
Effect of MUCOF alt-ez>nans on the persistence of DDT and dieldrin in
culture and in soil. J. Econ. Entomol. 63:1595-1599.
4. Barker, P. S., F. 0. Morrison, and R. S. Whitaker. 1965. Conversion of
DDT to ODD by Proteus vulgar-is, a bacterium isolated from the intes-
tinal flora of a mouse. Nature (London) 205:621-622.
5. Batterton, J. C., G. M. Boush, and F. Matsumura. 1971. Growth response
of blue-green algae to aldrin, dieldrin, endrin and their metabolites.
Bull. Environ. Contam. Toxicol. 6:589~594.
6. Bixby, M. W., G. M. Boush, and F, Matsumura. 1971. Degradation of diel-
drin to carbon dioxide by a soil fungus Trichoderma koningi. Bull.
Environ. Contam. Toxicol. 6:491-494.
7. Bourquin, A. W., S. K. Alexander, H. K. Speidel , J. E. Mann, and J. F.
Fair. 1971- Microbial interactions with cyclodiene pesticides.
Dev. Indust. Microbiol. 13:264-276.
8. Chacko, C. I., J. L. Lockwood, and M. Zabik. 1966. Chlorinated hydro-
carbon degradation by microbes. Science 154:893-895.
9. Ewing, G. 1950. Slicks, surface films and internal waves. J. Mar.
Res. 9:161-187.
10. Focht, D. D. 1972. Microbial degradation of DDT metabolites to carbon
dioxide, water, and chloride. Bull. Environ. Contam. Toxicol.
7:52-56.
241
-------
Center for Wetland Resources3 LSU-SG-73-01, 1973
11. Focht, D. D., and M. Alexander. 1970. Bacterial degradation of diphenyl-
methane, a DDT model substrate. Appl. Microbiol. 20:608-611.
12. Frobisher, M. 1968. Fundamentals of Bacteriology, 8th ed. W. B. Saun-
ders Co., Philadelphia.
13. Guenzi, W. D., and W. E. Beard. 1968. Anaerobic conversion of DDT to
ODD and aerobic stability of DDT in soil. Soil Sci. Soc. Am. Proc.
32:522-524.
14. Gunner, H. B., and B. M. Zuckerman. 1968. Degradation of "Diazinon" by
synergistic microbial action. Nature (London) 217:1183-1184.
15. Horvath, R. S., and M. Alexander. 1970. Co-metabolism of m-chloro-
benzoate by an Arthrobacter. Appl. Microbiol. 20:254-258.
16. Kallerman, B. J., and A. K. Andrews. 1968. Reductive dechlorination of
DDT to DDD by yeast. Science 141:1050.
17. Ko, W. H., and J. L. Lockwood. 1968. Accumulation and concentration of
chlorinated hydrocarbon pesticides by microorganisms in soil. Canad.
J. Microbiol. 14:1075.
18. Lichtenstein, E. P., and K. R. Schulz. 1959. Breakdown of 1 indane and
aldrin in soil. J. Econ. Entomol . 52:118-124.
19- McRae, I. C., K. Raghu, and E. M. Bautista. 1969. Anaerobic degradation
of the insecticide 1 indane by Clostr-idium sp. Nature (London)
221:859-860.
20. Matsumura, F., and G. M. Boush. 1967. Dieldrin: Degradation by soil
microorganisms. Science 156:959*961.
21. Matsumura, G., G. M. Boush, and A. Tai. 1968, Breakdown of dieldrin in
the soil by a microorganism. Nature (London) 219:965-967.
22. Mendel, J. L., and M. S. Walton. 1966. Conversion of p,p'-DDT to
p,p'-DDD by intestinal flora of the rat. Science 151:1527.
23. Meyers, S. P., D. G. Ahearn, W. Gunkel, and F. J. Roth, Jr. 1967.
Yeasts from the North Sea. Mar. Biol. 1:118-123.
24. Miles, J. R. W., C. M. Tu, and C. R. Harris. 1969. Metabolism of hepta-
chlor and its degradation products by soil microorganisms. J. Econ.
Entomol . 62:1334-1338.
25. Patil, K. C., F. Matsumura, and G. M. Boush. 1970. Degradation of
endrin, aldrin, and DDT by soil microorganisms. Appl. Microbiol.
19:879-881.
26. Patil, K. C., F. Matsumura, and G. M. Boush. 1971, DDT metabol ized by
microorganisms from Lake Michigan. Nature (London) 230:325-326.
27. Patil, K. C., F. Matsumura, and G. M. Boush. 1972. Metabolic transfor-
mation of DDT, dieldrin, aldrin, and endrin by marine microorganisms.
Environ. Sci. Techno 1. 6:629-632.
28. Pfister, R. M., and F. Matsumura. 1972. Interactions of halogenated
pesticides and microorganisms: A review. In A. I. Laskin and H.
Lechevalier (eds.), Critical Reviews in Microbiology, 2:1-33. CRC
Press, The Chemical Rubber Co., Cleveland.
242
-------
THE MICROBIAL DEGRADATION OF OIL POLLUTANTS
29- Seba, D. B., and E. F. Corcoran. 1969. Surface slicks as concentrators
of pesticides in the marine environment, Pestic. Monit. J. 3:190-193-
30. Shin, Y. 0., J. J. Chodan, and A. R. Wolcott. 1970. Adsorption of DDT
by soils, soil fractions, and biological materials. J. Agric. Food
Chem. 18:1129.
31. Sweeney, R. A. 1968. Metabolism of lindane by unicellular algae. Proc.
12th Conf. Great Lakes Research.
32. Wedemeyer, G. 1966. Dechlorination of DDT by Aerobaoter aerogenes.
Science 152:64?.
33- Wedemeyer, G. 1968. Partial hydrolysis of dieldrin by Aerobacter
aerogenes, Appl. Microbiol. 16:661-662.
3*». Williams, P- M. 1967. Sea surface chemistry: organic carbon and organic
and inorganic nitrogen and phosphorus in surface films and subsurface
waters. Deep-Sea Res. 14:791-800.
35. Zhukova, A. R. 1963. On the quantitative significance of microorganisms
in nutrition of aquatic invertebrates. In C. H. Oppenheimer (ed.),
Symposium on Marine Microbiology, pp. 699-710. Charles C. Thomas,
Springfield, 111.
243
-------
CONTRIBUTION NO. 167
-------
Reprinted from COPEIA, 1973, No. 1, March 5
pp. 140-141
Made in United States of America
A LARVAL TARPON, MEGALOPS AT-
LANTICUS, FROM PENSACOLA, FLOR-
IDA.—A larval tarpon, Megalops atlanticus,
an early Stage II as designated by Wade
(1962), was collected in the upper reaches of
East Bay, about 45 km from the Pensacola
Inlet, Florida on 20 October 1970. It repre-
sents the second and most northern record
of a larval tarpon from the Gulf of Mexico,
and suggests late spawning in the Gulf.
Eldred (1968) reporting on the first capture
of a tarpon larva (Stage I) in the Gulf, about
117 km due west of Sanibel Island, Florida
on 6 July 1967, postulated that its presence
could have resulted either from spawning in
the Gulf or from being carried northward by
transport currents from a more southerly
spawning. Eldred's (1967) study on catch
records of young indicated that tarpon spawn
in the Florida Straits, the Gulf Stream, and
the Caribbean Sea during spring and summer.
The occurrence of the present early larva in
the northern Gulf provides evidence of
spawning in a Gulf locality.
The specimen from East Bay was collected
unusually late in the year. This may indicate
that spawning in the northern Gulf is later
than in southern regions. Except for a larva
-------
ICHTHYOLOGICAL NOTES 14]
taken 12 November 1921 off French Guiana,
all Stage I and II larvae have been captured
from 17 May to 1 October (Wade, 1962;
Eldred, 1967, 1968).
The larva was collected in a plankton tow
near the entrance of East Bay River in water
1.5 m deep. Surface salinity of the water was
7.3 fc bottom, 11.5. Surface water temper-
ature was 20.1° C; bottom, 21.6. Measure-
ments (in mm) of the specimen are 28.5 total
length, 25.8 standard length, and 2.6 head
length. It has a total of 56 myomeres (39
predorsal, 41 preanal), 13 dorsal rays, and
20 anal rays.
I thank Dr. Thomas W. McKenney of the
National Marine Fisheries Service Southeast
Fisheries Center, Miami, Fla. for checking the
measurements and meristic counts of the
leptocephalus.
LITERATURE CITED
ELDRED, B. 1967. Larval tarpon, Megalops at-
lanticus Valenciennes, (Megalopidae) in Florida
waters. Fla. Bd. Conserv. Mar. Lab., Leaf.
Ser., IV, Pt. 1, No. 4:1-9.
. 1968. First record of a larval tarpon,
Megalops atlanticus Valenciennes, from the
Gulf of Mexico. Ibid. IV, Pt. 1, No. 7:1-2.
WADE, R. A. 1962. The biology of the tarpon,
Megalops atlanticus, and the ox-eye, Megalops
cyprinoides, with emphasis on larval develop-
ment. Bull. Mar. Sci. Gulf Carib. 12:545-622.
MARLIN E. TAGATZ, National Marine Fish-
eries Service, Atlantic Estuarine Fisheries
Center, Beaufort, North Carolina 28516.
Present address: Environmental Protection
Agency, Gulf Breeze Laboratory, Gulf Breeze,
Florida 32561.
-------
CONTRIBUTION NO. 168
-------
RESIDUES IN FISH, WILDLIFE,
AND ESTUARIES
Mirex Residues in Selected Estuaries of South Carolina—June 1972 1
P. W. Borthwick,- G. H. Cook,= and J. M. Patrick, Jr."
ABSTRACT
Estuarine sediments, crabs, shrimps, and fishes were collected
in June 1972 at eleven stations two years after aerial ap-
plications of mirex bait for control of fire ants in coastal
areas near Charleston, S.C. These stations had previously
been monitored (October 1969 to June 1971) when levels
of mirex in animal samples were: crabs, 0-0.60 ppm; shrimps,
0-1.3 ppm; and fishes, 0-0.82 ppm.
The recent study showed that mirex was present in three
species of fishes (white catfish, 0.021 ppm; bluegill, 0.047
ppm; carp, 0.12 ppm) and blue crabs (0.026 ppm) at two
freshwater stations. However, mirex was not detected in 36
animal samples, most of which were taken from nine saline
stations in the estuaries after a period of restricted use of
the pesticide. Analysis of bottom sediment samples at all
stations detected no mirex. The lower limit of detection for
mirex was 0.01 ppm.
Introduction
In June 1972 samples of estuarine sediments, crabs,
shrimps, and fishes were collected at 11 stations near
Charleston, S.C., where mirex fire ant bait had been
applied aerially to coastal areas from October 1969 to
December 1970. The United States Department of
Agriculture supervised two applications of mirex, by
fixed-wing aircraft, to several hundred thousand acres
in the Charleston area. Treatments were terminated
approximately 24 months before the June 1972 collec-
1 Contribution No. 168 from the Gulf Breeze Environmental Research
Laboratory, United States Environmental Protection Agency, Gulf
Breeze, Florida 32561; Associate Laboratory of the National Environ-
mental Research Center, Corvallis, Oregon.
" Gulf Breeze Environmental Research Laboratory, Gulf Breeze, Flor-
ida 32561.
144
tions. However, 18 months lapsed since special applica-
tions were made by helicopter to 1200 acres at Toogoo-
doo Creek: Stations A, B, C, and D; and by hand seeder
around a one-acre pond at Riverland Terrace: Station 1
(Fig. 1).
Since 1970 less extensive applications have been made
for control of nuisance populations of fire ants. During
the 1971-72 cooperative Federal State Control Program,
mirex bait was applied aerially in South Carolina and
seven other Southeastern states at property owners'
requests.
FIGURE 1—Map of study area showing location of mirex
sampling sites, South Carolina—June 1972
PESTICIDES MONITORING JOURNAL
-------
The use of mirex decreased when the U.S. Environ-
mental Protection Agency (U.S. EPA) issued orders
(1, 2) to cancel the registration of products containing
mirex, pending relabeling. The orders required that no
mirex be applied aerially near estuaries and other
aquatic areas, wildlife refuges, or heavily forested areas.
The present study was implemented to determine how
much mirex remained in the estuarine fishes and
crustaceans following a period of restricted use of the
pesticide.
The stations (Fig. 1) and analytical techniques were
identical to those used in a more comprehensive study
of the area (3). Samples were collected and analyzed by
the United States Environmental Protection Agency,
Gulf Breeze Environmental Research Laboratory, Gulf
Breeze, Florida.
Results and Discussion
In June 1972, 24 months after large-scale aerial treat-
ment of inland areas, mirex residues greater than 0.01
ppm were found in only 4 of 40 animal samples (Table
1). All four samples were collected at the two freshwater
stations located on the Ashley and Cooper Rivers
(Stations 3 and 4, Fig. 1) which drain watersheds
within the treated area. Three species of freshwater
fishes and blue crabs contained the following amounts
of mirex (ppm):
white catfish
bluegill
carp
blue crabs
0.021
0.047
0.12
0.026
Except for the bluegill, these animals are omnivorous
bottom-dwellers. Blue crabs are euryhaline; they oc-
casionally enter brackish and fresh waters of estuaries.
Mirex was not detected (Table 1) in the remaining 36
animal samples, most of which were taken at 9 stations
located on tidal creeks in salt marsh areas that support
populations of finfish and crustaceans. Many of these
animals are transient and spend only a portion of their
lives in the estuary. No mirex was detected in bottom
sediments sampled at each location.
The lower limit of detection for mirex with the method
employed (3) was 0.01 ppm.
Between October 1969 and June 1971 mirex residues in
economically important members of the estuarine food
chain varied as follows: crabs, 0-0.60 ppm; shrimps,
0-1.3 ppm; and fishes, 0-0.82 ppm. Levels of mirex in
these species diminished to less than 0.01 ppm over a
period of 18 to 24 months after the last aerial broadcast
treatments to coastal South Carolina.
LITERATURE CITED
(1) Ruckelshaus, W. D. 1972. Products containing the in-
secticide mirex; determination and order of the Admin-
istrator. Fed. Regist. 37(106): 10987-10988.
(2) Ruckelshaus, W. D. 1972. Products containing the in-
secticide mirex; determination and order. Fed. Regist.
37(130): 13299-13300.
(3) Borthwick, P. W., T. W. Duke, A. J. Wilson, 1. 1. Lowe,
1. M. Patrick, Jr., and J. C. Oberheu. 1972. Accumula-
tion and movement of mirex .in selected estuaries of
South Carolina, 1969-1971. Pestic. Monit. J. 7(1): 6-26.
TABLE 1.—Whole body mirex residues, ppm, in estuarine animals, and sediments of South Carolina—June 1972
STATION LOCATION:
SPECIES STATION IDENTIFICATION
CRABS
CalUnectes sapldus (blue crab)
Uca pugilator (sand fiddler)
SHRIMPS
Penaeus aztecus (brown shrimp)
Palaemonetes pugio (grass shrimp)
FISHES
Leiostomus xanthurus (spot)
Bairdiella chrysura (silver perch)
Ictalurus catus (white catfish)
Cyprinus carpio (carp)
Lepomis macrochirus (bluegill)
MOLLUSKS
Crassostrea virginica (oyster)
Mercenaria mercenaria (hard
clam)
SEDIMENT
TOOGOODOO CREEK
A
i
—
—
—
B
—
—
—
—
C
—
—
—
—
—
—
D
—
—
—
—
RIVER-
LAND
TERRACE
POND
1
—
—
—
—
—
STONO
RIVER
2
—
—
—
—
UPPER 2
ASHLEY
RIVER
3
—
—
0.021
0.047
—
COOPER2
RIVER
4
0.026
—
—
—
0.12
—
—
LOWER
ASHLEY
RIVER
5
—
—
—
—
WANDO
RIVER
6
—
—
—
—
SOUTH
SANTEE
RIVER
7
—
—
—
—
1 — indicates <0.01 ppm mirex.
2 Freshwater stations.
VOL. 7, No. 3/4, MARCH 1974
145
-------
CONTRIBUTION NO. 169
-------
Short-term Effects of Organophosphate Pesticides on
Cholinesterases of Estuarine Fishes and Pink Shrimp
by
DAVID L. COPPACE and EDWARD MATTHEWS
U. S. Environmental Protection Agency
Gulf Breeze Environmental Research Laboratory
Sabine Island, Gulf Breeze, Fla. 32561
The esterase-inhlbiting insecticides (organophosphates and
carbamates) are now produced and enter the environment in greater
quantities than the chlorinated hydrocarbon insecticides (ANONY-
MOUS, 1972; ANONYMOUS, 1971). These pesticides act as nerve
poisons by blocking synaptic transmission in the cholinergic
parts of the nervous system (HEATH, 1961; KARCZMAR et al. 1970;
KOELLE, 1963; METCALF, 1971; O'BRIEN, 1967). The disruption of
nerve impulse transfers is caused by excessive accumulation of
the neurotransmitter acetylcholine (ACh) which is normally
broken down by the enzyme acetylcholinesterase (AChE, EC 3.1.
1.7 acetylcholine acetyl-hydrolase). The organophosphates and
carbamates bind to the active site of the AChE and prevent
breakdown of ACh (ALDRIDGE, 1971; FUKUTO, 1971; KOELLE, 1963;
METCALF, 1971). AChE inhibitors probably cause death in
higher vertebrates by blocking neurotransmission in the respira-
•tory center of the brain or neuromuscular junctions of the
respiratory apparatus (DeCANDOLE et al. 1953; HEATH, 1961;
KOELLE,. 1963), but this has not been confirmed for fish. In-
hibition of AChE is also believed to be the mode of action of
these pesticides on arthropods (HEATH, 1961; KOELLE, 1963;
O'BRIEN, 1960; O'BRIEN, 1967).
The possible hazards of AChE inhibiting pesticides in the
aquatic environment should not be ignored. Over one hundred
AChE inhibiting pesticides are produced and over 200 million
pounds are manufactured annually in the United States (CASIDA,
1964; ANONYMOUS, 1972; ANONYMOUS, 1971). Aquatic organisms
show a broad range of response to organophosphate pesticides,
depending on the compound, exposure time, water conditions, and
species (EISLER, 1970a). Short-term lethal concentrations in
water range from a few parts per trillion to several parts per
million (EISLER, 1970b; ANONYMOUS, 1963; ANONYMOUS, 1970).
Gulf Breeze Environmental Research Laboratory Contribution
No. 169
2
Associate Laboratory of the National Environmental Research
Center, Corvallis, Oregon
483
Bulletin of Environmental Contamination & Toxicology,
Vol. 11, No. 5 © 1974 by Springer-Verlag New York Inc.
-------
The organophosphate insecticides, with which we are concerned in
this report, generally degrade more rapidly in the environment
than do chlorinated hydrocarbon insecticides they are replacing.
But, their presence and effects in the environment may be greater
than expected because it may be necessary to apply the organo-
phosphates more frequently and in greater quantity to control
pests. The cholinesterases of vertebrates may remain inhibited
for several weeks after exposure because of irreversible in-
hibition by extremely small quantities of dealkylated oxygen-
analog metabolites of thiophosphates (COPPAGE and DUKE, 1972;
HEATH, 1961; KOELLE, 1963; MACEK et al. 1972; O'BRIEN, 1960).
Cumulative reduction of AChE by repetitive exposure has been
demonstrated in some vertebrates (HEATH, 1961; KOELLE, 1963),
and this may happen to fish subjected to similar repetitive ex-
posure in the environment (HOLLAND and LOWE, 1966; ANONYMOUS,
1965; WEISS, 1958).
Recent studies have indicated AChE measurements are probably
the best general index of organophosphate poisoning of fish in
the environment (COPPAGE, 1972; MACEK et al. 1972; COPPAGE and
DUKE, 1972). Also, if one considers the number of organophosphate
compounds and the difficulty in detecting their highly toxic
oxygen-analogs (McCULLY, 1972; PARDUE, 1971), AChE measurements in
animals from the environment are probably the best general indi-
cator of serious organophosphate pesticide pollution. The diffi-
cult task of detecting and interpreting residues alone in terms of
effects on organisms is eliminated by measuring AChE in animals
taken directly from the environment. A field study of three
species of estuarine fishes from an area sprayed with organophos-
phate pesticide showed brain AChE inhibition was correlated with
mosquito control operations with malathion (COPPAGE and DUKE,
1972). Brain AChE of fresh water fishes in ponds was also in-
hibited by application of Dursbany^(MACEK et al. 1972). In-
hibition of AChE in fish brains has been found below river out-
falls of pesticide plants (COPPAGE, unpublished data; WILLIAMS
and SOVA, 1966). Also, concentrations of malathion lethal to
commercial shrimp may exist during mosquito control operations
(CONTE and PARKER, 1971).
'Trademark: Dursban, Dow Chemical Co., Michigan. Mention of
commercial products does not constitute endorsement by the
U. S. Environmental Protection Agency.
484
-------
We need more information on the relationship of AChE in-
hibition to poisoning and deaths of estuarine animals to aid
in determining whether detrimental effects and "kills" in the
environment are caused by organophosphate pesticide exposure.
This report concerns AChE inhibitory effects of short-term
laboratory exposures of four species of estuarine fishes and a
commercial shrimp to lethal concentrations of malathion that may
be found in the environment (CONTE and PARKER, 1971). In
addition,. AChE inhibitory effects of lethal exposure to naled,
GuthionQy, and parathion are reported for two of the fish
species.
MATERIALS AND METHODS
Inhibition of AChE activity was used as an indicator of
poisoning in brains of spot (Leiostomus xanthurus; 65-150 mm
total length), pinfish (Lagodon rhomboides; 65-125 mm), Atlantic
croaker (Micropogon undulatus; 85-150 mm), and sheepshead minnows
(Cyprinodon variegatus; 45-70 mm), and in the ventral nerve cord
(VNC) of pink shrimp (Penaeus duorarum; 78-122 mm) . The acetyl-
choline hydrolyzing enzymes from fish brains were characterized
and assayed as previously described (COPPAGE, 1971). The assay
was carried out with a recording pH-stat at pH 7 and 22° C. We
mixed 2 ml of brain homogenate containing 5 mg of tissue per ml
with 2 ml of 0.03 M acetylcholine iodide and measured the acetic
acid liberated by enzymatic hydrolysis of ACh by titrating with
0.01 N NaOH. Shrimp VNC was assayed similarly, except tempera-
ture was 25° C and the homogenate contained 2 mg of VNC per ml.
AChE activity of both shrimp and fish was measured as micromoles
of ACh hydrolyzed per hour per mg of tissue in the reaction
vessel. Each AChE assay sample consisted of pooled organs from
4 to 6 animals that survived pesticide exposure at a designated
time.
In each test, 10 fish or shrimp were exposed in 3-5 replicates
to technical grade pesticide in 8-liter acrylic plastic aquaria
that received a mixture of flowing seawater (400 ml per minute)
and pesticide from a common source. The pesticide was dissolved
in acetone or benzene and infused into seawater by means of
syringe pumps. Solvent infusion never exceeded 2.5 parts per
million in the water and did not affect AChE activity. Pesti-
cide concentration in the water was expressed in theoretical
parts per billion (ppb), but was not verified by residue
analysis because our chosen criteria for toxic effects were only
death and AChE inhibition. In quadruplicate tests comparing
s
Trademark: Guthion, Chemagro Corp., Missouri.
485
-------
different solvent (acetone vs. benzene) carrying the same
quantity of malathion, there was no significant difference
(Student's t-test, P <0.05) in mortality or AChE inhibition.
Temperature range was 18-23° C and salinity was 23-29 parts
per thousand during the tests.
To determine the extent of AChE inhibition resulting from
a near median kill, we assayed survivors in tests in which 40-60
percent of the test population was killed. The shrimp assayed
had lost equilibrium (=moribund). Statistical comparisons of
AChE activities of exposed animals were made with unexposed
populations (Student's t-test, P <0.001).
RESULTS AND DISCUSSION
AChE inhibition was great in surviving fish and moribund
shrimp. Results of tests are summarized in Table 1.
TABLE 1.
AChE Inhibition in Fish and Shrimp by LC 40-60 of Organophos-
phates
Animal
Spot
Pinfish
Croaker
Sheepshead
Minnow
Pink Shrimp
(moribund)
Pesticide
Malathion
Naled
Guthion
Parathion
Malathion
Naled
Guthion
Parathion
Malathion
Malathion
Malathion
Theor-
etical
Cone.
(ppb)
1250
75
20
10
1000
75
10
10
1000
200
1000
Hours
Exposed
24
24
24
24
24
24
24
24
24
24
48
AChE Inhibition
Reduced (%) Significant
Mean
70
85
96
88
88
88
80
90
86
96
75
Range at t
65-82
82-89
93-98
87-89
87-89
88-88
77-84
88-92
79-90
90-99
72-82
0.001
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
486
-------
Relatively consistent levels of AChE inhibition occurred in
fishes even with different compounds and different species. The
survivors of populations of fish in which AO-60 percent were
killed by exposure to organophosphate pesticide had mean brain
AChE reductions of 70-96 percent (Table 1) . Mean AChE inhibitions
in fishes were near or exceeded the "lethal threshold" of about 82
percent reduction indicated in a previous study of sheepshead
minnows (COPPAGE, 1972), except inhibition of spot brain-AChE by
malathion, which differs by only 12 percent. These inhibitions
indicate that mean reductions in AChE activity of about 80 per-
cent are critical in short-term organophosphate poisoning of the
fishes tested and this may apply to fishes in general. Deaths may
occur even at mean inhibition values of 70 percent in some cases,
so the "lethal threshold" probably varies slightly among species.
These specific levels of reduction of AChE show that it is un-
necessary to rely on the dubious interpretation of residues alone
to determine poisoning and cause of "kills" in the environment.
Measurements of AChE activity and residue analysis or pesticide
usage data would be especially helpful in cause and effect studies.
Reduction of activity of ACh hydrolyzing enzymes in the VNC of
moribund shrimp was similar to that observed in fishes (Table 1).
The large reduction (75 percent) of enzyme activity in moribund
shrimp indicates that they too may be useful indicators.
REFERENCES
ALDRIDGE, W. N.: Bull. W. H. 0. 44, 25 (1971).
ANONYMOUS: U. S. Environ. Prot. Ag. Tech. Studies Report: TS-
00-72-04 (1972).
ANONYMOUS: U. S. Dep. Agric.: The Pesticide Review (1971).
ANONYMOUS: U. S. Fish Wildl. Serv. Circ. 167 (1963).
ANONYMOUS: U. S. Fish Wildl. Serv. Circ. 226 (1965).
ANONYMOUS: U. S. Bur. Sport Fish Wildl. Resour. Publ. 106 (1970).
CASIDA, J. E.: Science 164. 1011 (1964).
CONTE, F. S., and J. C. PARKER: Sea Grant Publ. No. TAMU-SG-71-
211 (1971).
COPPAGE, D. L.: Bull. Environ. Contain. Toxicol. £, 304 (1971).
COPPAGE, D. L.: Trans. Am. Fish. Soc. 101. 534 (1972).
487
-------
COPPAGE, D. L.: Unpublished data.
COPPAGE, D. L. and T. W. DUKE, Proc. 2nd Gulf Coast Conf. Mosq.
Suppr. Wildl. Manage. New Orleans, La., Oct. 20-22. 1971 (1972).
DeCANDOLE, C. A., W. W. DOUGLAS, K. E. V. SPENCER, R. W. TORRANCE,
and K. M. WILSON: Br. J. Pharmacol. 8_, 466 (1953).
EISLER, R.: U. S. Bur. Sport Fish. Wildl. Tech. Pap. 45 (1970a).
EISLER, R.: U. S. Bur. Sport Fish. Wildl. Tech. Pap. 46 (1970b).
FUKUTO, T. R.: Bull. W. H. 0. 44., 31 (1971).
HEATH, D. F.: Organophosphorous Poisons. Pergamon Press, N. Y.
(1961).
HOLLAND, H. T., and J. I. LOWE: Mosq. News 26_, 383 (1966).
KARCZMAR, A. G., S. NISHI, and L. C. BLABER: Acta Vitaminol.
Enzymol. 24-, 131 (1970).
KOELLE, G. B., ed.: Cholinesterases and Anticholinesterase Agents.
Springer-Verlag, Berlin. (1963).
MACEK, K. J., D. F. WALSH, J. W. HOGAN, and D. D. HOLTZ: Trans.
Am. Fish. Soc. 101, 420 (1972).
METCALF, R. L. : Bull. W. H. 0. 44^ 43 (1971).
McCULLY, K. A.: J. Assoc. Of. Anal. Chem. 55., 291 (1972).
O'BRIEN, R. D.: Toxic Phosphorus Esters. Academic Press, N. Y.
(1960).
O'BRIEN, R. D.: Insecticides. Academic Press, N. Y. (1967).
PARDUE, J. R. : J. Assoc. Of. Anal. Chem. 54_, 359 (1971).
WEISS, C. M.: Ecology 3£, 194 (1958).
WILLIAMS, A. K., and R. C. SOVA: Bull. Environ. Contam. Toxicol.
1, 198 (1966).
488
-------
CONTRIBUTION NO. 170
-------
Accumulation of Aroclor® 1254 in Grass Shrimp
(Palaemonetes pugio) in Laboratory and Field Exposures
by
D. R. NIMMO, J. FORESTER, P. T. HEITMULLER, and G. H. COOK
U. S. Environmental Protection Agency
Gulf Breeze Environmental Research Laboratory
Sabine Island, Gulf Breeze, Fla. 32581
Results of several experiments Indicate that aquatic Inverte-
brates accumulate total body concentrations of polychlorinated bi-
phenyls (PCB) thousands of times greater than that of the sur-
rounding water. For example, SANDERS and CHANDLER (1972) showed
that fresh water insects and crustaceans rapidly (1 day) accumu-
lated PCB (Aroclor 1254) up to 24,000 times greater than the
concentration in the water. Results of similar exposures con-
ducted with estuarine animals showed oysters concentrating 85,000
(LOWE et al. 1972), shrimp 10,000 (NIMMO et al. 1971a). and fish
30,000 (HANSEN et al. 1971) times the amount of PCB in the
water.
Although SANDERS and CHANDLER (1972) stated that PCBs
entering the aquatic environment are below concentrations acutely
toxic to invertebrates, we have noted that most of the accumu-
lation studies conducted thus far by the investigators cited in
the paragraph above have been at concentrations of 1.0 ug/£ and
above, i.e., concentrations demonstratively toxic to test animals.
Little is known about accumulation in marine invertebrates at
extremely low concentrations, and with one exception (NIMMO et al.
1971a), no one to our knowledge has placed PCB-free animals in a
natural environment known to have PCBs and followed accumulation
with time.
We report here the results of several experiments on chronic
toxicity of Aroclor 1254 to Palaemonetes pugio, an estuarine
grass shrimp, as well as concentration and loss of the compound
from the animals with time. We also exposed grass shrimp for up
to 3 months to Aroclor 1254-contaminated sediments in Escambia
Bay, near Pensacola, Florida.
METHODS AND MATERIALS
With one exception, all laboratory experiments were conducted
in 30-ml chambers supplied with flowing water from Santa Rosa
Gulf Breeze Environmental Research Laboratory Contribution
No. 170
2
Associate Laboratory of the National Environmental Research
Center, Corvallis, Oregon
303
-------
Sound. Three sets of 5 chambers each received test concentrations:
the fourth set was a control. Each chamber contained 4-10 shrimp,
the number depending on the size of the animals. Water flowed
continuously through each chamber at a rate of 1.0 1/hr. Aroclor
1254, dissolved in polyethylene glycol (mol. wt. 200), was metered
into each mixing tank with a syringe pump before the water entered
the test chamber. An equal amount of solvent was added to the
water flowing to controls. David J. Hansen of this laboratory,
found the sensitivity of a marine fish to Aroclor 1254 remained
unchanged when he varied concentrations of polyethylene glycol
used to deliver the toxicant (personal communication). The shrimp
were fed daily a commercial molly-rlake diet (<0.02 mg/kg organo-
chlorine compounds).
The experiment to determine the concentration and loss from
the tissues of f_. pugio was also conducted in a flowing-water
system. We constructed 18-liter aquaria with false floors of
nylon screen (1/4-inch mesh) to hold shrimp above the detritus
brought in with the water or produced by the animals. This modi-
fication was intended to prevent the animals from eating these
particles with adsorbed Aroclor 1254. Consequently, we assume
that shrimp obtained more of the chemical from the water by ab-
sorption through the gills rather than from ingestion of con-
taminated detritus. The shrimp were not fed during this experi-
ment.
Concentrations of Aroclor 1254 in tissues by gas chroma-
tography were determined using pooled samples of at least 10
shrimp each (NIMMO et al. 1971a).
P_. pugio were exposed to Aroclor 1254-contaminated sediments
in upper Escambia Bay from November 1971 to February 1972. The
shrimp in specially-constructed cages (HEITMULLER and NIMMO, 1972)
were exposed directly to the sediments. Average concentration of
Aroclor 1254 in the uppermost two inches of sediment in November
1971 was 5.0 mg/kg (dry weight).
RESULTS OF LABORATORY EXPOSURES
Tests conducted in flowing water showed P_. pugio to be sus-
ceptible to Aroclor 1254 (Table 1). In a 7-day exposure, 60%
died at 9.1 yg/£, but significant mortality did not occur at 0.17
and 0.62 jjg/£. In the second series of tests lasting 16 days,
4.0 and 12.5 yg/j. were toxic, but significant mortality did not
occur in l."3 yg/£.
At the conclusion of several one-week exposures to a range of
concentrations (0.17 to 9.1 yg/fc), surviving shrimp from each ex-
posure were analyzed for whole-body residues. Ambient concen-
tration of toxicant in the water and resultant residues in the
shrimp were correlated (r=0.91, Table 2). In some cases, dupli-
cate test concentrations produced biological accumulations that
differed by a factor of 2. Concentration factors ranged from
3,000 to 11,000. These ranges were similar to those found in
304
-------
TABLE 1. MORTALITY AMD ACCUMULATION OF AROCLOR 125U IN
Palaemonetes pugio*
Test Cone. Days Average Mortality Body Cone.
(yg/S,) Exposed (%)** (mg/kg)
Concentration
Factor
CONTROL 7 4(0-20) 0.1
0.17 7 8(0 - 40) 1.3
0.62 7 4(0 - 20) 5.4
9.1 7 60(20 - 80)*** 65.0
CONTROL 16 25(0 - 50) <0.1
1.3 16 40(0 - 100) 18.0
4.0 16 45(25 - 50)*** 27.0
12.5 16 55(50 - 75)*** 46.0
7600
8700
7100
14000
6700
3700
*A11 exposures were conducted in flowing seawater: salinity and
temperature ranges were 22 to 28X and 17 to 28° C.
**5 replicates per concentration: at least 4 shrimp per repli-
cation.
***Significant at P >0.05.
TABLE 2. ACCUMULATION OF AROCLOR 125^ BY Palaemonetes pugio*
Test Cone.
(yg/JO
0.17
0.62
1.0
2.3
2.7
3.2
3.2
5.2
5.3
5.3
9.1
Body Cone.
(mg/kg)
1.3
5.4
3.2
25.0
19.0
15.0
26.0
29.0
16.0
30.0
65.0
Concentration
Factor
7600
8700
3200
11000
7000
4800
8100
5600
3000
5700
7100
*7-day exposures conducted in flowing seawater at salinity and
temperature ranges of 22 to 28 and 17 to 28° C.
305
-------
TABLE 3.
AFTER EXPOSURES TO THE CHEMICAL IN WATER AT THREE CONCENTRATIONS
(Each value represents a composite sample of 10 animals)
Length of
Exposure
(hr/
0
1
2
3
4
8
12
16
24 /
36 /
48 /
72 /
96 /
154 /
336 /
504 /
672 /
840 /
1176 /
1512 /
days)
Control
0.04
Body Cone . Body
Cone. Factor Cone.
Cone.
Factor
(mg/kg) (mg/kg)
0.1 * 0.
1
1.5
2
3
4
6.5
14
21
28
35
49
63
_
_
-
-
_
-
-
-
-
-
0
-
0
0
0
0
0
* 0.
* 0.
* 0.
—
—
—
—
—
—
—
—
—
.1
—
.1
.14
.10
.1
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
1
1
1
1
1
1
1
1
1
1
1
1
1
1
13
15
17
21
PCB -
0.
.15 0.
1
1
*
A
*
*
*
*
*
*
*
*
*
*
*
1590
3250
3750
4250
5250
STOPPED
*
*
-------
tests using penaeid shrimp (NIMMO et al. 1971a), but were some-
what lower than those found by SANDERS and CHANDLER (1972), in
tests using several invertebrate species in fresh water.
There appeared to be no threshold below which levels of the
chemical added to water failed to produce residues in the tissues
(Table 3) in our tests. Whole-body concentrations produced after
5 weeks exposure to 0.04, 0.09 and 0.62 yg/j, ranged from 200 to
26,000 times the concentrations in the test water. Concentrations
did not reach equilibrium and from 60 to 90 percent of the Aroclor
1254 was lost from the shrimp within 4 weeks after exposure to the
chemical was stopped. Test concentrations of the chemical were
not significantly toxic to shrimp. Although accumulation
increased with increasing concentration of toxicant in this test,
this was not observed in earlier studies (see Tables 1 and 2).
Implications are to be discussed elsewhere.
RESULTS OF FIELD EXPOSURES
Average whole-body residue of Aroclor 1254 in P_. pugio after
1 month was 0.41 mg/kg (0.34 to 0.57); after 3 months, 0.42 mg/kg
(0.37 to 0.50). There was no evidence that significant mortality
occurred during the exposures of grass shrimp to contaminated
sediments.
DISCUSSION
Concentrations of Aroclor 1254 in P_. pugio. after exposure
to contaminated sediments for 3 months was equivalent to a
laboratory-exposure of 0.09 yg/jj, in water for 2 weeks (Table 3).
We expected residues to be higher in caged shrimp since we had
found that fiddler crabs exposed in the laboratory accumulated
residues equal to or greater than (wet-weight basis) that of the
contaminated substratum (dry-weight basis) after 30 days
(NIMMO et al. 1971b). Concentrations of Aroclor 1254 in caged
shrimp exposed to contaminated sediments appeared to reach a
plateau, but this was not the case in laboratory exposures
(Table 3) where an equilibrium was not reached. Therefore,
we believe that shrimp exposed to the sediments might have
obtained PCB from the water or food singly, but shrimp exposed to
Aroclor in the laboratory obtained chemical from two sources,
water and food. It might also be more available in the laboratory
than the field due to the carrier. In earlier laboratory studies
with penaeid shrimp, both water and food appeared to be sources
(NIMMO et al. 1971a).
No significant mortality was observed in caged shrimp and
none would be predicted since residues produced in the field were
similar to those found in shrimp after laboratory exposures which
caused no death.
Penaeid shrimp spend only a fraction of their life cycle in
an estuary, moving into oceanic waters after reaching maturity
307
-------
(PEREZ FARFANTE 1969), but grass shrimp are endemic in estuaries.
Therefore, in relation to time of exposure we would expect grass
shrimp to accumulate a pollutant from a contaminated estuary to a
greater degree than penaeid shrimps, nevertheless, this is not
true. In August 1968, penaeid shrimps (Penaeus duorarum, P_.
setiferus, and P_. aztecus) collected during a survey of Escambia
Bay, Florida, had whole-body residues of Aroclor 1254 as high as
14.0 mg/kg (NIMMO et al. 1971b). In that survey and in subsequent
collections, P_. pugio had a maximum residue of only 1.4 mg/kg.
Lower residues in P_. pugio from Escambia Bay may be due to
amounts of PCB in bay sediments and behaviorial patterns of the
animals. We noted earlier (NIMMO et al. 1971a) in species of
penaeid shrimp were related to higher concentrations of Aroclor
1254 in sediments that predominate in upper Escambia Bay. We
found that penaeid shrimp, as adults, usually are captured in
deeper waters and burrow into silty or sandy substrates. In
contrast, grass shrimp usually do not burrow, rather are found
along shallow sandy beaches and grass beds, where they obtain
food that is relatively uncontaminated with PCB.
REFERENCES
HANSEN, D. J., P. R. PARRISH, J. I. LOWE, A. J. WILSON, JR., and
P. D. WILSON: Bull. Environ. Contam. and Toxicol., 6_, 113 (1971),
HEITMULLER, P. T., and D. R. NIMMO: Prog. Fish-Cult., 34_, 120
(1972).
LOWE, J. I., P. R. PARRISH, J. M. PATRICK, and J. FORESTER: Mar.
Biol. (Berl.), 17_: 209 (1972).
NIMMO, D. R., R. R. BLACKMAN, A. J. WILSON, JR., and J. FORESTER:
Mar. Biol. (Berl.), 11_, 191 (1971a).
NIMMO, D. R., P. D. WILSON, R. R. BLACKMAN, and A. J. WILSON, JR.
Nature, 231, 50 (1971b).
PEREZ FARFANTE, I.: U. S. Fish Wildl. Serv., Fish. Bull., 67_,
461 (1969).
SANDERS, H. 0., and J. H. CHANDLER: Bull. Environ. Contam. and
Toxicol., 7, 257 (1972).
308
-------
CONTRIBUTION NO. 172
-------
ENVIRONMENTAL RESEARCH 7, 363-373 (1974)
Aroclor 1016: Toxicity to and Uptake by Estuarine Animals1-2
D. J. HANSEN, P. R. PAEBISH, AND J. FORESTER
U. S. Environmental Protection Agency, Gulf Breeze Environmental Research Laboratory,
Sabine Island, Gulf Breeze, Florida 32561 (Associate Laboratory of the National
Environmental Research Center, Corvallis, Oregon)
Received June 24, 1973
Bioassays were conducted to determine the acute toxicities of the polychlorinated
biphenyl (PCB) Aroclor 1016 in flowing sea water to American oysters (Crassostrea
virginica), brown shrimp (Penaeus aztecus), grass shrimp (Palaemonetes pugio),
and pinfish (Lagodon rhomboides), and to determine its chronic toxicity to, and
uptake and retention by pinfish. Acute 96-hour ECSO's or LCSO's were: oysters, 10.2
/i/liter; brown shrimp, 10.5 /ig/liter; grass shrimp, 12.5 /ig/liter. The PCB was not
toxic to pinfish at 100 /ig/liter for 96 hours, but significant mortality occurred when
pinfish were exposed to 32 /ig/liter of Aroclor 1016 for 42 days. Pinfish exposed to 1
/ig/liter for 56 days accumulated the chemical with maximum concentrations attained
in whole-fish by 21 to 28 days. Maximum whole-body residue (wet weight) was
17,000 X the nominal concentration in test water. Tissue alterations, such as severe
vacuolation in the pancreatic exocrine tissue surrounding the portal veins, occurred
in pinfish exposed to 32 /ig/liter of Aroclor 1016 for 42 days.
Polychlorinated biphenyls (PCB's) have been used industrially for over 40
years (Broadhurst, 1972) and recently there has been concern about their en-
vironmental impact. Because of this concern, manufacture and sale of most PCB's
were discontinued, and sales restricted to uses that are not likely to produce
environmental contamination. A new PCB, Aroclor 1016, is now manufactured
in the United States for sale to capacitor manufacturers as a substitute for all
other PCB's. This new PCB is similar to Aroclor 1242^;except that amounts of
isomers containing 5 or more chlorine atoms per bfphenyl group have been
considerably reduced. Domestic sales of Aroclor 1016 increased from about 3.3 X
106 pounds in 1971 to 20.9 X 106 pounds in 1972 (W. B. Papageorge, personal
communication3).
Our study was conducted to determine the acute toxicity of Aroclor 1016 to
the American oyster (Crassostrea virginica), brown shrimp (Penaeus aztecus),
grass shrimp (Palaemonetes pugio), and pinfish (Lagodon rhomboides) and to
determine its chronic toxicity to, and uptake and retention by pinfish.
1 Aroclor is a registered trademark, Monsanto Company, St. Louis, MO. Mention of com-
mercial products or trade names does not constitute endorsement by the Environmental
Protection Agency.
* Contribution No. 172, Gulf Breeze Environmental Research Laboratory.
' W. P. Papageorge, Monsanto Industrial Chemicals Company. 800 N. Lindbergh Boulevard,
St. Louis, MO 63166.
363
Copyright © 1974 by Academic Press, Inc.
All rights of reproduction in any form reserved.
-------
364 HANSEN, PABBISH AND FORESTER
MATERIALS AND METHODS
Test Animals
Test animals were collected near the Gulf Breeze Laboratory and acclimated
to laboratory conditions for at least 7 days before exposure. If mortality exceeded
1% in the 48 hours immediately preceding the test, or if abnormal behavior was
observed during acclimation, the animals were not used. Oysters tested were 35-
55 mm in height; brown shrimp, 11-26 mm rostrum-telson length; grass shrimp,
20-32 mm rostrum-telson length; and pinfisb 27-84 mm standard length. Animals
were not fed during acute toxicity tests but they could obtain plankton from the
unfiltered sea water. In the chronic exposures and the uptake and retention test,
pinfish were fed commercial fish food that contained no detectable PCB (<0.2
/•g/g)-
Acute and Chronic Tests
Acute toxicity to Aroclor 1016 was determined by exposing 10 individual
animals to 1, 10, or 100 /ig/liter for 96 hours in each of two 20 liter aquaria. Each
experiment was conducted twice. The PCB was dissolved in acetone or poly-
ethylene glycol 200 and metered at 30 or 0.1 ml/hour, respectively, into unfiltered
sea water that entered each aquarium at 75 liters/hour. Two control aquaria
received the same quantities of water and solvent. Temperature and salinity of
the water flowing into aquaria in replicated tests were similar (±10%).
Chronic toxicity of Aroclor 1016 to pinfish was determined in 3 experiments,
each lasting 42 days. In each experiment, 50 fish were placed in each 90 liter
aquarium that received 140 liters/hour of sea water. The PCB, dissolved in poly-
ethylene glycol, was metered into the water at 0.083 ml/hour in the first two
experiments, and 1.04 ml/hour in the third experiment. Control aquaria received
the same quantity of water and solvent.
The same exposure techniques that were used in the first two chronic toxicity
experiments were used to determine: (1) the rate of uptake and retention of
Aroclor 1016 in pinfish exposed to 1 fig/liter for 56 days and (2) the rate of
depuration of Aroclor 1016 by pinfish in PCB-free water for 56 days.
Effect of Aroclor 1016 was assessed by measuring percentage reduction in
shell growth of exposed oysters as compared to control oysters (Butler, 1962),
by determining mortality in shrimps and fish, and by pathological examination
of chronically exposed fish.
Histopathological Examination
Dr. J. A. Couch, pathobiologist at this laboratory, examined viscera from live
pinfish from the third 42-day exposure. Viscera were fixed either in 10% neutral
buffered formalin or in Davidson's fixative. Those fixed in Davidson's were stored
in 70% ethyl alcohol until processed for paraffin sections (7 /tm) and stained with
Harris hematoxylin and eosin (HHE) or Periodic Acid SchifFs (PAS). Viscera
fixed in 10% neutral buffered formalin were processed for frozen sections (12
and stained with oil Red O and hematoxylin.
-------
AHOCLOH 1016: TOXICTTY TO ESTUABINE ANIMALS 365
Chemical Analyses
Concentrations of Aroclor 1016 in water and animals were determined by
electron capture gas chromatography. Unaltered water samples from each con-
centration were analyzed once during the 96-hour exposures, and weekly during
longer exposures. Concentrations in animals that survived the 96-hour exposures
were determined as whole-body residues. At the conclusion of each chronic
exposure, surviving pinfish were dissected and PCB residues in flesh, flesh and
scaleless skin, and remaining tissue determined. Residues in all tissues were sum-
med to compute concentrations of Aroclor 1016 in whole fish. The same procedure
was followed in the uptake and retention study, except fish were removed for
analysis at selected intervals during exposure and depuration. Also, at the end
of the 56 day exposure, brain, gills, heart, and liver were removed from exposed
fish for residue analysis. All fish samples were composites of 10 individuals.
Tissue samples that weighed more than 5 g were prepared for analysis by
mixing them with anhydrous sodium sulfate in a blender. The mixture was ex-
tracted for 4 hours with petroleum ether in a Soxhlet apparatus. Extracts were
concentrated to approximately 10 ml and transferred in 3-4-ml portions to a 400 X
20 mm chromatographic column that contained 76 ml of unactivated Florisil. After
each portion settled in the column, vacuum was applied until all solvent was
evaporated. This was repeated with three 5-ml rinses. The residue was eluted
from the column with 70 ml of a 9:1 mixture (v/v) of acetonitrile and distilled
water. The eluate was evaporated to dryness and the residue transferred to a
Florisil column (Mills et al., 1963) with petroleum ether. Aroclor 1016 was eluted
with 6% ethyl ether in petroleum ether.
Tissue samples that weighed less than 5 g were analyzed by a modification
of the micromethod described in the Pesticide Analytical Manual, Volume III
(U. S. Food and Drug Administration, 1970). The samples were weighed into a
size 23 Duall tissue grinder and extracted 3 times with 5-ml portions of aceto-
nitrile. The acetonitrile was flooded with 15 ml of 2% (w/v) sodium sulfate in
distilled water and extracted with three 5-ml portions of hexane. The hexane was
evaporated to approximately 1 ml and transferred to a 9 X 200 mm Chromaflex
column with a 59 ml reservoir that contained 3.3 g of Florisil topped with 3.3 g
of anhydrous sodium sulfate. Aroclor 1016 was eluted with 20 ml of 5% ethyl ether
in hexane and adjusted to an appropriate volume for analysis.
Water samples were extracted with petroleum ether, the extracts dried with
anhydrous sodium sulfate, and evaporated to approximately 1 ml. The concen-
trates were transferred to a size 7 Chromaflex column containing 1.6 g Florisil
topped with 1.6 g anhydrous sodium sulfate. Aroclor 1016 was eluted with 20 ml
of 1% ethyl ether in hexane and the eluates were adjusted to an appropriate
volume for analysis.
All samples were analyzed by electron capture gas chromatography using a
15 X 3.2 mm glass column packed with 2% OV-101 on 100-120 Gas Chrom Q.
Nitrogen flow rate was 25 ml/min, the oven temperature was 190 °C, and the
injector and detector temperature was 210° C.
Aroclor 1016 was quantitated by comparing the total height of all peaks in the
-------
366
HANSEN, PARRISH AND FORESTER
sample with the total height of all peaks in a standard of known concentration.
Recoveries were greater than 80%; data were not adjusted for recovery. All tissue
residues were determined on a wet-weight basis.
RESULTS AND DISCUSSION
Acute (96-hr) Exposure
Aroclor 1016 was acutely toxic to the estuarine organisms tested (Tables 1 and
2). Shell growth in oysters was inhibited greatly by exposure to 100 jug/liter for
96 hours. Sensitivities of brown shrimp and grass shrimp were similar, and pin-
fish was the least sensitive species. Acute toxicities of Aroclor 1016 to oysters,
brown shrimp, and pinfish were similar to that of Aroclor 1242 to these species
(P. R. Parrish, unpublished data), and Aroclor 1254 to oysters, pink shrimp
(Penaeus duorarum), and pinfish (Duke et al., 1970).
All animals accumulated Aroclor 1016 (Table 1). The quantities accumulated
depended on the exposure concentrations and not on species. Whole-body con-
centrations in live animals ranged from 440 to 4200 X the nominal concentration
in test water and 1200 to 6700 X the measured concentration in test water.
TABLE 1
ACUTE TOXICITY TO AND UPTAKE OF AROCLOR 1016 BY AMERICAN OYSTERS
(Crassostrea virginica), BROWN SHRIMP (Penaeus aztecus), GRASS SHRIMP
(Palaemonetes pugio), AND PINFISH (Lagodon rhomboides)
IN 96-HouR EXPOSURES"
Species
Test concentration (/^g/liter)
Nominal
Measured
Effect
Whole-body residue
g, wet weight)
C. virginica
P. aztecus
P. pugio
L. rhomboides
Control
1
10
100
Control
1
10
100
Control
1
10
100
Control
1
10
100
ND'
0.6
7.2
58
ND
0.9
8.9
33
ND
0.4
9.4
38
ND
0.8
6.9
56
0
10
38
93
0
8
43
100
8
33
38
93
2
5
0
18
ND'
4.0
32
95
ND
3.8
42
—
ND
1.1
22
44
ND
2.2
21
65
" Effect is expressed as percent reduction in shell growth in oysters and death in shrimps and
fish. Whole body residues are from animals alive at end of exposure period.
6 ND, not detectable: <0.2 pg/liter in water; <0.2 Mg/g in tissue.
-------
AHOCLOE 1016: TOXICTTY TO ESTUARINE ANIMALS
367
TABLE 2
ACUTE TOXICITY OP AROCLOR 1016 TO AMERICAN OYSTERS (Crassostrea virginica),
BROWN SHRIMP (Penaeus azlecus), AND GRASS SHRIMP (Palaemonetes pugio)"
96-hour LC50 (/ig/liter)
Temperature (C)
Salinity (0/00)
Species
Nominal
Mean
Range
Mean
Range
C. virginica
P. aztecus
P. pugio
10.2
10.5
12.5
30
31
30
25-32
29-32
29-32
29
29
29
26-31
28-30
25-30
EC50:concentration expected to cause a 50 percent reduction in shell growth in oysters.
Chronic (42-day) Exposure
Toxicity of Aroclor 1016 to juvenile pinfish was greater in tests lasting 6 weeks
than in 96-hour exposures. Pinfish seemed unaffected by 10 /xg/liter or less, but
died in concentrations of 32 and 100 /xg/liter (Table 3). Mortality began in the
second week of exposure. Fifty percent mortality did not occur in the third ex-
posure, for example, until the 33rd day at 32 ,ug/ liter and the 18th day at 100 /tg/
liter. Delayed mortality of pinfish was also observed with Aroclor 1254 (Hansen
etal, 1971).
Most of the fish that died in the 42 day exposure exhibited symptoms of poison-
ing, such as changed appearance and behavior. Initially, their color darkened,
they stopped feeding, and they swam erratically with bodies inclined downward.
TABLE 3
TOXICITY AND UPTAKE OF AHOCLOK 1016 BY PINFISH (Lagodon rhomboides) EXPOSED
FOR 42 DAYS IN THREE SEPARATE EXPERIMENTS
Test concentration (pg/liter)
Mortality
Nominal Measured (%)
Concentration in fish (Mg/g, wet weight)
Flesh
Flesh and skin Whole fish
Control
0.1
1.0
10.0
Control
1.0
3.2
10.0
32.0
Control
10.0
32. 0'
100.0=
ND°
ND
0.8
3.0
ND
0.9
2.5
7.0
13
ND
6.8
21
59
36
16
48
38
12
16
16
28
445
6
6
50*
504
ND
0.7
5.1
60
0.5
4.0
34
63
140
ND
23
30
38
ND
0.8
6.3
90
0.6
6.0
39
76
180
ND
49
48
72
ND
2.4
11
166
0.5
17
65
170
620
ND
111
106
205
" ND, not detectable: <0.2 Mg/h'ter in water; <0.2 Mg/g in tissue.
* Mortality significantly greater than in control fish, a = 0.01.
c Exposure terminated and tissues analyzed when 50% of the fish died: 33 days at 32 jig/liter
and 18 days at 100
-------
368
HANSEN, PARRISH AND FORESTER
Difficulty in swimming progressed until the fish swam with their tails and dorsal
fins breaking the water surface. Finally, the fish lost equilibrium, swam upside
down, and died. Affected fish became vulnerable to attack by other pinfish in
the tank. In the third experiment, the majority of dying fish exposed to 32 and 100
/ig/liter lost scales, skin and, finally, flesh in front of the dorsal fin. This formed
lesions sometimes as deep as the neural spine. This condition did not occur in fish
exposed to 32 jug/liter in the second experiment.
Hepatocytes in liver sections of 13 control fish showed no unusual characteristics
when stained with HHE and PAS or oil Red O. The hepatocytes from 6 fish
demonstrated only a moderate PAS-positive reaction, indicating moderate to light
glycogen reserves. In 7 other control fish, liver sections stained with oil Red O
also showed a broad range of lipid patterns. Structure of livers of control fish
was normal, being tubulosinusoidal in nature, with disseminated pancreas prom-
inent along the course of the portal vein (Fig. 1).
Eight fish exposed to 10 ^ig/liter of Aroclor 1016 for 42 days showed no
pathologic microscopic visceral characteristics distinguishable from control fish.
Half of the fish samples were paraffin-processed; half were prepared for frozen
sections.
Pinfish exposed to 32 ^.g/liter of Aroclor 1016 had several liver and pancreatic
alterations that distinguished them from control fish and fish exposed to 10 /ig/
liter. Tissues from 8 fish were examined; half were paraffin-processed and half
• j» ,scr *
Vfc^l* f \ *
-T^MPfct .*. vH, * •
a^ss^
FIG. 1. Section of normal liver from control pinfish. The vacuoles in hepatocytes are
results of extraction of lipid and/or glycogen during paraffin processing. Note the normal
pancreatic exocrine tissue containing normal secretory deposite (arrows). ( X450).
-------
AROCLOR 1016: TOXICITY TO ESTUABINE ANIMALS
369
rt»
W +
I^SiPfc*-*™
ffifr^Msf*
Jt v* *. i/>-s^':'
* ^ffiSs^
a.; 'r.jNrariSf^ai; £
««r ^.^,
FIG. 2. Section of liver from pinfish exposed to 32 /ig/liter Aroclor 1016 for 42 days. The
hepatocytes appear relatively dense with more prominent nuclei than in Fig. 1. Note Ihr lack
of lipid or glycogen vacuoles in hepatocytes. Small, abnormal vacuoles in pancreatic exocrine
tissue (arrows) distinguish fish exposed to 32 /ig/liter fiom control fish. (X-150).
were studied as frozen sections. Hepatocytes appeared slightly enlarged and more
basophilic than in the control fish (Fig. 2). Normal liver cord orientation was
somewhat altered and PAS-positive granules accumulated at the edge of the
pancreatic acinar tissue in the liver (Fig. 3). Less lipid material existed in the
livers of fish exposed to 32 jug/liter than in control iish. This contrasts with the
heavy, abnormal accumulation of lipid in the livers of spot exposed to 6 /ig/liter
of Aroclor 1254 for 30 days (Couch, 1973). The most remarkable alteration in the
pinfish was the occurrence of severe vacuolation in the pancreatic exocrine tissue
surrounding the portal veins (Figs. 2 and 3). This vacuolation was distinguishable
from normal secretory vacuoles and deposits in pancreatic tissue from control
fish because those in exposed fish were small, abundant and contained no
secretory granules (Fig. 1).
After the first exposure, 16 pinfish from each control or Aroclor 1016-con-
taminated aquarium were held in PCB-free water, and -the salinity reduced to
determine if ability of pinfish to survive osmotic stress had been impaired by
exposure to sublethal concentrations of Aroclor 1016. (Aroclor 1254 reduced the
ability of pink shrimp (Penaeus duorarum) to withstand simss of decreased
salinity, D. R. Nimmo. personal communication.4) Salinity was '•• -wered from an
D. R. Nimmo, Gulf Breeze Environmental Research. Laboratory, Gulf Breeze, Florida
32561.
-------
370
HANSEN, PARR1SH AND FORESTER
Fio. 3. Pigment deposition (PAS-positive) between pancreatic exocrine tissue and liver
parenchyma (arrow). Note nature of small vacuoles in pancreatic tissue. This tissue is from
fish exposed to 32 MR/Hter Aroclor 1016. ( x 1000).
initial 23 %„ by 50% on each of four consecutive days (14, 7, 3.5 and 1.8 %0). None
of the fish died until the salinity fell below 2 %0. At that salinity, mortalities of
control and PCB-exposed fish were similar.
Pinfish exposed for 42 days to concentrations of Aroclor 1016 between 0.1 and
32 fig/liter stored the chemical in proportion to the concentration in test water
(Table 3). Concentrations in whole fish ranged from 11,000 to 24,000 X the
nominal concentration in the test water and 14,000 to 55,000 X the measured
concentration in test water, whereas the concentration factor in spot (Leiostomus
xanthurus] exposed for 42 days to 1 /ig/liter of Aroclor 1254 was 30,000 X the
nominal concentration (Hansen et al., 1971).
The concentration of Aroclor 1016 in edible tissue was less than that in whole
fish. Concentrations in whole fish averaged 2.1 times those in flesh and scaleless
skin and 2.8 times those in flesh. Pinfish exposed to 1 //g/liter of Aroclor 1016
stored quantities that exceeded the Food and Drug Administration's provisional
action-level for all PCB's (5 «g/g) in edible tissues (Table 3).
Chronic (56-day) Exposure
Pinfish exposed to 1 /ig/liter of Aroclor 1016 for 56 days accumulated the
chemical, maximum concentrations in whole fish being attained in 21-28 days
(Table 4). In a similar study with Aroclor 12.54, whole-body concentrations of spot
stabilized at about the same time, 14-28 days (Hansen et a]., 1971). Maximum
-------
AROCLOR 1016: TOXICITY TO ESTUAHINE ANIMALS
371
TABLE 4
CONCENTRATIONS OF AROCLOR 1016 UG/G WET WEIGHT) IN PINFISH (Lagodon rhomboides)
EXPOSED TO 1 MG/LITER OP THIS PCB (EACH SAMPLE CONSISTED
OF TISSUES FROM 10 FISH)
Concentration
Days of exposure
0
3
7
14
21
28
42
56
Depuration
14
28
56
Flesh
ND"
0.8
1.6
2.3
3.2
3.0
4.0
5.9
4.1
3.5
2.2
Flesh and skin
ND
0.9
3.4
3.3
4.3
6.8
6.0
8.3
7.8
6.6
3.9
Whole fish
ND
1.6
3.9
6.5
9.7
25
17
17
13.5
9.3
6.6
0 ND, not detectable; <0.1 /*g/g.
whole-body residue in pinfish was 17,000 X the nominal concentration in test
water. Increases in concentrations in edible tissues were also rapid (Table 4),
but may not have reached a maximum even after 56 days of exposure.
The quantity of Aroclor 1016 accumulated by pinfish differed in the various
tissues and organs. Fish exposed to 1 /xg/liter for 8 weeks accumulated 17 /xg/g
whole-body residue. Concentrations (/tg/g) in other tissues or organs were:
gills, 23; skin, 19; liver, 16; brain, 8.7; muscle, 5.9; and remaining tissues, 22.
Aroclor 1016 was lost from the tissues after pinfish were placed in PCB-free
water (Table 4). After 56 days of depuration, concentrations in whole fish
decreased by 51%. In an earlier study, spot which had accumulated Aroclor 1254
lost 66% after 56 days in PCB-free water (Hansen et al, 1971).
We examined chromatograms to compare the proportions of 9 peaks of Aroclor
1016 in reference standards (Fig. 4) with those peaks from water and pinfish
tissue samples (Table 5). Chromatograms of standards, tissue spikes, and water
samples were similar, while chromatograms of standards and tissue samples were
dissimilar. Chromatograms of Aroclor 1016 from different pinfish tissues (flesh,
flesh and skin, and rest) were similar. Chromatograms from all tissues had
smaller early eluting peaks, numbers 1, 2, and 3. Reduction in early eluting peaks
of Aroclor 1254 from shrimp and fish tissues was noted by Nimmo et al. (1971).
The relative proportions of the 9 peaks found in chromatograms from pinfish
tissues early in the 56-day exposure, late in the exposure, and throughout the 56-
day depuration period were similar. We do not know whether differences between
chromatograms from reference standards and chromatograms from tissue samples
reflect alterations in Aroclor 1016 molecules, differential solubility, or other factors.
It is unlikely, however, that the PCB molecules were altered, because no change
in relative proportions of peaks was noted throughout the 112 day experiment.
-------
372
HANSEN, PARRISH AND FORESTER
FIG. 4. Chromatogram of 9 peaks of Aroclor 1016 reference standard. Operating con-
ditions: gas flow nitrogen 25 ml/min; injection and detector temperature 210°C; oven tem-
perature 190°C; SH electron capture detector; 152.4 X 0.32 cm glass column packed with 2%
OV-101 on 100-120 Gas Chrom Q.
TABLE 5
PERCENTAGES OF THE 9 MEASURED PEAKS FROM CHROMATOGRAMS OF AROCLOR 1016
REFERENCE STANDARDS AND FROM TISSUES OF PINFISH EXPOSED TO 1 ^G/LITER OF
AROCLOR 1016 FOR 56 DAYS AND THEN HELD IN PCB-FREE WATER FOR 56 DAYS
Percentage of peak"
Chro-
mato-
9 grams
Reference standard 12.4 11.3 10.0 23.5 13.3 6.4 8.1 8.8 6.2 8
All tissues through 14 days 2.4 5.3 1.8 39.6 7.7 10.3 12.1 9.4 11.4 7
of exposure
All tissues, days 21-56 of 2.5 5.0 2.1 33.2 7.8 11.9 11.3 10.3 15.9 12
exposure
All tissues during 56 day 1.0 1.7 0.3 38.2 2.8 15.0 12.4 11.9 16.7 7
depuration
peak height
- Determined as — , . r~ X 100.
sum of nine peaks
-------
AHOCLOR 1016: TOXICITY TO ESTAURINE ANIMALS 373
The potential environmental hazard of a chemical is dependent upon its likeli-
hood of entering the environment and its potential hazard to organisms. Aroclor
1016 is similar to other PCB's in its toxicity to, and uptake and retention by
estuarine animals. Therefore, its substitution for other PCB's reduces environ-
mental hazard only if the policy of restricting sales to uses not likely to produce
environmental contamination is continued.
ACKNOWLEDGMENTS
We thank Dr. ]. A. Couch for histopathological examination of the pinfish, S. S. Foss for
preparing illustrations, and the Monsanto Company for providing Aroclor 1016.
REFERENCES
BHOADHURST, M. G. (1972). Use and replaceability of PCB's. Environ. Health Perspect.
2, 81-102.
BUTLER, P. A. (1962). Reaction of some estuarine mollusks to environmental factors. In
"Biological Problems in Water Pollution" Third Seminar. U. S. Dept. of Health, Education
and Welfare, Public Health Serv. Publ. No. 999-WP-25, 1965: 92-104.
COUCH, J. A. (1973). Pathologic effects of pesticides and related chemicals on the livers
of fishes. Proc. Fish. Path. Symp. AFIP. Univ. Wis. Press. In press.
DUKE, T. W., LOWE, J. I., AND WILSON, A. J., JR. (1970). A polychlorinated biphenyl
(Aroclor 1254®) in the water, sediment and biota of Escambia Bay, Florida. Bull. Environ.
Contam. Toxicol. 5, 171-180.
HANSEN, D. J., PARRISH, P. R., LOWE, J. I., WILSON, A. J., JR., AND WILSON, P. D. (1971).
Chronic toxicity, uptake and retention of Aroclor® 1254 in two estuarine fishes. Bull. Environ.
Contam. Toxicol. G, 113-119.
NIMMO, D. R., BLACKMAN, R. R., WILSON, A. J., JR., AND FORESTER, J. (1971). Toxicity
and distribution of Aroclor® 1254 in pink shrimp, Penaeus duorarum. Mar. B:ol. (Berlin)
11, 190-197.
MILLS, P. A., ONLEY, J. F., AND GAITHER, R. A. (1963). Rapid method for chlorinated
pesticide residues in nonfatty foods. /. Assoc. Agric. Chem. 46, 186-191.
U. S. FOOD AND DRUG ADMINISTRATION. (1970). "Pesticide Analytical Manual." U. S. Dept.
of Health, Education and Welfare.
-------
CONTRIBUTION NO. 174
-------
AROCLOR® 1254, DDT AND DDD, AND
DIELDRIN: ACCUMULATION AND LOSS BY
AMERICAN OYSTERS (CRASSOSTREA
VIRGINICA)
EXPOSED CONTINUOUSLY FOR 56 WEEKS l
Patrick R. Parrish
U.S. Environmental Protection Agency
Gulf Breeze Environmental Research Laboratory
Sabine Island, Gulf Breeze, Florida
Separate populations of oysters were exposed
continuously for 56 weeks to 0.01 jug/1 of
Aroclor® 1254, p,p' -DDT and DDD, or dieldrin
and sampled at 8-week intervals for residues.
Maximum concentrations based on body weight
(pig/g) occurred after 8 weeks of exposure, but
maximum concentrations based on absolute
amount of toxicant accumulated (jag) occurred af-
ter 56 weeks of exposure. After 8 weeks, average
whole-body residues (wet weight) from five
oysters analyzed individually were: Aroclor 1254,
1.65 Atg/g, 4.6 /ig; DDT (and metabolites DDD and
DDE), 0.46 Mg/g, 1.0 ug; and dieldrin, 0.08 fig/g,
0.2 ng. After 56 weeks, residues were: Aroclor
1254, 0.89 /jg/g, 25.7 ng; DDT and metabolites,
0.37 /ug/g, 7.0 pg; and dieldrin, 0.03 /ug/g, 0.6 pg.
Seasonal patterns of accumulation and loss of the
three toxicants were similar. Residues based on
body weight (Mg/g) decreased 45%-81% in early
July and late October, apparently as the result of
spawning, and increased following these periods.
This shows that the life history of oysters must
be considered when evaluating residue data from
monitoring programs. Growth rate (height and
in-water weight) of exposed oysters was not dif-
ferent from that of control oysters (Student's t-
test; o = 0.01) Mortality was not significant in
any group.
1 Contribution No. 174, Gulf Breeze Environmental Research
Laboratory.
®
Registered trademark, Monsanto Company, St. Louis,
MO. Mention of commercial products or trade names does
not constitute endorsement by the Environmental Protec-
tion Agency.
Proc. National Shellfisheries Assoc.
64: 7. (1974).
-------
CONTRIBUTION NO. 175
-------
Effects of Aroclor® 1254 on Laboratory-Reared Embryos
and Fry of Sheepshead Minnows (Cyprinodon variegatus)
STEVEN C. SCHIMMEL, DAVID J. HANSEN AND JERROLD FORESTER
Made in United States of America
Reprinted from TRANSACTIONS OF THE AMERICAN FISHERIES SOCIETY
Vol. 103, No. 3, July 1974
pp. 582-586
-------
Effects of Aroclor® 1254 on Laboratory-Reared, Embryos
and Fry of Sheepshead Minnows (Cyprinodon variegatus)1
STEVEN C. SCHIMMEL, DAVID J. HANSEN AND JERROLD FORESTER
U. S. Environmental Protection Agency
Gulf Breeze Environmental Research Laboratory
Sabine Island, Gulf Breeze, Florida 32561
(Associate Laboratory of the National Environmental Research Center, Corvallis, Oregon)
ABSTRACT
Eggs of the sheepshead minnow (Cyprinodon variegatus) were artificially fertilized and main-
tained at temperatures from 15 to 35 C and in salinities from 0 to 35%0 to determine efficient
culture conditions. Fertilization was not affected by temperature or salinity ranges chosen, but
hatching success was greatest (x2; o = 0.01) at a temperature range of 24 to 35 C and a salinity
range of 15 to 30%».
Artificially fertilized sheepshead minnow eggs were exposed to logarithmic concentrations of
Aroclor 1254 (10.0 to 0.1 /xg/liter) in seawater averaging 30 C and 24%0 in a flow-through
bioassay. Fertilization was not affected but significantly fewer embryos developed in the 10.0
,ug/liter concentration, and fewer fry survived in concentrations greater than 0.1 /ig/liter. Fry
were more susceptible to Aroclor 1254 than were embryos, juveniles, or adults.
Polychlorinated biphenyls (PCB's) occur in
estuaries in many states (Butler 1973), and the
occurrence of one, Aroclor 1254, in nearby
Escambia Bay, Florida and its acute toxicity
to estuarine animals has been documented
(Duke, Lowe and Wilson 1970). Hansen,
Parrish and Lowe (1971) found 5 /xg/liter
of Aroclor 1254 toxic to the juvenile estuarine
fishes, pinfish (Lagodon rhomboides) and
spot (Leistomus xanthurus}, in 14- to 45-
day bioassays. Because Escambia Bay is a
nursery ground for many marine species of
fish, it is important to determine the effect
of Aroclor 1254 on the early life stages of
these fish.
The sheepshead minnow (Cyprinodon vari-
egatus) is found in brackish waters from Cape
Cod, Massachusetts to Brownsville, Texas
(Hildebrand 1917). It is important in
estuarine food chains as a voracious omni-
vore and as food for predators such as
croakers (Micropogon undulatus) and spotted
seatrout (Cynoscion nebulosus) (Darnell
1958). The size, hardiness, high fecundity,
and generation time of the sheepshead minnow
make it a nearly ideal laboratory test fish.
In this research, we determined water tem-
1 Contribution No. 175, Gulf Breeze Environmental
Research Laboratory.
® Registered trademark, Monsanto Co., St. Louis,
Mo. Mention of commercial products does not con-
stitute endorsement by the Environmental Protection
Agency.
peratures and salinities suitable for the culture
of embryos and fry of the sheepshead minnow
and observed the effect of Aroclor 1254 in
water on the various life stages of this fish.
METHODS AND MATERIALS
Adult fish were acclimated for 1 wk in
salt water averaging 25 C and 20%0. The
following week, acclimated female fish were
given three intraperitoneal injections of 50
I.U. human chorionic gonatrophic hormone
at 48-hr intervals to induce egg maturation.
Over 70% of the fish produced viable eggs.
Eggs were manually stripped and deposited
in 40 ml of filtered seawater. Only large,
round, clear eggs were selected for fertil-
ization. Testes from 7-10 acclimated males
were excised, macerated in 20 ml of filtered
seawater and mixed with eggs in a 100 ml
beaker. Thirty minutes were allowed for
fertilization. Each experiment lasted as long
as was required for embryos to hatch (4 to
15 days) plus an additional 2 wk to determine
survival of fry. Artemia salina nauplii, with
no detectable levels of PCB, were fed to the
fry daily.
We investigated the effect of temperature
on hatching by withdrawing 25 eggs from
the 100-ml beaker with a wide-bore pipette
and placing them in two 1-liter dishes con-
taining 25 C, 20%0 seawater. A pair of
dishes was partially submerged in a bath at
15, 20, 25, 30, or 40 C in one temperature
582
-------
SCHIMMEL—AROCLOR EFFECTS ON SHEEPSHEAD MINNOWS
583
FIGURE 1.—Dosing apparatus: A—oscillating
pump; a—pump micro-switch; B—mixing bottles;
C—vacuum lines; D—receiving bucket; E—solenoid;
e—solenoid micro-switch; F—injector lever appa-
ratus; G—SOcc. syringe; H—toxicant delivery tubing;
I—water delivery tube.
test and 22, 24, 26, or 28 C in another. One
hour later, when temperatures in the dishes
equaled the bath temperature, fertilization
was confirmed microscopically. The criterion
for fertilization was cleavage. Dishes were
checked daily during the 3-wk static tests
and dead or nonfertile eggs and larvae were
removed. The criteria for embryo death was
an opaque, white, fungal growth and for the
larvae, a white coloration of the trunk mus-
culature. Temperatures were monitored con-
tinuously and were within 1 C of the desired
temperatures. Salinity alterations due to
evaporation were compensated for daily by
addition of distilled water.
Fertilization procedures for the salinity
study were identical to those of the temper-
ature study except that the number of eggs in
each salinity varied from 35 to 75, depending
on egg availability, and the temperature was
30 C. Filtered seawater from Santa Rosa
Sound, Florida was diluted to give salinities
of 0, 5, 10, 15, 20, 25, 30, and 35%0. The
30 and 35%o concentrations were attained by
adding Rila® salts to the water from the
Sound. Salinities, checked daily with a TS®
refractometer, were within 2%o of the desired
level.
An Aroclor 1254, flow-through bioassay
® Registered trademark, Rila Products, Teaneck,
N.J.
® Registered trademark, American Optical Corp.,
Buffalo, N. Y.
FIGURE 2.—Distribution apparatus: J—distribution
box; K—egg/larvae trays; L—Petri dishes; M—
siphon.
was accomplished using culture conditions
determined suitable in the temperature and
salinity tests. Eggs were fertilized in control
water and placed in four Petri dishes, 20
eggs per dish, in each PCB concentration and
control. Fertilization was confirmed after
1 hr. Temperatures in both tests averaged
29 C (range 27-31 C) and salinity averaged
24,%0 (range 16-32%0). T1--; salinity fluc-
tuated with that of Santa Rosa Sound. The
toxicant dosing system used in this test was
a modification of the apparatus of Brungs
and Mount (1970). In our system, the toxicant
and carrier were injected into the delivery
tube leading to each exposure tank (Fig. 1).
Our apparatus allowed us to retain the ad-
vantages of the Mount and Brungs (1967)
dosing apparatus and to select any concen-
tration of toxicant while maintaining the same
concentration of carrier in each toxicant
concentration.
Seawater used in this bioassay was pumped
from Santa Rosa Sound into a constant head
box in the laboratory. An oscillating pump
(1A), regulated by a rheostat, pumped water
from the head box through a 20-^-pore poly-
propylene filter and into the compartments in
the dosing apparatus. After all compartments
were filled, the self-starting siphon in the last
compartment emptied into a receiving bucket
(ID). The weight of the bucket being filled
operated two micro-switches. One switch
-------
584
TRANS. AMER. FISH. SOC., 1974, NO. 3
TAISLE 1.—Ejjcct of temperature on fertility, time-to-
hatch, hatch success and survival oj fry for 2 wk
following hatching oj sheepshead minnows (Cy-
prinodon variegatus). Temperatures varied ± 0.5
C; salinity averaged 20r/,e, ± 1%,
TABLE 2.—Effect of salinity on fertility, time-to-
hatch, hatch success and survival of fry for 2-wk
following hatching of sheepshead minnow (Cy-
prinodon variegatus). Temperature 30 ± 2 C;
salinity varied ± !%„
per.iture
(°C)
15
••><)
O.T
°4
°5
°6
°8
30
35
40
Eggs Hatching
Num-
her
50
50
50
50
50
25«
50
50
50
50
Fertile
%
92
98
100
96
98
92
90
94
94
94
Days
#
No hatch
No hatch
15.0
9.0
7.0
7.0
6.0
4.5
4.0
4.0
Survival
%
0"
Ob
28. Ob
50.0
83.7
78.0
55.5
82 9
70.2
8.5b
Frv
try
survival
%
Ob
0"
100
100
80
100
92
90
100
50
••' Loss of 25 eggs due to spillage.
b Significantly less than the greatest fertility or survival
(X-; a = 0.01).
(la) shut off the oscillating pump and the
other (le) activated a solenoid (IE) that
raised the lever of the injection apparatus
(IF). This rising lever was connected to gears
that forced the barrels of six 50-ml syringes
(1G) equal distances. Mixing bottles (IB)
received injections from a control syringe
containing the carrier (polyethylene glycol
200).
Stock solutions of the carrier and Aroclor
1254 were mixed to give concentrations 0.1,
0.32, 1.0, 3.2 and 10.0 ^g of Aroclor per
liter of water. The water then flowed to the
distribution boxes (Fig. 2) containing one
large and four small compartments, each with
a standpipe or siphon. Each compartment
filled completely during each cycle and the
siphon delivered water to the aquarium in
which adults and juvenile fish were held.
Water remaining in the four small compart-
ments flowed slowly out through submerged
holes in the standpipes into each of four con-
tainers holding eggs or fry. Each container
received 100 ml of water per cycle. The num-
ber of cycles per day ranged from 100 to 140
and was sufficient to replace water in each egg
container at least three times each day.
Aroclor 1254 concentrations in test and
control water were determined weekly by
gas chromatography. Methods were the same
as those of Nimmo et al. (1971), except that
an OV-101 column was used and all peak
heights were averaged for PCB quantification.
Measured PCB concentrations in test water
Eggs
Salinity
%
0
5
10
15
^0
25
30
35
Number
60
35
75
35
75
35
75
35
Fertile
%
35
46
35
51
44
54
49
37
Hatching
Days
#
No hatch
7.0
7.0
5.5
6.0
5.5
5.5
6.0
Survival
%
0"
12"
35"
67
67
53
73
30"
Frv
rry
survival
%
0
50
89
92
100
90
93
50
" Significantly less than the greatest fertility or survival
were typically 35% to 60% of nominal con-
centrations; control water contained no de-
tectable PCB (<.03 ^g/liter). Recovery ef-
ficiency of Aroclor 1254 was greater than
60%. Measured concentrations were not cor-
rected for percentage recovery.
Dissolved oxygen was determined weekly
by the modified Winkler method (Strickland
and Parsons 1968). Concentrations seemed
adequate and above 50% saturation.
The chi-square (^2; a = 0.01) test was used
in the statistical analysis of the data. In tem-
perature and salinity studies the maximum
positive response was compared with all other
responses to determine the most efficient cul-
ture conditions. Data in the two temperature
experiments were analyzed separately. Probit
analysis was applied to the data to determine
the LC50 of the Aroclor 1254.
RESULTS AND DISCUSSION
Temperature influenced the number of days
required for hatching (time-to-hatch) and
affected the survival of embryos and fry of
the sheepshead minnow (Table 1). Sheeps-
head minnow eggs hatched at water tem-
peratures from 22-40 C but none hatched at
lower temperatures. Hatching success was
greatest at temperatures ranging from 24
to 35 C. Fry survival did not differ in the
range of 22 to 35 C. The longest time-to-
hatch was 15 days at 22 C, and decreased
rapidly, leveling off at about 4 days at tem-
peratures ranging from 30 to 40 C.
Salinity affected survival of embryos and
fry but did not affect time-to-hatch (Table
-------
SCHIMMEL—AROCLOR EFFECTS ON SHEEPSHEAD MINNOWS
585
TABLE 3.—Effect of Aroclor® 1254 on fertility, time-to-halch, hatch success and survival of fry for 2 vik fol-
lowing hatching of sheepshead minnow (Cyprinodon variegatus). Temperature averaged 30 C (range 27-
31 C); salinity averaged 24%c (range 16-32%c)
Concentration (#g/liter)
Nominal
Control
0.1
0.32
1.0
3.2
10.0
Measured
< 0.03 /Kg/liter
0.06
0.16
0.36
1.04
3.48
Eggs
Number
160
160
160
160
160
160
Fertile
86
86
86
91
85
91
Hatching
Days
#
7.0
6.5
7.0
7.0
7.0
6.5
Survival
%
79
69
73
82
75
57*
Frv
Survival
%
89
95
62"
63 «
40"
8"
" Significantly different from control fish (x=; a = 0.01).
2). Survival to hatching was greater at
salinities from 15 to 30%o and fry survival
did not differ in that range. Under natural
conditions, adult and juvenile sheepshead min-
nows occur in a wide salinity range (Simpson
and Gunter 1956). Our laboratory data show
that embryos and fry also can survive in a
wide salinity range.
Aroclor 1254 affected the survival of both
embryos and fry of the sheepshead minnow
but had no effect on time-to-hatch. Embryos
developed and hatched at all PCB concentra-
tions, but hatching succes at 10 //.g/liter was
significantly less than that of control eggs
(Table 3). After the eggs hatched, survival
of fry was significantly less than control fry
at all PCB concentrations except 0.1 /xg/liter.
Mortality increased with an increasing con-
centration of PCB. Three-week LC50 in ex-
posed embryos and fry was estimated at 0.93
jug/liter (S.E. = 0.17 ^g/liter). Fry did not
die immediately after hatching but were killed
over most of the 2-wk period of post hatch
exposure. Many of the dying fish developed
fin rot as previously described in other PCB
exposed fishes (Hansen et al. 1971).
Aroclor 1254 was more toxic to fry of
sheepshead minnows than it was to juveniles,
adults or to the fertilization of eggs (Table 4).
In 3-wk exposures to the same concentrations
of this PCB, mortality of the juveniles was
significantly greater (24%) in the 10 jug/
liter concentration. Adult fish were not killed
in a 3-wk exposure but became lethargic and
exhibited fin rot. The concentration of PCB
in the fry compared to that in the water (con-
centration factor) was nearly identical with
that of adult fish. Concentration factors in
fry ranged from 1.6 to 3.2 X 104 and those
for adults ranged from 1.1 to 3.2 X 104. A
24-hr static test in 10.0 /xg/liter PCB showed
no inhibition of fertilization of sheepshead
minnow eggs. The greater sensitivity of early
life stages of this fish to Aroclor 1254 stresses
the need for bioassays designed to assess the
effects of such chemical pollutants on these
stages.
The relationship between Aroclor 1254
contamination in Escambia Bay, Florida and
the effects of the PCB on various life stages
of C. variegatus in our bioassay is not clear.
Water and fish samples (12 species not in-
cluding C. variegatus) were taken 1 to 28 mo
after the reported spill that led to the Bay's
contamination. PCB residues, found in 10 of
the 37 water samples, ranged from non-
detectable (<0.03 ^g/liter) to 0.07 ^g/liter.
Concentrations in the Bay water were less
lhan those found lethal in our bioassays. Fish
in the estuary can accumulate the Aroclor
from the water, food, and sediment. Concen-
trations in the fish ranged from 0.29 to 20
,u,g/g (average 4.0 /xg/g) whole body wet
weight. Adult sheepshead minnows averaged
9.3 /ig/g when exposed to 0.32 /Ag/liter PCB
TABLE 4.—Relative susceptibility of various life
stages of sheepshead minnows (Cyprinodon vari-
egatus) to Aroclor® 1254 in it flow-through sys-
tem. Criteria are infertility of eggs and death of
embryos, fry, juveniles, and adults
Life stage
E«4K fertilization11
Embryos
Fry
Juveniles
Adults
Exposure
( days )
1
7
21
21
21
Concentration
Maximum
not affecting
10.0
3.2
0.1
3.2
10.0
(AH/liter)
Minimum
affecting
10.0
0.32
10.0
—
" 24-hr static test.
-------
586
TRANS. AMER. FISH. SOC, 1974, NO. 3
for 4 wk in the laboratory (Hansen, Schimmel
and Forester In press). Eggs produced by
these exposed fish were fertilized and although
adults seemed normal and embryos appeared
to develop normally, survival of fry in the
first week after hatching was significantly
reduced compared to that of unexposed fry.
ACKNOWLEDGMENTS
We thank Gary Cook and Dennis Knight
for chemical analyses of water samples from
the Aroclor 1254 test and Steve Foss for pre-
paring the illustrations.
LITERATURE CITED
BRUNCS, W. A., AND D. I. MOUNT. 1970. A water
delivery system for small fish-holding tanks.
Trans. Amer. Fish. Soc. 99(4): 799-802.
BUTLER, P. A. 1973. Organochlorine residues in
estuarine mollusks, 1965-1972. National Pesti-
cide Monitoring Program. Pestic. Monit. J.
6(4): 238-362.
DARNELL, R. M. 1958. Food habits of fishes and
larger invertebrates of Lake Pontchartrain,
Louisiana, an estuarine community. Publ. Inst.
Mar. Sci. Univ. Tex. 5: 354-416.
DUKE, T. W., J. I. LOWE, AND A. J. WILSON, JR.
1970. A polychlorinated biphenyl (Aroclor
1254®) in the water, sediment, and biota of
Escambia Bay, Florida. Bull. Environ. Contain.
Toxicol. 6: 171-180.
HANSEN, D. J., P. R. PARRISH, J. I. LOWE, A. J.
WILSON, JR., AND P. D. WILSON. 1971. Chronic
toxicity, uptake, and retention of Aroclor® 1254
in two estuarine fishes. Bull. Environ. Contam.
Toxicol. 6: 113-119.
, S. C. SCHIMMEL, AND J. FORESTER. In press.
Aroclor® 1254 in eggs of sheepshead minnows:
Effect on fertilization success and survival of
embryos and fry. Proc. 26th Annu. Conf. South-
east. Ass. Game Fish. Comm.
HILDEBRAND, S. F. 1917. Notes on the life history
of the minnows (Gambusia affinis and Cy-
prinodon variegatus). Rep. U. S. Comm. Fish.,
1917: 1-14.
MOUNT, D. I., AND W. A. BRUNCS. 1967. A sim-
plified dosing apparatus for fish toxicology
studies. Water Res. 1: 21-29.
NIMMO, D. R., R. R. BLACKMAN, A. J. WILSON, JR.,
AND J. FORESTER. 1971. Toxicity and distri-
bution of Aroclor® 1254 in the pink shrimp
Penaeus duorarum. Mar. Biol. (Berlin) 11(3):
191-197.
SIMPSON, D. G., AND G. GUNTER. 1956. Notes on
habitats, systematic characters and life histories
of Texas salt water cyprinodontes. Tulane Stud.
Zool. 4: 115-134.
STRICKLAND, J. D. H., AND T. R. PARSONS. 1968.
A practical handbook of seawater analysis. Fish.
Res. Board Can. Bull. 167: 21-26.
-------
CONTRIBUTION NO. 176
-------
PROCEEDINGS
OF THE
NATIONAL SHELLFISHERIES ASSOCIATION
OFFICIAL PUBLICATION OF THE NATIONAL
SHELLFISHERIES ASSOCIATION;
AN ANNUAL JOURNAL DEVOTED TO
SHELLFISHERY BIOLOGY
VOLUME 64
Published for the National Shellfisheries Association. Inc. by
Economy Fainting Co., Inc., Boston, Maryland
JUNE 1974
-------
Proceedings of the National Shellfisheries Association
Volume 64 1974
TRENDS IN PESTICIDE RESIDUES IN SHELLFISH
Philip A. Butler'
U.S. ENVIROMENTAL PROTECTION AGENCY
OFFICE OF PESTICIDES PROGRAMS
GULF BREEZE FLORIDA
ABSTRACT
The National Estuarine Monitoring Program, a cooperative effort between the
State and Federal Governments, collected and analyzed shellfish samples for per-
sistent synthetic pesticides at monthly intervals during the years 1965-1972 in 15
coastal states. The recently completed study of the 8000-plus analyses demonstrates
that: (1) the residues found, primarily DDT and its metabolites, were universally
too low to have human health significance, (2) areas of both high and low residues
were clearly defined geographically, (3) in some areas there has been a trend towards
a wider distribution of smaller residues, and (4) there has been a marked decline
generally in DDT residues since 1968 when peak levels in molluscs were detected.
INTRODUCTION
During the period 1965-1972, samples of oysters
and other bivalve molluscs were collected at month-
ly intervals at about 180 estuarine locations to
determine the incidence and magnitude of
pesticide residues along the Atlantic, Pacific and
Gulf of Mexico coasts. More than 8000 samples
were screened for the presence of 12 of the more
persistent chlorinated pesticides. In the later
years, chlorinated biphenyls or PCB's were in-
cluded in the analytical procedures. This report
briefly summarizes the implications of some of
the principal findings. A detailed report of the
sample collections and analyses has been
published recently (Butler, 1973).
BACKGROUND
Oysters exposed to varying concentrations of
pesticides under controlled conditions in the
laboratory demonstrate their sensitivity to these
pollutants. In aquaria with flowing unfiltered
1 Contribution No. 176, Gulf Breeze Environmental Research
Laboratory, U.S. Environmental Protection Agency, Gulf
Breeze, FL 32561.
seawater, for example, as little as l.O^g/kg (ppb)
of DDT inhibits oyster shell growth by about 20
percent in a 4-day period. One n g/g (ppm)
inhibits shell deposition completely at water tem-
peratures of about 17-20 C (62-68' F) (Butler,
1966).
Concentrations as high as these were not an-
ticipated in the natural environment and so it
was of importance in the development of a
proposed monitoring program to discover that
oysters were sensitive to the presence of DDT in
ambient water at levels as low as 10 x 10~12(10
parts per trillion). Exposure of oysters for 7 days
to this extremely low concentration led to the
formation of DDT residues in the tissues of about
70/ug/kg, a biological magnification of 70,000x.
DDT levels of this magnitude might be an-
ticipated in the marine environment since it is
less than the solubility of DDT in water. Further
laboratory experiments demonstrated that
oysters and other molluscs would be reliable as
biological tools to monitor estuarine ecosystems
because of this tendency to concentrate per-
sistent chemicals (Table 1).
Additional experimentation showed that con-
taminated oysters cleansed themselves of resi-
77
-------
78
P. A. BUTLER
TABLE 1. Uptake of DDT by eastern oysters maintained in flotving seawater. Exposure period
7-15 days in different tests. (Butler, 1968)
Concentration in water Residue in oyster Biological magnification
(ug/kg) or (ppb)
10.0
1.0
0.1
0.01
0.0001
control
te/g) - fan)
150.0
30.0
7.0
0.72
0.07
0.06
(xlOOO)
15
30
70
70
0
dues when returned to clean water. The disap-
pearance time or biological half-life of the resi-
dues in molluscs was short; a matter of days
as compared to months or years in fish and
other vertebrates. Consequently, when oysters
were sampled at about 30-day intervals, it was
possible to estimate when pollution entered
the estuary and thus gain some insight as to
its source.
FINDINGS
Analyses of monthly collections of oysters in
an estuarine complex near Pensacola, Florida
revealed a seasonal pattern of DDT residues
later found to be typical of estuaries in many
coastal areas. In the period February through
May there was a gradual increase in residue
magnitude to a seasonal high in late spring.
This was followed by a decline to background'
levels typical of the remainder of the year. It
seems reasonble to assume that this picture
results primarily from the occurrence of sea-
sonal rains and surface water run-off which
carry soil eroded from agricultural lands
through the river basin and into the estuary.
In contrast to this picture, there was a second
seasonal peak of DDT residues during the
winter months in samples from the South Texas
coast. This bimodal cycle probably reflected
the double cropping of farm lands and the
associated multiple applications of pesticides
in this sub-tropical area.
A more obvious result of the seasonal
agricultural use of DDT was indicated by
residues in oysters monitored in the Caloosahat-
chee River Basin in southwest Florida. Here,
peaks in DDT residues in oysters appeared soon
after the seasonal application of DDT to
maturing crops of sweetcorn and sugarcane. In
1967-68, the early spring residues were nearly
ten times the level of residues found during the
other months of the two-year monitor period
(Fig. 1). In some instances, seasonal and .annual
patterns of pesticide accumulation in estuarine
oysters could be associated with the dumping of
industrial effluents or with the control of
noxious insect populations. The declining use of
DDT in stable-fly control in northeast Florida,
for example, was clearly indicated by annual
decreases in DDT residues in local oyesters in the
period 1965-1968. DDT residues were no longer
-------
PESTICIDE RESIDUES IN SHELLFISH
79
FIG. 1. DDT residues in the eastern oyster
from the Caloosahatchee River Basin, Lee
County, Flo., by month of collection (Butler,
1973).
identified after the substitution of methoxy-
chlor, a less persistent compound, for fly control
in 1969. More importantly, methoxychlor was
not detected in the monitor samples in suc-
ceeding years.
The significaace of DDT residues in field
samples may be judged to some extent by the
magnitude of DDT residues observed in labora-
tory experiments. Market-size eastern oysters
were exposed to 1.0 n g/kg of DDT in flowing
seawater for a 10-day period and then 12 were
individually analyzed. The sum of DDT and its
metabolites found as residues ranged from a
low of 3.9 to a high of 23.2 p.g/g with an arith-
metic average of 10.1 Mg/g (ppm) for the group.
This value is about twice the largest DDT
residue observed (5.39 ppm) in all of the molluscan
samples collected in the 7-year monitor-
ing period. It should be noted further that DDT
residues were less than l.Opg/g in 99.5 percent
of the 8000+ monitoring samples analyzed. It
appears that despite the build-up of large resi-
dues in higher carnivores DDT pollution of
estuarine waters generally has been at levels
below 1 .Opg/kg (Fig. 2).
It must be emphasized that the observed
levels of DDT residues in molluscs were too low
to have human health significance or to have
demonstrable effects on the oysters themselves.
Only in isolated area were DDT residues high
enough to indicate that some elements of the
estuarine fauna might have been damaged by
20
10
DDT
ppm
INDIVIDUAL
VARIATION
EXPERIMENTAL
AVERAGE
ll
MONITORED
MAXIMUM
Jl
MONITORED
MAXIMUM
IN 99 5 %
CL
FIG. 2. DDT residues in experimental and field-
collected oysters in tHe period 1965-1972. See
text for explanation.
the magnification and accumulation of DDT
residues in the food web.
With these observations in mind, the overall
findings of the monitoring data may be sum-
marized geographically. The lowest average in-
cidences of DDT positive samples were found,
in order, in Washington, Georgia and Maine.
Highest incidence rates were observed in New
Jersey, Alabama, North Carolina and California.
However, the largest residues of DDT and its
metabolites were found in samples collected in
the estuaries of Florida, California and Texas.
There has been a well-defined but gradual
decline in both the incidence and magnitude of
DDT residues in oysters during the monitoring
period in most areas. In some coastal estuaries
this trend is obscured by the lack of uniformity
in the timing of sample collections or by
variations in the kind of mulluscs collected.
Despite erratic fluctuations in magnitude and
the fact that individual residues were never
very high, it is clear that DDT pollution in
estuaries was at peak levels'in 1966-1967 and
gradually declined thereafter. This 1966 peaking
in the magnitude of residue data parallels, not
unexpectedly, the findings of peak DDT levels
in fresh water monitoring samples in 1966
followed by sharp declines in 1967 and 1968
(Lichtenberg, et al., 1970).
Data demonstrating the overall decline in
the magnitude of DDT residues in estuarine
molluscs are summarized in Fig. 3. This dia-
-------
80
P. A. BUTLER
TIINOl IN rtlCINT OCCUIIIIMCI OF DDT MSIDUIS IN OTITID1
• ISIDUI RAMOI - ppm
1965-70 I 1971
1964-70 I 1971
1965-7O ; 1971
1965-70 ; 1971
FIG. 3. Percentage occurrence of DDT residues
in estuarine bivalves in the period 1965-1970
o.s compared to 1971. Data summarize about
7000 analyses of more than 75,000 animals.
See text for expla-nation.
gram shows that, in the period 1965-1970, 39%
of all samples contained negligible DDT resi-
dues, less than 0.01 ppm. while in 1971 this
value increased to 56%. Conversely, in these
same years the percentage of samples contain-
ing larger residues declined sharply. In Cali-
fornia and a few other isolated locations there
was an exception to this generalized picture
in that the number of samples with DDT re-
sidues in the 0.01-0.10 ppm range increased
during the monitoring period but the per-
centage of samples with high residues
decreased sharply as in other coastal areas.
Apparently in these drainage basins, there was
an increased cycling of DDT in the trophic web
accompanied by a diminution of the amount
present in individual animals. In other words,
DDT residues were distributed more thinly
among more members of the biota.
At ten monitoring stations in North Carolina,
where the continuity of sample collections was
especially good, the data provide a clear picture
of annual trends in DDT pollution levels. Fig.
4 shows the decline in the percentage of sam-
ples having measurable DDT residues as com-
pared with the approximate percentage decline
in the domestic use of DDT throughout the
United States after 1965. DDT supplies in that
year have been arbitarily designated as 100%
for the basis of this comparison (USDA, 1967-
100
eo
60
FIICINTACt OKLINi IN NOITM
CAIOtIHA OTSIII SAMKil
PIDCIMTACI DICLINI IN
DDT US! IN U.S.A.
(1964 = too*)
L
1970
FIG. 4. Percentage decline in DDT residues
of more than 10 n g/kg in North Carolina
oysters as compared to the decline in the con-
sumption of DDT in the entire United States
in the period 1965-1971.
72). These .data demonstrate the progressive
loss of residual DDT from "at least one segment
of an estuarine ecosystem following the general-
ized curtailment in the agricultural use of
DDT, and controvert the widespread belief
that environmental problems with DDT would
be longlasting regardless of how soon its use
was terminated.
SUMMARY
These monitoring data show that the domes-
tic use of DDT resulted in only nominal resi-
dues in estuarine molluscs in the United States
in the period 1965-1972. By extrapolation from
laboratory data, we may infer that these re-
sidues were too small to have a deleterious
effect on the growth and productivity of
estuarine bivalves. Despite the chemical
stability of DDT, curtailment in its use "was al-
most immediately reflected by declines in the
magnitude of residues in estuarine molluscs.
The data establish a baseline for levels of
DDT pollution in estuaries during the monitored
period, and suggest that despite the stability
of a synthetic organic compound it
may become biologically unavailable
soon after its widespread use is dis-
continued.
-------
PESTICIDE RESIDUES IN SHELLFISH
81
LITERATURE CITED
Butler, P. A. 1966. Pesticides in the marine en-
vironment J. Appl. Ecol. 3(Suppl.):253-259.
Butler, P. A. 1968. Pesticide residues in marine
molluscs. In Proc. Natl. Symp. Estuarine
Pollut., Stanford Univ., Stanford, Calif.
1967. p. 107-121.
Butler, P. A. 1973. Organochlorine residues in
estuarine molluscs, 1965-197?, — National
Pesticide Monitoring Program. Pestic.
Monit. J. 6: 238-362.
Lichtenberg, J. L., J. W. Eichelberger, R. C.
Dressman and J. E. Longbottom, 1970.
Pesticides in the surface waters of the
United States — a 5-year summary,
1964-68. Pestic. Monit. J. 4:71-86.
USDA, Agricultural Stabilization and Conserva-
tion Service. The Pesticide Review.
1967-1972. Washington, D. C.
-------
CONTRIBUTION NO. 177
-------
Reprinted from the proceedings of the 27th Annual Conference of the Southeastern Association of
Came and Fish Commissioners, 1973.
AROCLOR® 1254 IN EGGS OF SHEEPSHEAD
MINNOWS: EFFECT ON FERTILIZATION
SUCCESS AND SURVIVAL OF EMBRYOS AND FRY1
David J. Hansen, Steven C. Schimmel and Jerrold Forester
U. S. Environmental Protection Agency
Gulf Breeze Environmental Research Laboratory
Sabine Island, Gulf Breeze, Florida 32561
(Associate Laboratory of the
National Environmental Research Center, Corvallis, Oregon)
ABSTRACT
The effect of the polychlorinated biphenyl (PCB), Aroclor 1254, in eggs of the
sheepshead minnow, Cyprinodon variegatus, on fertilization success and
survival of embryos and fry was investigated. Adult fish were exposed for four
weeks to 0.1,0.32,1.0, 3.2 or 10.0 ug//of PCB, then injected twice with 50IU of
human chorionic gonadotrophin to stimulate egg production. The eggs were
fertilized, placed in PCB-free flowing seawater and observed for mortality.
Fertilization success was unimpaired by concentrations in eggs as high as 201
ug/g but survival of embryos and fry was reduced. Usually, fry from eggs con-
taining 7.0 ug/g or more began dying 24-48 hours after hatching. If this PCB
affects other species similarly, then populations of fish that presently have com-
parable concentrations in their eggs may be endangered.
INTRODUCTION
Polychlorinated biphenyls (PCB's) have been found frequently in estuarine
organisms from many states (Butler, 1973) and in an estuary near the Gulf
Breeze Laboratory (Duke et al., 1970). PCB's in seawater are toxic to and ac-
cumulated by juvenile shrimp, crabs, oysters and fishes (Nimmo, et al., 1971;
Lowe, et al., 1972 and Hansen, et al., 1971). The relationship between the
amount accumulated by fish and subsequent effects is poorly understood.
However, PCB's in eggs may decrease fertility and survival in early stages of em-
bryonic development in Atlantic salmon, Salmo salar (Johannsson et al., 1970),
and PCB's have been implicated in poor reproductive success of striped bass,
Morone saxatilis (Anonymous, 1971). Because reproductive success with both
fishes varied, the exact relationship of success to concentration of PCB in eggs
remains unclear.
Our study was conducted to determine the effect of one PCB, Aroclor 1254,
on fertilization success of eggs of sheepshead minnows, Cyprinodon variegatus,
and on survival of embryos and fry. Aroclor 1254 was selected because we found
eggs from striped bass that exhibited decreased reproductive success contained a
PCB whose chormatograms closely resembled Aroclor 1254. Sheepshead min-
nows were selected because they can be readily exposed in the laboratory and
reproductive success is excellent.
MATERIALS AND METHODS
Test fish
Adult sheepshead minnows were seined from ponds on laboratory grounds
and acclimated to laboratory conditions for four days before exposure. During
'Contribution No. 177, Gulf Breeze Environmental Research Laboratory. ®Registered trademark, Monsanto Com-
pany, St. Louis, Mo. Mention of commercial products or trade names does not constitute endorsement by the En-
vironmental Protection Agency.
420
-------
acclimation, mortality was less than 1% and no abnormal behavior was
observed. Females averaged 42.5 mm standard length, range 35-52 mm, and
males averaged 42.8 mm, range 35-52 mm. During acclimation and exposure,
fish were fed commercial fish food that contained no detectable PCB ( 0.01
ug/g).
Adult exposure
We exposed 20 female and 10 male fish in aquaria containing none, 0.1, 0.32,
1.0, or 3.2 ug//of Aroclor 1254 and exposed 25 females and 15 males to 10 ug//
for four weeks in an intermittent-flow bioassay. The apparatus used was a
modification of that of Brungs and Mount (1970). In our modification, Aroclor
1254 and carrier, polyethylene glyco! 200, were injected into seawater each time
the apparatus cycled. Each cycle siphoned water to six 807 test aquaria. The in-
jection device was operated by a solenoid that raised a lever each cycle turning
gears on six injectors and pushing the plungers of six50cc syringes. Each of the
approximately 150 daily cycles delivered 1.51 of filtered 30C seawater, 11 ug of
carrier and appropriate amounts of PCB to each aquarium. Water and carrier
without PCB were delivered to the control aquarium. Salinity of water averaged
17 o/oo, range 5 to 28 o/oo.
Egg fertilization, embryo and fry survival
The effect of Aroclor 1254 in eggs was determined by enhancing egg produc-
tion in exposed fish by hormonal injection, fertilizing the eggs artificially and
monitoring their development in flowing PCB-free seawater. Female sheep-
shead minnows were injected intraperitoneally with 50 I. U. human chorionic
gonadotrophic hormone on exposure-days 25 and 27. On day 28, eggs were
stripped manually from five females from each aquarium and those from each
female placed in individual beakers containing 40 ml of filtered 30C seawater.
Ninety-three of 96 females that survived produced eggs. Eggs from a female were
fertilized with excised macerated testes from a male from the same aquarium. In
addition, eggs from five control fish and two fish surviving exposure to 10 ug//
PCB were fertilized by males exposed to 1.0 ug//. Twenty-five eggs from each
fish were placed in Petri dishes to which a nine cm high collar of SOOunitex mesh
was glued. Dishes were submerged 7 cm in the 80/ aquarium which received ap-
proximately 2251 of filtered PCB-free seawater per day; average salinity was 18
o/oo, range 10-27 o/oo. Success of fertilization was confirmed by checking
microscopically for cleavage 1.5 hours after fertilization. Thereafter, dishes were
checked daily to determine survival of embryos and fry. Dishes remained in the
aquarium for 34 days. Fry were fed brine shrimp nauplii or dry commercial fish
food daily.
Chemical analyses
Concentrations of Aroclor 1254 in water, eggs and fish were determined by
electron capture gas-chromatography. Unfiltered water samples from each
aquarium were analyzed weekly during the four-week exposures of adults. At
the end of the adult exposure, concentrations were determined in the fertilized
eggs from each fish and in surviving adult males and females. Also, fry that hat-
ched from these eggs and survived for four weeks in PCB-free water were
analyzed for Aroclor 1254 content. Analytical methods for water, eggs and fish
were the same as those of Nimmo et al. (1971), except than an OV-101 column
was used and all peak heights were summed for PCB quantification. Recovery
efficiency of Aroclor 1254 exceeded 80%. Measured concentrations were not
corrected for percentage recovery.
Manufactured b> George Fra?er. 4528 Pitt Street. Duluth, Minn. 55804.
421
-------
Statistical analysis
Probit analysis was used to determine whether increasing concentration of
PCB in eggs increased the effect on fertilization success and on survival of em-
bryos and fry. The X2test for independent samples was used to compare data for
eggs from individual unexposed and exposed fish. Differences were considered
real at « = 0.05 for probit analysis and « = 0.01 for X2 tests.
RESULTS AND DISCUSSION
Aroclor 1254 in water was toxic to and accumulated by adult sheepshead min-
nows exposed for four weeks (Table 1). Mortality offish was negligible, except
in the aquarium receiving 10 ug//. Dying fish in this aquarium typically became
lethargic, ceased feeding, and some developed fin rot. Fish accumulated the
PCB in direct proportion to the concentration in the water and concentrations in
fish ranged from 15,000 to 30,000 X the nominal concentration in the water.
Concentrations in males and females were similar. Concentrations of the
chemical in eggs from exposed adult fish were proportional to the concen-
trations in the fish and concentrations in female fish were 1.8 to 2.3 times greater
than the concentrations in their eggs. The PCB exposure apparently did not alter
the percentage of females producing eggs or their fecundity.
Fewer embryos and fry from eggs of exposed fish survived than did embryos
and fry from eggs of control fish (Table 2). The percentage of the eggs fertilized
was not affected, but survival of embryos to hatching was less in eggs from fish
exposed to 10 ug//. Survival rate of fry in the first week following hatching was
less in eggs from fish exposed to 0.32 to 10.0 ug// than in eggs from unexposed
fish. The estimated LC50 was 6.1 ug/ g; 95 percent confidence limit equals 3.5 to
11.8 ug/g. Fry typically began to die one or two days after hatching, about the
time they started feeding. If fry survived the first week, there seemed to be no ad-
ditional mortalities related to PCB during three weeks of additional
observation. Concentrations of Aroclor 1254 in surviving fry were similar, 0.26-
0.56 ug/g, and not proportional to concentrations in eggs.
Embryo survival decreased at the highest concentration of PCB in eggs and
fry survival decreased with increasing concentration of PCB in eggs. The
amount in eggs was critical because it was the sole source of PCB for the em-
bryos and fry reared in PCB-free water. PCB in milt was probably not critical to
fertility and survival because when eggs from control fish were fertilized with
milt from either control or 1.0 ug/ / exposed males, survival rate of embryos and
fry was not altered (Table 2). Survival rate of fry hatched from eggs containing
7.0 ug/g or more of PCB was significantly less than the lowest survival rate of
eggs from any of the five control fish (Table 3).
If the effect of PCB in eggs of other fishes is similar to that found with sheep-
shead minnows — and we have no data to support this view — then variations in
published information concerning the chemicals relation to spawning success
could be explained. Atlantic salmon eggs containing up to 1.9 ug/ g of PCB had
decreased fertility and survival of early embryos, but survival of late embryos
and sac fry was unimpaired (Johannsson et al., 1970). Chesapeake Bay striped
bass eggs containing PCB's had decreased fertility and survival of newly hatched
fry (Anonymous, 1971). Our analysis of eggs from eleven striped bass from the
Eastern shore of Chesapeake Bay showed that the eggs contained about 2.5 to
8.7 ug/g of a PCB resembling Aroclor 1254. Because concentrations of PCB in
eggs of sheepshead minnows as high as 201 ug/g were not accompanied by
decreased fertility and only minimal embryo mortality, it seems unlikely that
decreased fertility and embryo survival in Atlantic salmon and striped bass
could be related solely to PCB in their eggs. Diminished survival of newly hat-
ched striped bass fry, however, could be PCB-related since concentrations in
422
-------
their eggs were similar to those in sheepshead minnow eggs which produced fry
whose survival was poor.
LITERATURE CITED
Anonymous. 1971. The striper — this century's dinosaur. Stripers Unlimited
1971 Directory and Guidebook, pp. 11-62.
Brungs, W. A. and D. I. Mount. 1970. A water delivery system for small fish-
holding tanks. Trans. Am. Fish. Soc. 99(4):799-802.
Butler, P. A. 1973. Organochlorine residues in estuarine mollusks, 1965-1972.
National Pesticide Monitoring Program. Pestic. Monit. J. 6(4):238-362.
Duke, T. W., J. I. Lowe, and A. J. Wilson, Jr. 1970. A polychlorinated biphenyl
(Aroclor 1254®) in the water, sediment and biota of Escambia Bay, Flori-
da. Bull Environ. Contam. Toxicol. 5(2): 171-180.
Hansen, D. J., P. R. Parrish, J. I. Lowe, A. J. Wilson, Jr. and P. D. Wilson.
1971. Chronic toxicity, uptake and retention of Aroclor® 1254 in two estu-
arine fishes. Bull. Environ. Contam. Toxicol. 6(2):113-119.
Johannsson, Nils, S. Jensen and M. Olsson. 1970. PCB-indicators of effects on
fish. In "PCB Conference, Wenner-Gren Center", Sept. 29, 1970. pp. 59-
68. Natl. Environ. Prot. Bd., Stockholm.
Lowe, J. I., P. R. Parrish, J. M. Patrick, Jr. and J. Forester. 1972. Effects of the
polychlorinated biphenyl Aroclor® 1254 on the American oyster (Cras-
sostrea virginica). Mar. Biol. (Berl.) 17(3):209-214.
Nimmo, D. R., R. R. Blackman, A. J. Wilson, Jr. and J. Forester. 1971. Toxic-
ity and distribution of Aroclor® 1254 in the pink shrimp (Penaeus duorar-
um). Mar. Biol. (Berl.) 11(3):191-197.
423
-------
Table 1. Toxicity and uptake of Aroclor® 1254 by adult sheepshead minnows (Cyprinodon variegatus) exposed for 28 days in an in-
termittent-flow bioassay. Thirty fish were tested per concentration. Residue analyses are for a minimum of 7 male and 17
female fish and eggs from 5 fish.
TEST CONCENTRATION
(ug//)
Nominal
Control
0.1
0.32
1.0
3.2
10.0
Measured
ND*
0.09
0.14
0.39
1.1
5.6
MORTALITY CONCENTRATION IN FISH
% (ug/g, wet weight)
7
13
7
10
3
95
Males
0.64
2.5
9.7
—
49.
—
Females
0.47
1.9
9.3
25.
49.
—
Eggs
0.52
0.88
5.1
11.
27.
170.**
FEMALES
GRAVID
100
89
100
100
94
100
AVERAGE
FECUNDITY
No.
97
121
110
127
152
138
*ND = not detectable, < 0.03 ug//
**Eggs from two fish.
-------
Table 2. Success of fertilization of eggs from sheepshead minnows exposed
to Aroclor® 1254 for four weeks, survival of embryos from fertile
eggs until hatching and survival of hatched fry. Eggs are from five
fish per concentration (except two fish from 10 ug//). Percentages
are in parentheses.
CONCENTRATION
Adult
Exposure
(ug//)
Control
0.1
0.32
1.0
3.2
10.0
Control F
and 1.0
ug//M
Eggs
Average
(ug/g)
0.52
0.88
5.1
11.
27.
170.
—
Tested
125
125
126
126
126
50
128
EGGS
Fertile
125 (100)
120 (96)
120 (95)
121 (96)
118(94)
46 (92)
119(93)
FRY
Hatched
116(93)
106 (88)
107(80)
118 (98)
100 (85)
33 (72)*
113 (95)
Survival Survival
Week 1 Weeks 2,3,4
1 1 1 (95)
103 (97)
82 (77)*
31 (26)*
23 (23)*
0(0)*
112 (99)
106 (96)
98 (95)
76 (93)
26 (96)
19 (83)
O(-)
1 1 1 (99)
'Significantly less than control hatching or one week fry survivals (X2;tt = 0.01).
-------
Table 3. Comparison of concentration of Aroclor® 1254 in eggs (wet weight)
from sheepshead minnows exposed to the PCB for four weeks and
success of fertilization of eggs and survival of embryos and fry.
Concentration
in Eggs
(ug/g)
0.41
0.44
0.45
0.53
0.57
0.76
0.84
0.91
0.98
1.1
3.7
4.1
5.4
5.4
7.0
7.1
9.5
10.8
13.2
13.3
23.6
25.7
27.9
28.6
28.7
145.
201.
Tested
25
25
25
25
25
25
25
25
25
25
25
26
25
25
25
26
25
25
25
25
25
25
25
25
26
25
25
EGGS
Fertile
25
25
25
25
24
25
23
25
23
25
23
26
24
24
23
23
24
25
25
24
23
24
25
25
21
23
23
FRY
Hatched
23
24
25
24
19
20
21
25
17
24
22
23
19
22
21
22
23
25
25
23
23
16
23
25
13
15
18
Adults
Week 1 Weeks 2,3,4 from
Aquaria
23
19
25
24
18
20
21
23
17
24
21
22
13
21
5
2
7
8
9
5
4
6
0
5
8
0
0
22
19
24
23
18
18
19
23
15
23
20
21
12
19
4
2
7
5
7
5
4
6
0
3
6
0
0
(ug//)
Control
"
"
"
0.1
Control
0.1
"
"
"
0.32
"
"
"
"
1.0
"
"
"
ft
3.2
"
"
"
"
10.0
"
426
-------
CONTRIBUTION NO. 178
-------
Reprinted from the proceedings of the 27th Annual Conference of the Southeastern Association of
Came and Fish Commissioners, 1973.
DIELDRIN: EFFECTS ON SEVERAL
ESTUARINE ORGANISMS1
Patrick R. Parrish,' John A. Couch, Jerold Forester,
James M. Patrick, Jr. and Gary H. Cook
U. S. Environmental Protection Agency
Gulf Breeze Environmental Research Laboratory
Sabine Islnad, Gulf Breeze, Florida 32561
(Associate Laboratory of the National Environmental
Research Center, Corvallis, Oregon)
ABSTRACT
Tests were conducted to determine (1) the acute toxicity of dieldrin in flowing
sea water to American oysters (Crassostrea virginica), pink shrimp (Penaeus
duorarum), grass shrimp (Palaemonetes pugio) and sheepshead minnows
(Cyprinodon variegatus) and (2) the rate of dieldrin uptake and depuration by
spot (Leiostomus xanthurus). Acute (96-hour) ECSO's were: oysters, 12.5 ug/1;
pink shrimp, 0.9 ug/1; grass shrimp, 11.4 ug/1; and sheepshead minnows 23.6
ug/1. Spot exposed to 0.0135, 0.075, 0.135, 0.75 or 1.35 ug/1 for 35 days ac-
cumulated the chemical with maximum concentrations attained in 11 to 18 days.
Maximum whole-body residue (wet-weight) was 6,OOOX the concentration in
test water. Spot contained no detectable dieldrin residues at the end of a 13-day
depuration period in dieldrin-free water. Tissue alterations, such as
subepithelial edema in gill lamellae and severe lysis and sloughing of the small
intestine epithelium, occurred in spot exposed to 1.35 ug/1 for four days.
INTRODUCTION
The effects of dieldrin on estuarine organisms were investigated because this
toxicant is present in most of this nation's estuaries. Dieldrin was the second
most commonly detected organochlorine compound in molluscs from 15 coastal
states during the period 1965-1972 (Butler, 1973).
Dieldrin, a chlorinated hydrocarbon insecticide, is acutely toxic to certain
non-target estuarine animals under field conditions (Harrington and
Bidlingmayer, 1958). Dieldrin is also acutely toxic to several estuarine animals
exposed for 48 hours under laboratory conditions (Lowe, personal com-
munication1).
This study was conducted to determine (1) the acute (96-hour) toxicity of
dieldrin to American oysters (Crassostrea virginica), pink shrimp (Penaeus
duorarum), grass shrimp (Palaemonetes pugio) and sheeshead minnows
(Cyprinodon variegatus) and (2) the rate of uptake and depuration in spot
(Leiostomus xanthurus).
MATERIALS AND METHODS
Test animals
All test animals except pink shrimp were collected near the Gulf Breeze
Laboratory and acclimated to laboratory conditions for at least ten days before
exposure. Pink shrimp were purchased from a local bait dealer and acclimated
similarly. If mortality in a specific lot of animals exceeded 1% in the 48 hours
immediately preceding the test or if abnormal behavior was observed during ac-
climation, those animals were not used. Oysters tested were from 24 to 43 mm in
'Contribution No. 178, Gulf Breeze Environmental Research Laboratory
'Jack 1. Lowe, Gulf Bree?e Environmental Research Laboratory, Sabine Island, Gulf Bree/e, FL 32561.
427
-------
height; pink shrimp, 52 to 81 mm rostrum-telson length; grass shrimp, 18 to
24 mm rostrum-telson length; sheepshead minnows, 11 to 14 mm standard
length; and spot, 22 to 38 mm standard length. Animals were not fed during
acute toxicity tests but they could obtain food (plankton and other particulate
matter) from the unfiltered sea water in which they were maintained. In the up-
take and depuration study, spot were fed commercial fish food that contained no
pesticide or polychlorinated biphenyl contaminants detectable by gas
chromatographic analysis.
Test Conditions
Acute toxicity of dieldrin was determined by exposing ten animals per
aquarium to different concentrations for 96 hours. Two 20 1 aquaria were used
for each concentration. Technical grade dieldrin (92% active ingredient) was dis-
solved in reagent grade acetone and metered at 0.14 ml/hr into unfiltered sea
water that entered each aquarium at 75 1/hr. Two control aquaria received the
same quantities of water and solvent but no dieldrin.
The rate of uptake and depuration of dieldrin by spot was determined by ex-
posing 35 animals per aquarium in duplicate 20 laquaria to 0.0135, 0.075,
0.135, 0.75, or 1.35 ug/1 for 35 days and then placing them in dieldrin-free
water for 14 days. Sea water flow rate was 751/hr per aquarium. Technical
grade dieldrin (92% active ingredient) was dissolved in reagent grade acetone
and metered at 15 ml/hr into the unfiltered sea water.
Effect of dieldrin was assessed by measuring reduction of shell growth of
oysters (Butler, 1962), by determining mortality in shrimps and fish, and by ex-
amining for pathological changes fish from the uptake and depuration ex-
posure.
Histopathological examination
Gills and viscera from live fish from the uptake and retention exposure were
examined. Tissues were fixed in Davidson's fixative, stored in 70% ethyl alcohol
and then processed for paraffin sections (7 u). Sections were stained with Harris
hematoxylin and eosin. Six fish from each concentration were removed for tis-
sue preparation after 4 days of exposure. Six fish from concentrations of 0.135,
0.075, and 0.0135 ug/1 were removed at the end of the 35-day exposure, and six
fish from concentrations of 0.135 and 0.075 ug/1 and control were removed after
the 13-day depuration in dieldrin-free water.
Chemical analyses
Concentrations of dieldrin in water and animals were determined by electron
capture gas chromatography. Unfiltered water samples from each concentration
were analyzed once during the 96-hour exposures and weekly during the uptake
and depuration exposure. Concentrations in animals that survived the 96-hour
exposures were determined as whole-body residures. In the uptake depuration
exposure, six fish were removed from each connentration after 4,11 18. 25 and
35 days and after 13 days in dieldrin-free water. Concentrations were deter-
mined for pooled samples of liver, muscle (all muscle above lateral line on left
side of fish with scalless skin), and remaining tissues. Results from the two
pooled samples of each tissue from each concentration were averaged. Resi-
dues in all tissues were summed to compute concentrations of dieldrin in
the whole fish.
Tissue samples that weighed more than 5 g were prepared for analysis by mix-
ing with anhydrous sodium sulfate in a blender. The mixture was extracted for 4
hours with petroleum ether in a Soxhlet apparatus. Extracts were concentrated
to approximately 10 ml and transferred in 3- to 4-ml portions to a 400 x 20 mm
chromatographic column that contained 76 ml of unactivated Florisil. After
428
-------
each portion settled in the column, vacuum was applied until all solvent was
evaporated. This was repeated with three 5-ml rinses. The residue was eluted
from the column with 70 ml of a 9:1 mixture (v/v) of acetonitrile and distilled
water. The eluate was evaporated to dryness and the residue transferred to a
Florisil column (Mills, et al., 1963) with petroleum ether. Dieldrin was eluted in
the 15% ethyl ether-in-petroleum ether fraction.
Tissue samples that weighed less than 1 g were analyzed by the micro method
described in the Pesticide Analytical Manual, Volume III (U. S. Food and Drug
Administration, 1970).
Water samples were extracted with petroleum ether, the extracts dried with
anhydrous sodium sulfate and evaporated to approximately 1 ml. The concen-
trates were transferred to a size 7 Chromaflex1 column containing 1.6 g Florisil
topped with 1.6 g anhydrous sodium sulfate. Dieldrin was eluted with 20 ml of
10% ethyl ether in hexane and the eluates were adjusted to an appropriate
volumn for analysis.
All samples were analyzed by electron capture gas chromatography using a
182cm x 2 mm ID glass column packed with 2% OV-101 on 100-120 mesh Gas
Chrom Q. Nitrogen flow rate was 25 ml/min, the oven temperature was 190° C,
and the injector and detector temperature was 210° C. Recovery exceeded 85%;
data were not adjusted for recovery. All tissue residues were determined on a
wet-weight basis.
Statistical analyses
Data from the acute (96-hour) exposures were analyzed statistically. Oyster
shell growth data were analyzed by unweighted least squares and shrimp and
fish mortality data were -analyzed by maximum liklihood profit analysis
(Finney, 1971).
RESULTS AND DISCUSSION
Acute (96-hr) exposures
Dieldrin was acutely toxic to the estuarine organisms tested (Tables 1 and 2).
Shell growth in oysters was appreciably inhibited by exposure to32 ug/1 for 96
hours. Pink shrimp were more sensitive to dieldrin than were grass shrimp, but
significant numbers of both these crustaceans died when exposed to concen-
trations in the low parts-per-billion (ug/1) range.
All animals accumulated dieldrin (Table 1). The quantities accumulated
depended on the species and the exposure concentration. In live oysters, whole-
body (meats only) concentrations ranged from 2,000 to 5,OOOX nominal concen-
trations in test water and 2,400 to 21,500X measured concentrations. In an
earlier experiment at this laboratory, oysters chronically exposed toO.Olmg/lof
dieldrin accumulated 8,OOOX the concentration in test water after 8 weeks ex-
posure (Parrish, 1973). In live pink shrimp, whole-body concentrations ranged
from only 240 to 250X nominal concentrations in test water and 280 to 420X
measured concentrations. In live grass shrimp, whole-body concentrations
ranged from 330 to 660X nominal concentrations in test water and from 470 to
750X measured concentrations. In live sheepshead minnows, whole-body
concentrations ranged from 2,000 to 4,OOOX nominal concentrations in test
water and from 3,500 to 7.300X measured concentrations.
'Mention of commercial products or irade names does nol constitute endorse men! by th« U. S. Emironmenlal Prelec-
tion Agency.
'Present Address: Bionomics Marine Laboratory. Route0. Bo.x 1002. Pensacola HI.32507.
429
-------
Uptake and depuration
Spot exposed to 0.0135,0.075,0.135,0.75 or 1.35 ug/1 of dieldrin for 35 days
accumulated the chemical, maximum concentrations being attained in 11 to 18
days (Table 3). Fish in some concentrations began to lose dieldrin after body
concentrations had peaked, even though the exposure continued and dieldrin
concentrations in test water remained constant (Table 4). Unlike our findings,
DDT concentrations in pinfish (Lagodon rhomboides) and Atlantic croaker
(Micropogon undulatus) exposed to 0.1 and 1.0 ug/1 increased for 14 days, then
remained relatively constant for 21 days (Hansen and Wilson, 1970).
Dieldrin was accumulated in greatest quantity in the liver of spot, where max-
imum concentration was 113,OOOX that in test water. Maximum concentration
in muscle was 11 ,OOOX that in test water and maximum concentration in whole-
body was 6,OOOX that in test water.
Spot lost all detectable dieldrin residues after a 13-day depuration period in
dieldrin-free sea water (Table 3). Pinfish lost 87% of DDT residues and Atlan-
tic croaker lost 78% of accumulated DDT when held in pesticide-free water for
56 days (Hanson and Wilson, 1970). Similarly, goldfish (Carassius auratus)
have been reported to eliminate l4C-dieldrin from various tissues more ra-
pidly than DDT (Grzenda et al., 1972). Thus, the flushing rate of dieldrin
in fish appears to be faster than that of DDT.
Fish exposed to 1.35 ug/1 showed degenerative changes in gill and visceral tis-
sue after 4 days of exposure. Gill lamellae from three of six fish exhibited
subepithelial edema (Fig. 1). A similar condition was observed in gills of cut-
throat trout (Salmo clarki) exposed chronically to endrin (Eller, 1971) and in
gills of goldfish exposed chronically to mirex(V;an Valihet al., 1968). Alteration
of visceral tissue included severe lysis and sloughing of the muco'sal epithelium
of the anterior small intestine (Fig. 1) and apparent inflamation of the underly-
ing lamina propria in three of six fish.
Fish examined at the end of the exposure (from concentrations of 0.135,
0.075, and.0.0135 ug/1) and at the end of the depuration (from concentrations of
0.135 and 0.075 ug/1) showed no significant differences from control fish.
Dieldrin is a persistent chlorinated hydrocarbon insecticide (Wurster,/1971)
and, as shown by our study, is acutely toxic to an estuarine mollusc, two crus-
taceans and a fish. Concentrations of dieldrin shown by our study to be acutely
toxic to estuarine animals, as well as concentrations which are chronically toxic,
should be kept out of the estuarine environment.
430
-------
Table I. Acute toxicity of dieldrin to and uptake by American oysters
(Crassostrea virginica), pink shrimp (Penaeus duorarum), grass
shrimp (Palaemonetes pugio), and sheepshead minnows (Cyprino-
don variegatus) during 96-hour exposures. Effect is expressed as
percentage reduction in shell growth for oysters and death for
shrimps and fish. Whole-body residues are from animals alive
at end of exposure.
SPECIES WATER CONCENTRATION EFFECT WHOLE-BODY
(ug/1) (%) RESIDUE
C. virginica
P. duorarum
P. pugio
C. variegatus
Nominal
Control
1.0
3.2
10.0
32.0
Control
0.01
0.32
1.0
3.2
10.0
Control
3.2
10.0
32.0
100.0
320.0
Control
1.0
3.2
10.0
32.0
100.0
Measured
<0.01
0.23
5.8
6.7
13.0
<0.01
0.014
0.19
0.9
2.5
11.4
ND'
2.8
7.1
27.1
57.4
65.7
<0.01
0.52
2.2
6.0
17.6
13.1
0
18
0
24
61
0
0
25
55
70
100
0
20
30
85
100
100
0
0
0
10
65
100
(ug/g, wet weight)
0.022
4.95
13.85
20.0
80.5
0.016
<0.01
0.08
0.25
0.76
0.09
2.1
3.3
1.1
3.8
12.8
34.0
62.4
'Not detectable;<0.005 ug/l.
431
-------
Fable 2. Acute toxicity of dieldrin to American oysters (Crassostrea
virginica), pink shrimp (Penaeus duorarum), grass shrimp (Palae-
monetespugio), and sheepshead minnows (Cyprinodon variegatus).
Effect is expressed as percentage reduction in shell growth for oy-
sters and death for shrimp and fish. Confidence limits (95%) are
in parentheses.
SPECIES
96-HOUR EC50 TEMPERATURE SALINITY
(ug/1) (°C) (o/oo)
Nominal Measured Mean Range Mean Range
12.50 31.20 16.6 14.5-19.0 30.8 30.0-32.5
C. virginica
P. duorarum
P. pugio
(4.80-20.2) 0.60-61.80)
0.93
0.70
(0:52-1.48) (0.39-1.15)
11.39
8.64
(7.47-16.71) 5.92-12.05)
C. variegatus 23.57
(17.47-32.03)
10.00
19.6 18.2-21.0 26.0 22.0-30.0
22.5 21.4-23.5 30.8 28.5-33.0
13.8 12.0-15.5 31.5-33.0
Table 3. Uptake and depuration of dieldrin by spot (Leiostromus xanthurus)
exposed to 0.135, 0.075, 0.135, 0.74 or 1.35 ug/1 in flowing sea
water. Residue concentrations (wet-weight) are the average of two
samples of pooled tissue from three fish.
LIVER
MUSCLE
WHOLE-
BODY
DAYS
Exposure
4
11
18
25
35
Depuration
13
Exposure
4
11
18
25
35
Depuration
13
Exposure
4
11
18
25
35
Depuration
13
CONCENTRATION, ug/g
Control
NO
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Control
ND
ND
ND
ND
ND
ND
.0135
0.08
_b
0.42
0.15
0.31
0.029
_b
0.029
0.029
0.030
.0135
0.029
_D
0.031
0.033
0.045
.075
0.52
3.90
1.10
1.20
0.55
ND
0.07
0.44
0.12
0.11
0.15
ND
.075
0.43
0.15
0.07
0.15
0.12
ND
.135
0.98
15.3
2.0
1.4
0.47
ND
0.16
1.45
0.15
0.24
0.20
ND
.135
0.63
0.52
0.23
0.29
0.27
ND
.75
1.8
12.9
17.5
5,8C
0.81
1.40
1.20
0.81C
.75
1.2
1.9
2.0
0.6C
1.35
10.2
2.6
1.35
2.9
"Sample lost.
Analysis of one sample only.
432
-------
Table 4. Concentration (ug/1) of dieldrin in test water on days 4, 11, 18,
25 and 35 of uptake and depuration exposure of spot (Leiostomus
xanthurus).
NOMINAL
Control
0.0135
0.075
0.135
0.75
1.35
MEASURED
ND
0.015
0.075
0.11
0.55
0.70
ND
0.019
0.052
0.12
0.45
ND
0.010
0.070
0.13
0.50
ND
0.016
0.057
0.15
0.68
AVERAGE
ND
0.016
0.067
0.11
ND
0.015
0.064
0.12
0.55
0.70
O^^r.SL,^'.,-
Photomicrographs of tissues from spot (Leiostomus xanthurus)
exposed to dieldrin. 1: Normal gill tissue from fish exposed 35
days to 0.075 ug/1. (X450) 2: Gill tissue from fish exposed 4 days
to 1.35 ug/1. Note subepithelial edema in lamellae. (X450) 3.
Normal small intestine tissue from fish exposed 35 days to 0.075
ug/1. (X450) 4: Small intestine tissue from fish exposed 4 days to
1.35 ug/1. Note severe lysis and sloughing of mucosal epithelium.
(X450).
433
-------
LITERATURE CITED
Butler, Philip A. 1973. Organochlorine residues in estuarine mollusks, 1965-72
— National Pesticide Monitoring Program. Pestic. Monit. J. 6(4): 238-362.
1962. Reaction of some estuarine mollusks to environmen-
tal factors. In: Biological problems in water pollution. Third Seminar.
U. S. Dept. Health, Educ., Welfare, Public Health Serv. Publ. No.
999-WP-25, 1965:92-104.
Eller, Lafayette L. 1971. Histopathologic lesions in cutthroat trout (Salmo
clarki) exposed chronically to the insecticide endrin. Am. J. Pathol.
64(2): 321-336.
Finney, D. J. 1971. Probit analysis. (Third ed.) Cambridge Univ. Press,
Cambridge, England. 333 p.
Grzenda, A. R., W. J. Taylor, and D. F. Paris. 1972. The elimination and
turnover of I4C - dieldrin by different goldfish tissues. Trans. Amer. Fish.
Soc. 101(4): 686-690.
Hansen, David J. and A. J. Wilson, Jr. 1970. Significance of DDT residues
from the estuary near Pensacola, Fla. Pestic. Monit. J. 4(2): 51-56.
Harrington, R. W. and W. L. Bidlingmayer. 1958. Effects of dieldrin on
fishes and invertebrates of a salt marsh. J. Wildl. Manage. 22(1): 76-82.
Mills, P. A., J. F. Onley and R. A. Gaither. 1963. Rapid method for chlorinated
pesticide residues in non-fatty foods. J. Assoc. Offic. Agric. Chem. 46(2):
Parrish, Patrick R. 1973. AroclorR 254, DDT and ODD, and dieldrin: ac-
cumulation and loss by American oysters (Crassostrea virginica) exposed
continuously for 56 weeks. (Abstract) Proc. Natl. Shellfish. Assoc.
(In Press).
U. S. Food and Drug Administration. 1970. Pesticide Analytical Manual.
U. S. Dept. of Health, Educ., Welfare, Sect. H212.
Van Valin, Charles C., A. K. Andrews, and L. L. Eller. 1968. Some effects
of mirex on two warm-water fishes. Trans. Amer. Fish. Soc. 97(2):
185-196.
Wurster, Charles F. Aldrin and dieldrin. Environment 13(8): 33-45.
434
-------
CONTRIBUTION NO. 179
-------
Reprinted from MOSQUITO NEWS, Vol. 34, No. 3, September, 1974
EFFECTS OF GROUND APPLICATIONS OF MALATHION
ON SALT-MARSH ENVIRONMENTS IN
NORTHWESTERN FLORIDA1
M. E. TAGATZ, P. W. BORTHWICK, G. H. COOK AND D. L. COPPAGE
U. S. Environmental Protection Agency, Gulf Breeze Environmental Research Laboratory,
Sabine Island, Gulf Breeze, Florida 32561
(Associate Laboratory, National Environmental Research Center, Corvallis, Oregon)
ABSTRACT. Effects of thermal fog {6 wt.
oz/acre (420 g/ha)} and ULV aerosol spray
{0.64 fl. oz/acre (57 g/ha)} applications of mal-
athion 95 (o,o-dimethyl phosphorodithioate of
diethyl mercaptosuccinate) on salt-marsh environ-
ments near Pensacola Beach, Florida, were in-
vestigated. Studies were conducted on selected
plots after each of three treatments using a port-
able thermal fogger and three ultra low volume
(ULV) sprays with a truck-mounted generator.
The ULV sprays were typical of usual mosquito-
control operations. The loggings were on a small
scale and results should be considered as indica-
tive of what may occur under usual conditions.
Deaths due to malathion were not observed
among confined blue crabs, Callinectes sapidus;
grass shrimps, Palaemonetes vulgaris and P.
pugio', pink shrimp, Penaeus dttorarum; or
sheepshead minnows, Cyprinodon variegatus.
Brain acetylcholinesterase activity was not reduced
in confined C. variegatus exposed to one or more
treatments. Confined animals and the snail, Lit-
torina irrorata, contained no measurable malathion
at our limit of detectability. The chemical was
not detected in sediment, but concentrations as
high as 4.10 parts per million (ppm) were found
in Jttncus sp., trace amounts persisting as long as
14 days (>o.05 but o.i but
-------
310
MOSQUITO NEWS
VOL. 34, No. 3
MATERIALS AND METHODS
Each thermal fog or ULV spray was
applied near the time of low tide to per-
mit maximum settling and retention in
the marsh and near sunset, when sprays
are usually applied to coincide with the
gieatest activity of mosquitoes and with
optimum winds and temperatures which
provide for the spray to remain close to
the ground. Salinity, water temperature
and pH were measured at the time of the
second fogging and each of the ULV
sprayings.
Water, grass, sediment and animals
were analyzed by gas chromatography
with a flame photometric detector in the
phosphorus mode to determine concentra-
tions of malathion. Based on our levels
of detection, the terms nondetectable
(N. D.) and trace (Tr.) amounts of mal-
athion in the estuarine components sam-
pled are defined as follows: 1.5! water
sample, not above o.r parts per billion,
ppb (N. D.) and >o.i but o.o5
but o.2 but
-------
SEPTEMBER, 1974
MOSQUITO NEWS
made of nylon screen over acrylic frames;
crabs and fish, in large cages made of
hardware cloth over wooden frames. To
prevent cannibalism, shrimp and crabs
were confined in individual compart-
ments. After the first application, unac-
countably high mortality of grass shrimp
occurred in treated and control groups
after 7 days; therefore, juvenile pink
shrimp, Penaeus duorarum, were used in
the second treatment. Shrimp studies
were terminated 3 days after the second
fogging because of vandalism of some
pink shrimp cages. Data on mortalities
of grass shrimp were obtained only dur-
ing 7 days after the first fogging; and
data for pink shrimp, 3 days after the sec-
ond fogging. Mortalities among crabs
and fish were recorded prior to the second
and third treatments; survivors 6 hours
after the third treatment were analyzed
for malathion residues. Shrimp were not
analyzed for residues.
Sheepshead minnows (300 in each of
two cages) were held in the treated plot
for a study on activity of brain acetyl-
cholinesterase (AChE) in fish exposed to
one or more applications of the chemical.
Using procedures reported in Coppage
(1972), 25 fish (representing five sam-
ples) were used to measure AChE activity
at each of the following times: i, 4 and 7
days after the first fogging, 12 hours and
3 days after the second fogging, and 6 and
12 hours after the third fogging. Cop-
page (1972) found that reduction of
AChE activity in sheepshead minnows
below a specific level (about 18% of nor-
mal) indicates death or impending death
from organophosphate poisoning.
Samples of sediment, Juncus, marsh
water (at base of Juncus), and canal wa-
ter were analyzed for malathion at 6
hours, 12 hours, I, 3, 7 and 14 days after
each fogging operation or until malathion
was not detected. Except for canal water,
each type of sample was a composite of
material from three locations. These lo-
cations were selected randomly from a
grid pattern of 54 divisions per plot.
II. ULV AEROSOL SPRAYS. ULV mala-
thion (Cythion® Technical 95%) was ap-
plied to a Juncus-dominsited salt-marsh by
a truck-mounted Leco HD ULV cold
aerosol generator.4 Discharge was toward
the rear at an upward angle of 45°. Ac-
tual discharge rate was 0.5 gallon per
hour at 2% mph (1.9 1/hr at 4 km/hr)
which is equivalent to 2.0 gph at 10 mph
(7.6 1/hr at 16 km/hr), the maximum al-
lowable rate for ground ULV application
in Florida. Pressure in the insecticide
tank was 4 pounds/square inch (0.28 kg/
cm 2). Discharge rate for the flowmeter
setting used was calibrated for tempera-
ture. Volume discharged was 160 ml and
spray time was 5 minutes.
Three sprays were applied (May 15,
June ii and 25, 1973) to approximately
81/2 acres (3.4 ha) of marsh by employees
of the West Florida Arthropod Research
Laboratory, Panama City, Florida. The
rate was equivalent to 0.64 fl. oz/acre (57
g/ha) based on a swath of 330 feet (100.6
m). For the first and second treatments,
the Range Point marsh served as the con-
trol plot and a similar Juncus-dominated
marsh about 5 miles (8 km) east of Range
Point was the treated plot. Range Point
was selected as the treated area for the
third spray because of a more favorable
wind direction for chemical drift; the
marsh east thereof was the control. Both
sites were connected to Santa Rosa Sound
by inlets that allowed tidal exchange.
Wind velocities during the three spray-
ings averaged 6.3, 6.0 and 10.2 mph (10.1,
9.6 and 16.4 km/hr).
Prior to each spray, grass shrimp (adult
P. pugio), blue crabs (15-25 mm wide)
and sheepshead minnows (25-40 mm to-
tal length) in 18 in. (45.7 cm) diameter
polyethylene tubs containing 25 liters of
water were placed in the marshes. In
each marsh, tubs were positioned in two
rows of three adjacent tubs. The rows
were 50 ft. (15.2 m) apart, and animals
in each row consisted of 25 shrimp in
* Lowndes Engineering Company, Inc., Val-
ilosta, Georgia.
-------
312
MOSQUITO NEWS
VOL. 34, No. 3
one tub, 15 crabs in a second tub, and 20
fish in a third tub. Immediately after
treatment, screens were placed over tubs
to keep out predators. Mortalities were
determined i and 3 days after spray. One
day after treatment, duplicate living sam-
ples of 15 shrimp, 5 crabs or 10 fish were
removed for chemical analyses.
Seventy sheepshead minnows in a 5.4
sq. ft. (0.5 m2) polyethylene pool contain-
ing 91 liters of water were centered be-
tween the rows of tubs in the treated
marsh. AChE activities in the fish were
measured at 6 hours and i day after each
spray.
At selected intervals after treatment
samples of sediment, Juncus, water from
the marsh, water from tubs, and snails
(Litlorma irrorata collected from Juncus)
were analyzed for malathion. A com-
posite water sample (1.5 1) was obtained
from each of the two groups of tubs; com-
posite samples of marsh water and other
materials were obtained randomly in the
vicinity of the tubs.
To determine the effectiveness of our
ULV sprays for mosquito control, caged
mosquitoes were placed on 5-ft. (1.5 m)
poles in the marsh for the third spraying
by personnel of the West Florida Arthro-
pod Research Laboratory. One hundred
forty Aedes taeniorhynchus in six cages
and 131 Culex nigripalpus in six cages
were used in the treated plot, and as a
check, approximately the same numbers
of mosquitoes were held in 12 cages away
from the treated plot.
RESULTS
I. THERMAL Foe APPLICATIONS. Physi-
cal and chemical characteristics of canal
water in the treated and control plots for
the second fogging were: temperature,
24.5°-25.o° C; salinity, 27.5-28.0 ppt
(parts per thousand); and pH, 7.2-7.6.
No effects of malathion on caged ani-
mals were observed. Mortality of crabs
and fish did not differ greatly between
control and treated groups after the first
and second foggings (Table i). Treated
crabs (average width 83.2 mm, range 44-
115) and control crabs (77.0 mm, 41-113
mm) each molted seven times in the 28-
day period. In our limited shrimp studies,
single deaths of grass shrimp occurred in
each plot after 7 days, and no deaths of
pink shrimp occurred after 3 days. Fish
and crabs obtained 6 hours after the third
treatment contained no detectable mala-
thion. No decided inhibition of AChE
activity in brains of sheepshead minnows
was detected after any of the three treat-
rnents.
No deaths that could be attributed to
the treatments were observed among resi-
dent populations of shrimp, crabs, and
fish.
Malathion did not persist for long in
sediment, Juncus or water after each ap-
plication. The chemical was not detected
in sediment after 6 hours. However,
trace amounts occurred in samples . of
funcus after 14 days (Table 2). Mala-
thion was not detected in water after i
TABLE i. Mortality of confined animals in salt-marshes after thermal fog applications of malathion
95 at 6 wt. oz/acrc (420 g/ha).
Animal
Blue crabs
Control
Treated
Sheepshead minnows
Control
Treated
14 days after
ist treatment
No. dead
3
3
4
5
15
15
7
8
14 days after
2nd treatment
No. dead %
5 25
2 10
3 5
6 10
Total
No. dead
8
5
7
ii
%
4°
2=;
12
18
-------
SEPTEMBER, 1974
MOSQUITO NEWS
3*3
day. After 6 and 12 hours, residues of II. ULV AEROSOL SPRAYS. Ranges of
the toxicant ranged from .
3 days
o. 16
0.71
7 days
N.D.
Tr.2
Tr.
1 4 days
N.D.
Tr.
Tr.
1 N.D. (non detectable) =not above 0.05 ppm.
2Tr. (trace)=>o.05 but
5°
30
3°
40
40
50
5°
30
30
40
40
50
50
Deaths
o-i day
2
2
0
0
I
0
I
0
0
0
0
0
o
4
0
I
0
0
Number
I day1
18
18
20
20
'9
20
19
20
20
20
20
20
20
16
20
'9
20
20
Deaths
1-3 days
0
i
0
0
8
0
5
0
0
0
6
3
2
2
0
0
2
0
1 Number of animals remaining after i day upon removal of dead animals and living samples (10
crabs, 20 minnows or 30 shrimp) for residue analyses.
2 Most shrimp listed as dead were not found; some may have escaped or been eaten.
-------
MOSQUITO NEWS
VOL. 34, No. 3
animals. Few deaths occurred among
treated animals (Table 3). Some deaths
of crabs in both marshes were due to
cannibalism. The bodies of most shrimp
listed as dead were not found; some may
have escaped or been eaten. No deaths
of resident crabs, fishes, and shrimps were
noted after any treatment. Confined
crabs, fish and shrimp and free-living
snails obtained i day after each spray
contained no detectable malathion. AChE
activities in brains of sheepshead min-
nows were not altered by any of the three
sprays.
Residues of malathion in sediment, wa-
ter, or ]uncus 3 days following ULV ap-
plications generally were low or not de-
tected (Table 4). Malathion was not
detected in sediment i or 6 hours after
treatments. None was detected in water
or from Juncus after the first spray.
After the third spray, however, it per-
sisted at least 3 days at concentrations up
to 0.34 ppb in tub water and 0.28 ppm in
Juncus samples. Malathion was not de-
tected in control samples.
Deaths of caged mosquitoes after the
third spraying were 100% treated and
0.1% check for A. taeniorhynchus, and
89.3% treated and o% check for C. nigri-
palpus. Mortalities were within the range
found in other field tests using caged
mosquitoes and ULV ground equipment
(Rathburn and Boike, 1972).
DISCUSSION
No adverse effects of malathion on con-
fined animals or on the salt-marsh envi-
ronment were observed under the condi-
tions of these studies. In addition, no
deaths were noted among resident crabs,
fishes and shrimps after any of the
treatments. Malathion was not detected
in animals or sediment. In general, when
found in plant samples or water, concen-
trations were low and did not persist. We
found trace amounts of the chemical in
Juncus samples for as long as 14 days
after treatment. Although Bender (1969)
found that carp, Cypnnus carpio, accu-
TABLE 4. Malathion in samples of tub water
(from each of two rows of tubs), marsh water
and Juncus from salt-marshes after ULV
sprays of malathion 95 at 0.64 fl.
oz/acre (57 g/ha).
Sample and
time elapsed
Tub water I
i hour
6 hours
12 hours
i day
3 days
Tub water 2
i hour
6 hours
12 hours
i day
3 days
Marsh water
6 hours
12 hours
i day
3 days
litncus
6 hours
12 hours
i day
3 days
Spray i
N.D.1
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.2
N.D.
N.D.
N.D.
Spray 2
Tr.1
Tr.
Tr.
Tr.
N.D.
Tr.
N.D.
N.D.
N.D.
N.D.
0.49 ppb
Tr.
N.D.
Tr.2
Tr.
N.D.
N.D.
Spray 3
1.52 ppb
0.58 ppb
u . 73 PPb
0.48 ppb
Tr.
0.32 ppb
Tr.
o . 36 ppb
u . 32 ppb
0.34 ppb
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
0.41 ppm
0.28 ppm
1 N.D. (non detectable) =not above o.io ppb;
Tr. (trace) =:>o.10 but <0.30 ppb.
2 N.D. (non detectable) —not above 0.05 ppm;
Tr. (trace) =>o.05 but
-------
SEPTEMBER, 1974
MOSQUITO NEWS
with truck-mounted equipment over
larger areas.
ACKNOWLEDGMENTS
Mr. Arthur Peagler and Mr. Howard
Shinton, Escambia County Mosquito Con-
trol, Pensacola, Florida, together with Dr.
Andrew Rogers, Dr. Carlisle Rathburn,
Jr., and staff members, West Florida Ar-
thropod Research Laboratory, Panama
City, Florida, contributed outstanding co-
operation in the study. Mrs. Dana Tyler-
Schroeder and Mr. Terrence Hollister
aided in the field work. The American
Cyanamid Company kindly supplied
malathion.
References Cited
Bender, M. E. 1969. Uptake and retention of
malathion by the carp. Prog. Fish-Cult. 31(3):
I55-I59-
Conte, F. S. and Parker, J. C. 1971.' Ecological
aspects of selected Crustacea of two marsh em-
bayments of the Texas coast. Texas A & M
Univ., Sea Grant Publ. No. TAMU-SG-7I-
211: 184 p.
Coppage, D. L. 1972. Organophosphate pesti-
cides: Specific level of brain AChE inhibition
related to death in sheepshead minnows. Trans.
Amer. Fish. Soc. 101 (3) :534~536.
Coppage, D. L. and Duke, T. W. 1971 Effects
of pesticides in estuaries along the Gulf and
Southeast Atlantic Coasts. Proc. 2nd Gulf
Conf. Mosq. Suppr. Wildl. Manage., N. O. La.,
Oct. 20-22:24-31.
Darsie, R. F., Jr. and Cornden, F. E. 1959. The
toxicity of malathion to killifish (Cyprinodonti-
dae) in Delaware. J. Econ. Entomol. 52(4):
696-700.
Fultz, T. O., Jr., McDougal, M. L. and Thrift,
E. C. 1972. Observations on ground ULV
applications in Chatham County, Georgia.
Mosq. News 32(4) :5oi~504.
Guerrant, G. O., Fetzer, L. E., Jr. and Miles,
J. W. 1970. Pesticide residues in Hale
County, Texas, before and after ultra-low vol-
ume aerial application of malathion. Pestic.
Monit. J. 4(0:14-20.
Hill, E. F., Eliason, D. A. and Kilpatrick, J. W.
1971. Effects of ultra-low volume applications
of malathion in Hale County, Texas. III. Effect
on nontarget animals. J. Med. Entomol. 8(2):
I73-I79-
Rathburn, C. B., Jr. and Boike, A. H., Jr. 1972.
Ultra-low volume tests of malathion applied
by ground equipment for the corrtrol of adult
mosquitoes. Mosq. News 32(2) :l83-l87.
Taylor, R. T. and Schoof, H. F. .1971. The
relative effectiveness of malathion thermal aero-
sols and ground-applied ULV aaginst three spe-
cies of mosquitoes. Mosq. News, 3i(3):34O-
349-
-------
CONTRIBUTION NO. 180
-------
current marine science
Criteria for Determining
Importance and Effects of Pesticides
on the Marine Environment:
A Brief
Overview
Scientists continually emphasize the importance of pesticide
residues that occur in the marine environment by assigning these
residues high priority in terms of needed surveillance and research. In
most instances, the analytical technology for determining residues of
pesticides in the parts per trillion range is available, but we often do
not fully understand the significance of these levels in relation to
biological and ecological effects.
fish whose life
Much time and effort are being
devoted to developing meaningful cri-
teria for assessing the impact of pesti-
cides on organisms and their environ-
ment. One approach to developing
these criteria is to assess the effect of
pesticides through laboratory research,
experimental ecosystems, and environ-
mental studies:
I. Laboratory Research
1. Standard "Bioassay"
A. Acute
B. Chronic
C. Criteria for Effects
D. Indicator Organisms
E. Application Factor
II. Environment Ecosystems
1. Compartmental Analysis
2. Community Bioassay
III. Environmental Studies
1. Baselines
2. Impact of Pesticide Applica-
tion in the Environment
Laboratory Research
The first step in assessment after
subjecting a candidate pesticide and its
degradation products to chemical char-
acterization is to perform a "standard"
bioassay. Known amounts of the pesti-
cide are administered to selected test
organisms maintained in static or flow-
ing water systems for a given period of
time. Periodically, test organisms are
examined and compared with "con-
trol" organisms to determine if an
effect has occurred.
These tests are often termed
acute because they are conducted for a
short period of time in relation to the
life span of the organism. For ex-
ample, when fish whose life span is
normally a year or more are subjected
to a test for 96 hours, it is considered
an acute bioassay. The objective of
these tests is to provide the researcher
with information on toxicity levels
which subsequently can be used to
conduct more comprehensive bio-
assays. Chronic tests are actually an
extention of the acute tests and are
conducted through a reproductive
cycle or through some developmental
stage
time.
for a relatively long period of
In the past, mortality of the test
population or a part of the population
was considered the prime criterion for
an effect, but this is no longer con-
sidered adequate. Other criteria, in-
cluding oxygen evolution (by phyto-
plankton), changes in growth rate,
respiration rate, shell deposition,
blood protein configuration, inhibition
of enzyme systems and pathological
changes, give the researcher more com-
plete information on an effect. Efforts
are being made by investigators in the
field to compile acceptable procedures
for bioassay tests incorporating these
criteria so that a more flexible bio-
assay procedure will be available to
marine scientists.
Care Required
Special care must be taken in
performing even preliminary bioassay
tests. The organisms used should be in
good physiological condition and
representative numbers should be
analyzed for background levels of con-
taminants, especially the pesticide
being tested. The quality of the water,
including pesticide content, must be
known before it enters the test appa-
ratus. In addition, the amount of
THOMAS W. DUKE
pesticide in the water of the test
apparatus must be checked to insure
that the desired concentration is
present. Accurate chemical analysis of
the pesticide content of water and test
organisms is imperative.
Representative organisms from
several trophic levels should be em-
ployed in the bioassay test when pos-
sible and should include organisms
known to be sensitive to a specific
group of compounds. Crustaceans,
particularly shrimp and crabs, are
usually more sensitive to organochlo-
rines than are other marine organisms.
Larval and juvenile stages are generally
more sensitive than adult forms. Un-
fortunately, we are unable at the
present time to culture a wide array of
test organisms for saltwater bioassays,
and in many instances, cannot main-
tain in the laboratory sensitive life
stages of needed test organisms. As a
result, we often have data on the acute
effects of the chemical with adults
only and must extrapolate to deter-
mine what the effect might be over a
longer period of time on more sensi-
tive stages.
An application factor is useful to
those involved in setting standards for
pesticide levels in marine waters. An
application factor is a ratio of a safe
concentration of a pesticide to the
acutely lethal concentration. One can
estimate a value for an "acceptable"
level of a pesticide in marine waters by
multiplying the lethal concentration
determined in acute bioassays by the
appropriate application factor. In
many cases an arbitrary application
factor of 0.01 is used when the neces-
sary scientific data has not yet been
developed. Much more effort should
be devoted to obtaining necessary
January 1974
21
-------
scientific data for determining applica-
tion factors for specific pesticides.
Also, users of application factors
should be made aware that this ratio
does not include a safety factor as
such, but is only a fractional factor
applied to a lethal concentration.
Additional information on the de-
velopment of application factors is
available in the literature (1), (2).
Experimental Ecosystems
Pesticides entering into the
natural environment can affect not
only individual animals but com-
munities of animals and ecosystems.
The interactions of the various com-
munities with each other and with
their physical environment could be
affected by a pesticide. One approach
to quantify such effects is to construct
an experimental ecosystem in which
several species of organisms and their
substrates can be subjected to the
pesticide. Quantitative information on
rates, routes and reservoirs of accumu-
lation can be obtained. Bioaccumula-
tion data are easily obtained by resi-
due analysis of individual organisms in
such environments. These studies
could lead to predictive models on the
effect of pesticides.
Sophisticated equipment is not
necessarily required to obtain informa-
tion on the effect and movement of
pollutants in experimental environ-
ments (3). This investigator observed
how Aroclor 1254, a polychlorinated
biphenly, affected the composition of
communities of estuarine animals in
water that flowed through test
aquaria. Communities of planktonic
larvae that developed in control
aquaria and aquaria that received one-
tenth of a microgram of PCB per liter
in the water were dominated (greater
than 75 percent) by arthropods. The
number of arthropods decreased and
the number of chordates (tunicates)
increased as the concentration in-
creased from one to ten micrograms
per liter; over 75 percent of the
animals in 10 micrograms per liter
aquaria were tunicates. Although
species diversity was not altered, num-
bers of phyla, species and individuals
were decreased by this PCB.
Environmental Studies
Much more information is
needed on the manner in which an
"unstressed" marine system operates
before we can properly assess the
impact of a pesticide or other chem-
icals on such systems. We require
integrated scientific studies leading to
the development of predictive models
that could assess possible effects of
specific environmental stresses. Often,
it is necessary to make observations on
the routes, rates and reservoirs of
pesticides used in large scale applica-
tions in the environment after applica-
tion is made. Such studies should not
be termed "ecological" because time
will not permit baseline data to be
developed before the pesticide is
applied.
Surveillance and analysis of resi-
dues in and near the application area
can, however, give some insight con-
cerning effect of the pesticide on
non-target species. Such a study was
made in Louisiana during aerial appli-
cation of malathion to control
mosquito vectors of Venezuelan
equine encephalomyelitis (4). Fish
were collected from spray areas a short
time before, during and after the
spraying operation. Acetylcholine-
sterase (AChE) activity in the brains of
these fish was used as an indicator of
the occurrence of malathion in the
fish's environment. Levels of inhibi-
tion of AChE activity in fish from
Lake Prien, Louisiana approached
levels associated with death of fish in
laboratory bioassay studies. However,
the AChE level of this species of fish
returned to normal within 40 days
after the application of malathion.
This is an example of how laboratory
bioassays and field observations can be
used to better understand the impact
of a particular pesticide on the marine
environment.
Future Challenge
The kinds and amounts of pesti-
cides used in this country are changing
and the change is reflected in produc-
tion figures. Recently, production of
herbicides and organophosphate in-
secticides has exceeded that of organo-
chlorine insecticides. Pesticide appli-
cators are substituting organophos-
phates and carbamates for more per-
sistent chemicals, such as DDT. Also,
much effort is being devoted to de-
veloping an integrated pest control
procedure whereby biological and
other control methods will play just as
important a role as chemicals. Several
companies are developing biological
control organisms, such as viruses, and
isolating juvenile insect hormones to
be used in control programs for certain
agricultural pests. This commendable
strategy could result in a much
"cleaner" environment.
We must be prepared to assess
control methods before they gain
widespread usage in the environment.
In many instances, we can no longer
depend upon routine monitoring
methods to detect the presence of
these biological control agents and
new chemicals. Our concern must in-
clude the potential effect of the new
agents on an environment that already
contains residues of organochlorines
and other persistent chemicals. D
Gulf Breeze Contribution No. 180
References
Brungs, William A. 1969. "Chronic Toxicity
of Zinc to the Fathead Minnow,
Pimephales promelas Raflnesque."
Trans. Am. Fish. Soc., 98(2):
272-279.
Mount, Donald I. 1968, "Chronic Toxicity
of Copper to Fathead Minnows
Pimephales promelas Raflnesque.''
Water Research, 2(3): 215-223.
Hansen, David J. (In press — Contributions
in Marine Science, University of
Texas). Aroclor 1254: Effect on
Composition of Estuarine Animal
Communities in the Laboratory.
Coppage, D. L. and T. W. Duke. 1972.
Effects of Pesticides in Estuaries
Along Gulf and Southeast Atlantic
Coasts. Proceedings of the 2nd Gulf
Coast Conference on Mosquito Sup-
pression and Wildlife Management,
New Orleans, La., October 20-22,
1971.
THOMAS W. DUKE is presently director of
the Environmental Protection Agency's Gulf
Breeze Environmental Research Laboratory,
Gulf Breeze, Florida. The laboratory's mis-
sion is to study the effects of toxic organics,
particularly pesticides, on marine organisms
and their environment. Information devel-
oped at the laboratory is used in EPA's
pesticide registration process and in setting
water quality criteria for the marine envi-
ronment. Dr. Duke has been employed by
the Federal government since 1961. He
received his Ph.D. in Oceanography from
Texas A. & M. University and worked with
the A. & M. Research Foundation for a year
before joining the Federal government. Dr.
Duke's research interest is in the field of
estuarine ecology-particularly pollution
ecology which includes studies of the move-
ment of radioactive materials and pesticides
in the estuarine environment.
22
MTS Journal v. 8 n.l
-------
CONTRIBUTION NO. 181
-------
Avoidance of Aroclor® 1254 by Shrimp and Fishes1
by D. J. HANSEN, S. C. SCHIMMEL, and E. MATTHEWS
U.S. Environmental Protection Agency
Gulf Breeze Environmental Research Laboratory
Sabine Island, Gulf Breeze, Fla. 32561
(Associate Laboratory of the National Environmental
Research Center, Corvallis, Ore.)
The polychlorinated biphenyl (PCB) Aroclor 1254 was found in
Escambia River and Bay, which are parts of the estuary near our
laboratory (DUKE et al. 1970). In laboratory experiments this
chemical was toxic to certain mollusks (LOWE et al. 1972), arthro-
pods (NIMMO et al. 1971) and fishes (HANSEN et al. 1971). Some
invertebrates (HANSEN et al. 1973 and PORTMAN 1970) and fishes
(HANSEN 1969; HANSEN et al. 1972; and SPRAGUE and DRURY 1969)
possess the ability to avoid other toxic pollutants in water.
Because it could be an advantage to mobile organisms in the river
and bay if they could avoid toxic concentrations of Aroclor 1254,
we conducted laboratory studies to determine if pink shrimp
(Penaeus duorarum), grass shrimp (Palaemonetes pugio), pinfish
(Lagodon rhomboides), sheepshead minnows (Cyprinodon variegatus)
and mosquitofish (Gambusia affinis) could avoid water contaminated
with 0.001, 0.01, 0.1, 1 or 10 mg/J> of the PCB.
Methods and Materials
Test animals were collected from local waters not contaminated
by Aroclor 1254 and were acclimated to laboratory conditions for at
least seven days before testing. If mortality exceeded 5% or ab-
normal behavior was observed in the 48 hours before a test the
animals were not used. Pink shrimp were between 13 and 75 mm
rostrum-telson length; grass shrimp 10-40 mm rostrum-telson length;
mosquitofish and pinfish 20-50 mm standard length. Animals were
not fed for 24-hours prior to testing.
The ability of these animals to avoid Aroclor 1254 was tested
in a black plastic apparatus (HANSEN et al. 1972) in which animals
could move from a holding area into either (1) a section that con-
tained water with Aroclor 1254 or (2) a section that contained
water without the PCB. Water maintained at 20° C, entered the
upper end of each of the two sections at the rate of 400 ml/min and
flowed to the drain in the holding area. Freshwater was used to
test mosquitofish and 20 °/oo saltwater was used to test the other
species. PCB dissolved in acetone was metered through stopcocks
at 0.5 ml/min into the water entering one of the two sections. The
same amount of solvent without PCB was metered into the other section.
Each species was tested at each concentration at least four
times; twice with the PCB entering one section of the apparatus
Contribution No. 181, Gulf Breeze Environmental Research
Laboratory.
253
Bulletin of Environmental Contamination & Toxicology*
Vol. 12, No. 2 © 1974 by Springer-Verlag New York Inc.
-------
and twice with it entering the opposite section. For each of the
four replications, 50 animals (except pink shrimp, when 25 animals
were used for each replicate test) were placed in the holding area
with a closed gate located at the junction of the holding area and
the two sections. After 30 minutes the gate was opened, allowing
access of animals to both sections. One hour later the gate was
closed and the number of animals in each section was recorded.
The apparatus was covered with black plastic during each test to
shield the animals from external disturbances.
Concentrations of Aroclor 125A selected were not lethal to
any animal during the 1 1/2 hour avoidance study but 0.01 mg/5,
was toxic to pink shrimp, (NIMMO et al. 1971) grass shrimp,
(NIMMO et al. In press) sheepshead minnows (SCHIMMEL In press)
and pinfish (HANSEN et al. 1971) in chronic bioassays conducted
for longer periods of time at this laboratory.
Some of the concentrations in the sheepshead minnow and grass
shrimp tests were checked by chemical analysis. Methods of
chemical analyses were identical to those used by NIMMO et al 1971.
The ability of these animals to avoid Aroclor 1254 was
evaluated by the chi-square test on the assumption that if there
was no avoidance response to the PCB, animals that left the holding
area would enter each section with equal frequency. Avoidance was
considered significant if the probability that observed distri-
butions would occur by chance was 0.01 or less. Animals remaining
in the circular holding area after a test was completed were not
included in the statistical analyses.
Results and Discussion
Grass shrimp, pinfish and mosquitofish avoided at least one
concentration of Aroclor 1254 but pink shrimp and sheepshead
minnows did not avoid any of the concentrations tested (Table 1).
Grass shrimp and pinfish avoided 10 mg/Jt and mosquitofish 0.1, 1
and 10 mg/l of the PCB. In mosquitofish tests, PCB was added to
fresh instead of salt water. Therefore, it is not known if the
fish had a greater ability to avoid or if the response was
affected by the test water.
This study demonstrates that some animals can avoid Aroclor
1254 in laboratory tests but we can only speculate on the possi-
bility of avoidance of PCB's in the estuary. Concentrations of
Aroclor avoided by mosquitofish in the laboratory have been found
in the Escambia River near the source of a leak of this chemical
(DUKE et al. 1970). Concentrations measured in water from other
localities in the river and Escambia Bay never approached con-
centrations avoided by grass shrimp, pinfish or mosquitofish in
the laboratory. If animals avoid because they sense the PCB in
254
-------
TABLE 1
Capacity of aquatic animals to seek water free
of the polychlorinated biphenyl, PCB, Aroclor 1254
Test
Species
Palaemonetes
pugio
Penaeus
duorarum
Cyprinpdon
variegatus
Lagodon
rhomboides
Ganbusia
affinis
Aroclor 1254
Concentration
(mg/fc)
0.001
0.01
0.1**
1.0**
10.0**
0.001
0.01
0.1
1.0
10.0
0.001
0.01**
0.1**
1.0
10.0**
0.001
0.01
0.1
1.0
10.0
0.01
0.1
1.0
10.0
Number of
In PCB-Free
Water
60
55
65
57
91
22
34
41
33
37
51
42
48
49
43
66
55
67
121
84
38
66
66
43
Animals*
In Water
with PCB
64
64
56
62
51
33
34
31
36
32
44
40
48
48
55
76
48
69
119
44
34
27
26
13
Percentage Tn
PCB-Free Water
48.4
46.2
53.7
47.9
64.1***
40.0
50.0
56.9
47.8
53.6
53.7
51.2
50.0
50.5
43.9
46.5
53.4
49.3
50.4
65.6***
52.8
71.0***
71.7***
76.8***
* Does not include animals in holding area at end of test.
** Nominal Concentration • Average Measured Concentration:
10.0 = 5.7, 1.0 <= 0.48, C.I •= 0.054, 0.01 = 0.033.
*** Statistically significant; x2-test, a =0.01.
255
-------
water, it seems unlikely that contamination by this chemical
altered the movements of these animals except immediately adjacent
to the site of the leak. Animals may avoid because they sense the
toxic effect of a PCB and move to reduce this effect. If so,
concentrations that can be avoided may possibly be much lower than
shown in these tests where fish were in the PCB for a maximum of
only 1 1/2 hours.
References
DUKE, T. W., J. I. LOWE and A. J. WILSON, JR.: Bull. Environ.
Contain. Toxicol. _5, 171 (1970).
HANSEN, D. J.: Trans. Amer. Fish. Soc. 98, 426 (1969).
HANSEN, D. J., P. R. PARRISH, J. I. LOWE, A. J. WILSON, JR. and
P. D. WILSON: Bull. Environ. Contam. Toxicol. £, 113 (1971).
HANSEN, D. J., E. MATTHEWS, S. L. NALL and D. P. DUMAS: Bull.
Environ. Contam. Toxicol. £46 (1972).
HANSEN, D. J., S. C. SCHIMMEL and JAMES M. KELTNER, JR.: Bull.
Environ. Contam. Toxicol. £, 129 (1973).
LOWE, J. I., P. R. PARRISH, J. M. PATRICK, JR. and J. FORESTER:
Mar. Biol. r7_, 209 (1972).
NIMMO, D. R., R. R. BLACKMAN, A. J. WILSON, JR. and J. FORESTER:
Mar. Biol. 11, 191 (1971).
NIMMO, D. R., J. FORESTER, P. T. HEITMULLER and GARY H. COOK:
Bull. Environ. Contam. Toxicol. (In Press).
PORTMAN, J. E.: Marine Pollution and Sea Life, FAO 212 (1972).
SCHIMMEL, S. C.: Trans. Amer. Fish. Soc. (In Press).
SPRAGUE, J. B. and D. W. DRURY: Adv. Water Pollut. Res. Proc.
Fourth Int. Conf., 169 (1969).
'Registered trademark, Monsanto Company, St. Louis, Mo. Mention
of trade names does not constitute endorsement by the Environ-
mental Protection Agency.
256
-------
CONTRIBUTION NO. 183
-------
BIOMP:THICS 30, 547-551
SEPTF.MBKR 1974
Reprinted Jrum
BIOMETRICS Copyright © 1974
THE BIOMETRIC SOCIETY, Vol. 30, No. 3, September 1974
365: LESLIE MATRIX MODELS FOR FISHERIES STUDIES
A. L. JlCNSKN1
Environmental Protection Agency, Gulf Breeze Environmental Research laboratory,1 Sabine Island, Gulf
Breeze, Florida 32561, U.S.A.
SUMMARY
Two modifications of the Leslie matrix model are developed. In the first modification the egg stage
as Well as the age groups of a fish population are included in the vector of state. In the second modification
only the recruited members of the population are included in the vector of state.
1. INTRODUCTION
In the Leslie Matrix Model (Leslie [1945; 1948]) the vector of the number of individuals
of each age at time t, N, , is related to the vector of the initial number of individuals of
each age, N0 , by the equation,
N, = M'N0,
(1)
where M is the population projection matrix,
Bu B, B.2 ••• • B,,t B,
Pn 0 0 ••• - 0 0
0 P, 0 •• -0 0
M = 0 0 00
.000 •• 0 P,_, 0
(2)
In matrix M, 72, equals the number of females born to females of age .c in one unit of time
that survive to the next unit of time, P , equals the proportion of females of age x at time t
that survive to time / + 1, and c is the greatest age attainable. Equation (1) lias frequently
been applied in demographic and animal population studies (Keylitz [190NJ, Pielou [19G9],
Usher [1971]). Jensen [1971] has applied equation (1) to a lish population.
• Equation (1) is similar to the simple exponential model for population growth. Leslie
[19")9] proposed a modified matrix model to allow for the eflVet of population density on
population' growth. He divided each element in the population projection matrix by a
quantity that depended on the size of the current population and I he >ize of t he population
when the individuals were horn. Sevr.il other modi I ica lions of 1 lie matrix modi •! have been
proposed. Williamson [19">!)j and (ioodman
l!)<>'.)| modified the matrix model to
include both SCXPS. and Lefkoviteh | !'.)(>">] developed a modification for organisms grouped
rn«iMil :i.l'lrv«--
Si-lmnl nf N':itiir:il Hi-min-rs. I'mviT-iu ,.f MiHiii;:ii:. Ann ArLi.r. .MirliiL'aii ISKII
i >r\ "f tl.r Na1mn:il Kjivin.iiini'iiUvI Un-i-arcli CI-IIHT. < 'unalli.-. Ori ./.in
-------
548
B10MKTR1CS. SEl'TEAIIiKR. 1974
by life stages rather than age. Goodman [1<)(W| developed a general class of models that
can he applied to organisms grouped by life stages. Usher ]!%!)] modified the model for
study of forest trees which are classified by size rather than by age.
In this note two modifications of the matrix model are proposed: (1) a model for fish
populations in which individuals are grouped by a life stage as well as by age, and ('_') a
model for the recruited members of a fish population. Goodman's [190!)] general class of
models can also include both life stages and ago. A fish is recruited when it becomes large
enough to be vulnerable to fishing gear. This group of fish is of importance because often
data are available only for the age groups captured by a fishery.
A detailed analysis of fisheries data requires separation of males and females as in
actuarial science and demography, but this is not often practiced in fisheries science because
the limited data do not generally allow such a detailed analysis. It will be assumed that
the number of females equals the number of males and that growth and mortality rates
of males and females are equal, but the models also apply to either sex alone.
•J. ONE LIFE STAGE AND A(iE GROUPS
It is assumed that the eggs hatch in the time period after the adults spawn. For example,
if the time period is one year and the fish spawn in the fall, the eggs hatch the following-
spring. A similar model can be constructed for species in which tin1 adults spawn and the
eggs hatch during the same year.
The number of eggs produced at time t — 1 is proportional to the sizes of the age groups
at time t — 1, i.e.,
"7V(0, t - I)'
E(t - 1) = (hn ,
N(\, t - 1)
IN(V, t- 1
where some of the constants h, , i = 0, 1, 2, • • • , v, may be zero. The constant l<, is the
number of eggs produced by the population per individual of age ;. The size of the zero
age group at time t is a function of the number of eggs produced at time / — 1, and the
size of each nonzero age group at time t is a. function of the size of the preceding age group
at time t — 1, i.e.,
~N(Q, I) } r,S'(, 0 •
AT(1. 0
0 ,
-------
MATHIX MODEL FOR ]{KCRUITKI) POPl'LATKiX
,549
/ — 1 to age i. The survival functions may be functions of time. Mathematical forms of
these survival functions are discussed by Ricker [1954], Beverton and Holt [19."J7J, Paulik
and Greenough [1900], and Pcnnyeuick et al. [1908J.
Equations (3) and (4) can be combined into a single equation. Multiplication of the
matrices,
0
/>',
0
0 0 0~
1
0 0
0 0
i
0 0
0 S, Oj
h,, h,
1 0
0 1
0 0
0 0 •
_0 0 •
• • /!,-i h.
•• • 0 0
0 0
0 0
• 0 1 0
• 0 0 0 _
gives the matrix,
A =
~h0S,,
s,
0
0
Mo ••
0
52
0
• h..,Sn
0
0
0
h..tSn
0
0
0
M«"
0
0
0
,' °
! °
I o
0
0
0
0
0
0
S.
0
0
0
(6)
Applying ('(iiiation (0), the equation for population projection (equation 1) becomes,
N, = AX (7)
The above analysis shows that the matrix for population projection, A, is the product
of: (1) a survival matrix which represents movement of individuals out of the life stage
and among the age groups, and ('2) a reproduction matrix which represents input of new
individuals to the population.
Applying equation (7), the discrete age-discrete time equation for annual yield from
a fishery (Jensen [1971]) becomes,
where r is age in years, and .'/,(.!') is the .rtli element in the vector Y, ,
Y, = FWA'N,, (!))
In equation (9), F is a diagonal matrix with the age specific mortality rates /•'(.(•) on the
diagonal and W is a diagonal matrix with the age .-.pecilk' weights H '(•'') on the diagonal.
-------
550
BIOMETRICS, SEPTEMBER 1974
3. THE RECRUITED POPULATION
Equation (8) is of more practical value in fisheries studies if only age groups in the
recruited population are considered. The Leslie Matrix Model cannot be directly applied
to only the recruited population. The number of recruits at time t depends on the number
of eggs produced by the population at some previous time t — r, where ?• is the age of the
recruits. The sizes of all other age groups in the recruited population depend on the sizes
of the preceding age group in the previous unit of time. Hence, recruitment and mortality
of the recruited population must be separated.
Both Usher [1966] and Goodman [1969] have shown that the population projection
matrix is the sum of two matrices,
M = R + D.
(10)
The first matrix represents input of new members to the population and the second matrix
represents transition of members between the age groups. For application to a recruited
fish population, the square matrices R and D can be defined as:
(11)
R =
D =
hrSr hr + iSr • • • hfS
0 0 ••• 0
_ 0 0 ••• 0
" 0 0 ••• 0 0
Srt, 0 ••• 0 0
0 Sr>? 0
00 0
00 0
00 0 S.
r
o"
0
0
0
0
0.
(12)
The function Sr gives survival from the egg to recruitment, and the functions £,,,,? =
1, 2, • • , c, give survival from age /• + i - 1 to age r + i. Mathematical forms of tin-
survival function Sr are discussed by Rickcr [19.">4], Beverton and Holt [1!),">7|, and Paulik
and Greenough [1966].
Applying equations (10), (11), and (12) the vector of the number of individuals of
each age at time t for the recruited population becomes,
N, = RN,_r + DN,_, (i:j)
Applying equation (13), the discrete age-discrete time equation for annual yield from a
fishery becomes,
IV = Z //,'M
I - T
where ;//(z) is the .cth element in the vector Y,',
Y/ = FW(RN,_r + DN,.,).
(14)
(15)
-------
MATRIX MODEL FOR RECRUITED POPULATION 551
LES MODELES DE MATRICE DE LESLIE POUll LES ETUDES DE PECHERIES
RESUME
On decrit deux modifications au modele matriciel de Leslie. Dans la premiere on inclut dans le vecteur
d'etat le stade des oeufs aussi bien que les groupes d'age de la population de poisson. Dans la seconds on
ii'inclut dans le vecteur d'etat que les nouveaux membres de la population.
REFERENCES
Beverton, R. J. II., and Holt, S. J. [1957]. On the Dynamics of Exploited Fish Populations. H. M. S. O.,
London.
Goodman, L. A. [1968]. Stochastic models for the population growth of the sexes. Biomctrika 55, 469-87.
Goodman, L. A. [1969]. The analysis of population growth when the birth and death rates depend on several
factors. Biometrics Z5, 659-81.
Jensen, A. L. [1971]. The effect of increased mortality on the young in a population of brook trout, a theoret-
ical analysis. Trans. Amer. Fish. Soc. 100, 456-9.
Keyfitz, N. [1968]. Introduction to the Mathematics of Population. Addison-Wesley, Reading, Massachusetts.
Lefkovitoh, L. P. [1965]. The study of population growth in organisms grouped by stages. Biometrics 21,
1-18.
Leslie, P. H. [1945]. On the use of matrices in certain population mathematics. Biomctrika •?•/, 183-222.
Leslie, P. II. [1948]. Some further notes on the use of matrices in population mathematics. Hiomclrika 35,
21:5-45.
Leslie, P. H. [1949]. The properties of a certain lag type of population growlh and the influence of an external
random factor on a number of such populations. Physiological Zool. "2, 151-9.
Paulik, G. J., and Greenongh, J. W. [1906]. Management analysis for a salmon resource system. In Systems
Analysis in Ecology. Watt, K. E. F. (Ed.), Academic Press, New York.
Pielou, E. C. [1969]. An Introduction io Mathematical Ecology, Wiley-Interscience, New York.
Pennycuick, C. J., Compton, R. M., and Beckingham, L. [1968]. A computer model for .simulating the growlh
of a population, or of two interacting populations. J. Theorct. Biol. 18, 316-29.
Ricker, W. E. [1954]. Stock and recruitment. J. Fish. Res. Bd. Canada. 11, 559-623.
Usher, M. B. (1969). A matrix model for forest management. Biometrics 35, 309-15.
Usher, M. B. [1971]. Developments in the Leslie Matrix Model. In: Mathematical Models in Ecology. Jeffers,
J. N. R. (Ed.), Blackwell Scientific, London.
Williamson, M~. II. [1959). Some extensions of the use of matrices in population theory. Bull. Math. Kioplii/s.
27, 13-7.
Received February 1973, Revised October 1973
Key Words: Population dynamics; Leslie matrix models; Deterministic models; Recruited fish populations.
-------
CONTRIBUTION NO. 184
-------
NOTES 1669
Predator-Prey and Competition Models with State Variables:
Biomass, Number of Individuals, and Average
Individual Weight
A. L. JENSEN1
Environmental Protection Agency
Gulf Breeze Environmental Research Laboratory, Gulf Breeze, Fla. 32561, USA2
JENSEN, A. L. 1974. Predator-prey and competition models with state variables: bio-
mass, number of individuals, and average individual weight. J. Fish. Res.
Board Can. 31: 1669-1674.
Applying the identity that biomass equals number of individuals multiplied by average
individual weight, simultaneous equations for change with respect to time in biomass,
number of individuals, and average individual weight are obtained for Kostitzin's predator-
prey equations and for the Lotka-Volterra competition equations. By the same procedure
applied here, simultaneous equations for these three variables can be obtained for other
predator-prey and competition equations These equations can be applied to determine
the biomass, number of individuals, and average individual weight of interacting fish
populations under different rates of exploitation.
JENSEN, A. L. 1974. Predator-prey and competition models with state variables: bio-
mass, number of individuals, and average individual weight. J. Fish. Res.
Board Can. 31: 1669-1674.
A partir du postulat que la biomasse est egale au nombre d'individus multiplie par
leur poids moyen, on obtient des equations simultanees representant les changements dans
le temps de la biomasse, du nombre d'individus et du poids moyen des individus pour les
equations predateur-proie de Kostitzin et les Equations de Lotka-Volterra relatives a la
concurrence. Une procedure semblable, appliquee ici, permet de formuler des equations
simultanees pour ces trois variables applicables a d'autres modeles pr6dateur-proie et
concurrence. Ces equations peuvent servir a determiner la biomasse, le nombre d'individus
et le poids individuel moyen de populations de poissons agissant les unes sur les autres,
lorsque celles-ci sont soumises a des taux d'exploitation differents.
Received January 29, 1973 Recu le 29 Janvier, 1974
Accepted July 10, 1974 Accepte le 10 juillet, 1974
ALL species that are fished, live in close association interacting species are all combined in a common
with other species, and studies of interactions expression. Larkin's studies were done using the
among fish species are important to fisheries manage- number of individuals of each species as the vector
ment (Larkin 1963, 1966). Larkin (1956, 1963, of state. The decision to construct models for fish
and 1966) has applied predator-prey and competi- population interactions in terms of number of
tion models to determine the theoretical result of individuals rather than in terms of biomass is an
exploiting either or both of a pair of competing important decision. Jensen (1972) has shown that
species and either or both of a predator-prey pair, because biomass, B, number erf individuals, N, and
Application of the classical predator-prey and average individual weight, W, are related by the
competition models to fisheries studies results in identity,
surplus production models for yield in which
growth, reproduction, and natural mortality of the = —
1 Present address: School of Natural Resources, .
Natural Resources Buildina, University of Michigan, where ' ls tlme; independent models cannot be
Ann Arbor, Mich. 48104, USA. simultaneously constructed for these three variables.
2Associat'e Laboratory of the National Environmen- Jensen (1972) has derived simultaneous equations
tal Research Center, Corvallis, Orcg. for biomass, number of individuals, and average
individual weight implied by Schaefer's surplus
Printed in Canada (J2819) production equation. He showed that if change
Imprime au Canada (J2819) with respect to time in biomass of a population is
-------
1670
J. FISH. RES. BOARD CAN., VOL. 31(10), 1974
described by the logistic equation, then change in
number of individuals with respect to time can be
described by the logistic equation only if there is
no change with respect to time in average individual
weight or if change in number of individuals is
independent of change in average individual weight.
These are not realistic assumptions for fish popula-
tions.
In this note simultaneous equations for biomass,
number of individuals, and average individual
weight are developed for the Lotka (1956) and
Volterra (1928) competition equations, and for
Kostitzin's (1939) predator-prey equations. Larkin
(1963) applied the Lotka-Volterra equations to
study interspecific competition and exploitation,
and Larkin (1966) applied Kostitzin's predator-prey
equations to study exploitation in a predator-prey
situation. Larkin (1966) concluded that Kostitzin's
predator-prey equations are more realistic for fish
populations than the Lotka-Volterra predator-prey
equations. Fish prey on a variety of organisms and
extinction of a single prey species may not lead to
extinction of the fish population. In the Lotka-
Volterra predator-prey model extinction of the
predator results in exponential growth of the prey
and extinction of the prey results in extinction
of the predator. In Kostitzin's predator-prey equa-
tions the population density of the prey has an
upper limit in the absence of the predator, and the
population density of the predator has a lower limit
in the absence of the prey. Kostitzin's predator-prey
equations are nearly identical in mathematical form
to the Lotka-Volterra competition equations. The
method illustrated here to obtain equations for
biomass, number of individuals, and average in-
dividual weight can be applied to obtain similar
equations for other predator-prey and competition
models.
Mathematical symbols are listed below; the first
species is the prey species or the first of two com-
peting species, and the second species is a predator
species or the second of two competing species.
coefficients of interaction between first
and second species
6,, b2
Bt, B2 = biomass of first and second species
Fj, F2 = fishing mortality coefficients for bio-
mass of first and second species
F3, F4 = fishing mortality coefficients for num-
ber of individuals of first and second
species
Fs, F6 = fishing mortality coefficients for average
individual weight of first and second
species
gi< ff2 = coefficient of growth in average indivi-
dual weight of first and second species
hi, h2 = coefficient of intraspecific interaction
for first and second species
ki = coefficient of increase in number of
individuals of first species
k2 = coefficient of increase in number of
individuals of second species in com-
petition model and coefficient of
mortality of predator in predator-
prey model
number of individuals of first and
second species
coefficients of intraspecific interaction
for first and second species
coefficients of interaction between first
and second species
coefficient of biomass increase of first
species
coefficient of biomass increase of se-
cond species in competition model and
coefficient of negative feedback for
biomass of predator in predator-prey
model
coefficient of interaction between first
and second species
Wi, W2 = average individual weight of first and
second species
Relation among biomass, number of individuals,
and average individual weight — Differentiation of
equation (1) with respect to time gives the equation,
Nj, N2
Pi> P2
1i> #2
HI,
dB
dt
= N
(2)
If equations are specified for dW/dt and dN/dt,
the equation for dB/dt is implicitly specified by
equation (2). If equations are constructed indepen-
dently for both dW/dt and dN/dt, changes in aver-
age individual weight and number of individuals
are independent. Such equations imply two inde-
pendent upper limits —one that determines the
maximum number of individuals and one that deter-
mines the maximum average individual weight
(Jensen 1972). If an equation relating growth to
environmental carrying capacity is constructed for
biomass there is a single upper limit, termed the
environmental carrying capacity. An equation with
-------
NOTES
1671
a single upper limit and mutual dependence of
number of individuals and average individual weight
provides a more realistic description of fish popula-
tions than a model with two upper limits and mutual
independence of average individual weight and num-
ber of individuals. Therefore, the Lotka-Volterra
competition equations and Kostifzin's predator-
prey equations are developed in terms of biomass;
then mutually dependent equations, corresponding
to the biomass equations, are developed for number
of individuals and average individual weight.
The equations for change in number of individuals
and the equations for change in average individual
weight are not uniquely determined by equation
(2) and the equation for biomass change. To develop
equations for change in number of individuals and
equations for change in average individual weight
it is necessary to make additional assumptions.
These assumptions concern: 1) the form of the
equations for number of individuals, 2) the mutual
dependence of average individual weight and
number of individuals, and 3) the patterns of alge-
braic signs in the equations for change in average
individual weight. Many different systems of simul-
taneous equations, which correspond to different
assumptions, can be derived for biomass, number of
individuals, and average individual weight. The
systems of equations derived in this note are simple
systems in which the mathematical form of the
equations for dB/dt, dN/dt, and dW/dt are similar,
dW/dt does not equal zero, and NI, N2, Wt, and
W2 are all interrelated.
It is noteworthy that the equations for dN/dt
and dB/dt have been testedjvith empirical observa-
tions. The equations for dW/dt have not previously
appeared in the literature, and these equations have
not been tested with empirical observations. Al-
though the form of the equations for dW/dt result
from the form of the equations for dN/dt and dB/dt,
the equations for dW/dt must be considered as
untested hypotheses until they are tested with
empirical observations.
Predator-prey and competition models have been
applied interchangeably to biomass and number of
individuals. Therefore, it appears reasonable when
developing simultaneous equations for biomass,
number of individuals, and average individual
weight, to assume that the equations for change
in number of individuals are of the same mathemati-
cal form as the equations for change in biomass.
For change in average individual weight and change
in number of individuals to be mutually dependent
both the equation for change in number of indivi-
duals and the equation lor change in average indivi-
dual weight must contain terms for average indivi-
dual weight and number of individuals. The above
assumptions, together with equation (2), uniquely
determine the equations for change in number of
individuals. For example, if biomass is defined
by the logistic equation, dB/dt = a(B-<32B2, then,
to be of the same mathematical form as the biomass
equation, contain both number of individuals and
average individual weight, and satisfy equation (2),
the equation for change in number of individuals
must be dN/dt = ijN-^N'W.
When the equations for dB/dt and dN/dt are
specified, the form of the equation for dW/dt is
implicitly specified to within a narrow degree, and
only a small amount of biological information is
necessary to complete its specification. For example,
if for the logistic equation biomass and number
of individuals are described by the equations given
above, to satisfy equation (2) average^ individual
weight must be given byV W/<# = 0, dVJ/dt = ± c
W, or dW/dt = ± ci W ± c2W2N. Only the last
equation for dW/dt satisfies the condition that N
and W are mutually dependent. It is biologically
realistic to assume that input to average individual
weight is proportional to average individual weight
and that an increase in number of individuals
results in a more intense intraspecific competition
and a lower rate of change in average individual
weight. Therefore, the equation for chang£ in
average individual weight is dW/dt = CjW-c2W2N.
Kostitzin's predator-prey equations — Under the
assumptions : 1) the equations for change in number
of individuals are of the same form as the equations
for change in biomass, 2) the variables NI, N2,
Wj, and W2 are interrelated, and 3) selection of
algebraic signs in the equations for change in
average individual weight is correct, application
of equation (2) shows that Kostitzin's predator-prey
equations for change in biomass, number of indivi-
duals, and average individual weight are:
dBi/dt
dB2ldt
- F2B2
- ?iNiB2 -
dW2/dt =
_ (3)
± KiWiB2 - FjWi
- /*2W2B2 - F6W2.
The constants are related by the equations: rt =
ki + gi, r2 = k2-g2, al=pl + hl: a2 = p2 + h2,
bi =
-------
1672
J. FISH. RES. BOARD CAN., VOL. 31(10), 1974
equations (3) for Bj shows the following additional
constraints on the constants,
F2 + r2
F3-kj
-fli
-Pi
F4
± «lB2 Ffi -
«2
where B2 = (-r2di-b2Fi + r162-F2«i)/(«i«2 + 61*2)-
In equations (3), the biomass equations are
identical to the equations obtained when Kostitzin's
predator-prey equations are applied to biomass
alone. But the equations for change in number of
individuals are different from the equations that
would be obtained if Kostitzin's equations had
been applied to number of individuals alone. Both
the equation for change in number of prey and the
equation for change in number of predators con-
tain terms for prey biomass and predator biomass.
In the equation for change in number of prey,
the birth rate of the prey is proportional to the
number of prey. Mortality of the prey depends on
both the product of the number of prey with prey
biomass and the product of the number of prey
with predator biomass.
In the equation for change in number of preda-
tors, the birth rate of the predator depends on the
product of the number of predators with prey
biomass. Mortality of the predator depends both
on the number of predators and the product of
the number of predators with predator biomass.
These equations for change in number of prey
and for change in number of predators are bio-
logically realistic. Birth rate of the prey is propor-
tional to the number of prey. Mortality of the prey
increases with either an increase in the number of
prey or with an increase in prey biomass. Higher
prey biomass and a larger prey population density
both result in more intense intraspecific competi-
tion. Mortality of the prey also increases with
predator biomass. A larger predator biomass
requires a larger amount of food.
For both natural and experimental fish popula-
tions it has been established that average individual
weight is related to the number of individuals
(Nikolskii 1969; Swingle and Smith 1941). And
the carrying capacity of an environment for fish
appears to be related to the biomass of the fish
population rather than to the number of individuals
(Swingle and Smith 1941). Equations for change in
number of individuals that are a function of biomass
as well as a function of number of individuals are,
therefore, more complete and realistic than equa-
tions that do not contain terms for biomass.
If Kostitzin's equations had been applied to
number of individuals alone, mortality of the prey
would have been a function of neither predator
biomass nor prey biomass. But if average individual
weight of the predator and prey are not constant,
the number of prey does not accurately determine
the amount of food the prey supply the predator
and the number of predators does not accurately
determine the food requirements of the predators.
In equations (3), the equations for both biomass
and number of individuals are similar to the equa-
tions obtained when Kostitzin's equations are
applied to each of these variables alone, and the
equations for each variable alone have been tho-
roughly investigated both biologically (Gause 1964)
and mathematically (Goel et al. 1971). Equations
for change in average individual weight of a predator
and its prey have not been previously proposed,
and they must be considered more carefully than
the equations for change in biomass and number
of individuals.
The equations for change in average individual
weight cannot be developed independently of the
equations for biomass and number of individuals.
The equations for change in average individual
weight are developed by: 1) specifying the equations
for change in biomass and the equations for number
of individuals, 2) the requirement that the variables
be mutually dependent, and 3) selection of algebraic
signs based on biological assumptions.
The mathematical form of the equations for
change in average individual weight is the same as
the mathematical form of the equations for biomass
and number of individuals, but the algebraic signs
are different. Equations (3) indicate that an increase
in the prey biomass results in more intense intra-
specific competition and a slower change in average
individual weight of prey. The equation for change
in average individual weight of the prey also con-
tains the term ± w1W1B2. The sign of this term is
positive if predators select larger prey, the sign is
negative if predators select smaller prey, and this
term is zero if on the average predators select all
size-groups of prey equally often.
Change in average individual weight of the
predator depends on both average individual weight
of the predator and the product of average individual
weight of the predator with biomass of prey; this
second term realistically indicates that change in
average individual weight of the predator depends
on the biomass of food available. Loss in average
individual weight of the predator depends on the
product of the average individual weight of the
predator with predator biomass. This term realisti-
cally indicates that as predator biomass increases,
-------
NOTES
1673
intraspecific competition increases and change in
average individual weight decreases.
Lotka-Volterra competition equations — Under
the assumptions applied to obtain equations (3),
application of equation (2) shows that the Lotka-
Volterra competition equations for change in
biomass, number of individuals, and average
individual weight are:
= riB1-aiBi-6iB1B2-FiBi
= AriN1-p1BiN1-?1N1B2-F3N1
_
= 5-iWi-/iiWiBi-HiWiB2-FsWi
= g-2W2-/!2W2B2~H2W2Bi-F6W2.
The constants are related by the equations: rj =
^1 + ffi, r2 = k2 + g2, « i = Pi + hi, a2 = p2 + h2,
b\ = q\ + «i, 62 = 2B2N2. These
terms indicate that the intensity of intraspecific
competition depends on both the number of in-
dividuals and the biomass of the species. The equa-
tions for change in population density of the first
species contains the interspecific competition term
<7iNiB2 and the equation for change in population
density of the second species contains the inter-
specific competition term q2N2fti. These terms
indicate that the intensity of interspecific competi-
tion depends on the number of individuals of the
species and on the biomass of the competing species.
In equations (4), the change in average individual
weight of both competing species is proportional
to their average individual weight. Loss in average
individual weight of each species depends on the
biomass of both species; as the biomass of both
species increases, both intraspecific and interspecific
competition increase. Equations (4) are more
realistic for fish populations than the equations
obtained when the Lotka-Volterra equations are
applied to number of individuals alone. Changes in
number of individuals are related to population
biomass. Equations (4) contain terms for biomass,
whereas equations obtained when the Lotka-Vol-
terra model is applied to number of individuals
alone do not contain ternis for biomass.
Discussion — Relations among biomass, number
of individuals, and average individual weight are
important in fisheries biology. For fish populations
the environmental carrying capacity is the maximum
biomass that the environment can sustain, and the
same resources can support large populations of
small fish or small populations of large fish of the
same species (Nikolskii 1969; Swingle and Smith
1941). Fishing a population decreases the number
of fish and, by selection of older and larger indivi-
duals, decreases the average individual weight.
Expanding surplus production models to include
number of individuals and average individual weight
as well as biomass may, therefore, increase the use-
fulness of these models for fisheries management.
The expanded models can be applied to determine
number of individuals, biomass, and average in-
dividual weight for predator-prey and competing
species that result from different exploitation rates.
For example, for Kostitzin's predator-prey model
at equilibrium under average environmental con-
ditions,
Bi =
B2 =
Ni
-r\a\a2-r2aibi-a\b\P 2
N2 = — =:
_
1 ~
W2 =
The above equations are as easily applied as the
surplus production equations for biomass alone,
but data are needed for number of individuals and
average individual weight as well as for biomass.
-------
1674
J. FISH. RES. BOARD CAN., VOL. 31(10), 1974
Accurate estimation of the constants in equations
(3) and (4) is difficult. A linearization and curve
fitting procedure similar to the one applied by
Schaefer (1957) to estimate the constants in the
Schaefer surplus-production model can be applied
to estimate the constants in equations (3) and (4).
The equations are linearized by estimation of the
derivatives with the two point formula (Schaefer
1957; Hamming 1962). Schaefer's method of data
partition does not produce unique estimates of the
constants (Pella and Tomlinson 1969). Least squares
can be applied to the linearized equations to estimate
the constants as illustrated by Pella and Tomlinson
(1969). The two point formula for linearization
results in underestimation of the derivative (Ham-
ming 1962; Pella and Tomlinson 1969) and as a
general rule numerical integration should be applied
instead of numerical differentiation whenever poss-
ible (Hamming 1962). Pella and Tomlinson (1969)
have developed a method for approximation of the
general surplus-production model in which numerical
integration is applied instead of numerical differen-
tiation. Equations (2) and (3) cannot, however, be
solved in closed form and the method of Pella and
Tomlinson cannot be applied.
In summary, biomass, number of individuals,
and average individual weight are mutually depen-
dent variables. For most fish populations the en-
vironmental carrying capacity is the maximum
biomass sustainable by the environment rather than
the maximum number of individuals. Population
models for fish should, therefore, be constructed in
terms of biomass or in terms of biomass and number
of individuals. Surplus production models that are
constructed for number of individuals alone are
not more easily constructed than models for bio-
mass alone, and they ignore the interrelations
among average individual weight, biomass, and
number of individuals.
Data for application of competition and predator-
prey equations to natural or experimental fish pop-
ulations do not appear to exist. This lack of data
limits application of models for interactions of
exploited fish populations to theoretical considera-
tions such as those of Larkin (1956, 1963, and
1966). Continued development of models for inter-
action among species is necessary for theoretical
studies, and the models can also serve as guides for
the design of studies on experimental and natural
fish populations.
Acknowledgments — I thank J. J. Pella for useful
criticism of an earlier version of this note, for indicating
several errors, for indicating the need to discuss the
development of the equations in more detail, and for
indicating the restrictions on the parameters. W. W.
Fox Jr. gave helpful criticism of a manuscript closely
related to this one.
GAUSE, G. F. 1964. The struggle for existence.
Hafner, New York, N. Y. (Originally published 1934)
GOEL, N. S., S. C. MAITRA, AND E. W. MONTROLL.
1971. On the Volterra and other nonlinear models
of interacting populations. Academic Press, New
York, N.Y. 145 p.
H/nfMTNG, R. W. 1962. Numerical methods for
scientists and engineers. McGraw-Hill, New York,
N.Y.
JENSEN, A. L. 1972. Population biomass, number of
individuals, average individual weight, and the
linear surplus-production model. J. Fish. Res.
Board Can. 29: 1651-1655.
KOSTTTZTN, V. A. 1939. Mathematical biology. Har-
rap, London. 411 p.
LARKIN, P. A. 1956. Interspecific competition and
population control in freshwater fish. J. Fish. Res.
Board Can. 13:327-342.
1963. Interspecific competition and exploitation.
J. Fish. Res. Board Can. 20: 647-678.
1966. Exploitation in a type of predator-prey
relationship. . J. Fish. Res. Board Can. 23: 349-356.
LOTKA, A. J. 1956. Elements of mathematical
biology. Dover Publications, New York, N.Y.
(Originally published 1925)
NncoLSKn, G. V. 1969. Theory of fish population
dynamics. Translated by J. E. S. Bradley. Oliver
and Boyd Ltd., Edinburgh, Scotland. 323 p.
PELLA, J. J., AND P. K. TOMLINSON. 1969. A gener-
alized stock production model. Inter-Amer. Trop.
Tuna Comm. Bull. 13: 421-496.
RICKER, W. E. 1958. Handbook of computations
for biological statistics of fish populations. Bull.
Fish. Res. Board Can. 119: 300 p.
SCHAEFER, M. B. 1957. A study of the dynamics of
the fishery for yellowfin tuna in the eastern tropical
Pacific Ocean. Inter-Amer. Trop. Tuna. Comm.
Bull. 2: 245-285.
SWINGLE, H. S., AND E. V. SMITH. 1941. Experi-
ments on the stocking of fish ponds. Trans. N.
Am. Wildl. Nat. Resour. Conf. 5: 267-276.
VOLTERRA, V. 1928. Variations and fluctuations of
the number of individuals in animal species living
together. J. Cons. Int. Explor. Mer 3: 1-51.
-------
CONTRIBUTION NO. 190
-------
Accumulation of Mirex-14C in the
Adult Blue Crab (Callinectes sapidus)
by WlLHELM P. SCHOOR
V.S. Environmental Protection Agency
Gulf Breeze Environment Research Laboratory
Sabine Island, Gulf Breeze, Fla. 32561
(Associate Laboratory of the National Environmental
Research Center, Corvallis, Ore.)
Carrier-solubllized mlrex is absorbed from a disperse aqueous
system by juvenile (Lowe et al. 1971), and larval stages (Bookhout
et al. 1972) of the blue crab. Since in both cases only whole-body
residues were determined, it was thought to be of interest to
establish the actual distribution of mirex in the tissues.
Experimental
Adult blue crabs were exposed to mirex-^C (Mallinckrodt Inc.)
having a specific activity of 6.34 mCi/mM in a final concentration
of 0.05-0.25 ppb and 0.3% polyethylene glycol 200 in filtered sea
water that was diluted with distilled water to give 10 ppt salinity.
All tests were conducted in battery jars containing 3& of solution
at 25°C. Exposure time ranged from 15 minutes to 16 hours.
Tissue samples were counted in the following manner. Hemo-
lymph serum was obtained by centrifuging the clotted hemolymph.
About 0.5 g of hepatopancreas , 0.2 g brain and thoracic ganglion,
0.5 g muscle, and 1.0 ml of hemolymph serum were each added to
2.0 ml Soluene 100 (Packard Instrument Co.) and digested at 40°C
overnight. Ten ml of scintillation fluid (5.5 g PPO, 0.1 g
dimethyl POPOP, 667 ml toluene, and 333 ml triton X-100) were
added with 1.0 ml hexane for clarification, and the amount of
mirex-14c determined with a Packard Tri-Carb Scintillation Spectro-
meter. Any quenching was compensated for by means of an internal
standard .
Results and Discussion
Uptake of mirex-^-^C by organs from solutions that contained
0.22 ppb (measured) of mirex-l^C was as follows:
Hemolymph serum (10 crabs) 0.24 - 0.69
Muscle (2 crabs) 0.65 - 1.1 yg/kg
Brain and thoracic ganglion 0. 75 - 19 yg/kg
(5 crabs)
Hepatopancreas (6 crabs) 1.6 - 31 yg/kg
Mention of commercial products or trade names does not constitute
endorsement by the U. S. Environmental Protection Agency.
136
Bulletin of Environmental Contamination & Toxicology,
Vol. 12, No. 2 © 1974 by Springer-Verlag New York Inc.
-------
Mirex-^C is, I believe, absorbed through the gills because
the hemolymph serum showed traces of mirex-l^C after 5 minutes of
exposure, the hepatopancreas after 15 minutes. No difference was
noted between male and female crabs. Response to the toxicant
usually progressed through Increased aggressiveness to decreased
aggressiveness; followed by loss of equilibrium and death, although
some crabs recovered. Crabs in 1% Carbowax 200 solutions showed no
behavioral differences from those in solutions without it.
The rate of uptake and the distribution of mirex-^C in the
blue crab is similar to that observed for pink shrimp (Penaeus
duorarum) exposed to Aroclor® 1254. (Nimmo et al. 1971).
References
BOOKHOUT, C. G., WILSON, A. J. JR., DUKE, T. W. and J. I. LOWE:
Water and Soil Pollution ±, 165 (1972).
LOWE, J. I., WILSON, A. J. JR. and J. M. PATRICK, JR. Unpublished
results, Gulf Breeze Environmental Research Laboratory, Gulf Breeze,
Florida 32561 (1971).
NIMMO, D. R., BLACKMAN, R. R., WILSON, A. J. JR. and J. FORESTER:
Marine Biology 11, 191 (1971).
Registered trademark, Monsanto Company, St. Louis, Mo.
137
-------
CONTRIBUTION NO. 191
-------
EPA-600/4-74-004
October 1974
PROCEEDINGS
OF
SEMINAR ON METHODOLOGY
FOR
MONITORING THE MARINE ENVIRONMENT
SEATTLE WASHINGTON
OCTOBER 1973
Program Element No. 1HA326
ROAP/TASK - PEMP/2
SPONSORED BY
OFFICE OF MONITORING SYSTEMS
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
-------
BIOLOGICAL PROBLEMS IN ESTUARINE MONITORING
P. A. Butler *
INTRODUCTION
Awareness of the extent of persistent organochlorine
pollution both in the physical environment and the biota has
made apparent the need for continuing surveillance programs
to assess the problems. During the period 1965-72, samples
collected in the National Estuarine Monitoring Program, as
well as in other studies, revealed some of the difficulties
involved in the interpretation of residue data. Bivalve
molluscs are efficient bioassay tools for identifying the
ebb and flow of pollutants in surrounding waters, and the
monthly monitoring of molluscan populations made obvious the
dynamic nature of organochlorine pollution in the estuary.
The image of polluted versus unpolluted estuaries was soon
modified by monitoring data that indicated instead the
movement of relatively discrete masses of clean and polluted
water through the estuary. Organochlorine residues in
molluscs fluctuated from month to month in response to the
sometimes transitory nature of the pollution and contrasted
sharply at times with residues observed in other elements of
the associated biota.
To gain increased understanding of the significance of
residue levels, sample collections in problem areas were
intensified in variety, frequency, and size; and additional
work was undertaken under laboratory conditions. The
experimental program demonstrated that the uptake and
retention of persistent residues varied unpredictably with
the environmental element sampled. It became clear that
surveillance or monitoring systems had to be carefully
designed if they were to provide answers to specific program
objectives.
*0ffice of Pesticides Programs
Gulf Breeze Environmental Research Laboratory,
Gulf Breeze, Florida
Gulf Breeze Laboratory Contribution No. 191
-126-
-------
A primary concern is the extent of environmental
degradation - with the implied intention of reversing or
at least halting harmful pollution trends. But individual
agency goals are much more specific. For expedience and
because of monetary restrictions, programs are usually
designed to clarify rather narrowly defined sectors of the
whole pollution picture. Often, the foremost question
concerns only the existence of a human health problem, and
if this is not likely to occur, then some environmental
pollution problems may go uninvestigated. The idea that
any changes harmful to the environment will eventually
affect man is still not generally accepted.
The basic needs for environmental surveillance require
programs designed to provide current data that will be
adequate to identify later deleterious changes. Monitoring
data should indicate existing problems as well as their
extent in time and space. To provide information of this
tvPej protocols must take into account the influence of
various physiological and ecological factors affecting the
substrate selected for study. Some of the anomalous residue
data acquired so far are understandable, but reasons for
others are less certain. The following discussion of
factors affecting persistent organochlorine residues
indicates some of the options available in selecting the
most informative sample types.
FIELD AND LABORATORY OBSERVATIONS
Residue differentials resulting from kind £f species
monitored:
The necessity for utilizating different species to
monitor pesticides in different coastal areas prompted the
conduct of laboratory experiments to determine the relative
sensitivity of molluscan species selected for their
diversity in salinity tolerance. A number of controlled
experiments have shown the relative uniformity of DDT
residue formation in the eastern oyster (Grassestrea
virginica) under varying estuarine conditions of salinity
and temperature. Observations of four other molluscan
species exposed simultaneously to a mixture of common
organochlorine pesticides indicated considerable variation
in the relative rates at which residues were acquired, then
lost when clean water was restored to them (Table 1) (3).
Studies were directed toward evaluating residue flushing
rates in the hard clam (Mercenaria mercenaria) because of
-127-
-------
Magnification in Percentage loss
Bioassay animal Body after 5 after 7 days
days exposure in clean water
Soft clam (Mya arenaria)
Eastern oyster (Crass ostrea
virginica)
Marsh clam (Rangia ctmeata)
Asiatic clam (Corbicula fluminea)
Hard clam (Mercenaria mercenaria)
3000
1200
700
600
500
74
50
50
30
75
Table 1. Average biomagnification and depuration rates of a mixture
of seven common chlorinated pesticides by molluscs exposed
in a flowing seawater system.
-128-
-------
the prevalence of this species in the New England area and
its observed poor performance in storing DDT residues. The
biological half-life of pesticide residues in molluscs under
the same conditions is of importance in determining the
movement of pollutants in the estuary. Clams and oysters
with DDT residues of about 40 ppm were placed in aquaria
with uncontaminated flowing seawater. The clams flushed <*ut
50 percent of their DDT residue within five days. At the
end of 15 days less than 21 of the original DDT burden
remained. In contrast, the oysters still contained 50% of
the residue at the end of the 15 days and significant amounts
of DDT were present after a month. Clearly, clams would have
to be sampled more frequently than oysters to determine
pollution inputs.
Species differentials in pesticide uptake are apparent
in populations monitored in Conscience Bay, New York (6).
During the first half of 1968, monthly mussel samples
(Mytilus edulis) from this bay contained about 50 ppb of DDT
and its metabolites. In the latter half of the year and
until April 1969, hard clam was substituted because it was
easier to collect. During this period no DDT residues were
detected. In April and thereafter, the mussel was again
utilized and DDT residues were found to be once more 50 ppb
or higher. In this case, the convenience of collecting hard
clam samples 'determined1 the presence or absence of DDT
pollution in the bay.
Similar discrepancies were observed in simultaneous
collections of spot (Leiostomus xanthurus) and oysters in a
South Carolina estuary in 1968. Residues of DDT occurred in
all monthly samples of the fish and levels fluctuated
between 100 and 300 ppb although the fish sampled were of
uniform size. Oyster samples showed DDT pollution was
present only during the first six months of the year. If
DDT pollution was present after that, it was not stored by
the oyster at levels chemically detectable. Numerous other
studies have demonstrated the retention and gradual increase
of organochlorine residues in fish, at least until their
first spawning period. Consequently, it is not possible to
determine from periodic fish analyses the seasonal
occurrence of DDT pollution in an estuary.
Unexpected variations occur in the sensitivity or
selectivity of the biota in the same ecosystem to
organochlorine pollution even though the species monitored
are presumed to occupy similar tropic levels. These
differences are well-illustrated in a study of monitoring
methods conducted in Virginia (12). Sample analyses
demonstrated the existence of residues of three different
polychlorinated biphenyl compounds (Aroclors 1242, 1254,
-129-
-------
and 1260) in different types of samples. All samples were
large enough to minimize individual variation. In March,
Aroclor 1242 was detected only in anchovies (Anchoa sp.);
Aroclor 1254 was present in silversides (Menidia sp.),
oysters and two series of plankton tows. A third series of
plankton samples contained only Aroclor 1260. Three months
later, Aroclor 1242 was present in both anchovies and
silversides while Aroclor 1254 was no longer found in
silversides but was still present in oysters and plankton.
Aroclor 1260 was not detected. These variations in residue
accumulations in a short period in one river system are not
easily explained (Table 2).
Aroclor 1242
March
Fish A I/
June
Fish A
Fish S
Aroclor 1254 Fish S 2/
Oysters Oysters
Plankton Plankton
Sediment Sediment
Aroclor 1260 Plankton
17 Anchovies
2/ Silversides
Table 2. Estuarine samples containing residues of PCB's at two
sampling periods
-130-
-------
Uptake and depuration rates in molluscs under
laboratory conditions must be accepted as relative figures
and extrapolated to field conditions with caution. In one
study, for example, oysters exposed to mercury accumulated
residues of about 28 ppm (8). Relatively small declines in
residues took place in the following 160 days of depuration.
The authors concluded from the data that mercury residues
acquired by oysters in nature might require years to decline
to acceptable levels. Their experimental conditions were
described as a 'natural system1 but the oysters were
supplied only 2 H clean water/animal per hour. This volume
of water should be compared to their well-known utilization
of up to 30 Z per hour even under experimental conditions
(11). It is probable that in this experiment, the mercury
was continuously recycled and the oysters never had
opportunity to adequately flush their tissues.
The mercury exposure experiment is perhaps comparable to
a situation monitored in Mecox Bay, Long Island (6). The
waters of this bay are typically isolated from the ocean by
sand bars, and only periodically do winter storms cut the
bars and permit flushing (10). In the period 1966-72, the
oysters in this bay were continuously contaminated with DDT
and at a maximum level higher than that observed in any of
the other 16 estuarine stations monitored in the New York
area despite apparently low use records of DDT in the area.
In my opinion, these oysters were continuously contaminated
with recycled DDT because of the inadequate supply of clean
water.
Past dependence on fortuitous samples (found dead,
happened to be caught in a net, etc.) to assess pollution
levels has resulted in an uneven if not confused picture of
general environmental contamination. Broad conmunity
studies frequently show persistent organochlorine residues
differing by an order of magnitude in species of similar
habit (9,20). Consequently, valid assessment of persistent
residues in any one species of a community requires not only
an understanding of its position in the trophic structure
but also its variability as compared to similar species.
Residue differentials resulting from age of individuals
monitored.
In fish and other vertebrates, the localization of
organochlorines in highly lipid tissues and their
persistence, at least in part, for long periods is well-
documented. For example, DDT tends to accumulate gradually
in pinfish (Lagodon rhomboides) and an approximately tenfold
increase from 0-to-l-year fish has been documented (14).
-131-
-------
There is an approximate doubling of DDT residues in lake
trout in the Great Lakes each year up to age 10 (17)»
Accumulation of dieldrin residues in these fish is
proportional to age but of lower magnitude. Approximate
doubling of mercury levels during the first few years
has been reported in salmon (Salmo salar)from rivers
in Sweden (18).
In contrast, organochlorine residues in molluscs do-
not persist from year to year in the absence of pollution
despite their affinity for lipid tissues. Residue levels
fluctuate widely depending on the input of pollutants to
the estuarine system and not on the age of the mollusc.
Residue levels can be correlated frequently with pesticide
usage in the drainage system when oysters of similar size
are monitored. However, oysters of different size do react
differentially to similar pollutant levels and, in general,
residues per gram of tissue are higher in small oysters than
in oysters of signficantly larger size. Although the data
are not at hand, I assume that this difference is a function
of the larger amount of water circulated (filtered) per gram
of tissue in small oysters compared to large ones. This
higher biological magnification of residues in small oysters
might be disastrous since, with a given amount of contaminated
tissue consumed, predators would ingest much more DDT from
small as opposed to large oysters.
This demonstrated increase in persistent residues with
age complicates interpretation of data in many instances
where the age of individuals making up the sample are
unavailable and difficult to ascertain.
Residue differentials resulting from variations among
individuals.
For obvious reasons, uniform populations or aggregates
of individuals are selected as often as possible as
indicators of environmental degradation. But both
laboratory and field studies demonstrate that there can be
much individual variation in levels of organochlorine
residues. DDT residue levels in a presumably uniform sample
of yearling pinfish in a Florida estuary ranged from 13.7
ppm to 0.5 ppm (14). The standard error in these data was
more than 230% of the arithmetic mean. The average of two
of the fish was 13.2 ppm while the average residue in the
other eight fish was only 1.5 ppm DDT. Not all groups
analyzed have shown this diversity but it must be anticipated
in data interpretations. Diversity in residue levels of
molluscan populations is less extreme and may be illustrated
by one group of mature oysters exposed to DDT under controlled
-132-
-------
conditions in flowing seawater for 96 hours. In the 10
individuals, total DDT residues ranged from about 4 to
25 ppm with a mean value of 11.6 ppm. In this case,
the standard deviation was only about 50% of the mean.
Much larger samples must be collected to obtain more
uniform data, but this is a costly and time-consuming
process. We undertook in 1971 an exploratory project to
examine the merits of larger sample size (12). Oysters, two
species of fish, plankton and sediment cores were collected
concurrently at two stations in two Virginia rivers at a
two-month interval. Ten samples of 15 oysters and 10
samples of 25 fish were collected at each station. In
general, PCB's were the principal residues found and the
spread of values was reasonably small. Standard errors, for
example, were about 15 to 30% of the arithmetic means. Data
on sediment cores were less uniform even though samples were
collected in a restricted area. In one series, for example,
only 3 of 10 cores had measurable residues of PCB. This
means that had only replicate cores been taken there would
have been about a 50-50 chance that one of the cores would
have contained a residue. These data are significant in
light of the fact that sometimes only a single sediment core
is collected to assess pollution in an entire estuary.
Numerous studies have shown that the magnitude of
persistent residue levels in sediments is usually inversely
proportional to grain size. In consequence, care must be
exercised in selecting representative sample sites, as well
as in analyzing an adequate number of samples.
Despite the mathematical pleasure in achieving uniformity
in sampling results, it must be emphasized that averaging data,
either in the electric blender or the calculator, may lead to
serious management errors. Animals, in general, are not
responsive to average pollution levels, they survive and
flourish as a result of environmental extremes. A single
high incidence of endrin in the environment can be satisfactorily
averaged away on paper, but at the time of its occurrence all
of the endemic animals may have been killed.
There is a further important consideration in assessing
the importance of organochlorine residues in aquatic biota.
It is axiomatic that, with a given level of environmental
pollution, the sensitive species and the sensitive
individuals of more tolerant species will be affected first.
•In one experiment, pinfish were fed a diet contaminated with
•about 4 ppm of DDT (4). At the end of the 14th day, the ten
surviving fish were sacrificed and found to have average
residues of about 4 ppm. The 25 fish dying during tba 2-
week period averaged about 0.6 ppm of DDT, less than 1/6 as
-133-
-------
much as the resistant individuals. Obviously, the magnitude
of residues occurring in apparently healthy populations is
not necessarily an indication of tolerable pollution levels.
Residue differential showing seasonal variations.
Monitoring programs and laboratory studies in which
periodic samples have been collected at sufficiently short
intervals may show clearly defined seasonal variations in
residue levels. Such cyclic changes in the presence of
relatively constant pollution loading are indicative of
basic physiological changes in the monitored species. For
example, studies of speckled seatrout in Texas showed a 75%
decrease in gonad DDT residues in mature fish in the late
fall (7). Lowe reported a more than 50% decline in DDT and
a 70% decline in toxaphene residues in oysters continuously
exposed to these pollutants (15). Oysters exposed
simultaneously to PCB, dieldrin and DDT in the laboratory
lost from 45 to 80% of the residues within a short period
(16). Whole body residues of mercury in oysters are also
reported to decline seasonally in a manner similar to the
organochlorine compounds (8).
In each case, the abrupt declines have been clearly
identified with the normal spawning period of the animal in
question. That there should be a significant percentage
loss of such residues on a seasonal basis is entirely
predictable in view of the localization, for example, of DDT
in oyster gametes (1).
Seasonal declines in levels of organochlorine residues
in oysters are also clearly associated with fluctuating
levels in the input of pollutants into the aquatic system.
In southwest Florida, the former agricultural use of DDT was
intensified just prior to the harvest of sweet corn and
sugar cane. Residue levels of DDT in oysters collected
monthly in an associated river basin reflected this
management practice; peak DDT residues in oysters were as
much as lOOOx higher than minimal residue levels in 1967-68
(6).
Fluctuating industrial discharges may also have
significant seasonal effects on organochlorine residues in
molluscs. Spring and fall manufacturing peaks in a pesticide-
producing plant, for example, were closely followed by more
than 10-fold increases in the pesticide residues in oysters
collected about 10 miles downstream from the discharge
pipes (5).
-134-
-------
Organochlorine residue patterns in estuarine biota
resulting from agricultural and industrial practices may be
masked by the effects of marked changes in river discharge
in the drainage basin because of rainfall variations.
Residue differentials affected by body region monitored.
Regardless of the mode of entry of organochlorine into
living tissues, there is a partitioning and partial
immobilization of these compounds in fatty tissues because
of their lipophilic nature. Such segregation is clearly
demonstrated in fish in which adipose tissues are highly
localized. In coho salmon, for example, DDT residues in a
mid-body fillet range from an average of about 65 ppm in
fatty tissues to less than 6 ppm in the muscle. The whole
body DDT residue of similar fish was about 12 ppm (17).
As discussed above, residue levels increase with age
in fish. In aquatic mammals the localization of residues
in older individuals may be even more striking. In a dead
porpoise (Tursiops truncatus) found near Pensacola, Florida,
total DDT residues ranged from about 1.5 ppm in blood, 7 ppm
in the muscle, 9 ppm in the brain to 33 ppm in the liver,
and more than 500 ppm in the blubber (13). Even in oysters,
where total body fat is only about 4%, there is a marked
localization of DDT residues in the digestive gland, and,
seasonally, in the gonad which contains more fat than other
organs.
In general monitoring or surveillance programs, the
localization of persistent residues in particular body
regions has little importance from the point of view of
either human health or resource protection so long as the
monitored species is small enough to be analyzed on a whole-
body basis. Residues large enough to warrant further
investigation will show up in such analyses regardless of
their location in the body.
It is quite another matter, however, if as in market-
basket surveillance programs, only the products, e.g.,
lobster and shrimp tails or tuna muscle, are examined. It
is conceivable then the edible portions would contain
negligible organochlorine residues while the discarded body
parts could contain amounts detrimental to the productivity
of the animal itself or to other animals preying on it in
nature.
-135-
-------
SUMMARY
Successful monitoring of the estuarine environment for
persistent organochlorine pollutants is dependent in large
measure on the collection of appropriately biased samples.
Statistically randomized sample collections are
unsatisfactory for the simple reason that pollution patterns
are not random. The transport of persistent residues is
dependent on a large number of biological factors which in
turn are modified by physical and chemical parameters of the
environment.
Monitoring programs have several basic functions and
requirements. First, they should record existing residues
of persistent pollutants that occur at significant trophic
levels in aquatic ecosystems. Sample collection protocols,
as well as analytical procedures, must be sufficiently
standardized to ensure the comparability of data, not only
from one area to another but also from year to year. It is
essential that monitoring programs collect comparable data
for sufficiently long periods of time so that pollution
trends can be identified. Finally, it should be stressed
that monitoring data must be transmitted on a timely basis
to action agencies. Agencies mandated to identify and
regulate pollution sources, agencies with resource
protection responsibilities, and agencies concerned with
human welfare must have clearly established communication
channels with environmental monitoring programs.
ACKNOWLEDGEMENTS
I wish to thank Alfred J. Wilson and Jerrold Forestor of
the Gulf Breeze Environmental Research Laboratory and Roy
Schutzmann of the Pesticides Monitoring Laboratory of the
Environmental Protection Agency for many of the chemical
analyses reported.
-136-
-------
LITERATURE CITED
1. Butler, Philip A., 1966. The problem of pesticides in
estuaries. Trans. Amer. Fish. Soc. Spec. Publ.
2. Butler, Philip A., 1966. Pesticides in the marine
environment. J. Appl. Ecol. 3(Suppl) :253-259.
3. Butler, Philip A., 1968. Pesticide residues in
estuarine mollusks. In Proc. Natl. Symp. Estuarine
Pollut., p. 107-121, Stanford University, Stanford,
Calif., 1967.
4. Butler, Philip A., 1969. The significance of DDT
residues in estuarine fauna. In Chemical Fallout,
p. 205-220. C. C. Thomas, Springfield, 111.
5. Butler, Philip A., 1969. Monitoring pesticide
pollution. BioScience, 19(10) :889-91.
6. Butler, Philip A., 1973. Organochlorine residues in
estuarine molluscs, 1965-1972 - National Pesticide
Monitoring Program. Pestic. Monit. J., 6(4):238-
362.
7. Butler, Philip A., Ray Childress, -and Alfred J. Wilson,
Jr. 1970. The association of DDT residues with
losses in marine productivity. In M. Ruivo
(editor), Marine Pollution and Sea Life, p. 262-266.
Fishing News (Books) Ltd. London.
8. Cunningham, P. A. and M. R. Tripp, 1973. Accumulation
and depuration of mercury in the American oyster
Crassostrea virginica. Mar. Biol. 20:14-19.
9. Duke, T. W. , J. I. Lowe and A. J. Wilson, Jr., 1970. A
polychlorinated blphenyl (Aroclor 1254) in the
water, sediment, and biota of Escambia Bay, Florida.
Bull. Environ. Contain. Toxicol. 5(2) : 171-180.
10. Foehrenbach, Jack, 1970. N. Y. State Dept. of
Environmental Conservation, personal communication.
11. Galtsoff, Paul S., 1964. The American oyster,
Crassostrea virginica Gmelin. U. S. Fish Wildl.
Serv. Bull. 64:480 p.
-137-
-------
12. Gillespie, J. R., Jr., 1972. Estuarine Monitoring
Program - Sample Evaluation Project. NMFS Contract
No. N-042-37-72(N). (unpublished)
13. Gulf Breeze Environmental Research Laboratory, Gulf
Breeze, Florida, unpublished data.
14. Hansen, David J. and Alfred J. Wilson, Jr., 1970.
Significance of DDT residues from the estuary near
Pensacola, Fla. Pestic. Monit. J. 4(2):51-56.
15. Lowe, Jack I., Paul D. Wilson, Alan J. Rick, and Alfred
J. Wilson, Jr., 1971. Chronic exposure of oysters
to DDT, toxaphene and parathion. Proc. Natl.
Shellfish Assoc., 61:71-79.
16. Parrish, Patrick R., 1973. Chronic effects of three
toxic organics on the American oyster, Crassostrea
virginica. Proc. Natl. Shellfish Assoc. Meeting.
June 1973. Abstract.
17. Reinert, Robert E., 1969. Insecticides and the Great
Lakes. Limnos Magazine, Great Lakes Foundation,
2(3):4-9.
18. Reinert, Robert E., 1970. Pesticide Concentrations in
Great Lakes Fish. Pestic. Monit. J. 3(4):233-240.
19. Westoo'i G. , 1973. Methylmercury as percentage of total
mercury in flesh and viscera of salmon and seatrout
of various ages. Science 181:567-568.
20. Woodwell, George M., Charles F. Wurster, Jr. and Peter
A. Isaacson, 1967. DDT residues in an east coast
estuary. Science, 156:821-824.
-138-
-------
CONTRIBUTION NO. 192
-------
Biological Sciences
OCCURRENCE OF SNOOK ON THE
NORTH SHORE OF THE GULF OF MEXICO1
NELSON R. COOLEY
U. S. Environmental Protection Agency, Gulf Breeze Environmental Research Laboratory,
Sabine Island, Gulf Breeze, Florida 32561
ABSTRACT: The known range of snook is extended about 100 miles westward to Santa Rosa Sound.
THE geographical range of Centropomus undecimalis (Bloch) has been
reviewed by Marshall (1958) and Martin and Shipp (1971). Snook are found in
tropical and subtropical estuarine waters of the eastern coast of the Americas. In
the United States, snook have been reported as far north as Georgia (Dahlberg,
1972) and the Carolinas (Lunz, 1953; Martin and Shipp, 1971), but are abundant
only in Texas and peninsular Florida. In Texas, the range rarely extends north of
Port Aransas, although Jordan and Gilbert (1882) reported the species from the
vicinity of Galveston. On the east coast of Florida, Marshall (1958) placed the
'Contribution No. 192 from Gulf Breeze Environmental Research Laboratory, Associate Laboratory of the
National Environmental Research Center, Corvallis, Oregon.
-------
No. 2, 1974] COOLEY—SNOOK OCCURRENCE 99
northern limit in the vicinity of Volusia County, noting that the species is oc-
casionally taken in Duval County and in the St. John's River. On the west coast of
Florida, he noted that the northern limit was in the vicinity of Hernando County,
the species appearing to be absent from the north shore of the Gulf of Mexico.
Subsequently, Yerger (1961) reported a single adult from the Gulf off Alligator
Harbor, Franklin County, Florida, but the first record from a northern Gulf coast
estuary appears to be a 351-mm snook caught in St. Andrew Bay, Bay County,
Florida in August 1963 (Vick, 1964). Vick also noted that local commercial
fishermen told him that two or three snook were caught in that bay each year,
usually in August.
This report extends the known range of the species approximately 100 miles
westward along the northern Gulf coast of Florida into a second estuary. I
identified an adult snook, 395 mm standard length, that was caught on 3
November 1973 in a gill net in Santa Rosa Sound off Woodlawn Beach, Santa Rosa
County, Florida by Mr. J. A. Briggs, a commercial fisherman. The specimen was
deposited in the museum of the Gulf Breeze Environmental Research Laboratory
as GBERL-1911. Water temperature and salinity were not taken at the collecting
site, but should have been similar to those recorded that day in the Sound at
Sabine Island, 10 miles west of Woodlawn Beach, namely, 21.0° to 22.0°C and
29.5 to 30.0%o.
The scarcity of snook along the northern Gulf coast is probably related to their
known sensitivity to cold (see Marshall, 1958, for review of temperature
tolerance). Nevertheless, during warm seasons, isolated specimens from endemic
populations along the southwestern coast of Florida could move out of their
nominal range into localities along the northwestern coast of the state.
LITERATURE CITED
DAHLBERC, M. D. 1972. An ecological study of Georgia coastal fishes. U. S. Dept. Commer., Natl. Mar.
Fish. Sen., Fish. Bull. 70:323-353.
JORDAN, D. S., AND C. H. GILBERT. 1882. Notes on fishes observed about Pensacola, Florida, and
Galveston, Texas, with descriptions of new species. Proc. U. S. Natl. Mus. 5:241-307.
LUNZ, G. R. 1953. First record of the marine fish Centropomus undecimalis in South Carolina. Copeia
1953:240.
MARSHALL, A. R. 1958. A survey of the snook fishery of Florida, with studies of the biology of the
principal species, Centropomus undecimalis (Bloch). Florida State Bd. Conserv. Tech. Paper
22, 37 p.
MARTIN, J. R., AND R. L. SHIPP. 1971. Occurrence of juvenile snook, Centropomus undecimalis, in
North Carolina waters. Trans. Amer. Fish. Soc. 100:131-132.
VICK, N. G. 1964. The marine ichthyofauna of St. Andrew Bay, Florida, and nearshore habitats of the
northeastern Gulf of Mexico. Texas A. & M. Res. Found., A. & M. Proj. 286-D, 77 p.
YERCER, R. W. 1961. Additional records of marine fishes from Alligator Harbor, Florida, and vicinity.
Quart. J. Florida Acad. Sci. 24:111-116.
Florida Sci. 37(2):98-99. 1974.
-------
CONTRIBUTION NO. 193
-------
Translocation of Four Organochlorine Compounds by
Red Mangrove (Rhizophora Mangle L.) Seedlings1
by GERALD E. WALSH, TERRENCE A. HOLLISTER, and JERROLD FORESTER
U.S. Environmental Protection Agency
Gulf Breeze Environmental Research Laboratory
Sabine Island, Gulf Breeze, Fla. 32561
(Associate Laboratory of the National Environmental
Research Center, Corvallis, Ore.)
Mangrove vegetation is common in tropical estuaries and
serves as a habitat and source of food for many animals (ODUM
1971). Because mangrove, both living and detrital, is eaten by a
variety of animals, we conducted studies to learn translocatlonal
patterns of four organochlorine compounds in seedlings to deter-
mine if these persistent compounds could be introduced into
estuarine food webs that receive contributions from mangrove.
Previous studies showed that mangroves and other plants
translocated certain toxicants from the soil to leaves. Seedlings
of the red mangrove (Rhizophora mangle L.) translocated the herbi-
cides 2,4-D and picloram from soil to roots, hypocotyls, stems,
and leaves (WALSH et al. 1973). Translocation of the insecti-
cides dleldrin by alfalfa and hay (MUMMA et al. 1966), dieldrin,
endrin, and heptachlor by soybeans (MASH and BEALL 1970), and
mirex by peas and beans (MEHENDALE et al. 1972) has been demon-
strated.
Insecticides have been found to be associated with mangrove
from natural stands. Dieldrin (0.021 ppm - parts per million)
and polychlorinated biphenyls (0.181 ppm) were found in red man-
grove leaves from St. John and St. Croix in the Virgin Islands2.
We found DDD in roots (0.022 ppm), hypocotyls (0.220 ppm), stems
(0.032 ppm), and leaves (0.019 ppm) of red mangrove seedlings
from Joyuda, Puerto Rico (unpublished data).
In the present study, we investigated translocation of the
insecticides dieldrin, methoxychlor^and mirex and the poly-
chlorinated biphenyl (PCB) Arodor (3P1242 by red mangrove seed-
lings in the laboratory.
METHODS
Seedlings 18.5 to 38.2 cm long were obtained from trees in
the Loxahatchee River near Jupiter, Florida, and planted in plas-
tic boxes that contained muddy sand and natural sea water from an
estuary near Gulf Breeze, Florida. Fifteen seedlings were planted
in each box. Salinity of the water that covered the sediment was
25 parts per thousand; pH of the sediment was between 6.3 and 6.7.
Air temperature was 20 - 23° C. Light was provided by overhead
Grow-LuxQ/ flourescent tubes in a regime of alternate 12-hr
periods of light and darkness.
Contribution No. 193, Gulf Breeze Environmental Research
Laboratory.
2
Philip A. Butler (Gulf Breeze Environmental Research Laboratory,
Gulf Breeze, Florida). Personal communication: Unpublished
data, EPA National Monitoring Program.
129
Bulletin of Environmental Contamination & Toxicology,
Vol. 12. No. 2 © 1974 by Springer-Verlag New York Inc.
-------
Technical grade dieldrin (l,2,3,4,10,10-hexachloro-6,7-
epoxy-1, 4, 4a_, 5,6,7,8, 8a-octahydro-l, 4-endo-exo-5,8-dimethano-
napthalene), methoxychlor (l,l,l-trichloro-2,2-bis (p_-methoxy-
phenyl) ethane), mirex (dodecachlorooctahydro-l,3,4-metheno-lH-
cylobuta (cd) pentalene), and Aroclor 1242 (a mixture of poly-
chlorinated biphenyl isomers), dissolved together in 10 ml of
acetone, were added to the surface of the water. The same amount
of acetone was added to eight control boxes. Application rates
were 0.06, 0.11, 0.28, 0.56, 1.12, 2.80, 5.60, and 11.20 kg/ha
(0.05, 0.10, 0.25, 0.50, 1.00, 2.50, 5.00, and 10.00 Ib/acre).
These rates were equal to concentrations of 0.038, 0.075, 0.150,
0.300, 0.600, 1.50, 3.00, and 6.00 ppm in the muddy sand.
Three boxes of seedlings were treated at each application
rate after one or two pairs of leaves had emerged from the stems.
Two or three seedlings were collected from each box (total of 8
or 9 plants per sample) each week for six weeks after application.
The seedlings were washed with tap water and then with acetone.
Roots, hypocotyls, stems, and leaves of plants from each concen-
tration and the controls were analyzed separately for organo-
chlorine residues.
Samples were homogenized in a blender with four times their
weight of anhydrous sodium sulfate, then extracted for four hours
with 10% ethyl ether in petroleum ether in a Soxhlet apparatus.
The extract was concentrated to approximately 15 ml and trans-
ferred to a florisil column (MILLS et al. 1965).
Methoxychlor, mirex, and Aroclor 1242 were eluted with 6%
ethyl ether in petroleum ether; dieldrin with 15% ethyl ether in
petroleum ether. Approximately 1 ml of metallic mercury was
added to approximately 5 ml of the extracts of roots and hypo-
cotyls to remove sulfur compounds that interfered with electron
capture gas chromatography.
The samples were analyzed with a model 2100 Varian Aerograph
gas chromatograph equipped with 182.8 cm X 2 mm I.D. glass columns
and electron capture detectors. Two columns were packed with 2%-
0V - 101, one with 3% 0V - 210, and one with a 1:1 ratio of 2%
0V - 101 and 3% 0V - 210, all on Gas Chrom Q. Quantitation was
made on the 0V - 101 columns. The other columns were used to
confirm the analyses. Carrier gas was pre-purified nitrogen.
Operating conditions were: injector, 210° C; columns, 195° C;
detectors, 215° C; gas flow, 25 ml/min.
Aroclor 1242 was quantified by measurement of total peak
height of the 12 major peaks, which were compared with heights
of the same peaks in a standard solution of known concentration.
The other compounds were quantitated by peak height.
Aroclor, Registered Trademark, Monsanto Co., St. Louis, Mo.;
Gro-Lux, Sylvania Electric Products Inc., Salem, Mass. Mention
of commercial products does not constitute endorsement by the
Environmental Protection Agency.
130
-------
Recovery rates were above 80% in quality-control samples to
which known amounts of the compounds had been added. Residue
data do not include a correction factor for recovery rate.
RESULTS AND DISCUSSION
Mangrove seedlings translocated the four organochlorine com-
pounds tested, but no visible effects of the compounds on the
seedlings were noted. None of the compounds was detected in con-
trol seedlings.
Dieldrin
Dieldrin was translocated to hypocotyls and leaves more
rapidly than were the other compounds. It was detected in hypo-
cotyls and leaves one week after exposure at all concentrations
and these plant parts contained more of the chemical than did
roots and stems (Table 1). Dieldrin was never detected in stems
and was not found in roots at exposure concentrations less than
0.28 kg/ha. Accumulation of dieldrin in leaves was not related
to length of exposure: concentrations were similar during the
entire exposure. Average residues in leaves ranged from 0.072
to 0.113 ppm but were not related directly to exposure concen-
trations .
TABLE 1
Average concentrations of dieldrin in roots, hypocotyls, stems,
and leaves of red mangrove seedlings during six weeks of exposure
to eight application rates
Application Rate Average Concentration, Parts Per Million
kg /ha
0.06
0.11
0.28
0.56
1.12
2.80
5.60
11.20
Roots
ND*
ND
Tr**
Tr
0.03
0.04
0.04
0.06
Hypocotyl
0.01
0.02
0.02
0.04
0.08
0.08
0.08
0.16
Stem
ND
ND
ND
ND
ND
ND
ND
ND
Leaves
0.07
0.11
0.11
0.07
0.11
0.07
0.09
0.10
* Not detected; ** Trace, present but not quantifiable. Limit
of detection = 0.01 ppm.
Concentrations of dieldrin in hypocotyls were often related
directly to application rates and to length of exposure. At
application rates of 1.12 kg/ha and above, concentrations in
hypocotyls increased as exposure time increased (Figure 1).
Concentrations of dieldrin detected in hypocotyls at the end of
the six-week exposures were less than concentrations of methoxy-
chlor^ mirex, and Aroclor 1242 in hypocotyls of seedlings exposed
at the same concentration rates.
131
-------
O.2B
O.24
I
ft
M 0.20
O
u
^ 0 16
*•
^
X
Z
z 0.12
•
O
_•
a aos
0.04
0
APPLICATION RATIS S
• • 1.12 k,/h. -,X^
o.—- ..a 2.M) •• v^
T.....T 5.60 '• /
v—v 11.2O /
/
/
/
/ -T
/ .'*
/ .*
/ s
1
' ' S
.-—--' / X-°*
/L »* / X-°
/ ^..° 0*
'^+ >>%
. /- 0.1
X
z
21 06
J
O
>:
Q4
M
X
_
0.2
0
f*^1 c ?^
TO 1 (B
APPLICATION BATIS ^ 9 $.
o— .. o 0.56 k»/h. *" 3 c
• • 1.12 •• IB a. IB
a— .D 2.»0 •• rt co
»••-••» 5.6O " rt IB h*
v— -v 11.20 •' =T ^ 3
IB (B
IB 1 S
O. H-> O
(U O
„ O rt O
/HI (B rt
p> . v^
rt H*
3" Cu CO
(B H-
>-( H-
v »• IB IB 3
/V X 0 O
.' -a rt i-i
X O !-• rt)
y co ^ to
» a ^ rt fB
_^- -v ^* ^* (D O O.
^^r ^* Q*
o^^ ^* ^^ s~** P^ f^
^ ri ^^•^•*— ^***WflwMCir? 'Tl *O J3*
X * *^^— ^*y 3^ ..••°'i* H- *a n
X / .•»**^'k" .•••'*il* OP M O
^T f ^P f ,»••*** C H1* C
/ mr «*rt^*** i-t o OQ
^^ 1 iiiiiuj"'11*1'"^ (0 (i) jj"
/ ^ jt *? n °
f %* *3f ^* f^ ^^* C
4**'* •*°/.X ^ 0 rt
^I^^^B^B^^T f^
9 ^t 3^
i i i i i &i m
01 2 3 « 5 * IB co
WIIKS Of IXPOSURI CD H-
Figure 2. Uptake of methoxychlor by hypo- o> *
cotyls of red mangrove . o IB
rt rt (B O
(B CO H
a. n
to IB e
t* rt 3 cu
3 (B p. co
CO
co t; is
rt O (B ri
(B Ml CO IB
3 en
en o rt (B
O NJ (!) rt
i-( 00 3
CO H-
M ?r M 3
IB TO O
&> -^ o 1-1
< 3" B> O
(B Cu rt O
CD IB rt
• 0) ft. CO
3
ft. rt to
O rt
(a
a1 p- rt
O ""$ zf
< "O IB
rt) O
O rt
-^ o :r
H rt ri
(U ^ (B
0- M (B
M CD
(B 3"
O H-
N3 Ml TO
• CO IB
fD CO
(B rt
M ft<
rt 1
rT
rt
JJ*
0
3
o
o
1-1
-------
TABLE 2
Average concentrations of methoxychlor In roots, hypocotyls,
stems, and leaves of red mangrove seedlings during six weeks
of exposure to eight application rates
Application Rate Average Concentration, Parts Per Million
kg/ha Roots Hypocotyl Stem Leaves
0.06
0.11
0.28
0.56
1.12
2.80
5.60
11.20
ND*
ND
ND
ND
ND
0.02
0.06
0.07
ND
ND
0.03
0.26
0.26
0.29
0.31
0.49
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
* Not detected. Limit of detection = 0.02 ppm.
Mirex
Mirex was detected in seedlings treated at 11.2.0 kg/ha but
not at lower application rates (Figure 3). Residues were greatest
in all plant parts after two weeks of exposure and decreased
rapidly thereafter. It was not found in leaves after six weeks of
exposure and only 0.03 ppm was detected in roots at that time.
Aroclor 1242
Aroclor 1242 was detected in roots of seedlings exposed at
application rates of 5.60 and 11.20 kg/ha but not in stems. It
TABLE 3
Average concentrations of Aroclor 1242 in roots, hypocotyls,
stems, and leaves of red mangrove seedlings during six weeks
of exposure to eight application rates
Application Rate Average Concentration. Parts Per Million
kg/ha
0.06
0.11
0.28
0.56
1.12
2.80
5.60
11.20
Roots
ND*
ND
ND
ND
ND
ND
0.10
0.10
Hypocotyl
ND
ND
ND
0.35
0.79
0.92
0.53
1.50
Stem
ND
ND
ND
ND
ND
ND
ND
ND
Leaves
ND
ND
ND
0.46
1.11
0.92
0.80
0.90
* Not detected. Limit of detection =0.1 ppm.
133
-------
Fig. 3
Fig. 4
0.44 r
ROOTS ••—••
HYPOCOTYLS * *
STIMS o——.o
HAVES A«^—A
TREATED WITH 11.2O kg/ha
2.2
0.04
234
WEEKS OF EXPOSURE
Figure 3. Mirex in roots, hypocotyls, steins and
leaves of red mangrove treated with 11.20 kg/ha.
&
Ml
O
I
x
1.8
1.4
1.0
§0.6
2
0.2
APPLICATION RATES
O ••••O 0.56 kg/ha
• • 1.12 "
o.—.0 2.80 "
v^^v 11.20 "
y°*%.
/'/
^-o*
x!.'1^
»*>•
^..o. 0 ...c
123456
WEEKS Of EXPOSURE
Figure 4. Uptake of Aroclor 1242 by hypocotyls
of red mangrove.
-------
was detected In hypocotyls and leaves at application rates of
0.56 kg/ha and greater (Table 3).
Concentrations of Aroclor 1242 in leaves did not change with
time at any treatment rate, but concentrations in hypocotyls in-
creased in relation to exposure time for at least five weeks
(Figure 4). In hypocotyls, concentrations of PCB were greater
than concentrations of each of the other three compounds.
SUMMARY
Mangrove seedlings from the field were found to contain DDD,
dieldrin, and PCBs.
In the laboratory, mangrove seedlings translocated dieldrin,
methoxychlor, mirex, and Aroclor 1242 (a PCB) from soil to various
plant parts. Dieldrin was detected in hypocotyls and leaves of
seedlings exposed to application rates of 0.06 kg/ha and above;
methoxychlor in hypocotyls at rates of 0.28 kg/ha and above;
Aroclor 1242 in hypocotyls and leaves at rates of 0.56 kg/ha and
above; and mirex in roots, hypocotyls, stems, and leaves only at
the highest treatment rate of 11.20 kg/ha.
The data show that these persistent organochlorine compounds
can be translocated to seedlings. If the compounds are present
in the natural mangrove environment, it is possible that they
could enter seedlings and pass to higher trophic.levels when
seedlings are eaten by estuarine organisms.
ACKNOWLEDGEMENTS
We thank Mr. Robert L. Goodrick of the Central and Southern
Florida Flood Control District for providing the seedlings used
in these studies. We also thank Dr. Philip A. Butler, EPA,
Gulf Breeze Environmental Research Laboratory, for redidue data
on mangroves from the Virgin Islands, and Dr. Seppo E. Koleh-
mainen, Puerto Rico Nuclear Center, for sending seedlings from
Puerto Rico.
REFERENCES
MEHENDALE, H. M., L. FISHBEIN, M. FIELDS, and H. B. MATTHEWS.
Bull. Environ. Contam. Toxicol. 8^, 200 (1972).
MILLS, P. A., J. F. ONLEY, and R. A. GAITHER. J. Assoc. Off.
Agric. Chem. 46_, 182 (1965).
MUMMA, R. 0., W. B. WHEELER, D. E. H. FREAR, and R. H. HAMILTON.
Science 152, 530 (1966).
NASH, R. G. and M. L. BEALL, JR. Science 168. 1109 (1970).
ODUM, W. E. Univ. Miami, Sea Grant Tech. Bull. No. 7. 162 p.
(1971).
WALSH, G. E., R. BARRETT, G. H. COOK, and T. A. HOLLISTER. Bio-
science 2ji, 361 (1973) .
J.OJ
-------
CONTRIBUTION NO. 195
-------
Reprinted from:
POLLUTION AND PHYSIOLOGY OF MARINE ORGANISMS
©1974
ACADEMIC PRESS, INC.
New York Son Francisco London
IMPLICATIONS OF PESTICIDE RESIDUES
IN THE COASTAL ENVIRONMENT
THOMAS W. DUKE and DAVID P. DUMAS
U. S. Environmental Protection Agency
Gulf Breeze Environmental Research Laboratory
Sabine Island, Gulf Breeze, Florida 32561
Residues of pesticides occur in biological and
physical components of coastal and oceanic environ-
ments and some of the residues have been implicated
in degradation of portions of these environments.
The presence of many pesticides can be detected at
the parts-per-trillion level, but the effects of
such levels of pesticides on the organisms and sys-
tems in which they occur are not clear in many in-
stances. Knowledge of these effects is especially
important when the residues occur in the coastal en-
vironment—a dynamic, highly productive system where
fresh water from rivers mingles with salt water from
the sea. The coastal zone interfaces with man's
activities on land and, therefore, is especially sus-
ceptible to exposure to acute doses of degradable
pesticides, as well as chronic doses of persistent
ones.
This paper briefly reports the state-of-the-art
of research on the effects of pesticides on coastal
aquatic organisms. For a comprehensive review of
recent literature in this field, see Walsh (1972b);
137
-------
THOMAS W. DUKE AND DAVID P. DUMAS
for a compilation of data, see the EPA Report to the
States (1973).
Patterns of pesticide usage are changing in this
country and these changes are reflected in amounts of
various pesticides produced annually. Smaller amounts
of the organochlorine pesticides are being applied
because of their persistence in the environment, the
capability of organisms to concentrate them (biocon-
centration) and their adverse effects on nontarget
organisms. For many uses, organophosphates and
carbamates have replaced organochlorines because
organophosphates and carbamates hydrolyze rapidly in
water and, therefore, are not accumulated to the same
extent as organochlorines. Some of the organophos-
phates, however, are extremely toxic to aquatic
organisms on a short-time basis (Coppage, 1972).
Much effort is being devoted to developing biological
control measures that will introduce viruses and
juvenile insect hormones into the environment as part
of an integrated pest control program. The integrated
pest control approach combines biological and chemical
methods to control pests in an effort to reduce the
amount of synthetic chemicals being added to the
environment. A list of several important pesticides
that are used currently or appear as residues in
marine organisms or both is presented in Table 1.
Samples collected in the National Estuarine
Monitoring Program and in other programs show that a
variety of pesticides occur in biota and nonliving
components of the marine environment. Pesticide
residues have been reported in whales from the Pacific
Ocean (Wolman and Wilson, 1970), fish from southern
California (Modin, 1969) , invertebrates and fish from
the Gulf of Mexico (Giam et al., 1972), fish from
estuaries along the Gulf of Mexico (Hansen and Wilson,
1970), fauna in an Atlantic coast estuary (Woodwell
et al., 1967), zooplankton from the Atlantic Ocean
(Harvey et al., 1972), and shellfish from all three
coasts (Butler, 1973). These residues indicate that
pesticides can reach nontarget organisms in the
marine environment and give some indications of
138
-------
TABLE 1
Toxic Organics Used as Pesticides or Appearing as Residues in
Marine Organisms or Both
GJ
Organochlorines
(Insecticides)
Organophosphates
(Insecticides)
Carbamates
(Insecticides)
Herbicides
Chlordane
DDT
Dieldrin
Endrin
Methoxychlor
Mi rex
Toxaphene
Diazinon
Guthion
Malathion
Naled
Parathion
Phorate
Carbaryl 2,4-D
Carbofuran Picloram
Triazines
Urea
-------
THOMAS W. DUKE AND DAVID P. DUMAS
biological reservoirs of pesticides in this environ-
ment. The information obtained in these monitoring
programs is invaluable to those interested in manag-
ing our natural resources, but care must be exercised
in interpreting monitoring data.
Biological problems that affect the interpreta-
tion of monitoring data were discussed recently by
Butler (1974). Factors affecting persistent organo-
chlorine residues include kind of species sampled,
age of individuals monitored, natural variations in
individuals, seasonal variation, and selection of
tissues to be analyzed. Laboratory experiments and
observations in the field have shown that filter-
feeding mollusks are good indicators of the presence
of organochlorine pesticides in estuarine waters.
These animals are sedentary, have the capacity to
concentrate the chemicals in their soft tissues many
times the concentration in the water and lose the
chemicals rather quickly when exposed to clean water.
Obviously, mollusks would be helpful in locating the
source of a particular organochlorine. Conversely,
pelagic fish might not be useful in locating a par-
ticular source because they could have accumulated a
residue some distance from the point of collection.
As patterns of pesticide usage change, techniques
for monitoring the occurrence of the pesticides aj.sc
must change. Occurrences of organophosphates, carba-
mates and biological control agents cannot be moni-
tored in the same manner as occurrences of organo-
chlorine and other more persistent chemicals. To
help identify the presence of a pesticide it may be
necessary to utilize changes in biological systems,
as opposed to routine chemical analyses of organisms
or other components of the environment. Also required
is a concomitant effort to understand the effect of
residues on the organisms and systems in which they
occur.
140
-------
POLLUTION AND PHYSIOLOGY OF MARINE ORGANISMS
CONCEPT OF EFFECTS
The implications of pesticide residues in the
marine and other environments depends upon the effect
of the chemicals on the component in which they occur.
A conceptual model of possible effects of pesticides
and other toxic substances on biological systems is
shown in Figure 1 (Dr. John Couch, Gulf Breeze Envi-
ronmental Research Laboratory, Gulf Breeze, Florida,
unpublished personal communication). The possible
impact of a stressor on a biological system is de-
scribed as the system changes from (1) a normal
steady-state to (2) one of compensation to (3) decom-
pensation to death. Accordingly, a pesticide could
be considered to have an adverse effect if it tem-
porarily or permanently altered the normal steady-
state of a particular biological system to such a
degree as to render the homeostatic (compensating)
mechanism incapable of maintaining an acceptable
altered steady-state.
CONCEPT Of POSSIBLE EFFECTS OF
TOXIC SUBSTANCES
s
T
A
T
E
OF
B
1
O
L
O
G
1
C
S
Y
S
T
E
M
NORMAL STEADY
STATE
ALTERED STEADY
STATE
(COMPENSATION)
m
DECOMPENSATION
PT. OF NO RETURN!
DEATH -
POST- MORTEM
CHANGE
,,% SUBSTANCE X
i
i
i
»
**» '
» N X •'
\ \ " '
\
\
\
\
\
\
\
:\
: \
% \
_ ^
• • "\
0 TIME
Figr. 1. Concept of possible effects of toxic
substances.
141
-------
THOMAS W. DUKE AND DAVID P. DUMAS
NORMAL STEADY-STATE
It has been said that the most consistent trait
of biological systems is their inconsistency. The
normal steady-state of a particular biological system,
therefore, is difficult to define. Each system, from
an estuarine ecosystem to a system within individual
organisms, has a natural range of variability in such
factors as population density, species diversity,
community metabolism, oxygen consumption, enzyme pro-
duction, avoidance mechanisms, osmotic regulation,
natural pathogens, and others. Obviously, much must
be known about the normal or healthy system before an
evaluation can be made of the effect of a pesticide
on the system.
In relation to this, the impact of pesticides on
ecosystems is poorly understood because often the
"normal" system itself is poorly understood. An eco-
system can be considered a biological component that
consists of all of the plants and animals interacting
in a complex manner with their physical environment.
The "normal" state of a dynamic coastal ecosystem no
doubt depends upon the characteristics of a particu-
lar ecosystem, and changes as the system matures.
The importance of symbiosis, nutrient conservation,
and stability as a result of biological action in an
estuarine ecosystem is pointed out by Odum (1969).
According to Odum, in many instances, biological con-
trol of population and nutrient cycles prevents
destructive oscillations within the system. There-
fore, a pollutant that interferes with these bio-
logical actions could adversely affect the ecosystem.
ALTERED STEADY-STATE (COMPENSATION)
An acute dose of a pesticide could cause a bio-
logical system to oscillate outside its normal range
of variation, yet with time, the system could return
to the normal state without suffering lasting effects.
An example of this phenomenon at the ecosystem level
was demonstrated by Walsh, Miller, and Heitmuller
142
-------
POLLUTION AND PHYSIOLOGY OF MARINE ORGANISMS
(1971), who introduced the herbicide dichlobenil into
a small pond on Santa Rosa Island. Applied as a wet-
table powder at a concentration of one part per
million, the herbicide eliminated the rooted plants
in the pond. As the benthic plants died, blooms of
phytoplankton and zooplankton occurred and a normal
oxygen regime was maintained. As benthic plants
returned, the number of plankters dropped. The pond
returned to a "normal" state in reference to the pri-
mary producers approximately 3 months after treatment.
A possible example of such compensation in an indi-
vidual organism was shown recently when spot,
Leiostomus xanthurus, were exposed to Aroclor ®a 1254
under laboratory conditions (Couch, 1974). Even
though in many fish no outward signs of stress were
present, the livers of the fish accumulated excess
fat during the tests. For a period of time, the
liver evidently was able to contend with excessive
fat accumulation, but eventually chronic damage lead-
ing to necrosis occurred; therefore, the fish entered
another biological state.
DECOMPENSATION TO DEATH
The effect of a stress can eventually reach the
point where the biological system can no longer com-
pensate and death results. In the instance in which
Aroclor 1254 was related to fat globules in the liver
of fish, continued exposure to the chemical caused a
necrotic liver. Eventually, the test organisms died
as a result of the exposure. In the past, most of
the data upon which criteria and standards were based
used death as the criterion for effect. Much time
and effort now are being devoted to developing other
criteria, such as effects of relative concentrations
of the chemicals on tissue and cell structure, enzyme
a© Registered trademark: Aroclor, Monsanto Co.
Mention of commercial products does not constitute en-
dorsement by the U. S. Environmental Protection Agency.
143
-------
THOMAS W. DUKE AND DAVID P. DUMAS
reaction, osmotic regulation, behavioral patterns,
growth and reproduction.
ASSESSMENT OF EFFECTS
The concept just presented is helpful in visual-
izing the manner in which pesticides can affect
coastal organisms and systems. However, quantitative
information must be developed in order to assess the
effect of a particular pesticide on the environment
or on a component of the environment. For example,
it is not enough to know that a pesticide causes an
altered steady-state in a fish and eventually causes
death. The level of pesticide in the environment
that causes the effect must be known and, perhaps
even more important, the level at which no effect
occurs must be known.
Much of the quantitative information available
on effects of pesticides on marine organisms is in
terms of acute mortality of individual organisms. In
many instances, these data were obtained through rou-
tine bioassay tests in which known amounts of pesti-
cides are administered to test organisms for a given
period of time. In routine bioassays, the test
organisms are examined periodically and compared with
control organisms. If conducted for a short time in
relation to the life span of the organisms, usually
96 hrs, the tests are considered acute. Longer tests
over some developmental stage or reproductive cycles
are termed chronic. (An excellent discussion of
bioassays and their usefulness is presented by
Sprague (1969, 1970).)
Often, it is necessary to estimate the effect of
a pesticide on the coastal environment from only a
minimum amount of data. Interim guidelines sometimes
must be issued on the basis of a few acute bioassays
while more meaningful data are being obtained. An ap-
plication factor is helpful in these instances. This
factor is a numerical ratio of a safe concentration
of a pesticide to the acutely lethal concentration
144
-------
POLLUTION AND PHYSIOLOGY OF MARINE ORGANISMS
(LC5g). An estimate can be made for an "acceptable"
level of a pesticide in marine waters by multiplying
the LC5Q determined in acute bioassays by the appro-
priate application factor. In many instances, an
arbitrary application factor of 0.01 is used when
necessary scientific data have not yet been developed.
For a discussion on obtaining the application factor
experimentally, see Mount (1968) and Brungs (1969).
Information obtained by various bioassay tests
on some toxic organics of current interest is shown
in Table 2. These results were compiled from the
literature and indicate the most sensitive organisms
tested against these pesticides and organochlorines.
The data give a general idea of the relative toxicity
of the various pollutants.
During the past few years, the need for data on
chronic or partial chronic exposures and on sublethal
effects of pesticides on marine organisms has become
evident. Chronic studies involve the exposure of
organisms to a pesticide over an entire life cycle,
and often are referred to as "egg-to-egg" studies. A
subacute chronic is conducted over part of a life
cycle. Sublethal studies are designed to determine
if a pesticide has an effect at concentrations less
than those that are lethal to the organisms and uti-
lize such criteria as growth, function of enzyme
systems, and behavior of populations of organisms.
EFFECT OF PESTICIDES ON GROWTH OF ORGANISMS
The effects of pesticides on marine phytoplank-
ton are often related to growth of the organisms.
The effects often vary according to the pesticide and
to the species of phytoplankton. For example, Menzel
et al. (1970) found that growth in cultures of marine
phytoplankton was affected by DDT, dieldrin and
endrin. Dunaliella apparently was not affected by
concentrations up to 1000 parts per billion. In
Cyclotella, cell division was completely inhibited by
dieldrin and endrin and DDT slowed division of the
cells. The authors suggested that estuarine species/
145
-------
TABLE 2
Toxicity of Selected Pesticides to Marine Organisms
Substance Tested
Insecticides
Organochlorines :
Chlordane
DDT Compounds
p,p'-DDT(l,l,l-
Trichloro— 2,2— bis
(p-chlorophenyl)
ethane
p,p'-pDD(prp'-
TDE) (1,1-
Dichloro-2 , 2-bis
(p-chlorophenyl)
ethane
p,p'-DDE (1,1-
Diehloro-2 , 2-bis
(p-chlorophenyl )
ethylene
Dieldrin
Endrin
Hethoxychlor
Mirex
Toxaphene
Formulation Organism Tested
100% Palaemon macrodactylus
Technical Penaeus duorarum
77%
99% Palaemon macrodactylus
Falco peregrinus
100% Anguilla rostrata
100% Mugil cephalus
100% Menidia menidia
89.5% Palaemon macrodactylus
Technical Penaeus duorarum
100% Gasterosteus aculeatus
Common Name
Korean shrimp
Pink shrimp
Korean shrimp
Peregrine falcon
American eel
Striped mullet
Atlantic silverside
Korean shrimp
Pink shrimp
Threespine
stickleback
Cone, (ppb i
Act. Ingred.)
18
(10-38)
0.12
0.17
(0.09-0.32)
2.5
(1.6-4.0)
0.9
0.3
0.05
0.44
(0.21-0.93)
1.0
7.8
Method
TL-50
TL-50
TL-50
eggshell
thinning
LC-50
LC-50
LC-50
TL-50
1OO% paralysis/
death in 11 days
TLH
Test Procedure
96-hr static lab
bioassay
28-day bioassay
lab bioassay
96-hr intermittent flow
lab bioaSsay
DDE in eggs highly
correlated with
shell thinning
96-hr static lab
bioassay
96-hr static lab
bioassay
96-hr static lab
bioassay
96-hr static lab
bioassay
Flowing water bioassay
96-hr static lab
bioassay
Reference
Earnest,
unpublished
Ninuno, et a 1 . ,
unpublished
Earnest,
unpublished
Cade et al..
Eisler, 1970
Eisler, 1970
Eisler, 1970
Earnest,
unpublished
Lowe et al . ,
Katz, 1961
1971
1971
1971
-------
TABLE 2—Continued
Toxicity of Selected Pesticides to Marine Organisms
Substance Tested
Insecticides
Organophosphates t
Diazinon
Guthion
Malathion
Formulation
Technical
Grade
93*
Technical
Grade
Technical
Grade
Technical
Grade
Technical
Grade
Technical
Grade
Technical
Grade
100*
Organism Tested
Cyprinodon variegatus
Gasterosteus acuJe-atus
Cyprinodon variegatua
Lagodon rhoaboides
Leiostomisf xanthurus
Cyprinodon variegatus
Lagodon rhomboides
Leiostonus xanthurus
Tbalassooa bifasciatua
Canton Name
Sheepshead minnow
Threespine
stickleback
Sheepshead minnow
Pinflsh
Spot
Sheepshead minnow
Pinfish
spot
filuehead
Cone, (ppb Method
Act. Ingred.) of
in Water Assessment
100 Mean inhibition
of brain AChE
(Result: >84%>
4.8 TLM
3 Mean inhibition
of brain AChE
(Result: 84%)
10 Mean inhibition
of brain AChE
(Result: 80%)
20 Mean inhibition
of brain AChE
(Result; 96%)
190 Mean inhibition
of brain AChE
(Result: >84%)
238 Mean inhibition
of brain AChE
(Resultt 88%)
238 Mean inhibition
of brain AChE
(Result: 70%)
27 LC-50
Test Procedure
Static bioassay.
48-hr LC 40-60
96-hr static lab
bioassay
Static bioassay.
72-hr LC 40-60
Plowing seawater bio-
assay, 24-hr LC 40-60
Flowing seawater bio-
assay, 24-hr LC 40-60
Static bioassay,
24-hr LC 40-60
Plowing seawater bio-
assay, 24-hr LC 40-60
Flowing seawater bio-
assay, 24-hr LC 40-60
96-hr static lab
bioassay
Reference
Coppage, 1972
Katz, 1961
Coppage, 1972
Coppage and
Matthews, 1974
Coppage and
Matthews, 1974
Coppage, 1972
Coppage and
Matthews, 1974
Coppage and
Matthews, 1974
Eisler, 1970
-------
TABLE 2—Continued
Toxicity of Selected Pesticides to Marine Organisms
00
Substance Tested Formulation
Insecticides
Organophosphates :
Haled Technical
Grade
Technical
Grade
Parathion Technical
Grade
, Technical
Grade
Technical
Grade
Methyl
Parathion 100%
Phorate Technical
Grade
Carbamates :
Carbaryl 100%
Technical
Grade
Organism Tested
Lagodon rhomboides
Leiostomis xanthurus
Cyprinodon variegatus
Lagodon rhomboides
Leiostomus xanthurus
Crangon septemspinosa
Cyprinodon vari egra tus
Palaemon macrodactylus
Lagodon rhomboides
Cone . (ppb
Common Name Act. Ingred.]
in Water
Pinfish 23
Spot 70
Sheepshead minnow 10
Pinfish 10
Spot 10
Sand shrimp 2
Sheepshead minnow 5
Korean shrimp 7.0
(1.5-28)
Pinfish 1333
Method
1 of
Assessment
Mean inhibition
of brain AChE
(Result: 89%)
Mean inhibition
of brain AChE
(Result: 85%)
Mean inhibition
of brain AChE
(Result: 84%)
Mean inhibition
of brain AChE
Mean inhibition
of brain AChE
(Result: 90%)
LC-50
Mean inhibition
of brain AChE
(Result: >84%)
TL-50
Mean inhibition
(Result: 81%)
Test Procedure
Flowing seawater bio-
assay, 72-hr LC 40-60
Flowing seawater bio-
assay, 24-hr LC 40-60
Static bioassay.
72-hr LC 40-60
Flowing seawater bio-
assay, 24-hr LC 40-60
Flowing seawater bio-
assay, 24-hr LC 40-60
96-hr static lab
bioassay
72-hr LC 40-60
96-hr intermittent
flow lab bioassay
Flowing seawater bio-
assay, 24-hr LC 40-60
Reference
Coppage and
Matthews, 1974
Coppage and
Matthews, 1974
Coppage, 1972
Coppage and
Matthews, 1974
Coppage and
Matthews , 1974
Eisler, 1969
Coppage, 1972
Earnest,
unpublished
Coppage,
unpublished
-------
TABLE 2—Continued
Toxicity of Selected Pesticides to Marine Organisms
Insecticides
Carbamates:
Herbicides
d rivatives
Pic oram
To don ® 101
(3 .6% 2,4-D
1 .3% picloram)
Triazines :
Ametryne
wash from
sand-coated
particle
formulation
Isochrysis galbana
Technical Chlorococcum sp.
acid
acid
acid
acid tricornutun
acid
Technical Chlamydomonas sp.
acid
Technical Monochrysis luthen
acid
Cone . (ppb
5 * 105
10
60
77
Method
of brain AChE
(Result: 84%)
50% decrease in
02 evolution3
50% decrease in
growth
62 evolution3
O2 evolution"1
02 evolution3
growth
50% decrease in
(>2 evolution3
50% decrease in
O2 evolution3
48-hr LC 40-60 unpublished
bioassay 1969
Walsh, 1972a
Measured as ABS. Walsh, 1972a
(525mp) after 10 daysb
(525my) after 10 daysb
Hollister and
Walsh, 1973
Hollister and
Walsh, 1973
-------
TABLE 2—Continued
Toxicity of Selected Pesticides to Marine Organisms
un
Herbicides
Triazines:
Atrazine Technical
acid
Technical
acid
acid
Urea:
Diuron
Technical
Technical
acid
Isochrysis galbana
PhaeodactBJu",
tricornutum
Protococcus sp.
Monochrysis lutheri
Chlorococcunt sp.
Jsocrtrysis galbana
Honochrysis lutheri
Cone, (ppb
e Act. Ingred.
in Water
100
100
500
500
0.02
0.02
10
10
290
Method
) of
Assessment
50% decrease in
O2 evolution3
50% decrease in
02 evolution3
50% decrease in
growth
50% decrease in
growth
0.52 OPT. DEN.
expt/OPT. DEN.
controlb
0.00 OPT. DEN.
expt/OPT. DEN.
controlb
50% decrease in
growth
50% decrease in
growth
0.67 OPT. DEN.
expt/OPT. DEN.
control5
Test Procedure
(525mu) after 10 daysb
Measured as ABS .
(525mM) after 10 daysb
10-day growth
10-day growth
10-day growth
lO-day growth
10-day growth
Reference
Walsh,
Walsh,
Walsh,
Walsh,
Ukeles
Ukeles
Walsh,
Walsh,
Ukeles
1972a
1972a
1972a
, 1962
, 1962
197 2a
197 2a
, 1962
aO2 evolution measured by Gilson differential respirometer on 4 mi of culture in log phase. Length of test 90 rain.
bABS. (525mp) = Absorbance at 525 millimicrons wavelength. OPT. DEN. expt/OPT. DEN. control « Optical density of experimental culture/optical density
of control culture. <
-------
POLLUTION AND PHYSIOLOGY OF MARINE ORGANISMS
such as Dunaliella, are perhaps less susceptible than
are open ocean forms, such as Cyclotella. Similar
studies on phytoplankton and PCBs by Fisher et al.
(1972) also suggested that coastal phytoplankton may
be more resistant to organochlorines than are those
found in open ocean. Isolates of diatoms from the
Sargasso Sea were more sensitive than clones from
estuaries and the continental shelf. Herbicides ap-
plied to four species of marine unicellular algae
adversely affected their growth (Walsh, 1972a). Urea
and triazine herbicides were the most toxic of the
formulations tested. In some instances, smaller
amounts of herbicides were required to inhibit growth
than to inhibit oxygen evolution. Interestingly,
Dunaliella was most resistant of the four species
tested, as occurred in Menzel's et al. studies (1970).
The effect of mirex and a PCB, Aroclor 1254, on
growth of ciliate, Tetrahymena pyriformis, was stud-
ied by Cooley et al. (1972). Both chemicals caused
significant reductions in growth rate and population
density and the ciliate accumulated both toxicants
from the culture media, concentrating mirex up to
193 times and Aroclor to approximately 60 times the
nominal concentration in the media. The authors pos-
tulate that if this ciliate encountered similar con-
centrations of these materials in nature, the results
would be a reduction of their availability as food
organisms and nutrient regenerators. Also, the
capacity of the organisms to concentrate mirex and
Aroclor could provide a pathway for entry of these
chemicals into the food web.
Growth rates of young oysters, Crassostrea
virginica, as indicated by height and in-water weight,
was significantly reduced in individuals exposed to
5 micrograms of Aroclor 1254 per liter (ppb) for
24 weeks, but growth rate was not affected in indi-
viduals exposed to 1 part per billion for 30 weeks
(Lowe et al., 1972). Oysters exposed to 1 part per
billion concentrated the chemical 101,000 times, but
less than 0.2 part per million remain&d after 12
weeks of depuration. The growth rate of the oyster
151
-------
THOMAS W. DUKE AND DAVID P. DUMAS
was a much more sensitive indicator, since no sig-
nificant mortality occurred in oysters exposed to
5 ppb.
The effects of mirex on growth of crabs, as
measured by the duration of developmental stages of
crabs as an indicator of their growth, is illustrated
by the work of Bookhout et al. (1972). The duration
of developmental stages of zoea and the total time of
development was generally lengthened with an increase
in concentration of mirex from 0.01 to 10.0 parts per
billion. Menippe did not demonstrate this effect,
but the percentage of the extra 6th zoeal stage in-
creased as concentrations of mirex increased. This
method of determining the effect of mirex on crabs
appears to be more sensitive than previous tests with
juvenile blue crabs reported by McKenzie (1970) and
Lowe et al. (1971).
EFFECTS OF PESTICIDES ON BEHAVIOR OF ORGANISMS
The behavioral activity of organisms is a sensi-
tive criterion for determining the effect of pesti-
cides on marine organisms. Dr. H. G. Kleerekoper has
successfully studied the interactions of temperature
and a heavy metal on the locomotor behavior of fish
in the laboratory (Kleerekoper and Waxman, 1973) and
will present data on the effect of pesticides on
marine fish later in this volume. Hansen (1969)
showed that the estuarine fish, Cyprinodon variegatus,
avoided water containing DDT, endrin, Dursban ®k or
2,4-D in controlled laboratory experiments, but the
fish did not avoid test concentrations of malathion
or Sevin ®.c Likewise, grass shrimp, Palaemonetes
pugio, an important forage food for estuarine organ-
isms, avoided 1.0 and 10.0 ppm of 2,4-D by seeking
" © Registered trademark: Dursban, Dow Chemical
Company.
c® Registered trademark: Sevin, Union Carbide
Company.
152
-------
POLLUTION AND PHYSIOLOGY OF MARINE ORGANISMS
water free of this herbicide, but did not avoid the
five insecticides tested (Hansen et al., 1973). The
capacity of coastal organisms to avoid water contain-
ing pesticides may enhance their survival by causing
them to move to an area free of pesticides. Avoid-
ance could be disastrous to a population if, by
avoiding the pesticides, the population is unable to
reach an area where spawning normally occurs.
EFFECTS OF PESTICIDES ON ENZYME SYSTEMS
Inhibition of the hydrolyzing enzyme, acetyl-
cholinesterase (AChE) , by organophosphate and carba-
mate pesticides can be used as an indication of the
effect of these chemicals on estuarine fish (Coppage,
1972). Evidently, esterase-inhibiting pesticides
bind active sites of the enzyme and block the break-
down of acetylcholine, which causes toxic accumula-
tion of acetylcholine. As a result, nerve impulse
transfers can be disrupted. Laboratory bioassays
with estuarine fish spot, Leiostomus xanthuius,
showed that lethal exposures of this fish to malathion
reduced the AChE activity level by 81%. Such informa-
tion developed in the laboratory is useful in evalu-
ating effects of pesticides applied in the field.
EFFECTS OF PESTICIDES ON ECOSYSTEMS AND COMMUNITIES
Few data are available concerning the effects of
pesticides at the ecosystem or community level of
organization. This is not surprising considering the
complexities of ecosystems and our lack of knowledge
of the structure and function of coastal zones.
Effects of pesticides could be masked by variations
in population densities and it would require several
years to evaluate such variations. However, it is
possible to design laboratory and field experiments
to yield information on this complex system.
An experimental community that received 10
micrograms per liter of a polychlorinated biphenyl,
Aroclor 1254, did not recover to a "normal" state in
153
-------
THOMAS W. DUKE AND DAVID P. DUMAS
terms of numbers of phyla and species after 4 months
(Hansen, 1974). Communities of planktonic larvae
were allowed to develop in "control" aquaria and
aquaria that received the Aroclor 1254. Communities
that received 10 micrograms per liter of the chemical
were dominated by tunicates, whereas controls were
dominated by arthropods. The Shannon-Weaver species
diversity index was not altered by Aroclor 1254, but
numbers of phyla, species and individuals decreased.
The capacity of a fish population to compensate
for the effect of a pesticide was suggested in a
recent study made in Louisiana, where malathion was
applied aerially to control mosquito vectors of
Venezuelan equine encephalomyelitis (Coppage and
Duke, 1972). Fish were collected from the coastal
area before, during and after the application of
malathion. Acetylcholinesterase (AChE) activity in
the brains of fish were used as an indicator of the
effect of malathion on the community of fish. Levels
of inhibition during and soon after spraying in one
lake approached levels that were associated with
death of fish in laboratory bioassay studies. The
AChE activity of the fish population returned to
normal within 40 -days after application of the
chemical.
CONCENTRATION FACTORS
The capacity of organisms to concentrate a pesti-
cide is another factor that must be considered when
evaluating the impact of these chemicals on a coastal
system. Many of the persistent pesticides are passed
through the food web through accumulation and bio-
concentration. Some question exists about the mechan-
isms involved in trophic accumulation of fat-soluble
hydrocarbons from water by aquatic organisms (Hamelink
et al., 1971). Whatever the mechanisms for accumula-
tion, many coastal organisms have the capacity to
concentrate pesticides many times more than the con-
centration occurring in the water around them. Con-
centration factors, the ratio of the amount of
154
-------
POLLUTION AND PHYSIOLOGY OF MARINE ORGANISMS
pesticide in the animal to that in the water, for
some specific organisms and pesticides determined by
investigators at the Gulf Breeze Environmental
Research Laboratory are shown in Table 3.
STATE OF THE ART
Concern about the occurrence of pesticides in
the marine environment is continually emphasized
because surveillance and research on these chemicals
are given high priority by knowledgeable scientists.
The analytical capability for determining residues of
some pesticides in the parts per trillion range is
available, but we often do not understand the bio-
logical or ecological significance of these residues.
We need more information on chronic exposures of
sensitive marine organisms during complete reproduc-
tive cycles and on effects of sublethal levels of
exposure. Also, information is required on the
structure and function of coastal ecosystems and
criteria for evaluating the stress of pesticides on
these systems. Laboratory microcosms and other kinds
of experimental environments no doubt will be useful
in this evaluation.
As mentioned previously, use-patterns of pesti-
cides in this country are changing. We must be pre-
pared to evaluate possible effects, on the environ-
ment, of integrated pest control procedures, whereby
biological control may be just important as chemical
control of pests. Viruses and juvenile-hormone
mimics are being tested for use as pesticides and
could inadvertently reach the coastal zone. The
research effort to evaluate the impact of these new
agents must take into account that the coastal
environment already contains residues of pesticides,
persistent organochlorines, and other pollutants.
155
-------
TABLE 3
Accumulation of Pesticides from Water by Marine Organisms3-
\— 1
en
cr>
Substance Tested Organism Tested
Insecticides
Organochlorines :
Chlordane pseudomcnas spp.
DDT Brachidontes recurvus
Mercenaria mercenaria
My a arenaria
Crassostrea gigas
Penaeus duorarum
Lagodon rhomboides
Dieldrin Mercenaria mercenaria
Endrin Mercenaria mercenaria
Methoxychlor Mercenaria mercenaria
Mirex Tetrahymena pyriformis W
Penaeus duorarum
Common Name
Hooked mussel
Hard-shell clam
Soft-shell clam
Pacific oyster
Pink shrimp
Pinfish
Hard-shell clam
Hard-shell clam
Hard-shell clam
Pink shrimp
Exp
10
1
1
0
1
0
0
p.
0
0
1
0
0
. Cone.
ppm
ppb
ppb
.1
.0
.14
•1,
pb
.5
.5
.0
.9
.1
ppb
ppb
ppb
1.0
ppb
ppb
PPb
ppb
ppb
Cone . Factor Time
0.83
24,000
6,000
8,800
20,000
1,500
10,600
38,000
760
480
470
193
2,600
10 days
1 week
1 week
5 days
7 days
3 weeks
2 weeks
5 days
5 days
5 days
1 week
3 weeks
Special Details
Mixed culture of
four species
Whole body residues
(Meats)
Whole body residues
(Meats)
Whole body residues
(Meats)
Whole body residues
(Meats)
Whole body residues
Whole body residues
Whole body residues
(Meats)
Whole body residues
(Meats )
Whole body residues
(Meats)
Axenic cultures
incubated at 26°C;
concentration
factor on dry
weight basis
Whole body residues
Reference
Bourquin , unpublished
Butler ,
Butler,
Butler,
Butler,
1966
1966
1971
1966
Nimmo et al. , 1970
Hansen
1970
Butler ,
Butler,
Butler ,
Cooley
Lowe et
and Wilson,
1971
1971
1971
et al., 1972
: al., 1971
-------
TABLE 3—Continued
Accumulation of Pesticides from Water by Marine Organisms3-
in
•vj
Substance Tested Organism Tested
Insecticides
Organochlorines :
Mirex Rhi thropanopeus
harrisii
Callinectes sapidus
Thalassia testudinum
Halogenated Hydro-
carbon:
Polychlorinated
biphenyl IfCB)
Aroclor 1254 Tetrahynena pynformis I
Palaemonetes pugio
Common Name Exp. Cone. Cone. Factor Time Special Details
Mud crab (larvae) 0.1 ppb 1,000 7 weeks Static culture bowl
method with a
change to fresh
medium + chemical
each day
Blue crab 0,1 ppb 1,100-5,200 3 weeks Whole body residues
(juveniles)
Turtle grass 0.1 ppb 0 leaves 10 days Plants exposed to
0.36 rhizomes chemical through
rhizomes ; concen-
tration factor on
wet weight basis
incubated' at 26°C;
concentration
factor on dry
weight basis
Grass shrimp 0.62 ppb 2,069 1 week Whole body residues
(Meats)
26,580 5 weeks Whole body residues
(Meats)
Reference
Bookhout et al . , 1972
Lowe, unpublished
Walsh and Hollister,
unpublished
Cooley et al. , 1972
Nimmo and Heitmuller,
unpublished
Nimmo and Heitmuller,
unpublished
-------
TABLE 3—Continued
Accumulation of Pesticides from Water by Marine Organisms3
in
00
Substance Tested Organism Tested
Insecticides
Halogenated Hydro-
car bom
Polychlorinated
biphenyl (PCB)
Aroclor © 1254 Penaeus duorarura
Lagodon rhomboitjes
Leiostomus xanthurus
Thalassia testadinum
Herbicide
Tordon® 101 Rhlzophora mangle
(39.6% 2,4-D;
14.3% picloram)
Thalassia testudlnum
Common Name Exp. Cone.
Pink shrimp 2.5 ppb
Pinfish 5 ppb
Spot 1 ppb
5 ppb
Turtle grass 5820 ppb
Red mangrove 14.4 ppb
Turtle grass 5 ppm
Cone. Factor
1,800
7,600
2,800-21,800
17,000-27,000
9,200-30,400
0 leaves
0 rhizomes
Stems
1.28 (2,4-D)
0.64
(picolinic
acid)
Leaves
0 (2,4-D)
0 (picolinic
acid)
Time
2 days
9 days
2-15 weeks
4-8 weeks
3-6 weeks
10 days
20 days
10 days
Special Details
Whole body residues
Whole body residues
Whole body residues
Whole body residues
Whole body residues
Plants exposed to
chemical through
rhizomes j concen-
tration factor on
wet weight basis
Seedlings treated
when two pairs of
leaves were present;
concentration factor
on wet weight basis
Plants exposed to
chemical through
rhizomes ; concen-
tration factor on
wet weight basis
Reference
Nimmo et al. , 1971
Ninmo et al. , 1971
Hansen et al. , 1971
Hansen et al., 1971
Hansen et al. , 1971
Walsh and Hoi lister,
unpublished
Walsh et al., 1973
Walsh and Hollister,
unpublished
Information developed at the Environmental Protection Agency's Gulf Breeze Environmental Research Laboratory,
-------
POLLUTION AND PHYSIOLOGY OF MARINE ORGANISMS
ACKNOWLEDGMENTS
We thank C. M. Herndon for assistance in prepar-
ing the manuscript; N. R. Cooley, D. J. Hansen and
M. E. Tagatz for their constructive criticism of the
manuscript; and the investigators at this laboratory
for freely supplying data (some unpublished) for the
tables.
LITERATURE CITED
Bookhout, C. G., A. J. Wilson, Jr., T. W. Duke, and
J. I. Lowe. 1972. Effects of mirex on the
larval development of two crabs. Water, Air and
Soil Pollution 1:165-80.
Bourquin, A. W. Unpublished data. Environmental
Protection Agency, Gulf Breeze Environmental
Research Laboratory, Gulf Breeze, Florida.
Brungs, W. A. 1969. Chronic toxicity of zinc to
the fathead minnow, Pimephales promelas
Rafinesque. Trans. Am. Fish. Soc. 98:272-79.
Butler, P. A. 1966. Pesticides in the marine
environment. J. Appl. Ecol. 3:253-59.
. 1971. Influence of pesticides on marine
ecosystems. Proc. Roy. Soc. London B 277:321-29.
. 1973. Organochlorine residues in
estuarine mollusks, 1965-1972—National Pesti-
cide Monitoring Program. Pestic. Monitor. J.
6:238-62.
. 1974. Biological problems in estuarine
monitoring. Proceedings of Seminar on Standardi-
zation of Methodology in Monitoring the Marine
Environment held in Seattle, Washington,
October 16-18, 1973. In press.
Cade, T. J., J. L. Lincer, C. M. White, D. G. Roseneau,
and L. G. Swartz. 1971. DDE residues and egg-
shell changes in Alaskan falcons and hawks.
Science 172:955-57.
159
-------
THOMAS W. DUKE AND DAVID P. DUMAS
Cooley, N. R., J. M. Keltner, Jr., and J. Forester.
1972. Mirex and Aroclor ® 1254: effect and
accumulation by Tetrahymena pyriformis Strain W.
J. Protozool. 19:636-38.
Coppage, D. L. Unpublished data. Environmental
Protection Agency, Gulf Breeze Environmental
Research Laboratory, Gulf Breeze, Florida.
. 1972. Organophosphate pesticides:
specific level of brain AChE inhibition related
to death in sheepshead minnows. Trans. Am.
Fish. Soc. 101:534-36.
and T. W. Duke. 1972. Effect of pesti-
cides in estuaries along the Gulf and southeast
Atlantic Coasts. In: Proc. Second Gulf Conf.
on Mosquito Suppression and Wildl. Manage.,
pp. 24-31.
and E. Matthews. 1974. Short-term
effects of organophosphate pesticides on
cholinesterases of estuarine fishes and pink
shrimp. In press.
Couch, J. 1974. Histopathologic effects of pesti-
cides and related chemicals on the livers of
fishes. Proc. of Symposium on Research of
Fishes sponsored by the Registry of Comparative
Pathology. University of Wisconsin Press.
In press.
Davis, H. C. and H. Hidu. 1969. Effects of pesti-
cides development of clams and oysters and on
survival and growth of the larvae. Fish. Bull.
67:393-404.
Earnest, R. Unpublished data. Effects of pesticides
on aquatic animals in the estuarine and marine
environment. In: Annual Progress Report 1970.
Columbia, Mo.: Fish-Pesticide Research Labora-
tory, Bur. Sport Fish. Wildl., U. S. Dept.
Interior.
Eisler, R. 1969. Acute toxicities of insecticides
to marine decapod crustaceans. Crustaceans 16:
302-10.
160
-------
POLLUTION AND PHYSIOLOGY OF MARINE ORGANISMS
1970. Acute toxicities of organo-
chlorine and organophosphorus insecticides to
estuarine fishes. Bur. Sport Fish. Wildl.
Tech. Paper No. 46.
Environmental Protection Agency. 1973. Effects of
pesticides in water—a report to the states.
Fisher, N. S., L. B. Graham, E. J. Carpenter, and
C. F. Wurster. 1973. Geographic differences in
phytoplankton sensitivity to PCBs. Nature 241:
548-49.
Giam, C. S., A. R. Hanks, R. L. Richardson, W. M.
Sackett, and M. K. Wong. 1972. DDT, DDE, and
polychlorinated biphenyls in biota from the Gulf
of Mexico and the Caribbean Sea. Pestic.
Monitor. J. 6:139-43.
Hamelink, J. L., R. C. Waybrant, and R. C. Ball.
1971. A proposal: exchange equilibria control
the degree chlorinated hydrocarbons are biolog-
ically magnified in lentic environments. Trans.
Am. Fish. Soc. 100:207-14.
Hansen, D. J. 1969. Avoidance of pesticides by
untrained sheepshead minnows. Trans. Am. Fish.
Soc. 93:426-29.
. 1974. Aroclor © 1254: effect on com-
position of developing estuarine animal communi-
ties in the laboratory. Contributions in Marine
Science, University of Texas. In press.
, P. R. Parrish, J. I. Lowe, A. J. Wilson,
Jr., and P. D. Wilson. 1971. Chronic toxicity,
uptake, and retention of Aroclor 1254 in two
estuarine fishes. Bull. Environ. Contain, and
Toxicol. 6:113-19.
, S. C. Schimmel, and J. M. Keltner, Jr.
1973. Avoidance of pesticides by grass shrimp
(Palaemonetes pugio) . Bull. Environ. Contain.
and Toxicol. 9:129-33.
and A. J. Wilson, Jr. 1970. Significant
of DDT residues in the estuary near Pensacola,
Florida. Pestic. Monitor. J. 4:51-56.
161
-------
THOMAS W. DUKE AND DAVID P. DUMAS
Harvey, G. R., V. T. Bowen, R. H. Backus, and G. D.
Grice. 1972. Chlorinated hydrocarbons in open-
ocean Atlantic organisms. In: The Changing
Chemistry of the Oceans, pp. 177-88, ed. by
D. Dyrssen and D. Jagner. Stockholm: Almquist
and Wiksell.
Hollister, T. A. and G. E. Walsh. 1973. Differential
responses of marine phytoplankton to herbicides:
oxygen evolution. Bull. Environ. Contain, and
Toxicol. 9:291-95.
Katz, M. 1961. Acute toxicity of some organic
insecticides to three species of salmonids and
to the threespine stickleback. Trans. Am. Fish.
Soc. 90:264-68.
Kleerekoper, H. and J. B. Waxman. 1973. Interaction
of temperature and copper ions as orienting
stimuli in the locomotion behavior of the gold-
fish (Carassius auratus). J. Res. Bd. Canada
30:725-28.
Lowe, J. I. Unpublished data. Environmental Protec-
tion Agency, Gulf Breeze Environmental Research
Laboratory, Gulf Breeze, Florida.
, P. R. Parrish, J. M. Patrick, Jr., and
J. Forester. 1972. Effects of the poly-
chlorinated biphenyl Aroclor ® on the American
oyster Crassostrea virginica. Mar. Biol. 17:
209-14.
, , A. J. Wilson, Jr., P. D.
Wilson, and T. W. Duke. 1971. Effects of mirex
on selected estuarine organisms. Trans. 36th
N. Am. Wildl. and Natural Resources Conf.,
March 7-10, 1971, Portland, Oregon, 171-86.
McKenzie, M. D. 1970. Fluctuations in abundance of
the blue crab and factors affecting mortalities.
South Carolina Wildlife Resources Dept., Marine
Resources Division Technical Report 1.
Menzel, D. W., J. Anderson, and A. Randike. 1970.
Marine phytoplankton vary in their response to
chlorinated hydrocarbons. Science 167:1724-26.
162
-------
POLLUTION AND PHYSIOLOGY OF MARINE ORGANISMS
Modin, J. C. 1969. Chlorinated hydrocarbon pesti-
cides in California bays and estuaries. Pestic.
Monitor. J. 3:1-7.
Mount, D. I. 1968. Chronic toxicity of copper to
fathead minnows (Pimephales promelas, Rafinesque).
Water Research 2:215-23.
Nimmo, D. R., R. R. Blackman, A. J. Wilson, Jr., and
J. Forester. 1971. Toxicity and distribution
of Aroclor ® 1254 in the pink shrimp, Penaeus
duorarum. Mar. Biol. 11:191-97.
and P. T. Heitmuller. Unpublished data.
Environmental Protection Agency, Gulf Breeze
Environmental Research Laboratory, Gulf Breeze,
Florida.
, A. J. Wilson, Jr., and R. R. Blackman.
1970. Localization of DDT in the body organs of
pink and white shrimp. Bull. Environ. Contain.
and Toxicol. 5:333-41.
Odum, E. P. 1969. The strategy of ecosystem
development. Science 164:262-70.
Sprague, J. B. 1969. Measurement of pollutant
toxicity to fish. I. Bioassay methods for acute
toxicity. Water Research 3:793-821.
. 1970. Measurement of pollutant toxicity
to fish. II. Utilizing and applying bioassay
results. Water Research 4:3-32.
Ukeles, R. 1962. Growth of pure cultures of marine
phytoplankton in the presence of toxicants.
Appl. Microbiol. 10:532-37.
Walsh, G. E. 1972a. Effects of herbicides on photo-
synthesis and growth of marine unicellular algae.
Hyacinth Control J. 10:45-48.
. 1972b. Insecticides, herbicides and
polychlorinated biphenyls in estuaries. J. Wash.
Acad. Sci. 62:122-39.
, R. Barrett, G. H. Cook, and T. A.
Hollister. 1973. Effects of herbicides on
seedlings of the red mangrove, Rhizophora mangle
L. BioScience 23:361-64.
163
-------
THOMAS W. DUKE AND DAVID P. DUMAS
and T. A. Hollister. Unpublished data.
Environmental Protection Agency, Gulf Breeze
Environmental Research Laboratory, Gulf Breeze,
Florida.
, C. W. Miller, and P. T. Heitmuller. 1971.
Uptake and effects of dichlobenil in a small
pond. Bull. Environ. Contain, and Toxicol. 6:
279-88.
Wolman, A. A. and A. J. Wilson, Jr. 1970. Occurrence
of pesticides in whales. Pestic. Monitor. J. 4:
8-10.
Woodwell, G. M., C. F. Wurster, and P. A. Isaacson.
1967. DDT residues in an east coast estuary:
a case of biological concentration of a persist-
ent insecticide. Science 156:821-24.
164
-------
CONTRIBUTION NO. 196
-------
(17)
Effects of the Polychlorinated Biphenyl,
Aroclor®1016, on Estuarine Animals
PATRICK R. PARRISH, DAVID J. HANSEN, JOHN N. COUCH,
JAMES M. PATRICK, JR., AND GARY H. COOK
U.S. Environmental Protection Agency
Gulf Breeze Environmental Research Laboratory
Acute toxicity and rate of uptake and depuration of
the polychlorinated biphenyl (PCB), Aroclor® 1016,
were determined for certain estuarine animals in flow-
ing sea water bioassays. Ninety-six hour ECSO's were:
American oyster (Crassostrea virginica), 10.2 /tg/1; brown
shrimp (Penaeus aztecus), 10.0 /ig/1; and grass shrimp
(Palaemonetes pugio), 9.1 /ig/1. Pinfish (Lagodon rhom-
boides) did not die when exposed to 100 /tg/1 for 96
hours, but significant mortality occurred when pinfish
were exposed to 32 /ig/1 for 42 days. Further, altera-
tions in the pancreatic exocrine tissue surrounding the
portal veins occurred in pinfish from the 42-day exposure.
Maximum whole-body residue (wet-weight) in pinfish
was 17,000 X the nominal concentration in test water
and whole-body residue after a 56-day depuration period
in PCB-free water decreased 61%. Oysters exposed to
10 /ig/1 for 84 days accumulated the chemical 13,000 x
the concentration in test water and no PCB residue was
detectable after a 56-day depuration period. — ® Regis-
tered trademark, Monsanto Company, St. Louis, MO.
Mention of commercial products or trade names does not
constitute endorsement by the Environmental Protection
Agency. — Contribution No. 196, Gulf Breeze Environ-
mental Research Laboratory.
Reprinted from The ASB Bulletin,
Vol. 21, No. 2, April 1974, p. 74.
-------
CONTRIBUTION NO. 198
-------
Re printed from
AMD PHYSIOIOGY OF IUIINE 9IGANISHS
©1974
ACADEMIC PRESS, INC
New York Son Francisco Londei
SOME PHYSIOLOGICAL CONSEQUENCES OF
POLYCHLORINATED BIPHENYL- AND
SALINITY-STRESS IN PENAEID SHRIMP
D. R. NIMMO and L. H. BAHNER
Environmental Protection Agency
Gulf Breeze Environmental Research Laboratory
Gulf Breeze, Florida 32561
Estuaries are dynamic environments where there
are many factors that fluctuate, such as temperature,
salinity, currents, hydrostatic pressure, and oxygen
or carbon dioxide concentrations. Unfortunately,
domestic sewage (nutrients), oils, industrial chemi-
cals, pesticides, metals, or altered temperatures are
an influence in estuaries. The combined effects of
the natural and man-introduced factors are largely
unknown. In contrast, these interactions could
adversely affect the biota of an estuary before such
a trend was recognized. Therefore, one of the major
problems facing us today is understanding and pre-
dicting the interactions of pollutants and natural
stresses.
It is common knowledge that the commercial
shrimps along the Gulf Coast undertake distinct
euryhaline migrations. After adult shrimp spawn
in the open Gulf from spring to fall, the post-
mysids and juveniles migrate into the fresher waters
of bays where they grow rapidly to adulthood before
returning to the Gulf. Obviously, these stages of
427
-------
D. R. NIMMO AND L. H. BAHNER
shrimp must be able to adjust to the changing salini-
ties encountered in the estuary, and any factor
diminishing the ability of the shrimp to adjust physi-
ologically to these changes would have a detrimental
effect on them.
One group of chemicals introduced by man that
has recently been of concern to many ecologists is the
PCBs, or polychlorinated biphenyls. In 1969, a PCB,
identified as Aroclor ® 1254a was discovered as a con-
taminant in water, sediment, and fauna of Escambia Bay,
Florida (Duke et al., 1970). An early survey indicated
that whole body residues of the chemical in feral
shrimp were as high as 14 mg/kg whole body (Nimmo
et al., 1971a). Subsequent toxicity tests on juvenile
pink shrimp (Penaeus duorarum) revealed that about
1.0 pg/& in the water would kill 50% of the experi-
mental animals within 15 days (Nimmo et al., 1971b).
While conducting bioassays at our laboratory we
noted on several occasions that salinity appeared to
affect toxicity. In one instance, adult pink shrimp
were exposed chronically to a sublethal concentration
of the chemical (about 1.0 pg/£). The purpose of the
test was to determine whether structural damage might
occur in gill tissue. On day 27 of exposure at which
time we had recorded no previous deaths from the PCB,
the salinity of the incoming water decreased front
20 o/oo to 11 o/oo within 4 hrs due to rain, tides and
wind. As a result, ten experimental shrimp died before
the salinity had returned to 20 o/oo. During the next
2 days, the salinity was lowered again by aberrant
tides and climatic conditions and more experimental,
but not control, shrimp died. We, therefore, became
interested in the possible interaction of Aroclor ©
1254 and environmental stress, particularly the effect
of PCB on the ability of shrimp to regulate osmotically
and ionically at reduced salinities.
Mention of commercial products does not consti-
tute endorsement by the U. S. Environmental Protection
Agency.
428
-------
POLLUTION AND PHYSIOLOGY OF MARINE ORGANISMS
MATERIALS AND METHODS
Adult brown shrimp (Penaeus aztecus), 11.5 to
13.7 on, rostrum to telaon, were captured near Gulf
Shores, Alabama, and used in our studies. Approxi-
mately equal numbers of both sexes were used and the
methods of exposure to the Aroclor ® 1254 were similar
to those reported previously (Nimmo et al., 1971a)
except that the shrimp were maintained at 30 ± 1 o/oo
S, 25 ± 2°C, and the exposures to the chemical were
"sublethal" and lasted but 7 days. Three ug/£ were
chosen as the test concentration because previous
tests with adult brown shrimp, as well as adult pink
shrimp (P. duorarum), demonstrated that this concen-
tration would cause 50 o/oo mortality within 30 days.
Following exposure to PCB, equal numbers of PCB-
exposed and control shrimp were transferred to sepa-
rate aquaria. The experimental procedure is shown in
Figure 1. Since the possibility existed of physiolog-
ical stress from handling or inherent in the experi-
mental design, both PCB-exposed and control shrimp
were analyzed for osmotic and ionic concentrations
after being subjected to the procedure without ex-
ternal salinity change (30 o/oo). While temperature
was kept constant, the salinity in each aquarium was
gradually lowered during 8 hrs to a predetermined
level. For the first group, the salinity was main-
tained at 30 p/oo for 8 hrs; the second, salinity was
lowered from 30 o/oo to 22 o/oo; the third, from
30 o/oo to 10 o/oo; and the fourth, from 30 o/oo to
7 o/oo. Although there was a time differential
between groups of shrimp, and, therefore, a possible
difference in test animals due to a slight loss of
PCB, analyses for the chemical revealed no significant
difference in whole body concentrations among groups
(Table 1)- As in earlier studies, there was a wide
range in individual concentrations of PCB (Nimmo
et al., 1971b).
429
-------
D. R. NIMMO AND L. H. BAHNER
CONTROL SHRIMP
Carrier only
Held for 7 days In flowing
watar at 30 + 1 °/oo
salinity
EXPERIMENTAL SHRIMP
Aroelor® US*
(3 ug/t) + carrier
30 "too to 10 °/oo \3O loo to 7 "loo
30 "too 30 "loo to 22 "loo
Salinity acres*: flowing watar with the capacity of controlled flux
of 8-hr duration
Blood sample drawn from
perlcardlal »inu«, placed
in glass tuba, allowed to
clot and oaaotlc concen-
tration determined
. Shrimp saved
for residua
analysis
-------
POLLUTION AND PHYSIOLOGY OF MARINE ORGANISMS
TABLE 1
Whole Body Concentrations of Aroclor ®1254 in PCB-
Exposed Shrimp*
Salinity
0/00
30
22
10
7
mg/kg
Average
9.6
7.8
7.1
8.5
Range
3.0-13.7
1.9-18.8
3.9-14.0
2.8-15.0
Concentration of Aroclor © 1254 in the test
water was 3 ppb; length of exposure, 7 days. Control
shrimp had less than 0.1 ppm of Aroclor ®1254.
osmotic pressure was measured on whole blood or on
serum, but an individual analysis showed no signifi-
cant difference in osmotic concentrations. Replicate
determinations of 10 separate aliquots of pooled sera
from several shrimp yielded a standard error of 1.2
(mean concentration ^ 629 mOs).
Analyses of ions were performed on ashed sera.
To prepare the sample, the clot contained in the
osmometer tube war squeezed with a small glass rod,
the clot was removed and 0.2 ml of the serum was
transferred to a small crucible. The crucible was
placed in an oven and the contents ashed at 480°C for
6 hrs, cooled and the ash was dissolved in 2N I^SO^.
Analysis of chloride was performed with a Buchler-
Cotlove Chloridometer ® and cations were determined
on a Model 403 Perkin-Elmer © atomic absorption
spectrophotometer equipped with a deuterium arc-
background corrector and an HGA-70 heated graphite
atomizer. Cations in standard solutions were in the
same proportions as those in the sera. As a check on
our methods, an analysis of a single aliquot of serum
431
-------
D. R. NIMMO AND L. H. BANNER
by emission and atomic absorption yielded identical
results for potassium. Five replicates of pooled
sera from several shrimp yielded a standard error of
0.24 mEq/1 for Cl (mean = 257 mEq/1). Replicate
analyses on serum aliquots yielded standard errors
in mEq/1 of 8.26 for Na (mean = 324.9) , of 0.04 for
Mg (mean = 16.0), of 0.07 for K (mean = 8.1), of 0.19
for Ca (mean = 4.74), and of 0.10 for Cu (mean = 2.84).
The 95% confidence interval was used to evaluate
significance of differences in the data. The 95%
intervals are indicated in the graphs by vertical
bars on each datum and are listed in each table.
Since it is sometimes difficult to relate "osmolality"
or "osmotic concentration" to the environment, we
expressed the concentration of the environment as
salinity. The relationship between mOs and salinity
is indicated along the X axis in Figure 2.
15OO
llOOO
X
D
at
soo
Our irudy co»«i«d th«
ran§« of 7 - 3O °/M> salinity.
SOO 1OOO
27 3O SALINITY °fc«
ENVIRONMENT
1SOO (mOs)
Fig. 2. Osmotic concentration: Serum-
environment in Penaeus aztecus (after
McFarland and Lee, 1963). Our study
covered the range of 7 o/oo to 30 o/oo
salinity.
432
-------
POLLUTION AND PHYSIOLOGY OF MARINE ORGANISMS
Concentrations of Aroclor ® 1254 were determined
on individual shrimp by gas chromatography, using
procedures summarized earlier (Nimmo et al., 1971b).
RESULTS
The most significant result of this study was the
discovery that a sublethal concentration of Aroclor
1254 at constant salinity for 7 days became lethal
when the species was subjected to a gradual decrease
in salinity over an 8-hr period. Usually the shrimp
exhibited increased swimming activity while the salin-
ity changed from 30 o/oo to 20 o/oo; this was the only
observed behavioral aberration. Experimental shrimp
began to die at a salinity of about 12 to 13 o/oo.
After 8 hrs of exposure to 10 o/oo and 7 o/oo salinity,
mortality of experimental shrimp was nearly 50%. When
50% of the experimental shrimp had become moribund or
had died, living PCB-exposed shrimp were taken for
analyses of osmotic concentrations and ion determina-
tions on sera.
The results of these analyses indicated that
concentrations of most major ions in the sera of PCB-
exposed shrimp became significantly less as the ambi-
ent salinity decreased, the sum of major ions (e.gr.,
Na, Ca, Mg, K, Cu, and Cl) was 18% less after ambient
salinity reached 10 o/oo or 7 o/oo (Fig. 3). Of this
total, sodium was 16% less (Fig. 4), chloride, 19%
(Fig. 5) and calcium, 25% (Fig. 6). There was some
indication that magnesium iecreased, although the
loss was not statistically significant (Table 2}. No
apparent differences in potassium (Table 3), or
copper (Table 4) were noted.
Data for iron (Table 5) are not included in the
totals in Figure 3 because we could not distinguish
the divalent from the trivalent form.
Despite significant alterations in the major ion
complement or in some major ions, osmotic concentra-
tion was not significantly affected by PCB and salin-
ity stress (Table 6). Seemingly, osmotic pressure
was less in PCB-exposed shrimp at 10 o/oo or 7 o/oo
433
-------
D. R. NIMMO AND L. H. BANNER
salinity/ but individual variation was too great to
show a significant difference from controls.
MOO
SOO
10 SALINITY <%o) 22
Fig. 3. Total ions in brown shrimp
serum in relation to salinity.
600
soo
0400
300
700
SALINITY (%o)
30
Fig. 4. Serum sodium in brown
shrimp in relation to salinity.
DISCUSSION
Knowledge of interactions between toxic com-
pounds and environmental factors is essential for
predicting their effects on ecosystems or species.
Examples of this need have been demonstrated in both
fresh and marine investigations. In fresh water.
434
-------
POLLUTION AND PHYSIOLOGY OF MARINE ORGANISMS
600
2001
10 SALINITY (%o>
Fig. 5. Serum chloride in brown
shrimp in relation to salinity.
40
1J26
w
19
12
10 SALINITY Wbo)
22
30
Fig. 6. Serum calcium in brown
shrimp in relation to salinityf
low-level chronic exposure of the darter (Etheostooia
nigrum) to dieldrin greatly affected its ability to
survive thermal stress (Silbergeld, 1973). The sub-
lethal effects of mercury on fiddler crabs (Vca
pugilator) reduced survival times when crabs were
placed under temperature and salinity stress
(Vernberg and Vernberg, 1972). Mortality of fiddler
crabs that were previously exposed to cadmium was
greatest at high temperatures and low salinities
(O'Hara, 1973).
435
-------
D. R. NIMMO AND L. H. BAHNER
TABLE 2
Average Serum Concentrations of Magnesium in Brown
Shrimp in Relation to Salinity
Salinity
o/oo
30
22
10
7
mEq/1
Control
20.1
16.3
14.8
15.0
95% Conf .
Interval
16.7-23.5
14.2-18.4
12.4-17.2
11.7-18.3
Experi-
mental
17.3
14.6
13.0
11.5
95% Conf.
Interval
12.7-21.8
12.3-16.9
11.0-15.0
8.9-14.2
TABLE 3
Average Serum Concentrations of Potassium in Brown
Shrimp in Relation to Salinity
Salinity
0/00
30
22
10
7
mEq/1
Control
14.2
11.7
10.4
9.6
95% Conf.
Interval
13.5-14.9
11.1-12.3
9.8-11.2
8.4-10.7
Experi-
mental
14.7
12.1
10.1
10.0
95% Conf.
Interval
13.6-15.7
11.2-12.9
8.7-11.4
8.4-11.6
In our studies Aroclor ® 1254 possibly inter-
fered with the adenosine triphosphatase (ATPase)
activity in gills of shrimp. ATPase activity is
associated with active ion transport (Tanaka,
Sakamoto, and Sakamoto, 1971). Polychlorinated in-
secticides and the related polychlorinated biphenyls
436
-------
POLLUTION AND PHYSIOLOGY OF MARINE ORGANISMS
have been shown by several "in vitro" assays to
inhibit ATPase in the tissues of fishes (Davis and
Wedemeyer, 1971a, 1971b; Davis, Friedhoff and
Wedemeyer, 1972; Cutkomp et al., 1972; Yap et al.,
1971) and in the nerves of lobsters (Matsumura and
Narahashi, 1971). The need for greater efficiency
TABLE 4
Average Serum Concentrations of Copper in Brown
Shrimp in Relation to Salinity
Salinity
o/oo
30
22
10
7
Control
4.7
4.0
5.2
4.8
mEq/1
95% Conf. Experi-
Interval mental
3.6-5.9
3.4-4.6
4.0-6.4
3.9-5.7
5.3
5.9
4.6
5.7
95% Conf.
Interval
4.4-6.3
4.8-7.0
3.8-5.3
5.0-6.4
TABLE 5
Average Serum Concentrations of Iron in Brown Shrimp
in Relation to Salinity
Salinity mM/1
Control 95% Conf. Experi- 95% Conf.
o/oo Interval mental Interval
30
22
10
7
.23
.28
.20
.24
.17-.29
.15-.35
.15-.25
.14-.33
.31
.18
.26
.33
.15-.48
.14-.22
.17-.35
.24-.41
437
-------
D. R. NIMMO AND L. H. BANNER
TABLE 6
Average Serum Osmotic Concentrations in Brown Shrimp
in Relation to Salinity
Salinity
o/oo
30
22
10
7
Control
749
687
551
547
Milliosmoles
95% Conf . Experi-
Interval mental
728-771
665-708
508-593
521-572
752
688
523
516
95% Conf.
Interval
726-779
658-718
500-546
495-537
or capacity of ATPase in marine organisms can be
inferred from the results of Pfeiler and Kirschner
(1972), who showed that gill ATPase activity of
rainbow trout adapted to salt water, was greater than
in fish adapted to fresh water.
Polychlorinated hydrocarbons have interfered with
either osmo- or ionic-regulation in aquatic animals
(Eisler and Edmunds, 1966; Grant and Mehrle, 1970;
Kinter et al. , 1972; Nimmo and Blackman, 1972). The
physiological relationship of ionic effects to that
of ATPase activity was first reported by Kinter et al.
(1972), who postulated that lipophilic agents such as
DDT and PCBs, might interact with the phospholipid-
activating components of the lipoprotein enzyme. The
effect of dieldrin on ion movement in the nervous
system of cockroaches showed that dieldrin inhibited
binding of calcium to the phospholipid moiety of the
enzyme, thus inhibiting the movement of calcium
across the nerve membrane (Hayashi and Matsumura,
1967). Calcium salts in fresh water greatly increased
the ability of marine and euryhaline animals to sur-
vive in that medium. (Black, 1957). Toxic symptoms of
DDT poisoning in freshwater fish could be alleviated
by the addition of calcium salts (Keffler, 1972). In
438
-------
POLLUTION AND PHYSIOLOGY OF MARINE ORGANISMS
our studies calcium was lowered 25% in the sera of
PCB-exposed shrimp at 7 o/oo salinity, and it may be
that this reduction was responsible in part for the
observed decrease in sodium, chloride, and other ions.
Field observations of juvenile and subadult brown
shrimp by several investigators indicate that those
shrimp tolerate a wide range of salinities:
Salinity Location Observer
0.22-0.36 o/oo St. Lucie estuary, Gunter and Hall
Florida (1963)
0-1.0 o/oo Mobile Bay, Ala. Loesch (In Gunter
et al., 1964)
0-2.0 o/oo Choctawhatchee Bay, Nimmo (unpubl.
Florida data)
69 o/oo Laguna Madre, Texas Simmons (1957)
Gunter, Christmas, and Killebrew (1964) found
that in Texas bays young brown shrimp were most
abundant at 10 to 30 o/oo, with greater abundance
above 20 o/oo. Zein-Eldin and Aldrich (1965) found
that postlarval brown shrimp withstood a wide range
of salinity-temperature combinations.
It is evident that adult shrimp are osmoregula-
tors at all but extremes of salinity (Fig. 2).
Williams (1960) gives the isosmotic point of shrimp
hemolymph at 26.5 o/oo (788 mOs), as compared to our
calculation of 23.4 o/oo (694 mOs) in control shrimp.
Nevertheless, there was no significant difference
between PCB-exposed and control shrimp (Table 6).
Obviously, a slight change in environmental osmotic
pressure would not be critical to the osmoregulatory
ability of the animals at isosmoticity, but was
critical in the dilute environment. Therefore, the
salinities where the PCB exerted its greatest effect
in the laboratory (as judged from mortality of shrimp)
were well within the range in which brown shrimp
occur in nature.
Although there was no appreciable difference in
osmotic concentration between control and PCB-exposed
shrimp, there was significant difference in major
ions in the sera of PCB-exposed shrimp (Fig. 3).
439
-------
D. R. NIMMO AND L. H. BANNER
McFarland and Lee (1963) found that the point of con-
vergence of the total ions in the hemolymph of feral
shrimp to that of the environment was 27 o/oo
(800 mOs). In our study, by extrapolation, conver-
gence occurred at 34 o/oo (1000 mOs) in controls,
whereas in PCB-exposed shrimp showed no convergence
and total ions paralleled unity.
In surveys conducted soon after the PCB was
first discovered in the Pensacola estuary, the distri-
bution of shrimp in relation to salinity seemed to be
related to the amount of the chemical in the animals
(Nimmo et al., 1971b). Of three species captured,
brown shrimp had the highest whole-body residues
(14 ppm) although most samples were lower. Seemingly,
a concentration of 14 ppm PCB in feral shrimp would
have been lethal if the animals were subjected to
salinity stress such as imposed by our experimental
procedure. We have unpublished data that suggest
existence of a threshold in average whole-body con-
centration of PCB (5.6 to 7.8 mg/kg) in pink shrimp
(P. duorarum) that would be lethal when superimposed
on salinity stress caused by our procedure. However,
a recent survey of feral shrimp from the Penacola
estuary showed that young adult shrimp now have only
a fraction of PCB concentrations found in 1969/70
periods. For example, a sample taken from Escambia
Bay in August 1973 had a whole-body concentration of
only 0.1 mg/kg.
Future studies should include research on inter-
action of PCB and salinity on juvenile and postlarval
shrimp since chronic toxicity tests have shown that
these stages were more susceptible to the chemical
(Nimmo et al., 197la). In addition, studies by Dana
Beth Tyler-Schroeder, of the Gulf Breeze Laboratory,
have shown the susceptibility of larvae of grass
shrimp (Palaemonetes pugio) to the PCBs, Aroclors ©
1016 and 1242, decreases with age (personal communi-
cation) . Also, the "in vivo" effect of PCBs on
ATPase activity in shrimp should be fully investigated.
440
-------
POLLUTION AND PHYSIOLOGY OF MARINE ORGANISMS
ACKNOWLEDGMENT
This work was supported by Contribution No. 198
of the Gulf Breeze Environmental Research Laboratory.
LITERATURE CITED
Black, V. S. 1957. Excretion and osmoregulation.
In: Physiology of Fishes, pp. 163-99, ed. by
M. E. Brown. New York: Academic Press.
Cutkomp, L. K., H. H. Yap, D. Desaiah, and R. B. Koch.
1972. The sensitivity of fish ATPases to poly-
chlorinated biphenyls. Environ. Health Perspect.
1:165-68.
Davis, P. W., J. M. Friedhoff, and G. A. Wedemeyer.
1972. Organochlorine insecticide, herbicide and
polychlorinated biphenyl (PCB) inhibition of Na,
K—ATPase in rainbow trout. Bull. Environ.
Contam. Toxicol. 8:69-72.
and G. A. Wedemeyer. 197la. Inhibition
by organochlorine pesticides of Na+, K4"—
activated adenosine triphosphatase activity in
the brain of rainbow trout. Proc. West.
Pharmacol. Soc. 14:47.
and . 197 Ib. Na+, K+—activated
ATPase inhibition in rainbow trout: a site for
organochlorine pesticide toxicity. Comp.
Biochem. Physlol. 40B:823-27.
Duke, T. W., J. I. Lowe, and A. J. Wilson, Jr. 1970.
A polychlorinated biphenyl (Aroclor 1254 ®)
in the water, sediment, and biota of Escambia
Bay, Florida. Bull. Environ. Contain, and
Toxicol. 5:171-80.
Eisler, R. and P. H. Edmunds. 1966. Effects of
endrin on blood and tissue chemistry of a marine
fish. Trans. -flm. Fish. Soc. 95:153-59.
Grant, B. F. and P. M. Mehrle. 1970. Chronic endrin
poisoning in goldfish, Carassius auratus.
J. Fish. Res. Bd. Canada 27:2225-32.
441
-------
D. R. NIMMO AND L. H. BAHNER
Gunter, G., J. Y« Christmas, and R. Killebrew. 1964.
Some relations of salinity to population distri-
butions of motile estuarine organisms, with
special reference to penaeid shrimp. Ecology 45:
181-85.
and G. E. Hall. 1963. Biological inves-
tigations of the St. Lucie estuary (Florida) in
connection with Lake Okeechobee discharges
through the St. Lucie canal. Gulf Res. Rep. 1:
189-307.
Hayashi, M. and M. Matsumura. 1967. Insecticide
mode of action: effect of dieldrin on ion
movement in the nervous system of Periplaneta
americana and Blatella germanica cockroaches.
J. Agric. Food Chem. 15:622-27-
Keffler, L. R. 1972. A study of the influence of
calcium on the effects of DDT on fishes. Ph.D.
dissertation, The University of Mississippi,
University, Mississippi.
Kinter, W. B., L. S. Merkens, R. H. Janicki, and
A. M. Guarino. 1972. Studies on the mechanism
of toxicity of DDT and polychlorinated biphenyls
(PCBs): disruption of osmoregulation in marine
fish. Environ. Health Perspect. 1:169-73.
Matsumura, F. and T. Narahashi. 1971. ATPase
inhibition and electrophysiological change
caused by DDT and related neuroactive agents
in lobster nerve. Biochem. Pharmacol. 20:825-37,
McFarland, W. N. and B. D. Lee. 1963. Osmotic and
ionic concentrations of penaeidean shrimps of
the Texas coast. Bull. Mar. Sci. Gulf Caribb.
13:391-417.
Nimmo, D. R. and R. R. Blackman. 1972. Effects of
DDT on cations in the hepatopancreas of penaeid
shrimp. Trans. .Am. Fish. Soc. 101:547-49.
, , A. J. Wilson, Jr., and
J. Forester. 1971a. Toxicity and distribution
of Aroclor ® 1254 in the pink shrimp, Penaeus
duorarum. Mar. Biol. 11:191-97.
442
-------
POLLUTION AND PHYSIOLOGY OF MARINE ORGANISMS
, P. D. Wilson, R. BlacJanan, and A. J.
Wilson, Jr. 1971b. Polychlorinated biphenyl
absorbed from sediments by fiddler crabs and
pink shrimp. Nature (London) 232:50-52.
O'Hara, J. 1973. The influence of temperature and
salinity on the toxicity of cadmium to the
fiddler crab, Uca pugilator. U. S. Fish. Wildl.
Serv. Fish. Bull. 72:149-53.
Pfeiler, E. and L. B. Kirschner. 1972. Studies on
gill ATPase of rainbow trout (Salmo gairdneri).
Biochim. Biophys. Acta 282:301-10.
Silbergeld, E. K/ 1973. Dieldrin: effects of
chronic sublethal exposure on adaptation to
thermal stress in freshwater fish. Environ.
Sci. Technol. 7:846-49.
Simmons, E. G. 1957. An ecological survey of the
upper Laguna Madre of Texas. Publ. Inst. Mai.
Sci., Univ. Tex. 4:156-200.
Tanaka, R., T. Sakamoto, and Y. Sakamoto. 1971.
Mechanism of lipid activation of Na, K, Mg—-
activated adenosine triphosphatase and K, Mg—
activated phosphatase of bovine cerebral cortex.
J. Membrane. Biol. 4:42-51.
Vernberg, W. B. and J. Vernberg. 1972. The synerges-
tic effects of temperature, salinity and mercury
on survival and metabolism of the adult fiddler
crab, Uca pugilator. U. S. Fish. Wildl. Serv.
Fish. Bull. 70:415-20.
Williams, A. B. 1960. The influence of temperature
on osmotic regulation in two species of estuarin<
shrimps (Penaeus). Biol. Bull. (Woods Hole) 117
560-71.
Yap, H. H., D. Desaiah, L. K. Cutkomp, and R. B. Koch
1971. Sensitivity of fish ATPases to poly-
chlorinated biphenyls. Nature (London) 233:
61-62.
Zein-Eldrin, Z. P. and D. V. Aldrich. 1965. Growth
and survival of postlarval Penaeus aztecus under
controlled conditions of temperature and
salinity. Bio2. Bu22. (Woods Hole) 229:199-216.
443
-------
CONTRIBUTION NO. 200
-------
(22)
A Salinity Controller for Flowing-Water
Bioassays 1
LOWELL H. BAHNER
U. S. Environmental Protection Agency
Gulf Breeze Environmental Research Laboratory
Salinity and temperature are rate determining factors
for many physiological functions and these variables
affect the toxicity of several pesticides to marine or-
ganisms. Because some compounds that alter or inter-
fere with osmoregulatory mechanisms in estuarine
organisms appear more toxic as salinity changes, it is
often desirable in estuarine bioassays (flow-through) to
adjust salinity to a constant level.
A salinity controller consisting of a sea-water hydro-
meter, photocell detector, and a relay controlled by an
electronic amplifier has been developed that monitors
and adjusts salinity continuously in flow-through sys-
tems. The controller regulates electrical pumps or
valves to adjust salinity within ±1% of the desired
level, and with minor modifications, can control tempera-
ture, water height, or light intensity.
1 Contribution No. 200, Gulf Breeze Environmental
Research Laboratory.
Reprinted from The ASB Bulletin,
Vol. 21, No. 2, April 1974, p. 37.
-------
CONTRIBUTION NO. 201
-------
From: Abstracts of the Annual Meeting American Society for Microbiology.
1973
P256 Degradation of Malathion by Estuarine Microbes. Al W. Bourquin*
and Gary H. Cook. U.S. Environmental Protection Agency, Gulf
Breeze Environmental Research Laboratory, Gulf Breeze, Florida 32561
Pathways for the biological degradation of malathion were characterized
using estuarine bacteria. Some correlation with microbial marshland
ecosystems was attempted. Bacteria were isolated from estuarine muds
previously untreated with malathion. Three strains were selected which
metabolized malathion in Zobell's 2216 marine medium. None of the organisms
used malathion as a sole carbon source. Early enzymatic hydrolysis products
were identified as the mono- and di-carboxylic acid derivatives of malathion.
Further microbial degradation products were characterized (including -^C02
liberated from the methoxy side chain) using l^C-labelled malathion. Inves-
tigations into the possible effects of light, temperature, pH, and salinity
on degradation of malathion were analyzed as a check on the biological system.
Degradation products were characterized in the same manner as the biological
samples. Chemical degradation of malathion increased rapidly with salinity
with an accumulation of the mono-carboxylic acid derivative.
Correlation of the microbial, chemical, and physical degradations of
malathion as it occurs in the environment was attempted using artificial
microcosms.
Gulf Breeze Contribution No.201
-------
CONTRIBUTION NO. 202
-------
Effects of Pesticides on Protozoa
Effets des Pesticides sur Protistes
NELSON R. COOLEY (U. S. Environmental Protection Agency, Gulf Breeze Environ-
mental Research Laboratory, Sabine Island, Gulf Breeze, Florida 32561, U. S. A
[Associate Laboratory, National Environmental Research Center, Corvallis,
Oregon])
Little is known about effects of pesticides and related compounds on proto-
zoa. Most studies have been performed on phytoflagellates, only a few on
ciliates. Ciliates are the most numerous animals in the estuarine benthos and
function as food for higher organisms and are important as regenerators of
nitrogen and phosphorus. Because of the importance of ciliates in estuarine
ecosystems and because estuaries often act as sinks for pesticides, my co-
workers and I have performed studies on effects of pesticides and polychlori-
nated biphenyls on these animals.
Tetrahymena pyriforrois W is sensitive to toxicants we have tested. The
ciliates accumulated and concentrated the toxicants from the medium. Signifi-
cant reduction in population growth rate and 96-hour population density
occurred at low toxicant concentrations:
Toxicant
Growth Rate
reduction
96-hr, population
density reduction
Accumulation
(X initial
concentration)
Mirex
Aroclor 1248
Aroclor 1254
Aroclor 1260
33% at 0.9 yg/£
18.9% at 1 mg/£
8% at 1 yg/fc
19.1 to 25% at
12% at 0.9 yg/J>
9.6% at 1 mg/4
10% at 1
13.6 to 22.4%
at 1 mg/fc
193 X
48 X
60 X
79 X
However, exploratory experiments suggest that ^T. pyriformis may be less
sensitive to malathion than to these chemicals.
The data suggest that pesticides and related toxicants that enter aquatic
ecosystems could reduce the availability of ciliates as food and as nutrient
regenerators, thereby disrupting nutrient cycles and perhaps altering species
composition of ciliate communities. The ability of responsive ciliates to
accumulate persistent compounds could permit translocation of the chemicals
through food chains. In this manner, effects of the chemicals could be
exerted at higher trophic levels.
Reprinted from Progress in Protozoology (Abstracts of papers read at
the Fourth International Congress of Protozoology,
Clermont-Ferrand, France, 2-9 September 1973), p. 91.
-------
CONTRIBUTION NO. 203
-------
In: proceedings of the First Microbiology
Seminar on Standardization of Methods.
March, 1974, EPA-R4-73-022, Environmental Monitoring Series. Washington, D. C.: U. S.
IMPACT OF MICROBIAL SEED CULTURES ON G°vt' Dinting Qffj
THE AQUATIC ENVIRONMENT*
Al W. Bourquin**
The tremendous use of oil for energy in the United
States has caused rapid increase in oil imports on large
cargo carriers. These large tankers, with capacities
equal to or greater than 100,000 dead-weight tons capacity,
and increased shipping, has enhanced greatly the danger
of major oil spills. With the impending danger o± cata-
strophic spills, technology of clean-up is extremely
limited. Present clean-up methods include adsorption
and recovery, chemical dispersion, and physical removal.
Each^technique has limitations due to quantity and type
of oil spilled, extent of the slick, and nature of the
environment where the spill occurred or where the slick
floated. Some authors believe no efficient and safe method
exists for clean-up of a spill in shallow estuaries (1,2) .
Extensive research is being conducted for the purpose
of^increasing microbial oil degradation by seeding oil
slicks with hydrocarbonoclastic microorganisms. It may
be possible that large quantities of selected microorganisms,
under proper environmental conditions, could hasten degra-
dation and ultimate removal of pollutant hydrocarbons (1).
The need for standardization of testing procedures for
commercially available microbial formulations was pointed
out at a recent international workshop held in Atlanta,
Georgia. Papers were presented to show that at least two
commercial products are completely ineffective or have
very little hydrocarbonoclastic activity—below that of
natural seawater (3). Other evidence, presented by EPA
representatives, demonstrated that at least one commercial
formulation contained at least four species of pathogenic
microorganisms (4).
A panel, "Environmental Considerations in Microbial
Degradation of Oil", at the Atlanta workshop recommended
that a committee be formed to study the problems of
effective and safe use of microbial seed cultures in the
environment. The committee should be composed of members
of a governmental agency, members of API—representing the
petroleum industry, and members of the academic community
who are active in oil pollution research (5).
* Submitted in writing for the record.
** Gulf Breeze Environmental Research Laboratory, Associate
Laboratory of NERC-Corvallis
140
-------
Microbial seed cultures are currently being studied
for application to the environment as microbiological
pesticides. Viruses have been isolated which attack
selectively the cabbage boll; a bacterium has been
isolated as a specific pathogen of mosquitoes; and
chitinoclastic bacteria have been proposed as agents
against plant predators in estuarine areas. The range
of impact on the aquatic environment by seed cultures
must be investigated adequately before they are used
on a large scale.
141
-------
CONTRIBUTION NO. 204
-------
PROCEEDINGS
OF
SEMINAR ON METHODOLOGY
FOR
MONITORING THE MARINE ENVIRONMENT
SEATTLE WASHINGTON
OCTOBER 1973
Program Element No. 1HA326
ROAP/Task - PEMP/2
SPONSORED BY
OFFICE OF MONITORING SYSTEMS
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
-------
METHODS AND PROBLEMS IN ANALYSIS OF PESTICIDES
IN THE ESTUARINE ENVIRONMENT
A. J. Wilson, Jr. and J. Forester
Gulf Breeze Environmental Research Laboratory,
Gulf Breeze, Florida
The presence of pesticides in the marine environment has been well
documented. Cox (4) reported DDT concentrations in sea water along the
Pacific Coast to range from 0.0023 parts per billion (microgram/liter)
off Oregon and Washington to 0.0056 parts per billion off Southern
California. Residues of DDT and dieldrin were detected in livers of
fishes by Duke and Wilson (6) from the Northeastern Pacific Ocean and in
gray whales by Wolman and Wilson (16). Other documentation of chlo-
rinated hydrocarbons in the marine environment is presented by Goldberg
etal. (7).
The Gulf Breeze Environmental Research Laboratory at Gulf Breeze,
Florida, an associate laboratory of the National Environmental Research
Center, Corvallis, Oregon, has been conducting research on the effects
of pesticides in the marine environment since 1958. Since that time the
laboratory has analyzed over twenty thousand samples for these pol-
lutants in water, sediment, oysters, crabs, fish, birds and mammals.
From 1965 until 1972 this facility analyzed over eight thousand samples
for the National Pesticide Monitoring Program as reported by Butler (3).
This report describes analytical methods employed by this Program, some
recent studies in water analysis, and the need for adequate analytical
quality control in marine monitoring.
NATIONAL PESTICIDE MONITORING PROGRAM
From studies at Gulf Breeze, bivalve mollusks appeared to be suitable
animals to use as indicators of estuarine pollution. Adult bivalve
mollusks are sessile, permitting repeated sampling of the same population.
In addition, experiments by Butler (2) indicated that bivalve mollusks
readily accumulate chlorinated hydrocarbons. Consequently, oysters,
mussels and clams were the primary indicator organisms in this program.
108
-------
Mollusks were collected at about 30-day intervals at 183 estuarine
sites in 15 coastal states. Approximately 15 individuals were taken
from each station by other agencies, prepared in their laboratories, and
shipped to Gulf Breeze for analyses. The rationale for all analyses
being conducted at Gulf Breeze was based on the premise that these
methods of analyses would be consistent from station to station and
month to month. This would eliminate variation in methodology and
permit a more reliable interpretation of seasonal and geographic trends.
In addition, it was not economically feasible at the outset to equip
several satellite laboratories and conduct a suitable inter-laboratory
quality control program.
Prior to the start of the program several preservatives were
evaluated to find a method that would allow shipment of samples
without dry ice. When samples were dehydrated by mixing them with a 9:1
mixture of anhydrous sodium sulfate and Quso, a micro fine silica, they
could be held at room temperature for up to 15 days without loss or
degradation of the chlorinated hydrocarbon. This procedure allowed
shipment of samples in aluminum foil by surface mail from the collect-
ing laboratory to the Gulf Breeze Laboratory.
Analytical Procedures
Mollusks were analyzed for aldrin, chlordane, o,p' and p,p' isomers
of DDT and its metabolites, dieldrin, endrin, heptachlor, heptachlor
epoxide, lindane, methoxychlor, mirex, and toxaphene.
Sample Preparation
The tissues of 15 individuals were shucked into a one-pint Mason
jar and thoroughly homogenized with an Osterizer blender. Approximately
30 g of the homogenate was added to a second Mason jar and blended with
a 9:1 mixture of sodium sulfate and Quso. By alternately chilling and
blending, a free-flowing powder was obtained. The blended sample was
wrapped in aluminum foil and shipped to Gulf Breeze. Upon receipt of
the sample, it was weighed and extracted in a Soxhlet apparatus for 4
hours with petroleum ether.
Sample Clean-Up
The extracts were then purified by concentrating and transferring
the extract to 250 ml separatory funnels. The extracts were diluted to
25 ml with petroleum ether and partitioned with two 50 ml portions
109
-------
DDE
DDD
DDT
PERCENTAGE RECOVERY OF PESTICIDES
FROM FORTIFIED OYSTER SAMPLES
:IDE
ACTUAL (PPM)
0.033
0.033
0.34
0.34
0.067
0.067
0.70
0.70
0.10
0.10
1.0
1.0
FOUND (PPM)
0.026
0.026
0.30
0.29
0.061
0.064
0.67
0.63
0.087
0.094
0.95
0.92
% RECOVERY
79
79
08
05
91
96
96
90
87
94
95
92
-------
of acetonitrile previously saturated with petroleum ether. The acetoni-
trile was evaporated to dryness and the residue eluted from a Florisil
column, Mills et al. (11). In this technique, increasing proportions of
ethyl ether to petroleum ether were used to elute fractions containing
increasingly polar insecticides.
Quantitation and Qualitation
The extracts were analyzed with Varian Aerograph electron capture
gas chromatographs. Extracts were injected into at least two 180 cm x 2
mm (ID) columns of different liquid phases. The following columns have
been used: DC-200, QF-1, DECS, OV-101, mixed DC-200/QF-1, and mixed 0V-
101/OV-17. Liquid phases and gas chromatographic parameters were adjusted
so that p,p' DDT would elute in approximately 12 minutes. The lower
limit of detection for a 30 g mollusc sample was 0.010 parts per million
(milligrams per kilogram). Residues were reported on a wet weight basis
without adjustment for recovery rates. Thin layer chromatography and
"p" values after Bowman and Beroza (1) were used for additional confirmation
of compound identity.
Extraction efficiencies were determined by re-extracting samples
for longer periods of time and with different solvent systems. Recovery
rates were determined by fortification of samples with known levels of
pesticides. Table 1 shows typical recovery rates of DDT and its metab-
olites from fortified oyster samples. The values were adjusted to
account for naturally-occurring DDT residues.
SEA WATER ANALYSIS
Prior to 1971, the Gulf Breeze Laboratory belonged to the Bureau of
Commercial Fisheries, United States Department of Interior. During
those early days, there was little known regarding the effects and
kinetics of pesticides in the marine environment. Consequently, in
addition to scientific publications, the laboratory frequently published
quarterly and annual progress reports containing provisional or pre-
liminary data.
In 1968 the author submitted the following report for inclusion in
a Progress Report of the Bureau of Commercial Fisheries (15).
STABILITY OF PESTICIDES IN
SEA WATER
We began preliminary studies to determine the
stability of pesticides in sea water. Three p.p.b.
of aldrin, p,p'-DDT, malathion, and parathion in
acetone were added separately to four clear glass,
111
-------
one-gallon bottles containing sea water (salinity
29.8 p.p.t.; pH 8.1). One chemical per bottle.
After an initial sample of the water was analyzed,
the bottles were sealed and completely immersed
in an outdoor flowing sea-water tank. Table 5
shows the concentration of the chemical at the
indicated time interval.
Although we used natural sea water in these
preliminary experiments, the tests will be
repeated with sterile artificial sea water so that
the relative stability of the pesticides can be
evaluated under standardized experimental conditions.
Because these studies showed a rapid loss of DDT in sea water, the
report received a great deal of attention. As stated, these were
preliminary studies but, unfortunately, many readers carried the data
beyond the scope of the experiment. Obviously, additional studies were
required to account for the rapid decline of these pesticides before any
conclusions could be made.
Since these studies were conducted, several investigators have
reported on the transport of pesticides in marine waters. Cox (4)
reported that adsorption of DDT is implicated in the uptake mechanism
for algal cells. His experiments also indicate that particles less
than l-2y diameter carry most of the DDT residues in whole water.
Working in the laboratory with six species of marine algae, Rice and
Sikka (13) found that all species concentrated DDT to levels many times
higher than the original concentration of the medium. Transformation of
DDT and cyclodiene insecticide took place in surface films, plankton,
and algae but not in water from the open ocean according to Patil et
al. (12).
Recently, experiments have been conducted to determine the cause of
loss of DDT in the 1968 studies at Gulf Breeze. The experiments were
repeated under similar conditions with the exception that duplicate
samples were analyzed. In 1968 the salinity was 29.8 ppt (parts per
thousand) and the incubation temperature averaged 29 C; in 1973, the
salinity was 24.0 ppt and the incubation temperature averaged 12 C.
Figure 1 shows the percentage recovery of DDT (including DDE and ODD)
during the two experiments. The results are similar except for the 17
day 1968 analysis.
112
-------
Taolo 5-—'-Stability of pesticides in natural sea water
(salinity 29.3 p.p.t.; pH 8.1)
Pesticide
M
H
uj
1
•D.t)'~DDT. ....
pjpf~DDE#. .
•n *n * «*• nnryjf .
I J e \J ^^ \J\JlJ'* 1 •
-i. «/ ^<
^\ | \ \ 1^ 1 II * > ** 9 t tf 41 df
Dieldrin^. .
Ilalathion. . . .
Parathion* « • .
Days after start of erperii'nent
0 6
P.p/b. P.p.b.
2.9 ,75
.096
2.6 .58
.74
3.0 <0.2
2.9 1.9
17
P.p.b.
1.0
.95
.081
.096
1.0
<0.2
1.25
24
P.p.b.
.27
.065
.041
<0.01
1.0
^
1.0
31
P.p.b.
.13
.034
.038
<0.01
.75
.—
.71
38
P»p-b*
-16
.037
-037
<0.01
.56
^
,37
>4fetabolites of parent compound.
•w-w-Frorn the seventeenth day onward, 2 unidentified peaks ap-
peared on the chromatographic charts after aldrin had eluted.
-------
Experiments were then performed to determine if DDT was adsorbed to
the walls of the test containers. Additional experiments were designed
to determine if DDT was converted to DDA, a water soluble metabolite of
DDT, which would no.; have been detected by the method of analyses used
in the initial study. These experiments showed that less than 1% of the
DDT was adsorbed to the walls of the glass bottles and furthermore that
there was no conversion to DDA.
Since petroleum ether was the solvent used for extracting the DDT
from sea water, the following studies were initiated to evaluate the
extraction efficiencies of other solvent systems. Duplicate one-gallon
bottles of clear glass, containing 3.5 liters of sea water or distilled
water, were fortified with 10.5 yg of p,p' DDT in 350 y£ of acetone to
yield a concentration of 3.0 ppb. Duplicate 500 ml samples were taken
from each bottle and extracted with one of the following solvents:
three 50 ml portions of petroleum ether, two 50 ml portions of 15% ethyl
ether in hexane followed by one 50 ml portion of hexane, or three 50 ml
portions of methylene chloride. All solvents were dried with sodium
sulfate, concentrated to an appropriate volume and analyzed by electron
capture gas chromatography. Just prior to extraction, all samples were
fortified with o,p'DDE to evaluate the integrity of the analyses. The
recovery rate of o,p'DDE in all tests was greater than 89%, indicating
no significant loss during analyses.
After initial sampling, the bottles were sealed and incubated at 20
C under controlled light conditions (12 hours light, 12 hours dark).
Duplicate samples of 500 ml were extracted at various time intervals.
Tables 2-5 show the average percentage recovery of p,p'DDT extracted
from duplicate sea water or distilled water samples up to 14 days after
initiation of the experiment. p,p'DDE was the only metabolite measured,
and since it never exceeded 2% of the parent compound it is not included
in the percentage recoveries. The sea water was collected adjacent to
the Gulf Breeze Laboratory in Santa Rosa Sound and the salinity ranged
from 16 ppt to 21 ppt.
Table 2 shows that immediately after the sea water (21 ppt) was
fortified with 3.0 ppb of DDT all solvent systems removed 93% of the
DDT. After six days of incubation this level of recovery was not observed
with any of the solvents tested. However, methylene chloride was more
efficient than petroleum ether or 15% ethyl ether in hexane. Part of
this experiment was repeated with sea water (16 ppt) and incubated for 4
days with similar results (Table 3).
114
-------
METAIitOLIYES IK1 1960-1973
Percentage Recovery of DDT and Metabolites in 1968 and 1973
-------
TABLE 2
PERCENTAGE RECOVERY OF P,P' DDT FROM SEA WATER
BY DIFFERENT EXTRACTION"SOLVENTS
J- -***•-*• •*'- *-•«•-*• • J-*>
DAY
0
EXTRACTION SOLVENT
PETROLEUM
ETHER
93
67
15% ETHYL ETHER
IN HEXANE
93
66
lUill d ii IH"I»T1
METHYLKNE
CHLORIDE
93
76
-------
TABLE 3
PERCENTAGE RECOVERY OF P,PV DDT FROM SEA WATER
BY PETROLEUM ETHER AND METHYLENE CHLORIDE
BAY
PETROLEUM ETHER
0
90
67
METffZLEHE CHLORIDE
95
85
-------
TABLE 4
PERCENTAGE RECOVERY OF P,PT DDT FROM SEA WATER AND
DISTILLED WATER BY PETROLEUM ETHER AND METHYLENE CHLORIDE
I
M
M
CO
I
DAY
0
7
14
SEA WATER
PETROLEUM
ETHER
90
58
46
METHYLENE
CHLORIDE
94
78
68
DISTILLED WATER
PETROLEUM
ETHER
90
90
94
L. .
METHYLEi;
CHLORIDl
91
91
92
-------
TABLE 5
PERCENTAGE RECOVERY OF PSP'DDT FROM SEA WATER
INCUBATED UNDER DIFFERENT LIGHT AND TEMPEDATUM CONDITIONS
DAY
12 HOUR LIGHT AND
12 HOUR DARK AT 20 C
DARK AT 5 C
vC
1
87
7
69
14
68
81
86
-------
An experiment was performed with sea water (21 ppt) and distilled
water using petroleum ether and methylene chloride. Table 4 shows
that immediately after fortification recoveries were greater than
90% for water and solvents.
After 14 days, similar recoveries were observed only in distilled
water. In sea water however, there was 49% and 28% reduction in
recovery with petroleum ether and methylene chloride respectively.
Since distilled water is devoid of particulate matter, this study
suggests that DDT may be absorbed or adsorbed to plankton or par-
ticulate matter in sea water and the sorbed material was not removed
resulting in low recoveries of DDT. This would explain the initially
high extraction efficiency of DDT followed by the decline in recovery
as DDT was associated with the particulate phase. Since methylene
chloride was the most polar solvent used, it would have a greater
affinity for removing the sorbed DDT.
In another test, duplicate bottles containing sea water (20 ppt)
and DDT were incubated under controlled lighting conditions at 20 C
and another set incubated at 5 C without light. Both were extracted
with methylene chloride at various time intervals. Table 5 shows
low recovery at 14 days under controlled lighting condition. However,
those samples incubated at 5 C in total darkness did not show a
significant decrease in recovery rate. Since the metabolic activity
of plankton was probably inhibited under these temperatures and light-
ing conditions, these results suggest that DDT may be absorbed rather
than adsorbed by plankton. However, Rice and Sikka (13) comparing
the uptake of DDT by living and dead algae found that cells accumulated
equal amounts of the pesticide.
Interaction of pesticides between water and particulate matter
are complex. Not only do light and temperature appear to alter
equilibria, but other physical and chemical factors have effects.
Evaluating liquid-liquid extraction techniques of herbicides from
river water, Suffet (14) observed that the isopropyl ester of 2,4-D
was adsorbed to particulate matter in river water and that the amount
changed by alteration of the pH of the water. Huang and Liao (10)
found that adsorption of DDT to clays was rapid but the amount differed
with the type of clay. A mixed culture of algae consisting mainly of
Vauchenia had a greater adsorption for DDT than bentonlte according to
Hill and McCarty (9). Cox (5) reported that in natural marine pop-
ulations virtually all of the DDT available for uptake was Incor-
porated onto phytoplankton, but this may only account for 10% of the
DDT residues recoverable from whole sea water.
120
-------
These experiments support the work of other investigators in
that DDT and other pesticides are extremely hydrophobic and can
easily be adsorbed or absorbed by suspended matter from liquid
solutions. The 1968 experiments at Gulf Breeze supports the concept
that physical or chemical transformations of pesticides altered the
extraction efficiences of the solvent and prevented complete recovery
of the compounds. Obviously, additional work needs to be done to
account for all of the chemical added to the test system.
It is difficult to relate laboratory findings directly to that
of the estuary or open oceans. However, the laboratory data illustrate
clearly some problems that could be encountered in monitoring sea water
for pesticide pollution. The conventional analyses of water samples
by liquid-liquid extraction techniques may provide Invalid data if
suspended matter is not considered. Standardized methods are needed
to analyze the water column and suspended material separately.
Recently, a synthetic resin, Amberlite XAD-2 was evaluated as an
adsorption medium for chlorinated hydrocarbons dissolved in sea water
by Harvey (8). This technique utilizes large volumes of sea water and
therefore permits greater sensitivity in analysis. In addition, the
method eliminates the problems encountered in transport of large samples
of water. Pollutants could be adsorbed on the resin at the sampling
site and shipped to the appropriate analytical laboratory for desorption.
Since the resin only removes the dissolved portion, additional samplings
would be required to determine the levels absorbed or adsorbed to
particulate matter.
ANALYTICAL QUALITY CONTROL
All pesticide residue laboratories should maintain an adequate
analytical quality control program. The program should include both
an intra-laboratory performance evaluation of personnel and method-
ology and an inter-laboratory sample exchange program. These programs
are time consuming but are essential to the generation of valid analyt-
ical data.
Table 1 shows the recovery rates of oyster samples fortified
with known concentrations of pesticides. Fortification techniques
provide data only on the recovery efficiency of the total analytical
procedure end not on the extraction step. Field residues may be
subject to physical and chemical transformation and therefore may
not be in the same physical or chemical state as the fortified sample.
Table 2 illustrates the errors that can result from fortified samples.
Extraction of water samples Immediately after fortification yields
121
-------
relatively high recovery efficiency with all solvent systems in sea
water. Analyses several days later show the relative inefficiencies
of the solvents systems used in sea water. Regardless of the analyt-
ical method used or the substrate being extracted, recovery data must
be obtained on the extraction efficiency and the total analytical pro-
cedure.
There are several other factors in residue laboratories which,
if ignored, may lead to inaccurate data. To name a few: (a) all
glassware must be clean and free of residues; (b) the purity of all
reagents used during analyses must be determined; (c) the accuracy of
analytical standards must be maintained; (d) the condition of all com-
ponents of the gas chromatograph must be optimized; and (e) laboratory
personnel should be thoroughly trained.
An area that needs further study is the use of internal standards
in marine pesticide monitoring. Currently, the Gulf Breeze Laboratory
is no longer affiliated with a large monitoring program. Almost all
samples submitted for analysis are of known identity. Most of these
samples are fortified with an internal standard, just prior to analysis.
The standard is usually a compound which behaves in an analytically
similar way to the compound of interest. This technique is valuable in
assessing the validity of the analysis. This same technique could also
be applied to marine monitoring samples if an appropriate compound could
be found that would not interfere with the monitored pesticides. It
would be extremely valuable in laboratories with large sample volumes
where close supervision of laboratory operations is not possible.
The analysis of marine samples for chlorinated hydrocarbon pesti-
cides is at times complicated by the presence of polychlorinated
biphenyl compounds (PCB). These compounds a.re industrial pollutants
and are produced in the United States under the trade name Aroclor.
They have chromatograph retention times similar to the organochlorine
pesticides and therefore complicate the analysis when both are present
in a sample. Several techniques have been described for the separation
of PCB from organochlorine pesticides. A. review of these methods were
presented by Zitto and Choi (17). These techniques are time consuming
and, in general, semiquantitative. In addition, differential absorption
or metabolism of the Aroclor isotaers in marine biota prevent accurate
analysis of the PCB's. la view of these facts aad the large number of
samples that could result from & global monitoring program, this are&
of analysis required further study and/or standardisation.
-------
REFERENCES
1. Bowman, M. C. and M. Beroza, 1965. Extraction p-Values of
Pesticides and Related Compounds in Six Binary Solvent
Systems. J_. Assoc. Agr. Chem. 48: 943-952.
2. Butler, P. A., 1966. Pesticides in the Marine Environment.
J. Appl. Ecol. 3 (Suppl): 253-259.
3. Butler, P. A., 1973. Organochlorine Residues in Estuarine
Mollusks 1965-1972 National Pesticide Monitoring Program.
Pestic. Monit. J. 6: 238-362.
4. Cox, J. L., 1971. DDT Residues in Seawater and Particulate
Matter in the California Current System. U. _S_. Fish.
Wildl. Serv. Fish. Bull. 69: 443-450.
5. Cox, J. L., 1972. DDT Residues in Marine Plankton. Residue
Reviews 44: 23-38.
6. Duke, T. W. and A. J. Wilson, 1971. Chlorinated Hydrocarbons
in Livers of Fishes from the Northeastern Pacific Ocean.
Pestic. Monit. .J. 5: 228-32.
7. Goldberg, E. D., P. Butler, P. Miller, D. Menzel, R. Risebrough
and L. Stickel, 1971. Chlorinated Hydrocarbons in the Marine
Environment. National Academy of Sciences, Washington, D. C.
8. Harvey, G. R., 1972. Absorption of Chlorinated Hydrocarbons from
Sea Water by a Crosslinked Polymer. Woods Hole Oceanographic
Institute, Woods Hole, Massachusetts. Unpublished manuscript.
9. Hill, D. W. and P. L. McCarty, 1967. Anaerobic Degradation of
Selected Chlorinated Hydrocarbon Pesticides. -T. Water Pollut.
Fed. 39: 1259-1277.
10. Huang, J. C. and C. S. Liao, 1969. Absorption of Pesticides on
Clay Minerals. Presented at the 1969 Missouri Academy of
Science Meeting, St. Louis, Missouri. rtted in Proc. of '25th
Ind. Waste Conf., Purdue Univ., Lafayette, Ind., 1970.
11. Mills, P. A., J. F. Onley and R. A. Gaither, 1963. Rapid Method
for Chlorinated Pesticide Residue in Non-Fatty Foods. .J.
Assoc. Agr. Chem. 46: 186-191.
12. Patil, K. C., F. Matsumura and G. M. Boush, 1972. Metabolic
Transformation of DDT, Dieldriu, Aldrin and Endrin by Marine
Microorganisms. Environ. Sci. Technol. 6: 629-632.
-------
13. Rice, C. P. and H. Sikka, 1973. Uptake and Metabolism of DDT
by Six Species of Marine Algae. :J. Agr. Food Chem.
21: 148-152.
14. Suffet, I. H., 1973. The p-Value Approach to Quantitative Liquid-
Liquid Extraction of Pesticides and Herbicides from Water.
3. Liquid-Liquid Extraction of Phenoxy Acid Herbicides from
Water. J_. Agr. Food Chem. 21: 591-598.
15. Wilson, A. J.t J. Forester and J. Knight, 1970. Chemical Assays.
1969 Prog. Rep., Center for Estuarine and Menhaden Research,
Gulf Breeze, Fla. U. J3. Fish Wildl. Serv. Circ. 335: 18-20.
16. Woiman, A. A. and A. J. Wilson, 1970. Occurrence of Pesticides
in Whales. Pestic. Monjt. J_. 4: 8-10.
17. Zitko, V. and P. Choi, 1971. PCB and Other Industrial Halogenated
Hydrocarbons In the Environment. Fish. Res. Board Can.
Technical Report No. 272, Biological Station, St. Andrews,
N. B.
-------
CONTRIBUTION NO. 207
-------
(16)
Effects of Aroclor® 1254 on Laboratory-Reared
Embryos and Fry of Cyprinodon variegtaus
STEVEN C. SCHIMMEL, DAVID J. HANSEN
AND JERROLD FORESTER
U.S. Environmental Protection Agency
Gulf Breeze Environmental Research Laboratory
Eggs of the sheepshead minnow (Cyprinodon varie-
gatus) were artificially fertilized (wet method) and
maintained at 15° to 30°C and 0 to 35 "/«, to deter-
mine efficient culture conditions. Fertilization was not
affected within the temperature or salinity ranges tested,
but hatching success was greatest (x2; a = 0.01) within
the 24° to 35°C range and 15 Voo to 30 Voo range.
Artificially fertilized sheepshead minnow eggs were
exposed to logarithmic series of concentrations of the
polychlorinated biphenyl (PCS), Aroclor 1254, (0.1 to
10 fig/1) in seawater averaging 30°C and 24 Voo in a
flow-through bioassay. Fertilization was not affected,
but significantly fewer embryos developed in the 10
nS/\. Fry were more susceptible to this PCB than were
embryos, juveniles or adults. — ® Registered trademark.
Monsanto Co., St. Louis, Mo. Mention of commercial
product does not constitute endorsement by the Environ-
mental Protection Agency. — Contribution 207, Gulf
Breeze Environmental Research Laboratory.
Reprinted from The ASB Bulletin,
Vol. 21, No. 2, April 1974, p. 81.
-------
CONTRIBUTION NO. 208
-------
EPA-660/3-74-013
September 1974
THEORETICAL MODEL AND SOLUBILITY CHARACTERISTICS
OF AROCLOR® 1254 IN WATER:
Problems Associated With Low-Solubility Compounds
In Aquatic Toxicity Tests
by
W. Peter Schoor
Gulf Breeze Environmental Research Laobratory
National Environmental Research Center
Gulf Breeze, Florida 32561
Program Element 1EA077
ROAP/Task No- 10AKC/18
National Environmental Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Corvallis, Oregon 97330
-------
ABSTRACT
A theoretical model of the behavior of substances having low water-
solubility is presented and discussed with respect to aqueous bioassay.
Ultracentrifugal techniques were used in an attempt to study size distribu-
tions of Aroclor 1254 aggregates in aqueous emulsions. Results indicate
strong adsorption from emulsion by surfaces and a water-solubility at 20 C
of less than 0.1yg/£ in distilled water and approximately 40% of that value
in water containing 30 g/£ NaCl. Implications with regard to aqueous bioassay
are discussed.
This report was submitted in fulfillment of Program Element 1EA077,
ROAP/Task No. 10AKC/18 by the Gulf Breeze Environmental Research Laboratory
under the sponsorship of the Environmental Protection Agency. Work was com-
pleted as of September, 1974.
11
-------
CONTENTS
Sections Page
I CONCLUSIONS 1
II RECOMMENDATIONS 2
III INTRODUCTION 3
IV THEORY 5
V MODEL 10
VI EXPERIMENTS WITH AROCLOR 1254 13
VII RESULTS 15
VIII DISCUSSION 27
IX REFERENCES 30
lii
-------
TABLES
No.
1. Effect of storage time on amount of Aroclor 1254 remaining 17
in the water phase.
2. Isomer distribution of Aroclor 1254 type II emulsion after 18
standing for various periods of time in 3£ glass bottle.
3. Adsorption of Aroclor 1254 type II emulsion on Polyallomer 21
centrifuge tubes on standing.
4. Adsorption of Aroclor 1254 on stainless steel centrifuge 21
tubes as a function of time and concentration.
5. Adsorption of Aroclor 1254 on stainless steel centrifuge 24
tubes.
6. Centrifugation of Aroclor 1254 in water of varying salinities 24
at 69,000 x g (max.).
7. Distribution of isomers of Aroclor 1254 type II emulsion 25
on standing in stainless steel centrifuge tubes.
8. Distribution of isomers in the absorbed fraction of Aroclor 26
1254 type III emulsion on standing in stainless steel
centrifuge tubes.
IV
-------
ACKNOWLEDGMENTS
The author thanks Messrs. D. Lamb and W. Burgess for assistance with the
analytical work and Dr. Ralph Birdwhistell, Dean, School of Chemistry,
University of West Florida, for reviewing the manuscript.
AroclorB 1254 is a registered trademark of the Monsanto Company, St. Louis,
Missouri.
-------
Section I
CONCLUSIONS
An extrapolation from the theory presented suggests that the use
of "carriers" be continued with caution, because of two independent
effects that may be present. One effect can most simply be described
as an alteration of the aggregate-solvent interactions by "carriers"
forming transition-like links between aggregates and solvent molecules.
In such a fashion, solute aggregates are surrounded by "carrier"
molecules, thus enhancing the ability of the aggregate to remain in a
stable emulsion by permitting greater solute-solvent interaction. This
can be illustrated graphically in Fig. 1 by enlarging region "B" over
a greater range of aggregate sizes since some aggregates previously
belonging to regions "A" and "C" now become more stabilized. It may
also be visualized by flattening the two curves in Fig. 2, thereby
extending their region of overlap. Thus, when added with a "carrier",
more of an insoluble compound may be introduced into a stable water
emulsion. The other effect may be due to possible interference with the
uptake of a test compound by an organism. Any such uptake must by neces-
sity be preceded by an adsorption to a surface of the organism such as
the gills in a fish. If at this time the "carrier" molecules, which are
located at the surface of the aggregate, affect the actual process of
adsorption in any way, there will be a resultant change in the rate of
transfer of the compound into the organism. If the rate of uptake is
related to toxicity, there will be a concomitant change in toxicity.
-------
Section II
RECOMMENDATIONS
This study shows, both theoretically and experimentally, that in
so far as physical interactions are concerned, emulsions differing in
degree of dispersion and stability can be formed, depending on the method
of preparation and subsequent treatment. Consequently, the following
questions should be answered before conducting bioassays in disperse
aqueous systems:
(a) What are the solubility characteristics of the compound
under investigation?
(b) To what extent are these characteristics related to
field conditions?
(c) How can the solubility characteristics and field
conditions be best simulated in the laboratory?
Such information would undoubtably result in more precise data on acute
toxicity as well as long-term effects regarding aqueous bioassay of
water-insoluble test compounds.
-------
Section III
INTRODUCTION
Laboratory experiments designed to determine the effects of
chemicals on aquatic organisms require that the tests be conducted
under conditions which reproduce those present in nature as closely
as possible. In order to accomplish this in a precise and scientific
fashion, the physical state of a compound in an aqueous dispersion
must be known. Convenience, time and other factors have in the past
often led to the use of techniques in the laboratory which do not take
into consideration that the solubility characteristics of a compound
may possibly affect the toxicity, necessitating extrapolation from an
apparent toxicity established in the laboratory to an expected toxicity
under field conditions. In many instances, the practice of using extra-
polation in scientific investigations is necessary and has proven to be
a valuable tool when certain conditions cannot be met. However, the
range through which the extrapolation is carried out must be chosen
with great care, because without sufficient experimental and theoretical
justification, a resulting extrapolation in this light may well prove to
be unrealistic. Since natural water conditions represent a multi-
component system, any attempt to quantitatively understand it must be
preceded by a study of the system under ideal conditions. While the
knowledge thus gained may or may not be of consequence in direct appli-
cation, it, nevertheless, provides a more precise scientific basis for
choosing valid limits for extrapolation.
The physical state of a compound in water is not a simple and
straightforward phenomenon, even given the idealized conditions of a
-------
two-component system - a single solute and a single solvent. A
definable system should, however, be the starting point of any
investigation aimed to scientifically arrive at data which lead to
a quantitative understanding of the behavior of a compound in water.
With this data a more precise attempt can be made to extrapolate from
a system employed in the laboratory to the obviously much more complex
system present in natural waters.
The purpose of this work is to provide a working theory on the
behavior of substances of low water solubility and to test this theory
by investigating the solubility characteristics of Aroclor 1254.
-------
Section TV
THEORY
To explain and predict the characteristics of"water-insoluble sub-
stances at low concentrations, an attempt is made here to redefine the
basic principles underlying a disperse system. No attempts have been
made to include in the definition the somewhat obsolete and often vague
definitions of emulsions, suspensions, colloids, etc. The characteristics
ascribed to each becoming readily apparent as the theoretical treatment
of the proposed model continues.
In this paper, an ideal or true solution is defined as a solute dis-
persed in a solvent so that any single molecule of solute is surrounded
by enough solvent molecules to insure that at any instant all solute mole-
cules are distributed statistically equidistant, assuming a dilution at
which interactions between solute molecules become negligible.
The ideal solution, under the conditions described, is represented
by the presence of single solute molecules. Solute aggregates consisting
of two or more molecules may represent a deviation from the ideal solu-
tion because, at least theoretically, these aggregates could consist of
any number of molecules whose behavior would not necessarily coincide
with that of a single molecule. For each solute and a single solvent,
there is assumed to exist amongst all aggregates a maximally stable
aggregate which, due to its nature, remains statistically equidistant
from all other aggregates for at least a certain period of time. The
stability of this aggregate depends solely on the molecularly char-
acterized interactions at the solute-solvent interphase and on tem-
perature.
-------
By definition, a single solute molecule in a disperse system
possesses a certain sphere of influence, the nature of which governs the
fate of the solvent molecules that surround it, which in turn affects the
behavior of the solute molecule, and thus determines the characteristics
of the solute molecule in the system. While precise information is lack-
ing, it is known, nevertheless, that the range of effect of a solute
molecule may extend through several layers of surrounding solvent molecules.
This means, of course, an orderly alignment involving either oppositely
charged polar regions or non-polar regions on the solute and the solvent
molecules. If this interaction between solute and solvent molecules is
of significance, the above defined ideal solution can be visualized, pro-
vided also that there is no competition among the solvent molecules belong-
ing to respective spheres of influence of two separate solute molecules.
The complexity of the situation is increased in cases where the
interactions between solute and solvent molecules (solute-solvent inter-
actions) become less pronounced, and, as a result, the interactions between
solute and solute molecules (solute-solute interactions) become more pro-
nounced. This implies that the sphere of influence around the solute
molecule is diminished with respect to the solvent molecules which are
now no longer attracted to the same degree. As two or more solute mole-
cules start to form aggregates, the factor of size of aggregates versus
their stability in a solvent becomes of utmost importance.
A generalized illustration of the size distribution of aggregates
that one might expect to find in a suspension is shown in Fig. 1.
-------
INCREASE
IN
RELATIVE
STABILITY
OF
EMULSION
REGION WHERE SMALL
AGGREGATES COALESCE
B
REGION OF MAXIMUM
STABILITY
REGION WHERE LARGE
AGGREGATES PRECIPITATE
INCREASE IN AGGREGATE DIAMETER
Figure 1. Theoretical relative stability of different sizes of aggregates in
an emulsion during a given time interval.
-------
Region "A" describes an area in which the aggregates are too small to
exist independently because interactions in the sphere of influence
at that point are such that solute-solute interactions, which have now
become aggregate-aggregate interactions, are more pronounced than the
aggregate-solvent interactions. Therefore, these aggregates are
expected to coalesce, moving them into region "B", which describes a
range of aggregate sizes of maximum stability. The aggregate-aggregate
interactions in this range are weaker than in region "A" for that size
of aggregate. Region "C" described aggregates which are too heavy to
remain in suspension for a given period of time and will settle out or
break into smaller, more stable aggregates. The exact shape of this
curve and especially that of region "B", depends on how tightly the sol-
vent is held within the sphere of influence of the solute aggregate, which
is a function of the molecular interactions between solute and solvent.
The distribution of different aggregate sizes in terms of molecularly
characterized interactions is shown in Fig. 2. The actual equilibrium
reaction taking place is described in a simplified manner at the top of the
figure. The two curves relate the hypothetical strength of interactions
of solute-solvent (aggregate-solvent) type and solute-solute (aggregate-
aggregate) type to aggregate size. The region where the curves cross
corresponds to a distribution of aggregate sizes of maximum stability.
-------
EQUILIBRIUM BETWEEN
SINGLE MOLECULE (A)
AND AGGREGATES
(B) AND (C)
B
RELATIVE
STRENGTH
OF
INTERACTION
AREA OF MAXIMUM
STABILITY
SOLUTE-SOLUTE
(AGGREGATE - AGGREGATE)
INTERACTIONS
SOLUTE - SOLVENT
(AGGREGATE - SOLVENT)
INTERACTIONS
AGGREGATE SIZE
Figure 2. Theoretical strength of interaction between solute and solvent.
-------
Section V
MODEL
Aroclor 1254 was chosen as a model compound because it has been
extensively used in bioassay at this laboratory (Duke _e_t al. , 1970;
Nimmo jit. auL., 1971a; Nimmo _e_t _al. , 1971b; Hansen e_t al_. , 1971; Lowe et
al., 1972; Walsh, 1972; Cooley et_ al. , 1972).
One approach to estimate quantitatively the solubility of Aroclor
1254 in water and the behavior of its aggregates is to use ultracentrifugal
analysis. This technique permits the selective removal of particles of
a certain size. For a spherical particle having a density of (p) and a
radius of (r) the molecular weight (M.W.) is represented by:
M.W. = 4/3irr3 NQ (1)
where N is Avogadro's Number.
Two opposing forces (f) which determine the fate of a particle in solution:
3
sedimentation f = 4/3ur (p-p )g, and (2)
buoyancy f = 6irrn> (3)
where (p ) is the density of the solvent, (g) is gravity, and (n) is the
viscosity of the solvent.
To remove a small particle from an emulsion at a reasonable rate, a
force larger than gravity must be applied. Using the ultracentrifuge, (g)
2
in equation (2) is replaced with (u x), the angular velocity of the
centrifuge rotor (w) times the distance of travel (x) of the emulsified
particle.
1 The equations used are normally found in any textbook on physical
chemistry, and their reproduction here is intended merely for the
convenience of the reader.
10
-------
The rate of sedimentation during centrifugation is described by:
dx = 2r2(p-p0)io2x
dt 9n
(4)
where (t) is time in seconds to reach equilibriuo. Integration yields:
. _ 2r2(p-p0)a)2t
In xo - In xi =
1 9n
The radius of a spherical particle is then given by:
(5)
where
r =*
w =
n =
p =
X =
t =
9n(ln x2-ln
2(p-p0)to2t
1/2
0.10472 (rpm)rotor
g/cm/sec
g/cm3
cm
sec
(6)
Knowing the radius of a particle or assuming a radius, the time necessary
to remove the particle from an emulsion is given by:
t =
x2-ln
2(p-Po)r2u2
(7)
11
-------
The following are particle size limits calculated uaj.ug
Q
(6) for given centrifugation times, with n = 8.94 x 10 g/sec/cm, x^ = 6.7 cm,
x2 = 15.3 cm, p - p = 0.508 g/cm3 at 25,000 rpm.
Time (hrs) Radius of particle (nm)
1 16.3
2 11.5
3 9.3
4 8.1
6 6.6
8 5.7
The following are particle size limits calculated using equation (6)
for given centrifugation times, with n = 8.94 x 10~3 g/sec/cm, x = 6.00 cm,
x2 = 10.73 cm, p - pQ= 0.508 g/cm3 at 45,000 rpm.
Time (hrs) Radius of particle (nm)
1 7.6
2 5.4
3 4.4
4 3.8 (208,000 g/mole1;
636 molecules)
6 3.1
8 2.7
12 2.2 (40,000 g/mole1;
124 molecules)
Average molecular weight Aroclor 1254 = 327 g/mole (Hutzinger £t al.,
(1972).
12
-------
Section VI
EXPERIMENTS WITH AROCLOR 1254
Wide-mouth jars, 30 cm high and 14 cm wide, were used to produce
3£ of Aroclor 1254 emulsion per batch. Mechanical considerations
concerning the proper physical agitation of Aroclor 1254 and water
made it necessary to use 250 ml of Aroclor 1254 in the jar to submerge
the blades of the stirrer. Agitation for 0.5 hr at 60°C and 1,800 rpm
produced a cloudy emulsion which was allowed to settle for 48 hrs, when
the range of concentration was found to be 1-20 mg/£ and the emulsion
became almost clear. This emulsion is referred to as type-I. A second
homogenization was carried out by transferring to a jar identical to the
one used previously volumes of type-I emulsion to produce emulsions of
10-300 yg/&, and stirring 1 hr at 25°C and 1,800 rpm. This emulsion is
referred to as type-II. Type-III emulsions were prepared by taking an
appropriate volume of type-I emulsion, adding it to a stainless steel
blender jar to make a total volume of 500 ml, and homogenizing at high
speed for 5 min.
All centrifugations were performed in a Beckman Model L3-50 ultra-
centrifuge at 20°C using SW 50.1 and SW 25.2 rotors.
The extraction procedure was that of Schoor (1973), with modifica-
tions of the ratio of water to hexane. Evaporation was carried out by
placing the hexane extracts in a water bath at 35°C and allowing a gentle
stream of air to blow across. This method was found superior to dis-
tillation in percentage recovery and time involved. When the extract
volumes had to be reduced to less than 10 ml, dried, pre-purified nitrogen
was used instead of air.
13
-------
A Hewlett-Packard Model 5700 gas chromatograph with a linear
electron-capture detector ( Ni) was used for quantitative determina-
tion of the Aroclor 1254. The linearity of this detector eliminated
use of different standards at each attenuation or reduction in volume
of the sample, either being very time consuming and subject to errors.
An OV-101 column (2% OV-101 on Gas Chrom Q, 100-120 mesh) was operated
at 195°C with the detector at 300°C and the argon-methane (10:1) carrier
gas at a flow rate of 25ml/min. Except where noted, quantitation was
performed by comparing total peak heights of sample and standard.
To determine the amount of Aroclor 1254 adsorbed on walls of
the 34 ml stainless centrifuge tubes, the water phase was decanted and
any adhering droplets removed with a disposable pipet. Since acetone
injected with the sample was detrimental to the chromatographic column,
a sonic probe and hexane were used for removal of Aroclor 1254 from the
walls of the tubes. This was necessary because the thin layer of water
remaining on the walls shielded the Aroclor 1254 and prevented it from
being desorbed into the hexane phase. Bonification emulsified the water
at the boundary layer, thus allowing the hexane to contact the adsorbed
Aroclor 1254.
14
-------
Section VII
RESULTS
A typical chromatogram of an Aroclor 1254 standard in hexane (A)
and a hexane extract of a type-II emulsion (B) is shown in Fig. 3.
Some of the 11 peaks indicated are multiple peaks. Only peaks 1-7
were used to calculate the "total" peak height on which all quantita-
tions were based. Peaks 8-11 were excluded, because they were often
too small to permit accurate calculations.
The effect of storage time on Aroclor 1254 emulsions of type-I
and type-II is shown in Table 1. There is a fairly rapid initial
decrease in Aroclor 1254 in all cases and it appears that a plateau is
reached at around 7 ug/&. This should not be interpreted to mean that
solubility is approached at that point, only that perhaps a stable
emulsion is reached at that point.
The hexane extract of type-II emulsion (chromatogram B) indicates
a relative reduction in peak height for the early eluting peaks. This
phenomenon is better described by the results shown in Table 2. For
comparison peak 7 was arbitrarily assigned a relative value of 100%.
The results indicate that on standing a type-II emulsion shows a reduc-
tion of the individual peaks, with the early eluting components, or less
chlorinated biphenyls (Zitko, 1970), being reduced much more than the late
eluting ones. The degree of reduction depends somewhat on the preparation
and initial concentration of individual type-II emulsions (Table 2).
Type-III emulsions of comparable "total" concentration show a relative
distribution of the isomers identical to that of the standard.
15
-------
AROCLOR 1254
STD-1 (O.4ng/jul)
X32
X16
AROCLOR 1254
Water Extract
5.4 jjl
X4
VT = 2O.Oml
Figure 3.
Typical gas chromatograms (see text for
detailed information.
-------
Table 1. EFFECT OF STORAGE TIME ON AMOUNT OF AROCLOR 1254 REMAINING
IN THE WATER PHASE
Time (days)
0
2
5
6
8
9
13
15
19
20
21
23
26
28
33
34
41
43
>^g/£ Aroclor 1254
Type I Type II
2300 301 50.2
286
115 23.6
113 11.3
112
123
97 98.5
502
483 87 54.7 6.7
48.1
44.5
78 7.1
6.5
428 7.7
355 7.4
350
15.5
280 6.8
17
-------
Table 2. ISOMER DISTRIBUTION OF AROCLOR 1254 TYPE II EMULSION AFTER
STANDING FOR VARIOUS PERIODS OF TIME IN 3£ GLASS BOTTLE
Time
(days)
2
9
13
19
20
21
41
21
33
38
Total
cone .
(ygM)1
286
123
98
54
58
44
15
13
3
1
(3
.5
.7
.1
.5
.5
.4
.6
.6
. 4 ppm)
% Peak Height1
2
Peak Numbers
1
76
79
79
72
64
61
37
16
12
9
(41)
2
93
78
79
75
70
65
41
27
21
10
3
95
89
93
85
80
82
56
44
39
(80)
4
95
94
98
93
89
90
70
55
45
31
5
98
98
99
91
92
99
77
76
64
46
(87)
6
104
99
96
95
94
93
88
87
82
63
(100)
7
100
100
100
100
100
100
100
100
100
100
Calculations are based on the relative height of peak 7 (see below)
2
Peak numbers are shown on the chromatogram in Fig. 1.
18
-------
The distribution of isomers in a hexane extract of the gill tissue
of a pink shrimp (Penaeus duorarum) exposed to 2.5 yg/Jl Aroclor 1254 for
20 days is shown in parentheses at the bottom of Table 2. Because peaks
2, 4 and 7 showed obvious contamination, peak 6 was assigned the arbitrary,
relative 100% value. The "total" concentration of 3.4 mg/kg was based on
the total height of peaks 1, 3, 5 and 6, and on the wet weight of gill
tissue (blotted to remove adhering water).
Filtration of type-I emulsion through 450 nm (0.45y) MilliporeR
filters revealed obstructed passage of Aroclor 1254 aggregates smaller
than 450 nm. Starting with a 1 mg/& emulsion and changing filters after
each filtration, less than 0.01 yg/& of the material remained in the water
after 15 passages. Since aggregates in the starting emulsion were most
likely smaller than 450 nm (calculations using equation 1 lead to roughly
10 times the average molecular weight of Aroclor 1254) , the Aroclor 1254
must have been adsorbed on the filter. This was also evidenced by the
fact that the filter paper turned slightly transparent after the first
passage during which about 95% of the material was removed from the
emulsion.
The first centrifugation experiments were carried out by centri-
fuging 180 ml of 42 yg/£ Aroclor 1254 type-II emulsion in 60 ml polyace-
tate centrifuge tubes for 60 min at 107,000 x g (max.).
19
-------
At an 85% total recovery the following distribution was found:
Acetone extract of tubes 66%
Hexane rinses of tubes 18%
Top 50 ml water phase 5%
Bottom 10 ml water phase 11%
The low recovery (85%) was probably due to incomplete extraction
of the tubes in spite of refluxing with acetone.
Polyallomer centrifuge tubes were tried next. When 180 ml
of 286 yg/Jl type-II emulsion were centrifuged in 60 ml Polyallomer
tubes for 60 min. at 107,000 x g (max.) the following distribution
was found:
Acetone extract of tubes
Hexane rinses of tubes 22%
Top 25 ml water phase .5%
Bottom 35 ml water phase .6%
These percentages were based on the total amount of starting material,
i.e., assuming 100% recovery instead of the 85% in the case of
the polyacetate tubes. Extraction of the Polyallomer tubes by reflux-
ing with acetone produced too many interfering peaks on the chroma-
togram, making complete recovery calculations impossible. Direct
adsorption on Polyallomer tubes was achieved by permitting type-II
emulsions to sit undisturbed in the tubes. Table 3 shows the outcome
for two different concentrations.
To permit recovery and study of the material adsorbed on surfaces
34 ml stainless steel centrifuge tubes were used for static tests,
20
-------
Table 3. ADSORPTION OF AROCLOR 1254 TYPE II EMULSION ON POLYALLOMER
CENTRIFUGE TUBES ON STANDING
Time (hrs) Aroclor 1254 (yg/£)
in water phase
0
3
72
0
1
3
125
86
3.3
45
35
27
Table 4. ADSORPTION OF AROCLOR 1254 ON STAINLESS STEEL CENTRIFUGE TUBES
AS A FUNCTION OF TIME AND CONCENTRATION
Aroclor 1254 type II emulsion
Time
(hrs)
0.5
1
2
16
1
2
(yg)
3.83
3.83
3.83
3.83
0.48
0.06
Total
(yg/i)
113
113
113
113
14
2
Water
(Mg) (yg/Ji;
3.63 107
3.31 97
3.20 94
3.14 92
0.35 10
0.03 1
S. S. tube
> (yg)
0.18
0.30
0.33
0.51
0.08
0.02
% adsorbed
5
9
13
16
23
67
Stainless steel centrifuge tubes.
21
-------
as well as for ultracentrifugal analysis. Table 4 shows the amounts
of Aroclor 1254 adsorbed on the wall of a stainless steel centrifuge
tube in relation to starting concentration and time. The amounts
adsorbed from the 14 yg/£ and 2 yg/£ emulsions were greater than that
adsorbed from the 113 yg/£ emulsion during the same time period. It
should be pointed out that 0.100 yg of Aroclor 1254 adsorbed as a
monomolecular layer per tube represents about 2% of the minimum area
available. The calculated inside area of a stainless steel centrifuge
2
tube was 60.8 cm . This area must be considered minimum because the
surface was assumed to be ideally smooth, which certainly is not the case.
However, for the approximations involved, this figure was used.
2
A simple calculation using equation (1) yields 0.613 nm for the
cross-sectional surface area of an average Aroclor 1254 molecule using
the average molecular weight of 327 (Hutzinger et ji^L., 1972), and
3
p = 1.505 g/cm (W. B. Papageorge, Monsanto Company, St. Louis,
Missouri, personal communication). Utilizing a molecular model with
the phenyl groups at right angles to each other and bond length
(Pauling, 1940) as the basis for calculations, a cross-sectional area
2 2
of 0.643 nm for the fully chlorinated and 0.356 nm for the unchlori-
nated or biphenyl molecule was obtained. Values falling between are
. 2
not linearly related to amount of chlorination. Using 0.613 nm as an
approximate, average cross-sectional area, 0.100 yg of Aroclor 1254
2
occupies 1.13 cm in the form of a monomolecular layer. This corresponds
to approximately 3 yg/2. in a 34 ml stainless steel centrifuge tube.
22
-------
It can be seen that even at 50% adsorption from a 3 \ig/i emulsion only
about 1% (maximum) of the available surface area is occupied, and
surface saturation was not a factor.
The amounts of Aroclor 1254 in the form of emulsions of type-II
and type-Ill adsorbed on the walls of the stainless steel centrifuge
tubes are shown in Table 5. There is a difference in adsorption of the
two different types of emulsion in the absence of NaCl. At least for
type-Ill emulsions, the introduction of 30 g/& NaCl appears to have no
effect on the amount of Aroclor 1254 adsorbed. However, centrifugation
reveals a difference in the size of the aggregates formed in the presence
of NaCl, as shown in Table 6.
In comparison with an Aroclor 1254 standard, the relative distri-
bution of the isomers in emulsions of type-II and III is quite different,
as shown in Tables 7 and 8. However, in all cases the adsorbed Aroclor
1254 had a higher percentage of early eluting (gas chromatography) isomers
than did that which remained in solution.
23
-------
Table 5. ADSORPTION OF AROCLOR 1254 ON STAINLESS STEEL CENTRIFUGE TUBES
Time
((hrs)
0.5
1.0
2.0
4.0
19
22
•pg Aroclor 1254^ adsorbed
Type II Emulsion Type III Emulsion
0 g/A NaCL 30 g/A NaCl 0 g/A NaCl
0.19 0.09
0.30 0.10 0.10
0.33 0.14 0.14
0.42 0.19
0.39
0.45
LData. adjusted to 4.00 yg total starting amount.
Table 6. CENTRIFUGATION OF AROCLOR 1254 IN WATER OF VARYING
SALINITIES AT 69,000 x g (MAX.).
Aroclor 1254
remaining in water phase
Time (hrs)
0.5
1.0
2.0
g/£ NaCl
0 15
13.9 7.1
12.5 6.6
7.2 4.6
30
6.0
4.9
2.9
-'-Started with 50 yg/Jl Type III emulsion.
24
-------
Table 7. DISTRIBUTION OF ISOMERS OF AROCLOR 1254 TYPE II EMULSION
ON STANDING IN STAINLESS STEEL CENTRIFUGE TUBES
Storage Hrs in
(days) tube yg/&
%
123
Peak heights
2
Peak number
4 5
6
0
310 water
93 90 98 99 98 100 100
0 115 water 53 71 73 91 98 98 100
2 97 water phase 49 67 69 83 100 100 100
2 12 adsorbed 96 106 103 127 119 100 100
0 112 water 51 67 71 82 96 97 100
2 102 water phase 48 66 68 79 98 98 100
2 8.0 adsorbed 69 82 85 104 107 100 100
13 0 97 water 47 64 68 81 97 98 100
2 86 water phase 43 59 66 78 92 96 100
2 6.1 adsorbed 47 68 77 94 101 98 100
-'-Compared to standard Aroclor 1254 (Fig. 1). Calculations are based
on the relative heights of peak 7.
r\
Peak numbers are shown on the chromatogram in Fig. 1.
-------
Table 8. DISTRIBUTION OF ISOMERS IN THE ABSORBED FRACTION OF AROCLOR 1254
TYPE III EMULSION ON STANDING IN STAINLESS STEEL CENTRIFUGE TUBES
% Peak heights1
2
Peak number
NaCl hrs in water phase adsorbed
(g/A) tube (yg/£) (yg) 12345
0 2 47.4 0.122 149 127 135 130 98 100
30 1 46.9 0.075 144 121 129 129 105 100
0 22 39.7 0.190 139 118 113 122 127 100
Compared to standard Aroclor 1254 (Fig. 1). Calculations are based
on the relative heights of peak 6.
2
Peak numbers are shown on the chromatogram in Fig. 1.
26
-------
Section IX
DISCUSSION
The original intent for conducting the work described was to find
the absolute solubility of Aroclor 1254 in fresh and salt water. This,
unfortunately, was not completely accomplished to any accurate degree,
because a series of significant problems occurred at the beginning of
the centrifugation experiments. Recovery of Aroclor 1254 after
centrifugation was low and, hence, led to the discovery that adsorption
occurred on the walls of the polyacetate centrifuge tubes as well as on
Polyallomer and stainless steel centrifuge tubes. Ultimately, only the
stainless steel centrifuge tubes were used in the adsorption and ultra-
centrifugal studies.
The apparent disappearance of early eluting isomers, such as shown
in Table 2, has been observed by others. It was found to occur in the
eggs of the double-crested cormorant and regarded as possibly due to
metabolic breakdown (Hutzinger ejt al., 1972). Similar behavior in the
carcasses of bobwhite quail after exposure to Aroclor 1254 was observed
and believed to be because of isomeric transformations (Bagley and
Cromartie, 1973). Application of Aroclor 1254 to different types of
soil showed a reduced recovery of the early eluting, lower chlorinated
biphenyls (Iwata et^ jil., 1973), and it was postulated that this may have
been due to evaporation from the soil. My studies did not substantiate the
observations by Zitko (1970) that when Aroclor 1254 emulsions are centri-
fuged the dissolved fraction is richer in the lower chlorinated biphenyls
than is the original preparation. However, the difference could be due
to the method of the preparation of his emulsion, which was similar to my
type-Ill emulsion. In both type-II and type-Ill emulsions the distribution
27
-------
of isomers in the water phase shows a loss of the lower chlorinated
biphenyls on standing (Tables 7 and 8). This loss was accounted for
in all cases by adsorption on the stainless steel centrifuge tubes,
the "lost" lower chlorinated biphenyls always being found in the
adsorbed fraction. Thus, at least from water emulsions of Aroclor 1254,
loss of the lower chlorinated biphenyls is due to their relatively greater
affinity for surfaces.
The published values for solubility of Aroclor 1254 in fresh and
salt water of 2-3 mg/£ and 1-1.5 mg/£, respectively (Zitko, 1970),
appear much too high. A conservatively high estimate based on my ultra-
centrifugal experiments indicates the average solubility of the isomers
to be less than 0.1 yg/£ for fresh water and approximately 0.04 ygA
(calculated from Table 6) in water containing 30 g/£ NaCl. It is extremely
difficult, in my opinion, to obtain an absolute value for the true solubility
of the average molecular weight isomer of Aroclor 1254. The problem lies
in the fact that at low concentrations, long centrifugation times (in excess
of 12 hrs at 243,000 x g (max.) theoretically are necessary to eliminate
aggregates from the emulsion. At the low concentrations necessary to
eliminate undesirable stirring back after completion of the centrifugation
(Bowman et al., 1960), adsorption on the walls of the stainless steel
centrifuge tubes (67% at 2 yg/4 for 2 hrs, Table 4) makes it all but
impossible to employ ultracentrifugation for extended periods of time.
It appears that at least in the case of type-Ill emulsions the adsorp-
tion from water emulsions containing 0 and 30 g/£ NaCl was the same
(Table 5), although the rate of sedimentation was quite different. The
28
-------
explanation for this lies in the fact that the size of the Aroclor 1254
aggregate is much larger in the presence of salt and, while this is not
apparent at 1 x g, the larger aggregates are removed more quickly from
the salt-containing emulsion during ultracentrifugation. This agrees
very well with my hypothesis that a larger aggregate is more stable
under the given conditions and in the presence of salt, which is conducive
to greater solute-solute (aggregate-aggregate) interaction.
29
-------
Section X
REFERENCES
/g)
Bagley, G. E., and E. Cromartie. Elimination Pattern of Aroclor 1254
Components in the Bobwhite. J. Chromatogr. Sci. ^75_: 219-226, 1973.
Bowman, M. C., F. Acree, Jr., and M. K. Corbett. Solubility of Carbon-14
DDT in Water. J. Agric. Food Chem. £(5):406-408, Sept. 1960.
Colley, N. R., J. M. Keltner, Jr., and J. Forester. Mirex and Aroclor
1254 : Effect On and Accumulation by Tetrahymena pyriformis Strain W.
J. Protozool. 19^(4): 636-638, 1972.
Duke, T. W., J. T- Lowe, and A. J. Wilson, Jr. A Polychlorinated
Biphenyl (Aroclor® 1254) in the Water, Sediment, and Biota of Escambia
Bay, Florida. Bull. Environ. Contam. Toxicol. 5/2):171-180, 1970.
Hansen, D. J., P- R. Parrish, J. I. Lowe, A. J. Wilson, Jr., and
P. D. Wilson. Chronic Toxicity, Uptake and Retention of Aroclor
125^5)in Two Estuarine Fishes. Bull. Environ. Contam, Toxicol.
113-119, 1971.
Hutzinger, 0., S. Safe, and V. Zitko. Polychlorinated Biphenyls.
Analabs Res. Notes. 12(2):1-11, July 1972.
Iwata, Y., W. E. Westlake, and F. A Gunther. Varying Persistence
of Polycblorinated Biphenyls in Six California Soils Under Laboratory
Conditions. Bull. Environ. Contam. Toxicol. j) (4) : 204-211, 1973.
Lowe, J. I., P. R. Parrish, J. M. Patrick, Jr.-^and J. Forester.
Effects of the Polychlorinated Biphenyl AroclorS^1254 on the American
Oyster (Crassostrea virginica). Mar. Biol. r7(3):209-214, Dec. 1972.
Nimmo, D. R., R. R. Blackman, A. J. Wilson, Jr., and J. Forester.
Toxicity and Distribution of Aroclor® 1254 in the Pink Shrimp (Penaeus
duorarum) . Mar. Biol. 3.1(3) : 191-197, Nov. 1971(a).
Nimmo, D. R., P. D. Wilson, R. R. Blackman, and A. J. Wilson, Jr.
Polychlorinated Biphenyl Absorbed from Sediments by Fiddler Crabs
and Pink Shrimp. Nature 231:50-52, May 1971(b).
Pauling, L. Nature of the Chemical Bond. Ithaca, Cornell University
Press, 1940. 164 p.
Schoor, W. P. In Vivo Binding of p,p'-DDE to Human Serum Proteins.
Bull. Environ. Contam. Toxicol. _9(2):70-74, 1973.
Walsh, G. E. Insecticides, Herbicides and Polychlorinated Biphenyls
in Estuaries. J. Wash. Acad. Sci. 6_2(2) : 122-139, 1972.
Zitko, V. Polychlorinated Biphenyls Solubilized in Water by Nonionic
Surfactants for Studies of Toxicity to Aquatic Animals. Bull. Environ.
Contam. Toxicol. .5(3): 219-226, 1970.
30
-------
TECHNICAL REPORT DATA
(i'li-asc read laanictions on the reverse before completing)
i. ni con i NO
EPA 660/3-74-013
4. TITLE AND SUUTITLE
Theoretical model and solubility characteristics of
ArocloP' 1254 in water: Problems associated with low-
solubility compounds in aquatic toxicity tests.
5. REPORT DATE
September 1974
6. PERFORMING ORGANIZATION CODE
7. AUTHOH(S)
W. Peter Schoor, Ph.D.
8. PERFORMING ORGANIZATION REPORT NO
3. RECIPIENT'S ACCESSION-NO.
9. PERFORMING ORG MMIZATION NAME AND ADDRESS
U. S. Environmental Protection Agency
Gulf Breeze Environmental Research Laboratory
Sabine Island
Gulf Breeze, Florida 32561
10. PROGBAM ELEMENT NO.
1 EA077 / 10AKC / 018
11. CONTRACT/GRANT NO.
17. SPONSORING AGENCY NAME AND ADDRESS
13 TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. ABSTRACT
A theoretical model of the behavior of substances having low water-solubility is
presented and discussed with re&pect to aqueous bioassay. Ultracentrifugal techniques
were used in an attempt to study size distributions of Aroclor^l254 aggregates in
aqueous emulsions. Results indicate strong adsorption from emulsion by surfaces and &
water-solubility at 20°C of less than 0.1 yg% in distilled water and approximately
40% of that value in water containing 30 (g/£ NaCl. Implications with regard to
aqueous bioassay are discussed.
17.
1.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
Solubility
Aroclor^ 1254
Theoretical Model
Water
Aquatic Toxicity Tests
Low-Solubility Compounds
Emulsion
•i i IP nil a
Adsorption
b.IDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
!••). tjl jrnlBUTfON'STATEMENT
Release to public
19. SECURITY CLASS (This Report)
Unclassif-ipH
21. NO. OF PAGES
20. SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPX
• -* a. S.^OVERNMENT PRINTING OFFICE: 1975-698-089(103 REGION 10
-------
CONTRIBUTION NO. 209
-------
Reprinted from Abstracts of the A nnual Meeting-1974
Copyright © 1974, American Society for Microbiology
G 207 Observations of Luminescent Bacteria in Continuous
Culture. L. KIEFER*, H. JANNASCH, K. NEALSON, and
A. BOURQUIN, Univ. W. Fla., Pensacola, FL; Marine Biol.
Lab., Woods Hole, MA; Environ. Prot. Agy., Gulf Breeze, FL.
Free-living marine luminous bacteria have never been
observed to luminesce in the open ocean. The absence of
this activity is postulated to be a result of an insuffi-
cent concentration of extracellular inducer substance
(responsible for auto-induction in batch culture) in the
environment.
To investigate this model, Photobacterium fisheri, str.
121, was cultured in a glycerol-limited chemostat appara-
tus. Light production was shown to be sustainable for
several days when a cell density greater than the induc-
tion density was maintained. Thus, a potential for con-
tinuous light emission was demonstrated.
After steady-state conditions were achieved at high
cell density, dilutions of the limiting substrate resulted
in proportional and predictable decreases in cell density.
Light emission, on the other hand, was proportional to
dilution only at or above the cell density of induction.
Thereafter, light emission was rapidly extinguished while
cell density remained at the predicted value, thus support-
ing the critical concentration model for inducer activity.
-------
CONTRIBUTION NO. 210
-------
GB 210
Reprinted from Abstracts of the Annual Meeting-1974
Copyright © 1974, American Society for Microbiology
G 264 Mlcrobial Response to Malathion Treatments in Salt
Marsh Microcosms. AL BOURQUIN*. L. KIEFER, and
S. CASSIDY. U.S. Environmental Protection Agency, GBERL,
Gulf Breeze, FL.
Battery jars (6.5)1) were filled with salt marsh mud and
water and placed at a constant temperature (28C) and a 12 h
diurnal light cycle. The slowly aerated microcosms were
stabilized for 1 week before treating with malathion at IX
and 10X field application rate. Application of the toxicant
was repeated every 10 days for 30 days. Sediment and water
samples were analysed at appropriate intervals for total
aerobic heterotrophs and malathion degrading organisms (sole
carbon source, SCS, and added growth substrate, MN). Var-
ience analysis of the MN data showed significant differences
between control and treatment levels for both sediment and
water samples. No significant differences were noted
between treatment periods. Numbers of MN organisms increas-
ed rapidly (7 days) after the first treatment, remaining at
or over 70% (sediment) or 80% (water) of the total hetero-
trophic community. Although numbers of SCS degraders ap-
peared to increase with malathion treatments and increase
over the control in both sediment and water, no statistical-
ly significant differences were noted, due to fluctuations
after treatments. No changes in total numbers of hetero-
trophs over the controls were noted.
No differences in populations of amylase, chitinase, lipase
and casease producers were observed between controls and
treated microcosms.
-------
CONTRIBUTION NO. 211
-------
JOURNAL OP INVERTEBRATE PATHOLOGY 23, 389-396 (1974)
Pathological Effects of Urosporidium (Haplosporida)
Infection in Microphallid Metacercariae1
JOHN A. COUCH
U.S. Environmental Protection Agency, Gulf Breeze Environmental Research Laboratory;
Sabine Island, Gulj Breeze, Florida 32561 (Associate Laboratory oj the National
Environmental Research Center, Coruallis, Oregon)
Received November 30, 1973
Extensive pathological changes occur in Megalophallus metacercariae as a result of
natural infections by the haplosporidan hyperparasite Urosporidium crescens. Infected
and uninfccted metaceriae, recovered from blue crabs from Chincoteague Bay, Mary-
land, were examined and compared histologically in regard to condition of metacercarial
cyst wall, tegument, and specialized parenchymal cells. Changes from normal found in
heavily infected metacercariae were (1) suppression and replacement of possible secretory
and parenchymal cells by the hyperparasite, (2) lack of reticulin slromata, polysaccharides,
and acid mucopolysaccharides, (3) reduction in thickness of cyst wall, tegumental, and
connective tissue structures, and (4) loss of mobility and resistance to mechanical pressures.
Though severe pathological changes occur in heavily infected metacercariae, most infected
metacercariae remain viable within the blue crab and thereby serve as a vector for
Urosporidium until the death of the blue crab. At the time of the crab's death and
disorganization, infected melacercariae rupture and release spores of the hyperparasite.
INTRODUCTION
DeTurk (1940) described a hyperpara-
site, Urosporidium crescens (Protozoa:
Haplosporida), in the metacercariae of a
species of microphallid trematode that
parasitized the blue crab, Callinectes
sapidus, in the Beaufort, North Carolina,
area. He identified the trematode as the
metacercaria of Spelotrema nicolli, which
has since been transferred to the genus
Microphallus by Baer (1943). During my
studies of a species of microphallid trema-
tode from blue crabs in Chincoteague Bay,
Maryland, I found metacercariae of the
genus Megalophallus^ parasitized by a spe-
cies of Urosporidium (Fig. 1) which ap-
1 Most of the- work reported here was done
at the National Marine Fisheries Service Labo-
ratory, Oxford, Maryland.
2 Contribution No. 211, Gulf Breeze Environ-
mental Research Laboratory.
31 wish to (hank Dr. R. M. Cable for valuable
aid in identifying the trematode genus.
pears identical to U. crescens. Sprague
(1966), in a detailed review of
haplosporidan parasites of trematodes, and
Sprague and Couch (1971) reported finding
similar infected metacercariae of a micro-
phallid trematode in blue crabs from Chin-
coteague Bay, Maryland.
The infected Megalophallut, meta-
cercariae are readily recognized because
they become enlarged, darkly pigmented,
and rupture upon application of slight me-
chanical pressure (coverslip pressure). The
uninfected and lightly infected metacer-
cariae are smaller, white to cream color,
and withstand relatively intense mechani-
cal pressure.
A histological study was undertaken to
examine the Megalophallus-Urosporidium
relationship.
MATERIALS AND METHODS
Blue crabs harboring uninfected and in-
fected metacercariae were collected monthly
389
Copyright © 1974 by Academic Press, Inc.
Ali rights of reproduction in any form reserved.
-------
JOHN" A. COUCH
I'Kis. 1 s. ( I'tispofitliitm frcsct'jis spores ami suctions of mctacciTariai
-------
PATHOLOGIC EFFECTS OF L'rosporilll II 111
391
from Chincoteaguc Bay in Maryland and
Virginia during 1968 and 1969. Most of
these crabs were caught in commercial crab
traps during a crab survey by the U.S.
National Marine Fisheries Service. Crabs
were prepared by removing the dorsal cara-
pace and inspecting all internal tissues and
organ surfaces for metacercariac. Muscle
and hepatopancreatic tissues were teased
apart under a dissecting microscope; unin-
fected and infected metacercariac were
fixed in Davidson's (Shaw and Battle,
1957), Bouin's (aqueous), or AFA fixatives.
Uninfected metacercariac were allowed
to excyst in pctri plates, filled with filtered
seawater, over periods of 24-36 hr at room
temperature. The complex male reproduc-
tive system was studied in living and
stained whole mounts (Semichon's aceto-
carmine) in order to make generic
identification.
Fixed, uninfected and infected mcta-
cercariae were carefully embedded together
in paraffin blocks, sectioned at 3^m, and the
sections stained by one of the following
methods. Total protein was studied with
the mercury bromophenol blue method of
Mazia et al. (1953), with the exception
that, instead of dehydrating with an aque-
ous cthanol series, a 1-butanol (2 changes)
dehydration was used' to prevent excessive
loss of stain. Sclcroproteins, i.e., reticulin
and collagenous materials, were studied by
Lillie's (1965) silver oxide reticulin tech-
nique. Acid mucopolytaccharidos were
stained by a modified alcain blue method
of Lison (19541 (pH 2.5; no counterstain)
using 1-butanol for dehydration. Periodic
acid-Schiff (PAS) methods with or without
0.5% malt diastase digestion were used to
compare total polysaccharide patterns and
to determine location and condition of cer-
tain cell types in uninfected and infected
metacercariac. Mallory's triple stain and
standard iron hematoxylin-cosin were used
for general histological study.
Measurements were made of 20 normal
and 20 infected metacercariae in sections,
and averages and ranges given are based
on these measurements.
RESULTS
Cysts of uninfected metacercariae (in
section) range from 189 to 266 /*m in cliam-
4 Pearse (1968; p. 607) recommends tertiary
butyl alcol.ol following staining by this method.
FIG. 1. Arrows point to immature ami fully developed, tailed spores of liyperparasite; round
sporoplasms with nuclei visible in most spores. X970.
FIG. 2. Oral sucker region of uninfected mctaoercaria stained with Hg-BPB; arrow points to
normal cyst wall with moderate staining. x430.
FIG. 3. Oral sucker region of heavily infected metaccrcaria; arrows point lo thin cyst wall
and Hg-BPB-positive pharynx; note complete replacement of oral sucker cells by sporocysls
of Uro-Sporidium, but lack of infection of pharynx. X430.
FIG. 4. Dense fibers of silver-positive stroma in uninfected worm (upper arrow) ; lack of
silver-positive stroma in infected worm (lower arrow) ; nole the .silver-positive walls of Uro-
xporidium spores as well as dense meslnvork of connective tissue slroma of crab host between
metacercarial cysts (Lillie's silver oxide stain). x430.
FIG. 5. Lack of reticulin-eollagen-positive material in clear, .single-layered cyst wall of unin-
fected worm (arrowheads) ; note dense layer of reliculin-posilive host material closely abutting
negative cyst wall (Lillie's silver oxide stain). X430.
FIG. 6. PAS-treated infected metacercariae (large specimen); note the more intense staining
of uninfected metacercaria (smaller specimen), and the difference in sizes of uninfected and
infected metacercariae. xlOO.
FIG. 7. PAS (treated with 0.5% diastase) section of infected and uninfected metacercariae
(compare with Fig. 6) ; nole PAS-positivc diastase-resistant cyst wall (arrowhead) and
tegument. XlOO.
FIG. 8. PAS-treated uninfected (arrow) and infected metacercariae; compare differences in
thickness and intensity of staining in tegument, and parenchyma; particularly note the
reticulated PAS pattern (arrow) in uninfecied metaccrcaria. X430.
-------
392
JOHN A. COUCH
TABLE 1
RESULTS OF SELECTED STAINING METHODS ON UNINFECTKD AND INFECTED METACERCAHIAK"
Type I
Staining method
Hg-BPB
Lillie's silver oxide
PAS
PAS (O.o% diastase)
Alcian blue (pH 2.5)
Cyst
U"
2
0
•)
2
0
wall
P
2
0
2
2
0
Tegument
U
3
3
3
3
3
I
3
1
1
1
0
Parenchyma
U
3
3
3
0
0
I
0,1
1
0,1
0
0
cells
U
0
2
2
2
3
I
0
0
0
0
0
Type II
cells
U
2
0
3
3
0
I
0,1
0
0
0
0
° Staining reaction scale: 0 = negative staining or absence of structure; 1 = light staining; 2 = mod-
erate staining; 3 = strong staining.
6 U = uninfected metaoereariae; I = infected metacercariae.
eter, whereas those of infected meta-
cercariae are usually 410 to 654 ^m in
diameter. The difference in size is a result
of enlargement of infected metacercariae
when they become filled witl-v-sp'orocysts of
Urosporidium (Figs. 6, 7, 10).
Encysted metacercariae, both uninfected
and infected, arc surrounded by a single-
layered cyst wall of apparent parasite ori-
gin (Figs. 2, 5, 7). Often a layer of host
tissue adheres to cysts removed from the
host. Usually, the major difference between
cyst walls of normal and infected Megalo-
phallus metacercariae is their relative
thicknesses, the latter often having a much
thinner wall (average thickness of normal,
2.3/irn; infected, 0.8 /mi).
The tegument of normal metacercariae
is spinous from the anterior end of the
worm to a point slightly posttesticular. It
is 1.7-4.5 /am thick, with an obvious under-
lying basement membrane. In sections of
heavily infected metacercaria, the tegu-
ment is thinner (<0.5 to rarely 2.0 /iin),
spines are less obvious, and the basement
membrane appears thinner than normal.
The parenchyma (cells and spaces be-
tween tegumental basement membrane and
internal organs) of normal worms is com-
pact in sections stained with iron herua-
toxylin and eosin, whereas the normal rela-
tionships of specific parcnchymal cells,
connective tissue element?, and organs are
altered by hypcrparasites in heavily in-
fected metacercariae.
Table 1 gives the results of selected
staining methods on specific tissues of unin-
fected and infected metacercariae. The tis-
sues studied were the cyst wall, tegument,
parenchyma, and certain specialized cell
types in the peripheral and central paren-
chymal areas.
The cyst wall surrounding the uninfected
metacercaria stains blue with mercury
bromophenol blue (abbreviated Hg-BPB
hereafter) (Fig. 2). In cysts of the infected
worm, the wall is reduced in thickness and
is often broken, (Fig. 3). However, the cyst
wall of heavily infected metacercariae,
though thinner than normal, stains with
Hg-BPB.
As noted earlier, the single cyst wall of
Megalophallus is intimately surrounded by
host tissue. The nature of the fine layer of
host material adjacent to the cyst wall be-
comes clear when one studies sections
stained with Lillie's silver oxide reticulin
stain. The fibrous material deposited
against the cyst wall stains black (Fig. 5),
and corresponds in stain affinity to reticulin
fibers in the vertebrates. The presence of
this fine reticulin layer, which contains
some fibroblastlike cells, is the only histo-
logical evidence of a host response to the
presence of metacercariae, aiul ^ay be con-
sidered a very slight encapsulation reac-
tion. There appears to be no difference in
the amount of reticulin-positive material
surrounding uninfected and infected cysts
(Fig. 4).
-------
I'vnroi.ocic KFKKCTS or I'ros
iv
12
Fie. 1). Distribution of cells uf type 1 (rijjlit unow) and i.vpe 11 (left arrow) in uuinfc-ctcd
metacercnria; note difference in stain (PAS-diastiiso livaled) intensity in iwo cell types.
X430.
J'lC. 10. Infected inetacercaiia ( PAS-diastase treated) ID lie compared with Fij: !l; paren-
cliyiiiii cumplelely iilled with sporocysis. X-130.
l''lc:. 11. Alci.'iu l)lne-tre:iled seel ion; middle allow poinis lo :ilci:in Illuc-stniuing material in
cr.'ih host, I issue (ground s'"1 .:., '. arrow on reader's lefi puinls lo aiad iiiiico]iol\sacrliaride-
posili\c te^umont and type I cells of nnmlected nielacen-ai la; arrow ;tt ntilit poinld to
comparable section of inl'eried wonn wliirh lacks :dcian bhli'-liosilivc malerial and t \ pe 1
cells. X »30.
l''i i amount.- often closely ahuttinii the outer
The cyst walls ot uninlected and infected surface of the cyst wall. The cyst Walls
nu'taci'i'cariae :ire 1'. \S-po.-it ive alter dia-- proper of unmlected and infcctril meta-
tase dip'.-tion i !• 'tii;s. 7. 1)1. Because of tt> eercariae are alcian bllK'-lH'fjativc. (Mien.
11^-Bl'B staininii and I".\S-posit ivcni1.-.-. small amorphous amounts of alcian lilnc-
diastase-resist aiiee. t he • single-layered wall positi\-e material are lound against the in-
proliably consist- of a earliohx'drate pro em ncr -urfacc of the cyst wall. Inn tin- male-
i'
-------
394
JOHN A. COUCH
Iii both uninfected and infected worms,
the tegument stains positively for protein
(Figs. 2 and 3], but the reduced thickness
of the tegument of infected worms indicates
less total protein (Fig. 3).
The basement membrane of the tegument
of uninfected worms apparently reduced
more silver (per unit length) than that of
the infected worms, thus showing a greater
concentration of basement membrane con-
stituents per unit length of membrane in
section. When treated with Lillie's silver
oxide method and other connective tissue
stains, i.e., Mallory's triple stain and Hg-
BPB, the tegumcntal basement membranes
of the infected worm appear to have under-
gone a thinning or "stretching," as had the
tegument and cyst wall. This is probably
a result of the remarkably enlarged condi-
tion of heavily infected metacercariae.
The tegument of uninfected and infected
worms is PAS-positive and diastase-resis-
tant. The tegumcntal layer, including the
basement membrane, of infected worms
shows less staining for PAS-positivc, dia-
stase-resistant material than docs that of
the uninfected worms (Figs. 9, 10).
The outer tegument of uninfected meta-
cercariae stains intensely with alcain blue
(Figs. 11, 12), indicating that a heavy con-
centration of acid mucopolysaccharides
exists in this layer. The basement membrane
is negative for aid mucopolysaccharides.
Heavily infected worms do not stain with
alcian blue (Fig. 11).
Hg-BPB-positive fibrous tissue between
parenchyma! cells gives an overall blue cast
to sections of uninfected metarcercariae
(Fig. 2). Comparison of the same general
regions in infected worms suggests a loss
of protein. Figure 3 shows the almost com-
plete replacement of host tissue by plasmo-
dia and spores of the hyperparasitc. Note
particularly the oral sucker of the unin-
fected (Fig. 2) and infected (Fig. 3) meta-
cercariae. In the heavily infected worm, the
only tissue that shows a significantly nor-
mal protein concentration is the pharynx
(arrow), the only organ not invaded by
I'rosporiilium.
In their parenchyma, uninfected worms
have fibers of acellular, reticulin-positive
material. This material (Fig. 4) may com-
pose part of an interstitial tissue similar to
that described by Tlireadgold and Gal-
lagher (1966) as ramifying among and be-
tween parenchymal cells of Fasciola. These
fibers appear to be identical with, and oc-
cupy the same space as, the Hg-BPB-posi-
tive fibrous tissue in the parenchyma. In
infected worms, this Hg-BPB-positivc and
reticulin-positive tissue is less obvious (Fig.
4) because masses of sporocysts and
plasmodia replace or occupy large spaces
in the parenchyma. In infected meta-
cercariae, there is no evidence of increase
in reticulin or other connective tissue acel-
lular elements concomitant with enlarge-
ment of the worm.
The parenchymata of uninfected worms
show much heavier positive PAS reactions
than do those of infected worms (Figs. 6,
8). The PAS-positivc substances in unin-
fected worms seem to be distributed along
the paths of the supportive stromata, rather
than in the general interstromal spaces oc-
cupied by parenchymal cells. This may give
the PAS-positive material a reticulated ap-
pearance (Fig. 8).
Certain cells found in the peripheral and
central parenchymata of metacercariae arc
designated here as types I and II. These
two cell types arc characterized by the
staining reactions of their cytoplasms
(Table 11. These cell types are used mainly
as ''markers'1 of normal conditions in unin-
fected metacercariae and to demonstrate
pathological changes that take place in in-
fected metacercariae. In this paper, these
cells are not compared with specific cell
types described for other larval and adult
Digcnca, e.g., Dixon (1966), Thakur and
Cheng (1968), because of the lack of back-
ground cytological studies of microphallid
metacercariae needed as a basis for
comparison.
-------
PATHOLOGIC EFFECTS OF UrOSpOTldium
395
TABLE 2
THREE PATHOLOGICAL CHANGES AND POSSIBLE RELATED EFFECTS IN METACEHCABIAE
INFECTED BY Urosporidium crescens
I
II
III
Pathological change
Final effect
Reduction in thickness of
cyst wall and in thick-
ness of tegument
Rupture of cyst wall
upon application of
pressure
Enlargement of metacer-
cariae without conco-
mitant increase in
supportive connective
tissue stromata (total
protein; reticulin)
Collapse of parenchyma
and release of hyper-
parasite; loss of mo-
bility of preencysting
metacercaria
Replacement and suppres-
sion of activity of cell
types I and II
Loss of possible protective
layer of acid mucopoly-
saccharides and rnuco-
proteins
Cell type I is found immediately beneath
the basement membrane, mostly in the an-
terior dorsal and ventral regions of the
parenchyma of uninfected worms (Figs. 9,
12). The absence of Hg-BPB staining and
the occurrence of a distinct PAS reaction
(diastase-resistant) and alcianophilia in
the cytoplasm of cells of this type indicates
that they possess neutral, nonglycogen
polysaccharides and acid polysaccharides.
Infected worms (Figs. 7, 8, 10) show a loss
of these cells or replacement of them by the
hyperparasite.
The degree of alcianophilia in a given
section of the tegument appears to be pro-
portional to the concentration of neutral
and acid mucopolysaccharide-positive cells
(type I) underlying that section (Fig. 12).
The tegument in the anterior half of unin-
fected metacercariae gives the strongest re-
action with alcian blue, and the largest
number of type I cells is found there. These
observations suggest a possible relationship
between the distribution of acid mucopoly-
saccharides in the outer tegumental layer
and a concomitant distribution of type I
cells in the anterior peripheral parenchyma
(see particularly Figs. 11 and 12). The
identity of staining reactions of these cells
with staining reactions (PAS+, alcian
blue-)-) of materials in the tegument of the
worm suggests that the type I cells may
secrete the tegumental mucopolysaccha-
rides. The concomitant loss of tegumental
alcianophilia and absence of cell type I in
parenchymata of heavily infected meta-
cercariae further strengthens this concept.
The presence of large amounts -of acid
mucopolysaccharides in the tegument (par-
ticularly around the oral sucker) could pro-
tect the worm from the definitive host's di-
gestive enzymens, a function suggested for
tegumental acid mucopolysaccharides in
helminths by Monne (1959) and Lee
(1966).
Cell type II, found in the anterior central
and marginal parenchyma (Figs. 7, 9), is
positive for protein and is intensely PAS-
positive following diastase treatment
(Table 1). These staining reactions suggest
the presence of mucoproteins. In infected
metacercariae, these cells are replaced or
destroyed by the hyperparasites (Figs. 7,
10).
Effects of Urosporidium crescens infec-
tion on metacercariae of Megalophallus sp.
are summarized in Table 2.
DISCUSSION
DeTurk (1940) was the first to note that
Urosporidium occurs in such large numbers
that the tissues of the trematode host are
often replaced. I have observed that even
though the trematode becomes intensely in-
vaded and most of its parenchyma is re-
placed by the hyperparasite, the enlarged
-------
396
JOHN A. COUCH
and heavily infected metacercariae retain
the ability to move slowly, thereby demon-
strating their viability. It is possible that
a small percentage of infected metacer-
cariae are destroyed in the crab. There is
only a slight host response to living unin-
fected and infected metacercariae in blue
crabs.
Death of infected metacercariae in the
tissues of the crab would result in a pre-
mature release of Urosporidium crescens
into a hostile environment, exposing the
hyperparasite to possible destruction by
crab hemocytes or other defense mechanisms.
Therefore, I suggest that, usually, fragile
infected metacercarie while in the living
crab host serve as a vehicle for the hyper-
parasite until the crab dies or is killed and
disorganized, at which time the spores of
Urosporidium are freed by the rupture of
the metacercaria.
The replacement, failure, or reduction of
supportive tissue complexes, e.g. reticulin,
and the specific loss of possibly protective
acid mucopolysaccharide layers could
greatly reduce the probability of any given
heavily infected metacercaria establishing
itself in a definitive host, even if it were
released intact from the crab. The suppres-
sion or elimination of possible secretory
cells in heavily infected metacercariae sug-
gests that the specific roles of these cells
could be studied further by using unin-
fected metacercariae and infected meta-
cercariae as experimental systems.
REFERENCES
BAER, J. G. 1943. Les trematodes parasites de
la musaraigne d'eau Neomys fodiens
(Schreb.). Bull. Soc. Neuchatel Sci. Natur.,
68, 34-84.
EteToBK, W. E. 1940. The occurrence and develop-
ment of a hyperparasite Urosporidium cres-
cens sp. nov. (Sporozoa, Haplosporidia) which
infests the metacercariae of Spelotrema nicolli
parasite in Callinectes sapidus. J. Elisha Mit-
chell Sci. Soc., 56, 231-232.
DIXON, K. E. 1966. A morphological and histo-
chemical study of the cystogenic cells of the
cercaria of Fasciola hepatica L. Parasitology,
56, 287-297.
LEE, D. L. 1966. The structure and composition
of the helminth cuticle. Advan. Parasitol., 4,
187-254.
LILLIE, R. D. 1965. "Histopathologic Technic and
Practical Histochemistry,'' 3rd ed. McGraw-
Hill, New York.
LISON, L. 1954. Alcian blue 8G with chlorantine
fast red 5B. A technic for selective staining
of mucopolysaccharides. Stain TechnoL, 29,
131-138.
MAZIA, D., BREWER, P. A., AND ALFERT, M. 1953.
The cytochemical staining and measurement
of protein with mercuric bromophenol blue.
Biol. Bull, 104, 57-67.
MONNE, L. 1959. On the external cuticles of various
helminths and their role in the host-parasite
relationship. A histochemical study. Ark.
Zool, 12, 343-385.
PEARSE, A. G. E. 1968. "Histochemistry, Theoreti-
cal and Applied," Vol. 1, 3rd ed., Little,
Brown, • Boston, Massachusetts.
SHAW, B. L., AND BATTLE, H. I. 1957. The gross
and microscopic anatomy of the digestive
tract of the oyster Crassostrea virginica
(Gmelin). Can. J. Zool., 35, 325-347.
SPRAGTJE, V. 1966. Haplosporidan parasites of tre-
matodes. Proc. Symp. Anim. Parasites In-
vertebr., Washington, D.C., sponsored by Am.
Soc. Zool. Ref. No. 66-100, Natur. Resour.
Inst., Univ. Maryland, Solomons, Maryland
(Mimeo.).
SPRAGUE, V. AND COTICH, J. A. 1971. An annotated
list of protozoan parasites, hyperparasites, and
commensals of decapod Crustacea. J. Proto-
zool, 18, 526-537.
THAKUB, A. S., AND CHENG, T. C. 1968. The for-
mation, structure, and histochemistry of the
metacercarial cyst of Philophthalmus gralli
Mathis & Leger. Parasitology, 58, 605-618.
THREADGOLD, L. T., AND GALLAGHER, S. E. 1966.
Electron microscope studies of Fasciola hepa-
tica. I. The ultrastructure and interrelation-
ship of the parenchymal cells. Parasitology,
56, 299-304.
-------
CONTRIBUTION NO. 213
-------
(Rfprinted from Nature, Vol. 247, No. 5438, pp. 229- 231, January 25. 1974)
Free and Occluded Virus, similar to
Baculovirus, in Hepatopancreas of
Pink Shrimp
VIRUSES or virus-like particles have been reported infre-
quently from cells of estuarine and marine organisms'"'.
Herpes-type viruses were described from an estuarine fungus'
and oysters'. Helical or rod-shaped viruses have not been
reported from most aquatic invertebrates, although rod-
shaped virus-like particles were reported from a micro-
annelid and aquatic beetle0.
Recently. I observed rod-shaped virus particles and related
inclusion bodies in cells of pink shrimp (Penac'iis ?* '•
'H >KJ;
•*—. • :• \- 1
• : *
-4*-;*., * • '
i (
k :. • --^y
*. *• . • V ,
& '" ' • /-;>*i
• -» . « • • •*, • -A
^^^^^^ \ -;;^'
'^•Pi "v;:L.
hJ? ''--jt-^;' : ' ••?y
• ••/ak^>*> ^ '-V-4-'
•^•-•.T-- l>i -^i-;.: -;• .-J, ,^ ^t^
^-«'- • :C*» > r. ••• - ^ - **'*** ;J>?
Fig. I Light micrograph of cross section of hepatopancreatic
tubule from pink shrimp exposed to PCB; note normal nucleus
of epithelial cell (one arrow), and crystalloid, tetrahedral inclu-
sion bodies in hypertrophied nucleus of afTected cells (two smaK
arrows).
Fig. 2 a, Hypertrophied nucleus in epithelial, absorptive cell
from hepatopancreas of shrimp exposed to PCB; non-occluded
rod-shaped virions in longitudinal and cross section; single
arrows point to intranuclear membranes associated with early
virion formation, double arrows indicate abnormal multi-
laminate nuclear envelope. Inset: high magnification of rod-
shaped virion in nucleus (bar = 0.l um); note single envelope
surrounding nucleocapsid. b. Moderately heavily affected
nucleus; virions distributed mostly around inner periphery of
nucleus; arrow points to degenerate nucleolus; double arrows
point to envelope membranes entering the cytoplasm. Note many
free ribosomes in cytoplasm (bar= 1 um).
-------
Fig. 3 Low magnification electron micrograph of section through
hepatopancreatic tubule from exposed shrimp Note profile of
epithelial cell and nucleus (one arrow) containing free virions and
intranuclear, crystalline occlusion body (two arrows); note
hypertrophy of affected nucleus (bar = 5 um).
(from 210 to 316 nm; average 288 + 31 nm, n = 30) but had
a relatively narrow range of diameters (from 44 to 64 nm;
average, 59 nm t 6 nm, « = 30). In cross section, the average
diameter of nucleocupsids plus their containing envelopes
was 75 nm^7.5 nm (;i = 30). The envelopes surrounding
the nucleocapsids (Fig. 5) seemed to be membranous with
spatial and structural relationship to the nucleocapsid similar
to that of envelopes m nuclear polyhedrosis virus (a Baculo-
virus) from Bombyx inorf.
Virions were distributed in most nuclei in greater numbers
near the inner nuclear membrane (Fig. 2a and />); however,
they were associated with or occluded in crystalline bodies
at the ultrastructural level in some nuclei (Figs 3 and 4).
These crystalline, occlusion bodies were always triangular
in section, paracrystalline in structure, and were the only
structures observed with the electron microscope that could
be related to the intranuclear, tetrahedral, crystalloid bodies
observed by light microscopy (Fig. 1). The occluded virions
were similar in dimensions and structure to the non-occluded
virions.
Evidence suggesting early stages of virion formation was
found in altered nuclei (20% of cell profiles from exposed
shrimp) that contained few or no completely formed virions.
U-shaped membranous arrays occurred in these hyper-
trophied nuclei (Fig. la}. These arrays and associated
immature virions were similar to certain stages in early
virogenesis of Rhabdionvirus oryctes in the rhinoceros
beetle7, and of the virus reported from the whirligig beetle6.
Ultrastructural cytopathic alterations were associated with
the presence of virions. Nuclei that contained virions in
large numbers were often 1.5-2 times greater (hypertrophied)
in profile area than normal nuclei (Fig. 3). Their cyto-
plasms, however, usually exhibited no parallel difference in
profile area. Heavily affected nuclei had a distinct loss of
heterochromatin (Fig. la and b), and some affected nuclei
had no heterochromatin. Nucleolar degeneration was
evident in nuclei that contained many virions (Fig. 2b). The
most remarkable change associated with the presence of
large numbers of virions was a proliferation of nuclear
membranes (Fig. 2a and b), and their eruption into the
cytoplasm to form a multimembranous labyrinth (Fig. 2b).
Cells whose nuclei contained many virions possessed cyto-
plasms filled with ribosomes, but without normal numbers
of mitochondria or endoplasmic reticulum vesicles (Fig. 2a
and b). Most of these changes have been observed in cells
of insects that were infected by Baculovirus. At present
it is not known if exposure of infected shrimp to PCB
enhances the cytopathic effects of the virus.
The following findings suggest a close relationship of the
shrimp virus to the Baculovirus group1" of invertebrate
viruses. The virions were found in nuclei of hepato-
pancreatic cells of an arthropod (these cells are analogous
in many aspects to fat body and midgut cells of insects).
They are rod shaped (bacilliform), enclosed by one to two
envelopes or capsids, found both free in nuclei and occluded
in crystalline bodies within nuclei10, and were 288 nm long
by 75 nm in diameter, thus falling within the size range of
Baculoviru£-w. Stromata which seemed to be virogenic were
present in affected nuclei, and cytopathic alterations of
affected cells and nuclei were similar to those reported for
Baculovirus infections in insects.
In summary, a rod-shaped, free and occluded virus exists
in a marine shrimp, indicating that marine Crustacea are
potential hosts for viruses similar to certain viruses infecting
insects and mites. So far, the virus has been found only
in shrimp taken from near Cedar Key and experimentally
Fig.
4 Higher magnification (EM) of occlusion body from
. H. Note virions occluded randomly within body. This
body has a two-dimensional triangular form of crystalloid
inclusion body observed with light microscopy in nuclei of
hepatopancreatic cells; compare with Fig. 1 (bar=l um).
Fig. 5 Cross section of free virion in nucleus near abnormal,
multimembranous (six membranes) nuclear envelope; note
outer envelope and capsid of the nucleocapsid of virion (arrows)
(bar = 0.1 um).
-------
exposed to the toxic chemical, Aroclor 1254 (PCB). The
virus probably is a natural parasite, however, previously
undetected, of estuarine and marine shrimp.
Mudies of possible interactions of the PCB and virus in
pink shrimp may provide valuable information needed to
clarity the relationship between natural infectious diseases
and pollutant chemicals in the aquatic environment.
I thank Dr D. R. Nimmo for collecting and exposing the
shrimp, Dr Jean Adams for advice, Drs John Briggs, C. Vago
and Max Summers for examining electron micrographs and
for suggestions. Mrs Leslie Cupp and Mr Steve Foss gave
technical assistance.
JOHN A. COUCH
US Environmental Protection Agency,
Gulf Breeze Environmental Research Laboratory,
Sabine Island, Gulf Breeze, Florida 32561
Received September 24; revised November 5, 1973.
1 Vago, C, Nature, 209, 1290 (1966).
2 Runnger, D., Rustelli, M., Braendle, E., and Malsberger, R. C.,
J. invert. Path., 17, 72 (1971).
3 Kazama, F. Y., and Schornstein, K. L., Science, N.Y., 177, 696
(1972).
* Farley, C. A., Banfield, W. G., Kasnie, jim., G., and Foster, W. S.,
Science, N.Y., 178, 759 (1972).
5 Dougherty, C., Ferral, J., Brody, B., and Gotthold, M. L., Nature,
198, 973 (1963).
6 Gouranton, J., /. ultrastruct. Res., 39, 281 (1972).
7 Huger, A. M., J. invert. Path., 8, 38 (1966).
8 Reed, D. K., and Hall, I. M., /. invert. Path., 20, 272 (1972).
9 Berghold, G. H., Insect Pathology: An Advanced Treatise, 1, 413
(Academic Press, New York, 1963).
10 Wildy, P., Monog. Virol., 5, 32 (1971).
1 * Nimmo, D. R., Blackman, R. R., Wilson, jun., A. J., and Forester,
J., Mar. Biol., 11, 191 (1971).
12 Shaw, B. L., and Battle, H. I., Can. J. Zool., 35, 225 (1957).
13 Hayat, M. A., and Giaquinta, R., Tissue and Cell, 2, 191 (1970).
-------
CONTRIBUTION NO. 215
-------
Reprinted from JOURNAL OF INVERTEBRATE PATHOLOGY, Vol. 24, No. 3, November 1974
Copyright © 1974 Academic Press, Inc. Printed in U.S^A.
An Enzootic Nuclear Polyhedrosis Virus of Pink Shrimp:
infrastructure, Prevalence, and Enhancement1
JOHN A. COUCH
U.S. Environmental Protection Agency, Gulf Breeze Environmental Research Laboratory,
Sabine Island, Gulj Breeze, Florida 32561 (Associate Laboratory oj the National
Environmental Research Center, Corvallis, Oregon)
Received March 28, 1974
A nuclear polyhedrosis virus exists in pink shrimp, Penaeus duorarum, from waters of
the northern 'Gulf of Mexico. This virus is rod-shaped, 269 nm long, and possesses an
outer envelope surrounding its nueleocapsid. The nucleocapsid is 50 nm in diameter. The
virus occurs in nuclei of host hepatopancreatic and midgut cells, and is both free in the
nucleus and occluded within pyramidal-shaped polyhedral inclusion bodies (PIB's). Histo-
chemically and ultrastructurally, the shrimp PIB's appear to be ribonucleoprotein and in
fine structure bear close resemblance to polyhedral inclusion bodies of Baculovirus species
from insects. However, the lattice line-to-line spacing is greater than that usually reported
for insect PIB's. Crowding and chemical stress of shrimp in aquaria may enhance and in-
crease the virus infection and prevalence. In limited experiments, shrimp fed heavily in-
fected hepatopancreatic tissues had much higher mortality than controls fed only fish. The
virus appears to be enzootic in pink shrimp in nature. Cytopathological changes in infected
cells of shrimp appear similar to those in insects infected with certain species of Baculovirus.
The name Baculovirus penaei n.sp. is proposed for the shrimp virus.
INTRODUCTION
In the last decade, several reports of virus
diseases, and of viruslike particles in estua-
rine and marine organisms have been pub-
lished (Vago, 1966; Hunger et al., 1971;
Bang, 1971; Bonami and Vago, 1971; Bon-
ami et al., 1971, 1972; Devauchelle and
Vago, 1971; Kazama and Schornstein,
1972; Farley et al., 1972; Couch, 1974).
The report by Couch (1974) was con-
cerned with a rod-shaped virus in pink
shrimp, Penaeus duorarum. This virus is
associated with polyhedral (tetrahedral)
inclusion bodies visible with the light mi-
croscope in nuclei of host hepatopancreatic
cells. The virions are free and occluded
within the polyhedral bodies which possess
a crystalline lattice fine structure similar
to that of nuclear polyhedral inclusion
bodies associated with Baculovirus (sub-
Contribution No. 215, Gulf Breeze Environ-
mental Research Laboratory.
group A) in insects. Therefore, the designa-
tion "nuclear polyhedrosis virus of pink
shrimp" appears appropriate. This was the
first virus recognized in a host of the crus-
tacean suborder Natantia, and the first
nuclear polyhedrosis virus reported in ani-
mals other than insects or mites (Wildy,
1971; Couch, 1974). Other viruses reported
to date from Crustacea have been icosahe-
dral viruses, all from portunid crabs as de-
scribed by Vago (1966), Bonami et al.
(1971), and Bang (1971).
The purpose of the present paper is to
confirm and enlarge upon original observa-
tions on the nuclear polyhedrosis virus in
pink shrimp and to present new evidence
for the enzootic nature, laboratory trans-
mission, and enhancement of the virus.
MATERIALS AND METHOD
Source oj virus. Polyhedral inclusion
bodies were first observed in hepato-
311
Copyright © 1974 by Academic Press, Inc.
All rights of reproduction in any form reser
.
rved.
-------
312
JOHN A. COUCH
pancreatic epithelial cell nuclei of shrimp
that had been taken originally from
Apalachee Bay near Cedar Key, Florida
(actually landed at Keaton Beach, Flor-
ida) . Prior to examination, the shrimp
had been exposed experimentally to
3-5 /Ag/liter of the polychlorinated bi-
phenyl (PCB), Aroclor 1254, by Nimmo
(personal communication). These shrimp,
examined by Couch (1974) and Couch and
Nimmo (1973), possessed pyramidal or
tetrahedral (fresh squash), and triangular
(sections) inclusion bodies in hypertro-
phied nuclei. Later, electron microscopy
(Couch, 1974) revealed the presence of rod-
shaped virions, both free and occluded in
and associated with the inclusion bodies.2
These inclusion bodies will be called poly-
hedral inclusion bodies (PIB's) in compari-
son with, and in the sense of similar, well-
studied inclusion bodies associated with
Baculovirus (subgroup A) in insects
(Wildy, 1971).
Subsequent to the initial findings, over
400 pink shrimp have been examined from
the Gulf of Mexico, taken from near the
following localities: Keaton Beach, Apa-
lachee Bay, Port St. Joe, and Pensacola, all
in Florida.
Histological Methods
Hepatopancreatic tissues were removed
from shrimp, and fresh squashes were pre-
pared immediately for microscopical study.
Hepatopancreas containing PIB's was fixed
in Davidson's fixative or in neutral,
buffered 10% formalin, processed, and em-
bedded in paraffin. Sections (5-7 /urn) were
stained with Harris hematoxylin and eosin,
mercury bromophenol blue (Mazia and
Brewer, 1953), periodic acid-Schiff (PAS)
* Dr. Max Summers has subsequently confirmed
the viral structures in infected shrimp cells
through his own EM studies of shrimp tissues sup-
plied by OUT laboratory. Certain electron micro-
graphs in t.hiH paper were prepared by Dr. Sum-
mers; he is gratefully acknowledged for this and
his generous and helpful comments on fine struc-
ture of the virus.
(with and without diastase digestion),
methyl green-pyonin (Luna, 1968), and the
Feulgen method. Normal hepatopancreas,
i.e., without PIB's, was processed as above
for comparison with infected tissue.
For electron microscopy (EM), hepato-
pancreas containining heavy concentrations
of PIB's was diced in 2.5% glutaraldehyde
in plastic Petri plates. Diced tissue was
fixed for 30 min in fresh 2.5% glutaralde-
hyde and postfixed in \% Os04 for 30 min
at 0-4°C. The fixed tissues were then pro-
cessed and finally embedded in Epon 812
according to the method of Hay at (1970).
Sections 50-100 nm thick were collected on
300 mesh, unsupported copper grids, and
stained with uranyl acetate and lead cit-
rate. Normal, uninfected hepatopancreatic
tissue was prepared similarly for electron
microscopy. Several thousand hepatopan-
creatic cell profiles from 30 pink shrimp
were examined during this study with a
Zeiss EM 9S2 electron microscope.
Prevalence and .Relative Concentrations of
PIB's
Prevalence of polyhedral inclusion bodies,
hence prevalence of patent virus infec-
tions,3 is herein expressed as the proportion
of any given sample of pink shrimp that
possesses PIB's demonstrable in fresh hepa-
topancreatic squash preparations or in
stained sections of hepatopancreas. To find
a single PIB in a squash preparation with
light microscopy is to diagnose the presence
of the virus in a shrimp. The certainty of
this conclusion is based on a 100% associa-
tion of virions with the PIB's in over 1000
PIB-containing cell profiles examined by
EM.
Known volumes of hepatopancreatic tis-
sues containing PIB's were diluted with
exact volumes of distilled water (usually
0.25 ml hepatopancreas in 5 ml of distilled
* Patent virus infection herein refers to the situ-
ation in which virus development has proceeded
to the point of polyhedral inclusion body produc-
tion, thus making infections detectable with the
light microscope.
-------
ENZOOTIC NPV OF PINK SHEIMP
313
water). This mixture was homogenized with
a sonic probe and relative concentrations
of PIB's in individual shrimp were deter-
mined by hemocytometer counts. These
counts provided an index of relative inten-
sities of patent infections in shrimp because
the number of PIB's/:mm3 of hepatopan-
creatic tissue is proportionate to the num-
ber of host cells/mm3 that have patent
virus infections (a single PIB per patently
infected nucleus is the dominant histologi-
cal and EM finding; see Figs. 4, 8, and 11).
PIB's can be harvested by homogeniz-
ing hepatopancreas and centrifuging the
homogenate at 2000-30000. The pellet thus
obtained can be resuspended and cleaner
pellets may be obtained with further
centrifugation.
Enhancement experiments
Pink shrimp samples that were collected
periodically from near Keaton Beach,
Florida, in the Gulf of Mexico, and from
Santa Rosa Sound, near Pensacola, Florida,
were examined by the fresh squash method
to determine base prevalence4 of PIB-con-
taining shrimp. These samples usually con-
sisted of 100 shrimp of which 40 were
directly examined to determine base pre-
valence. Fifty to sixty of the surviving
shrimp from these samples were placed in
100-gal aquaria in premixed artificial sea-
water of controlled salinity and tempera-
ture. These shrimp were held under crowded
conditions for 30-40 days and fed only
frozen fish purchased from a local seafood
market. To detect any increase over the
initial base prevalence found in the original
stock of shrimp, samples of shrimp were
removed at periodic intervals after 3-0 days
holding, and PIB prevalence in each sam-
ple was determined. Daily attention was
given to mortality in the shrimp held for
30_40 days. Dead or moribund shrimp,
'Base prevalence refers to the proportion of
shrimp in a sample taken directly from nature
and prior to laboratory holding that have patent
virus infections.
upon detection, were examined for presence
and relative intensity of PIB's in hepato-
pancreas.
Juvenile pink shrimp from a stock that
showed no patent infections were kept indi-
vidually in 1000 ml beakers for 20-40 days
and fed only hepatopancreatic tissues that
contained large numbers of PIB's (>1000
PIB's/mm3 tissue). Other shrimp from this
stock, kept as controls in like manner, were
fed only frozen fish. Shrimp that died were
immediately examined for the presence of
PIB's or hypertrophied nuclei in hepato-
pancreatic cells.
Chemical Exposure of Shrimp
Samples of pink shrimp from populations
in Apalachee Bay were exposed to the poly-
chlorinated biphenyl (PCB), Aroclor 1254,
in flowing seawater by Nimmo et al.
(1971). Samples of pink shrimp from near
Pensacola were exposed to the chlorinated
hydrocarbon insecticide, mirex, in flow-
ing seawater, laboratory experiments by
Tagatz (personal communication). Control
shrimp in both of the preceding experiments
were kept in toxicant-free flowing seawater.
After approximately 30 days, exposed and
control shrimp samples were examined his-
tologically for prevalence of PIB's.
RESULTS AND DISCUSSION
Light Microscopy and Histopathology
The hepatopancreas of pink shr'mp
(Fig. 1) is histologically similar to the
hepatopancreas of other Natantia (Crus-
tacea: Decapoda). Polyhedral inclusion
bodies (PIB's) occur in nuclei of epithelial
cells of the acini of the hepatopancreas in
infected shrimp (Figs. 2, 3). They may be
found in cells in proximal, medial, and dis-
tal epithelia of acini. Usually, in shrimp
with moderate to light patent infections,
PIB's occur in foci (Fig. 2). In heavily in-
fected shrimp, homoeeneous distribution of
PIB's is the rule. PIB's have been found in
midgut cell nuclei but not as commonly as
in hepatopancreatic cells.
-------
314
JOHN A. COUCH
FIGS. 1-6. Light micrographs of hepatopancreatic tissues, and polyhedral inclusion bodies
(PIB's) of virus.
-------
ENZOOTIC NPV OF PINK SHRIMP
315
FIGS. 7 and 8. Light micrographs of fresh squash hepatopancreatic preparations showing
characteristic pyramidal forms of PIB's.
FIG. 7. Heavy infection showing PIB's; note pyramidal forms (arrows). xlOOO.
FIG. 8. Single large PIB in epithelial fell nucleus, in vivo. X3000.
PIB's of the shrimp virus are tetrahedral,
or pyramidal in three-dimensional form.
They fall into the class of polyhedra that
have cubic symmetry. When measured with
light microscopy (LM), they range in size
from 0.5-20 /tin from pyramidal base to
peak (Figs. 7, 8), with a modal, vertical
length of 8-10 /»m.
The polyhedra, when sectioned, always
have two-dimensional shapes of triangles
(Figs. 3-6). They stain light to dark blue
with mercury bromophenol blue, and they
stain bright red with methyl green-pyronin,
indicating the presence of ribonucleopro-
tein. They are PAS negative, mostly Feu-
glen negative, and show variable basophilia
when hematoxylin stains are applied, but
no acidophilia when eosin is applied.
Neither the virus particles occluded
within the PIB's nor the nonoccluded virus
particles in hypertrophied nuclei are ob-
servable with LM.
The major histopathological effect asso-
ciated with the PIB's and observable with
LM, is the eventual growth of the poly-
hedral inclusion body to a size that sur-
passes the infected cell's capacity to retain
it. The cell then ruptures, is destroyed, and
releases the large PIB into the lumen of the
acinus (Figs. 4-6). In histological sections
of heavily infected shrimp, it is not unusual
to find hundreds of PIB-containing cell
FIG. 1. Cross section of hepatopancreas of pink shrimp; A, acinus; C, capsule; HD, hapato-
pancreatic ducts; MG, midgut. X40.
FIG. 2. Epithelial layers of acini in hepatopancfeas containing PIB's; arrows point to foci
of PIB's; E, epithelium X80.
FIG. 3. Cross section of acinus showing PIB's in nuclei of infected epithelial cells (single
arrows) ; normal nuclei (double arrows). X800.
FIG. 4. Large, single PIB in nucleus of hepatopancreatic epithelial cell (arrow). X800.
FIG. 5. Four triangular PIB's in epithelial cell nucleus (arrow) ; note normal nuclei. X1600.
Fin. 6. Histopathological effect of PIB's and virus on hepatopancreatic tissues; note rupture
and liberation of large PIB's from cells. x800.
-------
316
JOHN A. COUCH
MV
w?..iKRL\
eJ*M?-y -..-••'
^ .<^%g!*
' ~*-J •" i. • *' ' i*-,--^' •
•J'
&C
Fin. 9. Portion of normal epithelial cell of pink shrimp; note salient features of microvilli
(MV), absorptive region (AR), nucleus (N), Nucleolus (No), and nuclear envelope (NE).
X14,400.
profiles (patent cell infections) ready to
rupture, or disintegrating cells with poly-
hedra being extruded from them (Figs. 6,
7).
Infected shrimp show no gross lesions
that would indicate infection. However,
lethargic and moribund as well as dead
pink shrimp from nature and from labora-
tory experiments are often infected and
contain many PIB's in hepatopancreatic
tissues. To date, PIB's have not been found
in any tissues other than hepatopancreas.
and midgut, although gill, gonad, and
muscle have been examined.
Electron Microscopy and Cytopathology
The ultrastructure of virions, PIB's and
infected cells was determined following EM
-------
ENZOOTIC NPV OK PINK SHRIMP
317
FIG. lOa-e. Pertinent eharacteristi s of virus particles in shrimp cells, a. Virions in longi-
tudinal and cross sections; E, envelope; NC, nuclrocapsid. X66500. b. Longitudinal sections
of virions; note terminal protrusion of central virion (arrow), x79,800. c. Single virion in
nucleus; note envelope detail. X79,800. d. Cross section of virus rod; note outer envelope
(E), capsid (C), and nucleoid (Nu). x 185,000. e. Cross section of virus rod showing dense
central core of nucleocapsid (arrow). x66,500.
studies of normal uninfected hepatopan-
creatic cell profiles. The normal hepatopan-
creatic absorptive cell (Fig. 9), one of sev-
eral major cell types in hepatopancreatic
epithelial tissue, possesses organelles and
fine structures remarkably similar to those
of absorptive cells of higher animals. Ab-
sorptive cells appear to be the cells most
commonly infected in pink shrimp, al-
though embryonic and secretory cell pro-
files frequently have been found to contain
virions and PIB's.
The mature shrimp virion consists of a
bacilliform, enveloped nucleocapsid (Figs.
lOa-e, 14). Often, a dense central core is
visible within the nucleocapsid of develop-
ing or incomplete virus particles (Figs. lOc,
15, 22). Certain profiles reveal a protruding
structure at one extremity of some virions,
giving them a "bullet" form (Fig. lOb).
The functional significance of the protru-
sion is not known, but it may only reflect
an artifact of the envelope or it may serve
as an attachment organelle in mature vir-
ions during infectious processes.
The sizes of virions and their components
TABLE 1
AVERAGE SIZE, STANDARD DEVIATIONS, AND SIZE RANGES OP VIRIONS AND VIRION COMPONENTS"
Component
Length (nm)
Diameter (nm)
Thickness (nm)
1. Nucleocapsid
2. Capsid
3. Nucleocapid and envelope
4. Envelope
269.6 ± 20.7(228.1-320.2) 50.3 + 4.8(40.0-60.7)
74.5 ± 8.3(60.3-97.6)
4.0(3.7-4.2)
8.5(5.3-10.7)
n =50 for 1 and 3; n =2 for 2; n = 17 for 4.
-------
318
JOHN A. COUCH
-
m
\
.,
•v
•.
¥
• - c?'
•
#•'•
#
*
f :
V
•
FIG. 11. Polyliedral inclusion liody and virions in advanced infection. Note occluded
(arrows) and numerous small, round subunits in nucleoplasm (arrows). X28,500.
-------
ENZOOTIC NPV OF PINK SHRIMP
319
are given in Table 1. Nonoccludcd and oc-
cluded virions have simi'ar dimensions and
fine structure.
The fine structure of the shrimp virus,
particularly the spatial relationship of the
nucleocapsid and outer envelope (Fig. lOd)
is similar to that of the nuclear polyhedro-
sis virus (Baculovirus) from Bombyx mori
described by Berghold (1963). Berghold re-
ferred to the envelope of the B. mori virus
as the "developmental membrane," and to
the capsid as the "intimate membrane." In
this work, I have selected the presently
more acceptable terms "envelope" and
"capsid'' for these structures, to be used as
applied by Summers (1971) to similar
structures in granulosis virus.
Light and electron micrographs of thin
sections of polyhedral inclusion bodies in
tissue and those isolated by centrifugation
reveal characteristic triangular or semitri-
angular forms i,Fi;j;s. II, 12, 13). Occasion-
ally, multipointed structures are found
(Fig. 12), which appear to be the result of
fusion of several triangular PIB's. Virions
are fully occluded randomly in the PIB's,
Fin. 12. Unusual coalescence of PIB's in nucleus. Also, note edge of P1H in triangular
form caught in thin section (white arrow). Nuclear envelope alterations are olivious (NEA).
X 14,400.
-------
320
JOHN A. COUCH
13
13. Isolated PilVs; impure- ])rp]i;ir;ition; note Occluded virions (arrows). X28..500.
but partially occluded virions may be ob-
served (Fig. 14).
Rarely, more than one PIB (up to five
or six) is found per nucleus (Fig. 5). Often,
only small corners are cut from a large
FIB, producing, in thin sections, small tri-
angular bodies | Fig. 12).
The fine structure of the PIB, as revealed
by KM. lias some similarity to that of
PIB's from Bacttlowrus-infected insects.
However, certain featuns of the shrimp
virus 1MB have not been reported for insect-
virus PIB's.
The crystalline structure of the shrimp
virus PIB consists of a linear lattice made
up of round subunits, each approximately
11-20 nm in diameter (Fig. 15), spaced in
rows approximately 5 nm apart (Fig. 15).
Sections at right angles to the linear lattice
plane show a cubic lattice substructure
(Fig. 18). The subunits, which appear
round in Fig. 15, arc probably ribonucleo-
protein (RNP) bodies that are incorpo-
rated into peripheral assembly planes of
the PIB's from a large population of similar,
but larger subunits (12-21 nm) in the nu-
cleoplasm. Incorporation or assembly of
these subunits is strongly suggested in Figs.
-------
ENZOOT1C NPV OF PINK SHRIMP
321
'
14
Fi
-------
JOHN A. COUCH
.
. - -I' - - V
M. . rr ••, * ,
FIG. 15. Section in linear plane of 1MB lattice; linear arrangement (L) of round subunits
(S) in crystalline lattice cut at rifiht angles to the cubic lattice face (see Fig. 16); note
free Mihumts (8) in nudeoplasm particularly near linear lattice, X"0,000; Inset: higher mag-
nification of region near periphery of lattice; note that some subunits may have central core
(arrow). X210.000.
-------
ENZOOTIC N'PV OF PIXK SHRIMP
323
FIGS. 16 and 17. Different aspects of thin sections through PIB's.
Fiu. 16. Section in plane of cubic face of lattice at right angle to linear lattice plane (see
Fig. 15). X70,000.
Fit;. 17. Section through edge of PIB at an angle (hut reveals round subunits (S) and
partially occluded virions; note U- and C-shaped membrane profiles in nucleoplasm adjacent
to PIB (arrows), x70,000.
15 and 17. Berghold (1963) described simi-
lar subunits in polyhedra of Bombyx mori
nuclear polyhedrosis virus as "spherical
protein molecules" which are arranged in
a cubic system, but did not mention the
presence of RNP in the subunits. The possi-
bility that subunits in shrimp PIB's may
be RNP bodies is suggested by the'r size
(11-20 nm) and shape, which are close
to those of presumed nucleolar RNP bodies
and ribosomes in the cytoplasm of infected
shrimp cells. Further evidence supporting
the RNP nature of these subunits is the
strong affinity of the whole PIB for pyronin
-------
Fins. 18 and 10
FIG. 18. Amorphous inclusion body that does not possess crystalline lattice substructure;
note membrane profiles within body (arrows). X70000.
FIG. 19. Single large PIB in advanced infection of nucleus; note abnormal nuclear envelope
(NE), virogenic stromata in nucleoplasm (VS), and virions in vesicles in cytoplasm near
nucleus (VV). X 14,000. Inset: enlargement of virus particles in vesicle near nuclear envelope.
X 28,000.
324
-------
ENZOOTIC NPV OF PINK SHRIMP
325
(methyl green-pyronin method), and for
mercury bfomophenol blue, indicating the
presence of RNA and protein. Biochemical
characterization, however, is needed to sub-
stantiate this possibility.
Rarely, profiles of amorphous dense
bodies containing virions and membrane
profiles are found in remains of nuclei (Fig.
18). These bodies do not show lattice and
subunit substructure similar to those of
PIB's. The origins and significance of the
dense body are enigmatic, for there are
no comparable structures reported in in-
sect virus infections (Summers, personal
communication).
Affected shrimp possess cells that are in
different stages of the virus infective-repro-
ductive cycle; thus a spectrum of virus-cell
ultrastructural relationships can be ob-
served. For convenience, three recognizable
levels of cellular infections, characterized
by morphological changes, will be described
in the following order: (1) advanced or
patent infections, (2) intermediate infec-
tions, and (3) early and eclipsed infections.
Both stages (2) and (3) may be considered
by some to be latent, cellular infections.
In advanced, patent infections the pres-
ence of a single, relatively large PIB in the
nucleus is the usual cytological case (Figs.
11, 19). Advanced infections are further
characterized by abnormal, bizarre forms
of the nuclear envelopes of the affected cells
(Fig. 19). Usually, the nuclear envelope
membranes have proliferated, are multi-
laminate, and become widely separated,
creating large cisternae. Further, the PIB
may be large enough to completely distort
the nuclear and cellular profile. Membrane-
lined vesicles containing virions were found
occasionally in the cytoplasm adjacent to
nuclei with advanced infections (Fig. 19).
Intermediate infections may be charac-
terized by hypertrophied nuclei that, in
profile, contain from few to many nonoc-
cluded virions (Fig. 20). Striking cytopath-
ological changes occur within these cells.
Nuclei are hypertrophied, usually 1.5-2
times the profile area of normal cell nuclei.
Heterochromatin is lacking or reduced in
amount and distribution (Figs. 19, 20).
Aberrant stromatic patterns are present in
the nucleoplasm, and nucleoli are absent or
degenerate (Fig. 20). The most remarkable
change, however, is the proliferation of
nuclear envelope membranes, which begins
in intermediate infections and increases to
such an extent that, by the time advanced
infections are reached, one is unable to
recognize a normal nuclear envelope (Figs.
19, 20). The major result of the membrane
proliferation is the production of a mem-
branous labyrinth that has its origins in the.
nuclear envelope, but extends considerably
into the cytoplasm (Fig. 20). The function
of this labyrinth is unclear, but it resembles
the network of reticulum membranes in
cells of Gyrinus natator infected by a rod-
shaped, nonoccluded virus described by
Gouranton (1972), and that described by
Summers (1971) for granulosis virus-in-
fected cells of Trichoplusia ni.
The cytoplasms of cells with intermedi-
ate infections become filled with.free ribo-
some and contain less endoplasmic reticu-
lum and fewer mitochondria than normal
(Fig. 20).
Early and eclipsed infections are charac-
terized by nuclear hypertrophy, diminution
of heterochromatin, and obvious segrega-
tion of the nucleoplasm into regions of
granular and fibrillar stromata (Fig. 21).
No virions are apparent in the eclipsed
phase; however, during the early phase, a
few virions and early virogenic stages" are
apparent (Fig. 21). The latter are repre-
sented by the U- and C-shaped membrane
configurations associated with dense bodies
resembling viral cores or nucleoids (Figs.
21, 22). These variable-shaped membranes
(Figs. 14, 17, 19, 20, 22) in the nucleoplasm,
associated with virions and nucleoids of
virions at all stages of infection are strik-
ingly similar to stages of virogenesis of
Rhabdionvirus oryctes described by Huger
(1986), and to virogenic figures reported in
midgut cells of the whirligig beetle by
Gouranton (1972).
-------
JOHV A. COUCH
Fin. 'JO. Profile of hepatopancreatic cell at intermediate level of infection; note hypertro-
phicil nucleus, loss of liclcrochomMlin, membranous liil).viinth (MI/), and virions in micleo-
lila.sm. Five riliosomrs arc almtiilant in cytoplasm. Xl4,400.
-------
li ••• " . • *s$
I ^ ii r'-ffi
'^2? '.^.^_^:,;,:.^'.. * Jfw>
Fiiis. 21 and 22
FIG. 21. Xuc'leus at'early .sl.-i^c of infection; note diminution of dinmint in, nurlcar liyi
tni])hy and lil>nll;ir and granular slniniala in nucli-oplasin. Xl-t,)00.
FIG. 22. Numerous membrane profiles and early \ironime stages in nucleoplasma of infected
cell. X 70,000.
327
-------
328
JOHN A. COUCH
PREVALENCE
TABLE 2
PIB's IN PINK SHRIMP FROM NORTHERN GULF OF MEXICO (1970-1974)
Number Number
Year examined with PIB's
Month
Source (all in Florida)
1970
1971
1973
1974
40
1
42
14
14
10
20
20
42
40
28
30
53
20
12
1
0
7
0
0
0
0
12
6
10
0
14
4
June
August
July
August
September
October
June
August
October
November
November
January
January
February
Keaton Beach
Pensacola
Pensacola
Keaton Beach
Keaton Beach
Keaton Beach
Pensacola
Pensacola
Keaton Beach
Pensacola
Port St. Joe
Pensacola
Keaton Beach
Keaton Beach
TABLE 3
DIRECT-OBSERVATION ESTIMATES (FRESH SQUASH)
AND ACTUAL HEMOCYTOMETER COUNTS OF
PIB's IN THREE PINK SHRIMP
Shrimp
no.
1
2
3
Direct
estimate
(squash)
Light
Moderate
Heavy
Hemocytometer
counts"
50-500 PIB's/mm3
550-850 PIB's/mm'
1100-1650 PIB's/mm3
" Range of 12 counts from each animal.
Prevalence and Relative Concentrations oj
PIB's
The prevalence of polyhedral inclusion
bodies, hence patent viral infections, in
pink shrimp sampled from three points in
the northern Gulf of Mexico is presented
in Table 2.
To date, a regular seasonal pattern of
PIB occurrence in pink shrimp has not been
established. However, during 1973, PIB-
containing shrimp were collected only dur-
ing the fall and winter months, October
through January. Shrimp samples from
Apalachee Bay near Keaton Beach,
Florida, had higher PIB prevalence than
those taken near Pensacola, Florida.
More extensive sampling, presently un-
derway in the northern Gulf of Mexico,
should reveal more accurate and valid pat-
terns of prevalence and distribution of
PIB's in pink shrimp. However, the pre-
valence data presented in Table 2 demon-
strates an enzootic occurrence of PIB's in
feral shrimp from northeastern Gulf of
Mexico waters.
Brown shrimp, Penaeus aztecus, white
shrimp, Penaeus setiferus, and grass
shrimp, Paleomonetes pugio, examined dur-
ing the last three years, have not yet been
found to possess PIB's or virions similar to
those found in the pink shrimp. However,
large samples of these species have not been
examined thoroughly for PIB's.
Relative concentrations of PIB's in indi-
vidual shrimp have been determined by
hemocytometer counts. Counts for three
shrimp that showed considerable difference
in PIB concentrations are given in Table
3. These counts are compared to direct esti-
mates of PIB concentrations (made a priori
on fresh hepatopancreatic squashes) ex-
pressed as light, moderate, or heavy. The
a priori direct estimates were based on the
approximate numbers of PIB's per micro-
scopical field (X430) in fresh squashes,
and the general distribution of the PIB's
throughout the squash.
Since each PIB represents, on the aver-
age, a single infected cell, it is possible to
-------
ENZOOTIC NPV OF PINK SHRIMP
329
estimate with LM the minimum number of
cells/mm3 of tissue that are destroyed in
the infection up to the time of examination.
According to several counts, in heavily in-
fected shrimp, from 1100-1600 cells/mm3
are infected patently, and are therefore des-
tined to be lysed or destroyed by the
growth of the PIB and associated cyto-
pathic alterations. A quantitative relation-
ship between cell death and organismic
debilitation or death, however, has not been
established in pink shrimp.
Enhancement and Transmission
Apparent increase in prevalence of pa-
tent virus infections occurs when samples
of pink shrimp from natural populations
with enzootic levels of virus (Table 2) are
kept under crowded conditions in labora-
tory aquaria. Experiments were conducted
in which samples of pink shrimp from
Keaton Beach, Florida, showing no patent
levels of infection, i.e., absence of PIB's in
squash, but with probable latent infections,
were kept for 40 days in aquaria. Figure
23 shows the occurrence and increased pre-
valence of PIB's in samples examined after
30 and 40 days under crowded aquarium
conditions.
Shrimp are notoriously cannibalistic and
in aquaria show no hesitation to feed on
all organs of shrimp carcasses, including
hepatopancreas. Therefore, one explanation
for the 40-50% increase in PIB prevalence
(Fig. 23) could be that transmission of the
virus from shrimp to shrimp was facilitated
via cannibalism, and individuals were ex-
posed to levels of virus higher than nor-
mally encountered under less crowded con-
ditions in nature, where other scavengers,
i.e., fish, other invertebrates, quickly con-
sume moribund or dead shrimp. Two other
possible modes of enhancement of preval-
ence levels in aquaria are (1) physical
stress of shrimp due to abnormal crowding,
hence loss of resistance, and (2) gradual
increase of infective PIB's in sediments in
aquaria.
Five juvenile pink shrimp from a larger
K 60
20
DAY 1
N:40
DAY 40
FIG. 23. Prevalence of polyhedral inclusion
bodies (PIB's) in sample of pink shrimp held
under abnormally crowded conditions in an
aquarium for 40 days. The initial sample (N =
40) taken directly from nature showed no PIB's
in any shrimp at onset of holding (Day 1).
sample of shrimp showing no patent virus
infections were fed heavily infected hepato-
pancreatic tissues (1000-1600 PIB's/mm3).
Four of the five died after 20 days feeding.
Control shrimp from the same stock, fed
frozen fish, did not die. Examination of the
experimental shrimp after 20 days of feed-
ing showed that one of five had a patent
virus infection (PIB's present). None of the
controls showed patent infections. Each of
the experimental shrimp had some hyper-
trophied nuclei in hepatopancreatic cells,
whereas none of the controls had hyper-
trophied nuclei. It is difficult to determine
from this small experiment whether or not
the deaths of the experimental shrimp were
due to feeding heavily infected tissues, be-
cause it is possible that some experimental
shrimp had latent virus infections initially.
More intensive transmission feeding and in-
oculation experiments of virus and PIB's
are presently underway.
In separate experiments pink shrimp
were exposed to the polychlorinated bi-
phenyl (PCB), Aroclor 1254, and to the
commercial organchlorine pesticide, mirex.
Prior to their exposure to the chemicals,
these shrimp were not examined for PIB
prevalence. Following exposure for 30 days
to Aroclor 1254, 60% of the Aroclor-exposed
shrimp had developed patent infections,
-------
330
JOHN A. COTTCH
TABLE 4
PREVALENCE OF PATENT INFECTIONS (PIB's
PRESENT) IN PINK SHRIMP AFTER EXPOSURE
TO CHEMICALS FOR 30 DAYS IN
FLOWING SEAWATER
Patent infections
Exposed Controls
Aroclor 1254 (PCB)
(3 Mg/liter) 12/20° 0/20
Mirex
(0.01-0.23 Mg/liter) 6/15 1/15
0 Shrimp with PlB's/total number of shrimp
exposed.
whereas controls for this exposure had
none. (Table 4). Forty percent of the
Mirex-exposed shrimp had patent infec-
tions, and only 6.6% of the controls from
this group had patent infections (Table 4).
Results of these two experiments suggest
that stress of shrimp by certain toxic chem-
icals may facilitate transmission or enhance
the expression of latent viral infections.
Further chemical exposure experiments,
utilizing larger numbers of shrimp from
populations with enzootic levels of virus,
are underway.
CONCLUSIONS
The virus reported and described herein
appears by virtue of its size, structure, site
of reproduction, polyhedral body associa-
tion, and cytopathic effects to be very simi-
lar to viruses in the genus Baculovirus
(Subgroup A), that prior to 1974 were de-
scribed only from insects and mites (Wildy,
1971). Therefore, the name Baculovirus
penaei sp. n. is proposed for purposes of
identification and classification of the
nuclear polyhedrosis virus of pink shrimp,
Penaeus duorarum. The group name,
Baculovirus, is used here in the sense indi-
cated by the Invertebrate Virus Subcom-
mittee of the International Committee on
Nomenclature of Viruses (ICNV) (Wildy,-
1971). Further chemical and biological
characterization of the shrimp virus is
needed to confirm its relationship to Bacu-
lovirus. Presently, efforts are underway to
identify the nucleic acid content of the
shrimp virus. Since the ICNV has approved
the rule that the law of priority shall not
apply in virus nomenclature, future evi-
dence showing lack of relationship of the
shrimp virus to Baculovirus may be reason
enough to reject or change the name at
some later date.
It is not known at the present if Baculo-
virus penaei causes mortalities of shrimp
in nature. Small-scale transmission-feeding
experiments in the laboratory and mainte-
nance of shrimp under abnormally crowded
conditions, and under chemical stress ap-
pear to enhance and increase the prevalence
of infection in shrimp samples. Dead and
dying pink shrimp with heavily infected
hepatopancreatic tissues have been found.
However, the majority of shrimp found to
be infected with PIB's and virus appear
grossly to be healthy, and it is possible
that under normal conditions the virus is
enzootic and not prone to become epizootic.
This appears to be the case for the similar,
but nonoccluded rod-shaped virus in the
whirligig beetle reported by Gouranton
(1972).
Because mass mortalities of marine ani-
mals, particularly shrimp, are rarely de-
tected and studied in nature, it is difficult
at present to assess the role of the virus
in shrimp ecology. Mass rearing of pink
shrimp in aquacultural efforts may permit
future assessments of roles of viral agents
in shrimp health.
ACKNOWLEDGMENTS
Mr. Lee Courtney participated technically in
many phases of the work and is gratefully ac-
knowledged. Dr. Del Nimmo and Mr. Sam
Tagatz, both of the Gulf Breeze EPA Laboratory,
are thanked for their contributions of shrimp from
their experimental, toxic exposure studies.
E.EFEHENCES
BANG, F. B. 1971. Transmissible disease, probably
viral in origin, affecting the amebocytes of
the European shore crab, Cartirms maenas.
Infect. Immun., 3, 617-623.
BERGHOLD, G. H. 1963. The nature of nuclear-poly-
-------
ENZOOTIC NPV OF PINK SHHIMP
331
hedrosis viruses. In "Insect Pathology An Ad-
vanced Treatise," Vol. 1. (E. A. Steinhaus, ed.)
pp. 413-456. Academic Press, New York.
BONAMI, J. R., AND VAGO, C. 1971. A virus of a
new type pathogenic to Crustacea. Experien-
tia, 27, 1363.
BONAMI, J. R., H. GRISEL, C. VAGO, AND J. L.
DUTHOIT. 1972. Recherches sur une maladie
epizotique de I'huitre pelate, Ostrea edulis
Linne. Rev. Trav. Inst. Peches Marit.
BONAMI, J. R., C. VAGO, AND J. L. DUTHOIT. 1971.
Une maladie virale chez les Curstaces deca-
podes due a un virus d'un type nouveau.
Comp. Rend. Seanc. I'Acad. Sci., 272,
3087-3091.
COUCH, J. A. 1974. Free and occluded virus, similar
to Baculovirus, in hepatopancreas of pink
shrimp. Nature (London), 247, 229-231.
COUCH, J. A., AND NIMMO, D. R. 1973. Cytopathol-
ogy, ultrastructure, and virus infection in pink
shrimp exposed to the PCB, Aroclor 1254.
(Abstract) Program of the Joint Meeting of
The Society for Invertebrate Pathology and
International Colloquium on Insect Pathology
and Microbial Control, p. 105. Oxford Univer-
sity, England.
DEVAUCHELLE, G., AND C. \ _>. 1971. Particules
d'allure virale dans les cellules de 1'estomac
de la seache, Sepia officinalis. Compt. Rend.
Soc. Acad. Sci. Ser. D., 272, 894-897.
FARLEY, C. A., W. G. BANFIELD, G. KASNIC, JR.,
AND W. S. FOSTER. 1972. Oyster herpestype
virus. Science, 178, 759-776.
GOURANTON, J. 1972. Development of an intra-
nuclear nonoccluded rod-shaped virus in some
midgut cells of an adult insect, Gyrinus nata-
tor L. (Coleoptera). J. Ultr: , Mt. Res., 39,
281-294.
HAYAT, M. A., AND R. GIAQUINTA. 1970. Rapid fixa-
tion and embeding for electron microscopy.
Tissue and Cell, 2, 191-195.
HUGER, A. M. 1966. A virus disease of the Indian
rhinoceros beetle, Oryctes rhinoceros (Lin-
naeus), caused by a new type of insect virus,
Rhabdionvirus oryctes gen.n., sp.n. /. Inver-
tebr. Pathol, 8, 38-51.
KAZAMA, F. Y., AND SCHORNSTEIN, K. L. 1972. Her-
pestype virus particles associated with a
fungus. Science, 177, 696-697.
LUNA, L. G. (Ed.) 1968. Manual of Histologip
Staining Methods of the Armed Forces Insti-
tute of Pathology. 3rd ed., pp. 134-135. Mc-
Graw-Hill, New York.
MAZIA, D., BREWER, P. A., AND ALFERT, M. 1953.
The cytochemical staining and measurement
of protein with mercuric bromophenol blue.
Biol. Bull, 104, 57-67.
NIMMO, D. R., BLACKMAN, R. R., WILSON, A. J.,
AND FORESTER, J. 1971. Toxicity and distribu-
tion of Aroclor 1254 in the pink shrimp
Penaeus duorarum. Marine Biol., 11, 191-197.
RUNGGER, D., M. RASTELLI, E. BHAENDLE, AND R.
G. MALSBERGER. 1971. A virus-like particle as-
sociated with lesions in the muscles of Octo-
pus vulgaris. J. Invertebr. Pathol., 17, 72-80.
SUMMERS, M. D. 1971. Electron microscopic obser-
vations on granulosis virus, entry, uncoating
and replication processes during infection of
the midgut cells of Trichoplusia ni. J. Ultra-
struct. Res., 35, 606-625.
VAGO, C. 1966. A virus disease in Crustacea. Na-
ture (London), 209, 1290.
WILDY, P. 1971. Classification and nomenclature
of viruses. First report of the international
committee on nomenclature of viruses.
Monogr. ViroL, 5, 1-81.
-------
CONTRIBUTION NO. 216
-------
Vol. II No. 2, June 1974
MOLECULAR STRUCTURE OF PCBs
Ultrastructural Studies of Shrimp Exposed to
the Pollutant Chemical Polychlorinated
Biphenyl (Aroclor 1254)
John A. Couch, Ph.D., and Del Wayne R. Nimmo, Ph.D.
U.S. Environmental Protection Agency
Gulf Breeze Environmental Research Laboratory
Sabine Island, Gulf Breeze, FL 32561
Presented at the 63rd Annual Meeting of the International Academy of
Pathology, March 13, 1974, San Francisco, CA
Gradually increasing signs of disease and toxicity in freshwater
and marine ecosystems have increased the need for detailed
aquatic animal pathology. Among others, there are two areas
of investigation, utilizing pathobiological methods, that appear
promising in toxicology of aquatic animals. These areas of
inquiry are based on two questions:
1) Are there indicative sublethal cytopathic changes that
occur in aquatic animals exposed to low levels of
pollutant chemicals?,
and
2) Are there interactions between natural pathogens
(e.g. parasites) and pollutant chemicals in aquatic
animals that will produce harmful synergistic effects?
Answers to these questions should aid in determining
long-term and short-term effects of pollutants on aquatic
ecosystems.
Methods that we are using to attempt to answer these
questions are: (1) experimental exposure of selected aquatic
species to both sublethal and lethal concentrations of
pollutants, followed by, or concomitant with, (2) histological,
subcellular, and physiological investigations of exposed control
and feral specimens.
One of the most useful marine species for our studies has been
the pink shrimp (Penaeus duorarum). Pink shrimp are
commercially valuable Crustacea common in the South
Atlantic and Gulf of Mexico. We have been using this species
as a test animal in studies of effects of the widespread
pollutant chemicals, the polychlorinated biphenyls (PCB's),
(Figs. 1,2).
In this study, hepatopancreatic tissue was selected as the tissue
of choice for monitoring cellular effects because of the
functional significance of the hepatopancreas to Crustacea. The
hepatopancreas is a complex, gland-like organ that functions in
digestion, secretion, absorption, and storage of nutrients (Fig.
3). The hepatopancreas consists of masses of acini or digestive
tubules which branch off of two hepatopancreatic ducts that
X INDICATING THE POSSIBLE CHLORINE POSITIONS
Fig. 1. Polychlorinated biphenyl (Aroclor) molecule.
have their origin in the wall of the pyloric stomach. The
acinus, the functional unit of the hepatopancreas, consists of a
tubule whose wall is epithelium. This epithelium is made up of
several cell types which are distributed differentially along the
length of the acinus from a point proximal to the
hepatopancreatic duct to distal blind sacs at the end of the
acinus. The major cell types are: (1) embryonic (distal tips of
acini); (2) absorptive (adjacent and medial to the embryonic
cells); (3) secretory (medial and proximal region of acinus
relative to the hepatopancreatic ducts).
In several experiments, shrimp were exposed to 3-5 jug/I PCB
(Aroclor 1254) in flowing seawater. After 20-30 days, 50% of
these shrimp died,'whereas control shrimp experienced little or
no mortality. Exposed shrimp accumulated up to 500 mg/kg
PCB in hepatopancreatic tissues according to results of gas
chromatographic analyses (Fig. 4).
E
Q.
Q.
Lf)
CN
o:
O
_i
O
O
cr
400
300
200
HEPATOPANCREAS
10
20
25
EXPOSURE TIME (days)
Fig. 4. Graph of uptake of Aroclor
hepatopancreas over 25-day period.
1254 (PCB) by
17
-------
The Bulletin of the Society of Pharmacological and Environmental Pathologist*
Fig. 2. Pink shrimp with dorsal carapace of cephalothorax
removed to expose hepatopancreas, in situ (arrow).
Fig. 3. Cross-section of hepatopancreas of pink shrimp Fig. 6, 7. Advanced ER proliferation and whorls in
showing complex relationships of ducts and acini (X 50). hepatopancreatic cells of shrimp exposed to Aroclor 1254
(X 28,500).
Fig. 5. Electron micrograph (EM) of early endoplasmic Fig. 8. ER membrane whorls surrounding lipid droplets in
reticulum (ER) proliferation in shrimp cells exposed for
25 days to Aroclor 1254. Note both attached and free
ribosomes and dilated cisternae of ER (X 28,500).
shrimp cells from same shrimp as in Figs. 6, 7. (X 84,000).
II
-------
Vol. II No. 2, June 1974
Fig. 9. Hepatopancreatic cell from shrimp exposed to 3 jug/I Fig. 11b. Fresh squash preparation of hepatopancreatic tissue
Aroclor 1254 for 30 days. Note small vesicles in containing numerous tetrahedral (3-D) inclusion bodies (X
nucleoplasm and loss of cytoplasmic integrity (X 14,400). 3,000).
Fig. 10. Advanced nuclear degeneration in Aroclor-exposed
shrimp. Note large vesicles in nucleoplasm surrounded by
myelin-like sheaths. Note, also, the extreme modification
of nuclear envelopes and myelin-like structures in
envelope area (X 28,500).
Fig. 12. Electron micrograph of inclusion body in
hepatopancreatic cell nucleus from shrimp exposed to
Aroclor 1254. Note rod-shaped virions, both free and
occluded within body (X 28,500).
• d
I
c
v C
)
*
f.%
> •»r
\ j
Fig. 11a. Cross-section (light micrograph) of hepatopancreas
acinus from pink shrimp. Note triangular inclusion bodies
in some nuclei, and hypertrophied nuclei as well as normal
nuclei (X1.000).
Fig. 13. Electron micrograph of hepatopancreatic cell with
infected nucleus. Note hypertrophy of nucleus, loss of
heterochromatin, and cytoplasmic changes (X 14,400).
-------
The Bulletin of the Society of Pharmacological and Environmental Pathologists
Hepatopancreatic absorptive cells from shrimp surviving 30 5. Trump, B. F., and Arstila, A. V.: Cell injury and cell
days' exposure revealed the following departures from normal: death, in: Principles of Pat'hobiology. Oxford University
1) 30 to 50% of cells had increased or proliferated Press. New York, pp. 9-95, 1971.
endoplasmic reticulum associated with high numbers
of attached and free ribosomes (Figs. 5, 6, 7).
2) Eventual production of "membrane whorls" or
myelin bodies that often enclose lipid droplets (Fig.
8).
3) Nuclear degeneration characterized by the occurrence
of vesicles in the nucleoplasm. These vesicles are of
two classes: 20-50 nm in diameter, and 100-700 nm
in diameter (Figs. 9, 10).
The proliferation of endoplasmic reticulum in certain cells
(hepatocytes) of higher animals has been associated with toxic
responses to phenobarbitol, dilantin (5), dieldrin (3), and
carbon tetrachloride (4,5). This proliferation has been
correlated with detoxification of poisons and can, if the
poison persists, progress to the formation of membrane
whorls, abnormal myelin bodies, and death of the cell (5).
Since the hepatopancreas of shrimp performs some functions
similar to those of the liver of higher animals, it is not
unreasonable to suggest that the responses observed in the
shrimp absorptive cells may indicate toxic responses to the
PCB.
Another finding in the hepatopancreas of PCB-exposed shrimp
was the discovery of a new virus associated with unique
polyhedral inclusion bodies visible at the light microscope level
(Fig. 11). This virus and its polyhedral inclusion body were
found in higher prevalence in shrimp exposed to PCB than in
shrimp taken directly from nature or held as controls (1). The
virus is rod-shaped and may be free in the nucleus or occluded
in the polyhedral inclusion body in nuclei of absorptive cells
(Fig. 12). Therefore, it is very similar to Baculovirus that
infects and causes diseases in insects. Cytopathic effects
associated with the virus are shown in Fig. 13.
We are presently studying the interactions of the virus and
PCB in shrimp in the laboratory to determine if there are
harmful synergistic interactions between low levels of
pollutants (pesticides) and virus infections. The concept of
possible synergism between pollutants and natural pathogens
introduces a novel field of research with aquatic animals,
similar to that suggested by Friend and Trainer (2) for
terrestrial vertebrates infected by viruses.
References
1. Couch, J.: Free and occluded virus, similar to Baculovirus,
in hepatopancreas of pink shrimp. Nature, 247: 229-231,
1974.
2. Friend, M., and Trainer, D. O.: Polychlorinated biphenyl:
Interaction with duck hepatitis virus. Science, 170:
1314-1316, 1970.
3. Hutterer, F., Schaffner, F., Klion, F., and Popper, H.:
Hy pertrophic, hypoactive, smooth endoplasmic
reticulum: A sensitive indicator of hepatotoxicity
exemplified by dieldrin. Science, 161, 1968.
4. Smuckler, E. A., and Arcasoy, M.: Structural and
functional changes of the endoplasmic reticulum of
hepatic parenchymal cells, in: Inter. Rev. Exper. Path. 7:
305-418. Academic Press, New York and London, 1969.
20
-------
CONTRIBUTION NO. 219
-------
In: 1974 Proc. Gulf Coast Regional Symposium on Diseases
of Aquatic Animals. LSU-SG-74-05. Baton Rouge, La. 70803
DETECTION OF INTERACTIONS BETWEEN
NATURAL PATHOGENS AND
POLLUTANTS IN AQUATIC ANIMALS1
John A. Couch and D. R. Nlmmo
U. S. Environmental Protection Agency
Gulf Breeze Environmental Research Laboratory
Sabine Island
Gulf Breeze, Florida 32561
(Associate Laboratory of the National Environmental
Research Center, Corvallis, Oregon)
Hopefully, it is now generally accepted that the "germ theory"
of disease applies to aquatic vertebrates and invertebrates as well
as to terrestrial forms. That this has not always been the case is
best illustrated by the fact that in the past when mass mortalities
of aquatic animals occurred, the general ecologist often overlooked
the possibility that infectious pathogens might have been the etio-
logic agent. More often than not, every other possible avenue of
cause and effect was explored before a search for infectious disease
agents was launched.
Presently, in this age of environmental consciousness when mass
mortalities of aquatic animals occur, one of the first causes to
be searched for is pollution. This is justified, of course, based
on documented evidences that water quality and aquatic ecosystem
stability have been lowered significantly in the last three decades
by increasing industrialization, population growth, and water misuse
Thus, those of us who must consider health of aquatic species
have, on the one hand, infectious diseases and, on the other, pollu-
tion of waters, coming together as one complex of environmental
factors that affect health in aquatic ecosystems. Impinging upon
Contribution No. 219, Gulf Breeze Environmental Research Lab.
261
-------
262 DISEASES OF AQUATIC ANIMALS
the complex of aquatic disease and pollution are, of course, other
environmental factors such as temperature, pH, salinity, oxygen
content, available nutrients, and agents of mechanical trauma.
These too must be considered in evaluating the health of an aquatic
species.
Only recently has the possible interaction of infectious
diseases and pollutants, as environmental complexes, been seriously
consided as threats to aquatic life. Snieszko (1972) has recently
reviewed fish diseases that are heavily dependent upon environmental
interactions. His general summation could be applied to any group
of interacting environmental complexes, particularly infectious
diseases and pollution:
Modern epidemiology is based on the premise that epidemic
outbreaks are caused by an imbalance between the host, the
pathogens or other disease agents and the environment.
Aquatic cold-blooded animals are much more affected by the
environment than are the terrestrial homeotherms. Therefore,
outbreaks of various diseases of fish are strongly affected by
ecologic factors.
The purpose of this paper is to give two examples of the detec-
tion of possible interactions between natural pathogens and chemical
pollutants in selected Gulf of Mexico, estuarine animals. These
will include results of both experimental laboratory work and field
observations in the vicinity of Pensacola, Florida.
Nimmo et al. (1971) have used pink shrimp (Penaeus duroarum)
(Fig. 1) as test animals in toxicity studies for several years at the
Gulf Breeze EPA laboratory. Recently, Couch (1974a) described a new
virus in pink shrimp. It was observed during light and electron
microscope studies of the hepatopancreas of toxicant exposed, control
and feral shrimp (Figs. 2,3). The virus was found in hepatopancreatic
epithelial cell nuclei (Fig. 4), is rod-shaped, and is either free in
the nucleus or occluded in patent infections in crystalline inclusion
bodies that range from 0.5 ym to 20 ym in size (Figs. 5,6).
The shrimp virus, named Baculovirus penaei by Couch (1974b), is
considered to be a nuclear polyhedrosis virus because it shares many
characteristics with the NPV or Baculoviruses (subgroup A) of insects
(Wildy, 1971). It is the first Baculovirus reported in a host other
than insects. The virus occurs in feral pink shrimp and is enzootic
in populations of shrimp from Apalachee Bay, Florida and Santa Rosa
Sound, Florida. It has been found most prevalent during the fall
and winter months, occurring in from 0-30 percent of shrimp in given
samples.
Pink shrimp from natural populations with enzootic levels of
the virus have been used as subjects in toxicity tests of the PCB,
-------
Interactions Between Pathogens and Pollutants 263
AroclorR 1254, and the organochlorine insecticide, Mirex. Shrimp
exposed to 3 ug Aroclor/1 in_flowing seawater for from 30 to 50
da'ya accumulated'up to 500 mg Aroclor/kg in their hepatopancreatic
tissues. After approximately 30 days exposure, 50 percent of these
shrimp died (Nimmo et al., 1971). From these series of tests, sur-
viving, exposed shrimp were examined histologically and 60 percent
were found to be lightly to heavily infected with the nuclear poly-
hedrosis virus. Control shrimp were free of patent virus infections.
In another exposure test, conducted by Tagatz (personal communication),
38 percent of shrimp exposed to 0.01 to 0.23 yg commercial Mirex/1 of
flowing sea water for 30 days demonstrated patent virus infections.
Only 6.6 percent of control shrimp for this experiment showed patent
infection. Mortality of shrimp in the Mirex exposure experiment was
81 percent, whereas the controls had only a 9 percent mortality.
Thus, the prevalence of virus infections in the chemically exposed
and control shrimp corresponded to the level of mortalities in those
respective groups.
Another interesting finding has been that samples of pink shrimp
kept under abnormally crowded conditions for 30-40 days in aquaria
have shown 40-50 percent virus prevalence as compared to initial pre-
valence of 0-10 percent at onset of captivity (day one of holding
period). This strongly suggests that the stress and proximity of
crowding for a period of time enhances or facilitates the virus
infection. Transmission of the virus from individual to individual
via cannibalism in densely crowded aquaria or culture containers
should be expected. In nature, even on fertile fishing grounds,
shrimp are rarely as densely found as under aquacultural or aquarium
conditions. Further, in nature dilution of infective stages of the
virus by several factors (water volume, predation of infected shrimp
by non-shrimp predators, etc.) would be greater than in closed less
voluminous, artificial systems.
Therefore, as an example of a model system, we have the
apparent enhancement of the prevalence of a Baculovirus by certain
potential environmental factors, including chemical pollutant stress
(PCB's and Mirex) and crowding. A similar model system of inter-
actions between a toxicant chemical and a virus for higher vertebrates
was presented by Friend and Trainer (1970), whose research demonstrated
enhancement of duck hepatitis virus by polychlorinated biphenyls.
Numerous casual and careful observations of natural, aquatic
ecosystems have led us to believe that where one finds chronic low
level pollution (both natural nutrients and synthetic chemicals),
one also observes increasingly frequent epizootics of certain
diseases and a .gradual increase in prevalence of certain pathogens
in stressed hosts. This is not true, however, for every pathogen
since some parasites may be adversely affected by the pollution as
much as, or more than, their hosts. Each disease agent-pollutant
-------
264 DISEASES OF AQUATIC ANIMALS
complex must be considered separately as well as part of more compli-
cated, larger systems.
Over the last 10 years, we have observed in Escambia Bay, Florida,
high prevalence of fin rot syndrome associated with mortalities in
croakers (Micropogon undulatus) and spot (Leiostomus xanthurus) during
periods of warm weather and oxygen depletion. Escambia Bay has been
contaminated with the PCB, Aroclor 1254, for several years (Duke et-al. ,
1970) and is a rapidly eutrophying system. Although we have not
attempted to isolate pathogens from healthy or moribund fish, the
patterns of disease and their seasonal occurrences strongly suggest a
bacterial etiology. In this regard, Schwartz (1974) found that the
bacteria Aeromonas and Pseudomonas, representative well-known fish
pathogens, were found in higher prevalences in fish from Clear Lake,
Iowa, in warmer seasons than in cooler seasons. In the laboratory
during warming months of the year (April through June), we have been
able to induce fin rot syndrome, identical to that in fish from
Escambia Bay, in up to 90 percent of spot exposed to 3-5 yg/1 of
Aroclor 1254 (Couch, 1974c). This fin rot was associated with high
mortality (80 percent), but again, no attempts were made to isolate
a bacterial pathogen.
Thus, there is strong empirical and circumstantial evidence
which suggests that interactions between natural pathogens and pollu-
tants probably occur (Snieszko, 1974). Further research must determine
the extent of the threat of such interactions to aquatic life and
ecosystems. The Gulf of Mexico and its northern and eastern estuaries
provide numerous natural sites for study of such interactions.
LITERATURE CITED
Couch, J. 1974a. Free and occluded virus, similar to Baculovirus,
in hepatopancreas of pink shrimp. Nature 247:229-31.
Couch, J. 1974b. An enzootic nuclear polyhedrosis virus of pink
shrimp. I. Ultrastructure, prevalence, and enhancement. J.
Invert. Path, (in press).
Couch, J. 1974c. Histopathologic effects of pesticides and related
chemicals on the livers of fishes. In The Pathology of Fishes.
Univ. of Wisconsin Press (in press).
Duke, T., J. I. Lowe, and A. J. Wilson, Jr. 1970. A polychlorinated
biphenyl (AroclorR 1254) in the water, sediment, and biota of
Escambia Bay. Florida Bull. Environ. Contam. and Toxic. 5:171-80.
Friend, M., and D. 0. Trainer. 1970. Polychlorinated biphenyl: inter-
action with duck hepatitis virus. Science 170:1314-16.
-------
Interactions Between Pathogens and Pollutants 265
Nimmo, D. R., R. R. Blackman, A. J. Wilson, and J. Forester. 1971.
Toxicity and distribution of Aroclor 1254 in the pink shrimp
Penaeus duorarum. Marine Biology 11:191-97.
Schwartz, J. J. 1974. Prevalence of pathogenic pseudomonad bacteria
isolated from fish in a warmwater lake. Trans. Amer. Fish. Soc.
Vol. No. 114-16.
Snieszko, S. F. 1972. Progress in fish pathology in this century.
Symp. Zool. Soc., London 30:1-15.
Snieszko, S. F. 1974. The effects of environmental stress on out-
breaks of infectious diseases of fishes. J. Fish Biol. 6:197-208,
Wildy, P. 1971. Classification and nomenclature of viruses. First
report of the international committee on nomenclature of viruses.
Monographs in Virology 5:1-81.
-------
266
DISEASES OF AQUATIC ANIMALS
iewpf pink shrimp (Penaeus duorarum)
Sal cut
Figure 1. Dorsal view
with dorsal cuticle removed to show hepato-
pancreas in situ (arrow).
^L ~ •*
%**
. -
*
\
*.'
%
Figure 2. Cross section of shrimp hepatopancreatic tubule
or acinus showing epithelial cell nuclei con-
taining triangular baculovirus inclusion
bodies (usually one per nucleus if cell is
infected; arrows); normal nuclei are small
with prominent nucleoli (820X). Feulgen,
picromethyl blue stain.
-------
Interactions Between Pathogens and Pollutants
267
Figure 3. Fresh squash preparation of end of hepatopancreatic
acinus; note single patently infected cell with
large inclusion body that has the form of a
tetrahedron (820X). No stain.
f -
,
•v
Figure 4. Section of hepatopancreatic epithelium showing cell
with viral inclusion body in nucleus (arrow).
(1230X)
-------
DISEASES OF AQUATIC ANIMALS
'N
\
,v,y
ri^fis^f
jtf&fty^ ' ;";_- ,/ *
*••. ," ^ x
": |^- r-
1 -,:;,X •
/
Figure 5. Electronmicrograph of virus infected hepato-
pancreatic epithelial cell from PCB-exposed
shrimp; note triangular section of virion-
containing inclusion body in infected cell;
note virions free in nucleoplasm (arrows).
Compare normal cell (nc) with infected cell
(7800X).
*
Figure 6. Higher magnification of inclusion body in shrimp
cell. Note virions in longitudinal and in cross
section embedded within matrix of characteristic
triangular inclusion body (15,400X).
-------
CONTRIBUTION NO. 220
-------
Ultrastructural and Protargol Studies of
Lagenophrys callinectes (Ciliophora: Peritrichida)
JOHN A. COUCH
Copyright 1973 by the Society of Protozoologists
Reprinted from THE JOURNAL OF PROTOZOOLOGY.
J. PROTOZOOL., 20(5), 638-647 (1973).
Made in United States of America
-------
J. PROTOZOOL. 20(5), 638-647 (1973).
Ultrastructural and Protargol Studies of Lagenophrys callinectes*
(Ciliophora: Peritrichida)
JOHN A. COUCHJ
U.S. Environmental Protection Agency, Gulf Breeze Environmental Research Laboratory,§
Gulf Breeze, Florida, U.S.A. 32561
SYNOPSIS. Ultraotructural and protargol studies reveal that the trophont of Lagenophrys callinectes, though highly spe-
cialized, generally conforms to the basic peritrich structural pattern.
Features described for L. callinectes trophonts which are unique for the genus are the fine structure and arrangement of
the lorica and lips, the attachment organelle of the peristomial cytoplasm, its attachment to the loricastome walls, and the
arrangement of the aboral kinetosomes of the trophont. Lack of a distinct scopularized region, and of a ventral lorica wall
also characterize L. callinectes trophonts.
The 4-row terminal peniculus, as revealed by protargol staining, differs from the 6-row terminal peniculus of L. nassa
suggesting that the patterns of infundibular structure, as revealed by protargol, should be useful in future taxonomic studies
of Lagenophrys species.
Index Key Words: Lagenophrys callinectes; ultrastructure; protargol; peritrich; morphology; infraciliature; lorica; kineties.
RESULTS of electron microscope (EM) studies have been
published on the trophonts of the following genera of ses-
siline, stalked peritrichs: Campanella (7); Opercularia and
Vorticella (1, 16); Zoothamnium (1, 8); Epistylis (8, 16);
and Carchesium (8, 16, 23). Work on members of the stalk-
less, but sessiline genus Scyphidia was recently completed (13),
having been preceded by work on a possible scyphidian species
in the genus Termitrophrya (14). Nonsessiline, free-swimming
peritrichs studied by EM have been Opisthonecta henneguyi
(1) and Telotrochidium sp. (14).
* Much of this work was done while the author was employed
with the National Oceanic and Atmospheric Administration, Na-
tional Marine Fisheries Service, Biological Laboratory, Oxford,
Maryland.
* Great appreciation is here expressed to Dr. Frank Perkins for
his generous advice and guidance in the use of the electron
microscope. Drs. R. B. Short, John Corliss, and Eugene Small
read and constructively criticized portions of the paper.
§ Associate Laboratory of the National Environmental Research
Center, Corvallis, Oregon.
Protargol-silver staining studies of representative species of
peritrichs have been reported by earlier workers (4-6, 12, 23,
24). To date, however, transmission EM studies of the highly
specialized, loricate, peritrich genus Lagenophrys have not been
published while the protargol studies of this genus have been
brief (4, 5, 12).
The purpose of the present paper is to describe aspects of
the ultrastructure of Lagenophrys callinectes Couch, 1967, a
commensal on the marine blue crab, and to give results of
further protargol studies on the trophont of this species.
MATERIALS AND METHODS
The blue crab (Callinectes sapidus Rathbun, 1896), host of
Lagenophrys callinectes, was collected by means of traps, nets,
and trawls. Blue crabs are euryhaline and are usually found
in estuarine and coastal waters ranging from fresh-water to
oceanic salinities (22). I have found L. callinectes on the gills
of blue crabs from Chesapeake and Chincoteague Bays in Mary-
land and Virginia, coastal estuaries of North and South Carolina,
-------
EM AND PROTARGOI. STUDIES OF A PERITRICH
639
All figures are of Lagenophrys callinectes.
Figs. 1-8. [Hematoxylin (Figs. 1, 2) and protargol (Figs. 3-8) preparations of trophonts.] 1. Solid line represents long axis of
organism; broken line represents plane of asexual division. Hi, hiatus: PC, peristomial cytoplasm, x 1000. 2. Longitudinal section
on both surfaces of gill lamella of blue crab. L, lorica. X 1000. 3. Lips and lorica outline. A, lorica aperture; L, lorica; LA, lips
around lorical aperture. X 1000. 4. Trophont with important cytoplasmic structures. AK, aboral kinety; C, cytostomc; CVO,
contractile vacuole orifice; Cy, cytopharynx; Inf. infundibulum: Ma, macronucleus; Mi, micronucleus; My, myoneme. X 1600. 5, 6.
Mirror image views of adoral kineties of trophont. AK, aboral kinety; H, haplokinety; PO, oral polykinety. X 1400. 7. Polykineties
(PI, P2) and aboral kinety (AK). X 2000. 8. Peniculus (Pn); note 4 terminal kineties. X 2000.
-------
640
EM AND PROTARGOL STUDIES OF A PERITRICH
" —PC
Fig. 9. Scheme of L. callinectes infraciliature. Mirror image of
Figs. 1, 4, 5, 7, 8. Same orientation as Fig. 6. Protargol. AK,
aboral kinety; C, cytostome; Cy, cytopharynx; CyT, cytopharyngeal
tube; ED, epistomial; GK, germinal kinety; H, haplokinety; Inf,
infundibulum; My, myoneme; PI, P2, P3, polykineties one, two,
and three; PC, peristomial cytoplasm; PO, oral polykinety; PS,
peristomial space; S, "S-belt" of Lom.
Georgia, and Florida; and the Gulf of Mexico near New Orleans,
Louisiana and Pensacola, Florida. Salinities of collection sites
ranged from 10% to 35%*.
To prepare L. callinectes for microscope studies, the dorsal
carapace of the host was removed; branchiae were removed and
individual lamellae cut from them. For short-term study,
lamellae were placed in drops of seawater on slides with No. 1
coverslips directly applied. For long-term observation the
lamellae were placed in seawater under No. 1 coverslips mounted
on fragments of coverslips to prevent crushing the ciliate.
Infested gill material was fixed in Bouin's or Davidson's
(20) fixative for treatment with protargol silver, modified
method (10), and subsequent study of the morphologic and
morphogenetic aspects of the infraciliature of different stages
of L. callinectes. Lom (12) reported that the protargol method
is far superior to any other (including Klein, Chatton-Lwoff)
for the difficult job of impregnating the well protected in-
fundibular infraciliature of peritrichs. Materials fixed as above
were also stained with Heidenhain's iron hematoxylin for general
cytology. Infested gill lamellae fixed in Schaudinn's fluid were
Feulgen stained.
For EM studies heavily infested pieces of lamellae were
teased apart into portions less than 1 mm2 These pieces were
fixed in 2.5% (v/v) glutaraldehyde for 1 hr, washed in 0.2
M Millonig's phosphate buffer for 45 min (3 changes) and
postfixed for 4 hr in 1% (w/v) OsO4 buffered in 0.4 M
Millonig's phosphate buffer. The lamellar fragments were then
dehydrated in alcohol and embedded in Durcupan ACM. Sec-
tions were cut with an LKB ultramicrotome, mounted on
Formvar-coated grids, and stained in Reynolds' lead citrate
(17) for 15 min and 2% (w/v) aqueous uranyl acetate for
4-5 min. The stained sections were examined and photo-
graphed in a Hitachi HU-11B electron microscope.
Many terms used in this work concerning ciliate taxonomy,
biology, and structure are defined by Corliss (2) and Kane
(11).
All measurements are in micrometers unless otherwise
stated.
RESULTS
General Form.—Lagenophrys callinectes lives in a colorless,
transparent, hemispherical lorica attached to the gill lamellar
surface of the blue crab. The lorica wall is ~0.2-0.4 thick and
48-57 wide (Figs. 1, 2). The aperture of the lorica is 9.1-11.4
wide and is surrounded by 2 anterior and 2 posterior lip ele-
ments; the anterior ones are very unequal in size, whereas the
posterior ones are equal in size (Fig. 3). These lips are easily
seen since they are opaque in the living as well as in the fixed,
stained animal. The form and relative size of the lip elements
comprise the most important features available for identification
of L. callinectes. The living animal is able to flex its peristomial
cytoplasm, opening and closing the aperture by pulling the
posterior lips together and then apart.
The alimentary complex (Figs. 4-9) of L. callinectes in con-
tracted specimens begins in the peristomial space, which as used
here is the region surrounded by the contracted peristomial
cytoplasm of fixed specimens. This space includes what some
would call the buccal cavity, but it does not include the in-
fundibulum (Figs. 9, 15). The floor of the peristomial space
is made up of the epistomial disc surrounded by the oral kinetics
(Figs. 5, 6). These kinetics lead to the infundibulum (Fig.
9) which spirals down to the cytostome, which opens into the
ampulla-like cytopharynx.
The relatively long cilia that surround the protrusible epi-
stomial disc are visible in the living animal both when it is
feeding with an extruded adoral apparatus and when the disc
is retracted with the aperture closed. Individual feeding
trophonts have been observed in salinities of 17%c, forming
food vacuoles in the region of their cytopharynx at the ra.te of
one every 2 min for up to 8-10 min, after which feeding may
cease for long periods.
The cytoplasm of living trophonts may contain a few or
many food vacuoles. In trophonts stained with iron hema-
toxylin, food vacuoles often appear basophilic and when ex-
amined closely appear to be filled with small basophilic rods.
These are probably bacteria upon which the peritrich feeds.
The contractile vacuole is associated with the side of the
cytopharynx immediately beneath the base of the epistomial
disc in both living and fixed, stained specimens. The vacuole
empties into the ampulla of the cytopharynx (Fig. 4).
Portions (rarely all) of the macronucleus are visible in living
-#
Fig. 10. Anterior and slightly lateral section of trophont; note the myonemal mass that makes up one of the lateral shoulders of
peristomial cytoplasm. ALsW; anterior loricastome wall; LA, lips around lorical aperture; Ls, loricastome; Mt, mitochondria- My
myoneme; PC, peristomial cytoplasm; PLSW, posterior loricastome wall. X 11.250. ' '
Fig. 11. Posterior or aboral region of lorica; arrow indicates point where lorica material disappears; note remnants of prokaryotic
organisms (bacteria?) within lorica (L). AL, amorphous layer. X 12.500.
-------
EM AND PROTARGOL STUDIES OF A PERITRICH
641
- ^--v.
V*
' •-*-
..'•-
M
•
-------
642
KM AND PROTAROOL STUDIKS OF A PERITRICH
ECu Cu
#*! :**'
12
N^ftfefapjiM
13
Figs. 12, 13. [Cuticle of uninfested (Fig. 12) and infested
(Fig. 13) crah gill lamellae. Cu. crab gill cuticle; ECu, crab
gill epicuticle. I 12. Note absence of lorica above epicuticle layer.
X 47,500. 1.3. Lorica is absent above epicuticle layer and beneath
trophont. Dark bodies above epicuticle are remnants of bacteria.
X 64,750.
iriiplionls. The micronucleus usually cannot be observed with
bright field microscopy in living animals. In iron-hematoxylin-
stained specimens macronuclei vary from short rod! ike and reni-
fonn to L, U. or C shapes. The small micronucleus is usually
niuiul to ellipsoid, from 1.7-3.4 in diameter, and usually rests
along the concavity of he curved mncronucleus (Fig. 4).
A dense basopliilic rric'shwork is distributed evenly throughout
ihe macronucleus, interspersed with lacunae that contain round
basophilic inclusions. In Feitlgen preparations the evenly dis-
tributed material, corresponding to the basophilic moshwork,
is positive for DNA, whereas the basopliilic inclusions arc
Feulgen negative and not visible.
Lorica,—Ultrathin sections were cut parallel to the long axis
of the soft body and lorica of L. ctiUhicclcs (see Figs. 1, 2, 10,
11 for orientation). The lorica wall is slightly variable in
thickness (0.1-0.4), being thickest inid-dorsally and at its point
of attachment to the cuticle of the host (Fig. 11). The lorica
does not appear to extend beneath the soft body of the ciliate
to' form a complete ventral wall, as described in light micro-
scope studies for certain species of Lagenophrys, like /.. mcta-
ftauliadis (3). Instead, the inner portion of the lorica wall
where it meets the host cuticle, appears to extend inward over
the host cuticle for a few jj.m, gradually decreasing in thick-
ness, until it becomes imperceptible see arrows Figs. 11, 19).
The rigid portion of the laminated gill cuticle of the crab host
is divided into 2 major layers: a very thin epicuticle (0.1-
0.3, Fig. 12) and a much thicker layer (beneath the epicuticle)
that may range 1-1.5 (Fig. 12). These layers are illustrated from
sections of gill cuticle both free of and infested by L. callincctcs
:. Figs. 12, 13). Therefore, the nature of the gill epicuticle
layer with and without L. callincclis can be determined. It
appears that the area of host cuticle midvcntral to attached
L. callinectes has no discernible layer on the epicuticle that
could be ciliate-contributed (Fig. 13).
An amorphous, granular layer of material was found be-
neath the lorica wall (Fig. 11). Toward the point of attach-
ment of the lorica, where the wall thickens considerably, there
appears to be an increase in density of the amorphous layer
and a gradual integration of the granular material into the
thickened peripheral buttress of the wall where it attaches to
the host cuticle (Figs. 10, 11).
The1 borders of the aperture of the lorica arc formed by the
specialized lorica extensions previously called lips (Figs. 3,
10). Uneven folds in the lorica are thrown up in the regions
anterior, lateral, and posterior to the lips (Fig. 10). From the
region of these folds, the lorica wall is expanded upward around
the aperture to reach electron-opaque crests (the lips) that are
apparent in thin sections (Fig. 10). The wall of the lorica
plunges down from the apex of the crests to form the inner
tube or loricastome of Kane (10) (Fig. 10). The walls of the
loricastomc extend downward to contact the pcristomial cyto-
plasm of the trophont. Electronmicrographs of sections of the
lips obtained in this study reveal little about the actual topog-
raphy (Fig. 3) of the different lip elements.
The' relationship of the trophont of L. callincctcs to its
lorica is schematized in Figure 19.
Pcristomial region and pellicle.—The posterior loricastome
wall is folded immediately before making contact with a
specialized dorsal rim of peristomial pellicle and cytoplasm of
the trophont (Figs. 15, 16, 19). This specialized pellicular-
cytoplasmic organelle appears to be of paramount importance
in the attachment of the trophont to its lorica. I can find no
comparable peritrich organelle described in the literature; there-
fore it is designated here as the attachment organelle. The
structure is 1 /xm or less wide (in section) and extends out-
ward and upward as a semi-V form of folded pellicle filled
with cytoplasm that is continuous with the peristomi.il cyto-
plasm (Fig. 16). Partial reconstruction (composite) of the
posterior loricastome-peristomial complex from available serial
sections suggests that the extremity of the posterior loricastome
wall is attached to the1 organelle along the entire dorsal width
of the peristomial region of the trophont (Fig. 15), forming
a firm but elastic relationship between the trophont and its
lorica.
The anterior loricastome wall attaches to the left and right
shoulders of the peristomial cytoplasm (Figs. 10, 19), but only
with ils extreme left and right edges. Thus a small anterior
hiatus (Figs. 1, 15, 19) exists between the left and right
shoulders of the peristomial region, anterior to the frontal
portion of the anterior loricastome wall edge, and the oral
opening of the trophont. This hiatus is apparent with light
microscopy (Fig. 1) and provides the telotroch an escape
hatch to the lorica aperture.
In the peristomial c ytoplasmic region is a mass of muscle-
like fibers lhat correspond in their fine' structure to the- con-
tractile myofibrillar system or "myoi'de" described for other
peritrichs (I, 7, 13). These protargol-positive myonemes are
concentrated mostly in the right or left shoulders of peri-
stoinial cytoplasm (Fig. 10). However, they extend across
the whole dorsal width of the' trophont as a band, subtend-
Fig. 14. Section through infundibulum (Inf) and ncphridial region or spongiome;
projecting crests (Cr) of infundibular wall. CV. contractile vacuole: NS. nephridial s
note bacteria (Ba) in infundibulum, and
projecting crests (L:r) ol mtundihular wall. L.v. contractile vacuole: IN a. nepnricuai system. X 19,375.
Fig. 15. Longitudinal section of aboral region, pcristomial space (PS) attachment organelle (AO), and epistomial disc (ED).
H. haplokincty; Hi. hiatus: Ls. loricastome: PO, oral polykinety. X 9.350.
-------
EM AND PROTARGOL STUDIES OF A PERITRICII
643
-------
644
KM AND PKOTARCOL STUDIES OF A PERITRICH
ALsW
Fig. 16. Higher magnification of adoral region and attachment organelle. Compare relatively small region of myonemes (my)
subtending attachment organelle (AO) in this figure to larger myonemal structure in lateral shoulder of peristomial cytoplasm in
Fig. 10. ALsW, anterior loricastome wall; Ls. loricastome; PLsW. posterior lorieastome wall. X 38,250.
ine; hut not entering the attachment organdie (Fit;. 16).
The myonemes have a fine slnicture and system of canals
("laciinos") similar to that possessed by tlie "myoi'des cndo-
plasmiques'' described for the sphincter collar of ]'nrlicclln
campanula (7). The peristomial myonemes of L. callincclcs are
organized into such a specialized subpellicnlar organelle, and
probably function as a muscle to open and close the aperture
of the lorica as well as a sphincter to close the oral opening
of the trophont. This myonemal organelle corresponds topo-
graphically to the contractile portion of the sphincter collar
described with light microscopy for Pyxicola nolandi (9).
The oral opening of the contracted trophont through which
adoral cilia are protruded with the epistomial disc when the
animal is feeding, lies at I he bottom of the tube formed by the
loricastome walls (Figs. 15, 16, 19).
The pellicle of the trophont of L. callincctcs consists of 3
unit membranes (''3-ply" pellicle, Refs. 13, 15). The arrange-
ment of the pellicle layers of L. callincctes differs from that of
Scyphidia ubiqniiti (13), but somewhat resembles the arrange-
ment reported for S. inrlinata (13). The 3 membranes of the
pellicle of L. callincctcs overlie one another closely so that no
well developed, rigid, pellicular alveoli are apparent between
the outer membranes and the inner one and its thin under-
lying epiplasm (Fig. 17). Thin spaces do exist between the
outer and the inner membranes, but these spaces are apparent
only along certain regions of the trophont's body surface.
Fig. 18. Higher magnification of adoral region.
polykinety. X H.375.
Note myonemes (My) beneath epistomial disc (ED). H. haplokinety; PO, oral
-------
EM AND PROTARGOI. STUDIES OF A PF.RITRICH
645
.
*.,..
Fig. 17. Dorsal surface with 3 unit membranes (I, II, III) of
pellicle, the epiplasm (Ep) underlying membrane III, and the
alveolar space (Al) between membranes II and III. X 152,500.
There are no kinetosomcs directly and intimately associated
with the outer body (nonoral) pellicle of L. caltinectcs trophonts,
nor is there an obvious arrangement of the pellicle into spe-
cialized scopnlar areas as reported for Scyphidia (13, 14), and
stalk forming peritrichs (6). A single row of barren kineto-
sonies dues exist, however, in an aboral ventral snbpellicular
position corresponding to the infraciliaturc of a trochal band
or peitinellar fringe in other peritrichs (1, 13). Lacking are
subpcllicular nnuue.nes that have been commonly reported in
oilier works <>n perinich nltrasiriicture (7, 8).
PcristinninI tpace, inlunilihuhim and cyti>/>li and 18 arc from a longitudinal section of the
tropbiint, and show the natnie of the perislomial space and
adjacent structures. At the base of the peristomial space lies
I hi' epistoniial disc surrounded by the1 haplokiiietv and poly-
kinety (Fig. 18).
Underlying the epistoniial disc is a large but discrete mass
of myonemes (Fig. 18). Their ultrastructural detail is similar
to the "myoi'de endoplasmique retracteur" of the epistoniial
disc of O/ifirtilaria articulala (7). The' disc myonemes appear
to be associated closely with ciliary rootlets of the oral ciliature
(Fig. 18). It is probable that these myonemes function to ex-
trude or retract the epistoniial disc and buccal ciliature in 71,.
callhiectcs as they were thought to do in O. articultila (7). This
epistoniial myonenie organelle appears to surpass in develop-
ment any comparable structure in .SVv/'/n'c/m, Epistylis or O/ii.\-
thonccta.
The haplokiiietv and polykinety are visible in section on
either side of the cpistomial disc as they make their complete
-------
646
EM AND PROTARGOL STUDIES OF A PERITRICH
ALsW
Hi
19
Fig. 19. Scheme of relationships-of lorica, trophorit, and blue
crab gill cuticle. Arrows indicate approximate points where
ventral lorica material ends. A, lorica aperture; ALsW, anterior
loricastome wall; Hi, hiatus; Ls, loricastome; PC, peristomial
cytoplasm; PLsW, posterior loricastome wall.
turns (360°) around the disc before descending into the in-
fundibulum. They run parallel at the base of the peristomial
space (Figs. 9, 18). The haplokinety is composed of 2 in-
timately associated rows of kinetosomes external to the poly-
kinety. The inner row of the haplokinety is always barren.
Thus, the ultrastructure of the adoral haplokinety of L. cal-
linectes resemble closely that described in Termitophrya (14)
and Opisthonecta (1). The adoral polykinety consists of 3
closely associated rows of kinetosomes (Fig. 18), thus also fitting
the general peritrichan plan. A row of "germinal" kinetosomes
running parallel to the haplokinety in the infundibular regions
of other peritrichs (12), was observed in L. callinectes with
light microscopy but was not precisely identified in the present
ultrastructural study.
At the infundibular entrance the paths of the adoral haplo-
kinety and polykinety separate (Fig. 9), the former running
down the wall of the infundibulum 100-180° away from the
latter until the cytostome is reached. This was determined
from light microscope studies of protargol-treated specimens
in conjunction with EM observations. The continuity of these
infundibular kinetics is best discerned by examining figures of
both protargol-treated specimens (Figs. 5-8) and electron-
micrographs (Figs. 15, 18). The adoral polykinety (PI), con-
sisting of 3 rows of kinetosomes at the entrance of the in-
fundibulum, is joined by parallel polykinety (P2) of 3 kineties
just within the infundibulum (Figs. 7, 9). These 2 sets form
the middle peniculus. Unfortunately, no electronmicrographs
of the terminal portion of the peniculus were obtained. There-
fore, the following description of its structure comes from
the study of protargol-treated specimens with the light micro-
scope. Near the cytostome, in the lower quarter of the in-
fundibulum, P2 terminates and PI is joined by a very con-
densed polykinety (P3) to form the end of the peniculus. The
exact number of rows of kinetosomes making up P3 is un-
known. However, P3 appears, at its origin (with light micros-
copy) to consist only of 2 rows of kinetosomes. At the very
end of the peniculus, P3 gives the appearance of a single row
(Figs. 8, 9). Thus, the number of distinguishable rows of
kinetosomes making up the end of the peniculus for L. cal-
linectes appears to be 4.
The infundibular haplokinety terminates at the cytostome
at a point close to, but separate from the end of the peniculus
(Fig. 9). Sometimes visible is a protargol-positive beltlike
structure that runs downward, through the infundibulum,
parallel to the course of the haplokinety (Fig. 9). This cor-
responds to the structure described for L. nassa which Lorn
termed the "impregnable structure" or "S-belt" (12).
Higher magnifications of sections of the infundibulum reveal
folds of the wall projected inward toward the lumen (Fig.
14). These folds correspond to the infundibular "crests" de-
scribed for Epistylis (8) and Opisthonecta (1). Often sub-
tending these crests are masses of fibers that appear identical
to the fibers of the "filamentous reticulum" described first in
the peritrich Campanella unbellaria (18), and subsequently
found in Epistylis, Vorticella, Termitophrya, arid Opisthonecta.
According to other investigators (1, 15) the reticulated fibers
beneath the infundibular crests of peritrichs served to strengthen
or reinforce the infundibular wall. The crests and associated
fibers probably correspond, to the impregnable structure of the
"S-belt" of Lorn (as revealed by protargol, Fig. 9).
No electronmicrographs of the cytopharynx and the cyto-
pharyngeal tube of L. callinectes were obtained. However, these
organelles can be observed in some detail in good protargol
preparations in the light microscope. The cytostome proper is
at the approximate level of the end of the peniculus (Figs.
4, 9). The cytopharynx begins there as an ampulla-shaped
vesicle whose walls are heavily protargol-positive (Fig. 4). In
certain protargol-treated specimens the ampulla region is greatly
distended, probably as a result of food vacuole formation at the
time of fixation. From the ampulla a long narrow cyto-
pharyngeal tube extends posterior and to the left (dorsal view)
and then curves anteriorly forming a U shape. This tube nar-
rows gradually until it disappears in the cytoplasm beneath the
peristomial region of the trophont, ending presumably in a
cytoproct or cytopyge.
Bradbury (1) illustrated a section through the cytopharynx
(tube) of Opisthonecta henneguyi in her ultrastructural study.
She observed that the wall of the tube was made up of, or
surrounded by, a bundle of fibers, which were not silver-positive.
As reported above, an analogous structure (the wall of the
cytopharyngeal tube) was found to be heavily protargol-positive
in L. callinectes (Fig. 4). The difference here probably resulted
from the different silver methods used. Bradbury used the Chat-
tqn-Lwoff method, whereas I used the protargol method which
stains the deep alimentary structures of peritrichs (12).
The presence of rod-shaped bacteria in the deep infundibulum
and cytopharynx of some L. callinectes trophonts was observed
(Fig. 14). The bacteria within the cytopharynx were consistent
in size (averaging 3 X 0.5) and larger than other prokaryotes
often observed within the lorica cavity of both living and fixed
trophonts (Fig. 14). The most probable explanation for the
presence of these bacteria in the alimentary system of L. cal-
linectes is that the trophont feeds on bacteria and was prepar-
ing to ingest those at the time of fixation.
Cytoplasmic organelles.—The very granular cytoplasm of L.
callinectes contains many mitochondria. Mitochondria are most
abundant in the peripherial cytoplasm of the trophont, except
in the peristomial region where large numbers are found close
to the myonemal mass (Fig. 10).
A complex system of tubules and canals is found adjacent
to the infundibulum (Fig. 14). The smaller tubules of this
system range 19-20 nm diameter and run into larger canals that
range 70-80 nm in diameter (Fig. 14). There is little doubt that
the complex is identical to the spongiome or nephridial (ex-
cretory) system described in Epistylis anastatica (8), Para-
mecium caudatum (19), and P. aurelia (19). The tubules and
canals of the nephridial system of L. callinectes empty directly
into the lumen of the contractile vacuole (Fig. 14).
Aboral infracilialure.—Asi aboral kinetal structure was found
in all protargol-treated trophonts of L. callinectes. In the vege-
tative trophont (nondividing) this structure appeared as a
curved row of protargol-positive bodies, each of which is 1.0
-------
EM AND PROTARGOL STUDIES OF A PERTTRICH
647
X .05 (Figs. 7, 9). These bodies appear, in the light and elec-
tron microscopes, to be in a ventral, subpellicular position,
extending (dorsal view) in a single row from the upper in-
fundibular region posterior to the macronuclear-micronuclear
region and then curving anterior to terminate just below the
myonemal mass of the epistomial disc (Fig. 9). The bodies
that make up the structure are here designated as barren kineto-
somes of the aboral kinety (= infraciliature anlage of trochal
band or aboral ciliary girdle). Though homologous aboral
kinetics have been reported for other peritrich trophonts (1, 6,
13) they have not been reported for any species of Lagenophrys
to date. The evidence that these ab'oral bodies in the trophont
are indeed barren kinetosomes of a trochal band anlage conies
from protargol, and morphogenetic studies (5). The role that
these aboral kinetosomes play in the morphogenesis of L. cal-
linectes is of paramount importance, particularly in the forma-
tion of the locomotor girdles of the telotroch and microconjugant
stages (5).
DISCUSSION
The trophont of Lagenophrys callinectes, though highly spe-
cialized, essentially conforms to known peritrich form and
ultrastructure. However, the present study has revealed interest-
ing variations on the peritrich plan as well as several cytoplasmic
structural modifications heretofore not reported.
Organelles possessed by L. callinectes and found in other
well-studied peritrichs are: 3-unit-membrane pellicle; peri-
stomial sphincter collar; myonemes of the peristomial region
plus retractor myonemes beneath the epistomial disc; haplo-
kinety, consisting of an inner barren row of kinetosome and
an outer ciliated row; polykinety, consisting of 3 parallel rows
of ciliated kinetosomes all within the outer haplokinety; in-
fundibular crests subtended by a filamentous reticulum; and
nephridial system or "spongiome" associated with the con-
tractile vacuole.
The ultrastructure and arrangement of the lips of the lorica
aperture, the attachment organelle, the loricastome walls, and
the arrangement of aboral kinetosomes are unique for the genus
Lagenophrys as revealed in the trophont of L. callinectes.
The lorica of L. callinectes appears to be a hemispherical,
elastic, protective house for the trophont. It does not have a
complete ventral shelf, and the soft body of the trophont lies
directly above the gill lamellar cuticle of the host. The chemical
composition of the lorica is unknown. The necessity for elas-
ticity of the aperture region of the lorica is demonstrated by
the ultrastructural relationships of the lips of the lorica aper-
ture, the loricastome walls, the attachment organelle, and the
fact that the lips of the aperture are movable in living animals
(opening and closing motions). The attachment organelle of
the dorsal peristomial cytoplasm appears to be a unique modi-
fication of the Lagenophrys pellicle region for the attachment
of the trophont to its lorica.
The number of rows of kinetosomes making up the terminal
peniculus varies among different peritrichs. Although Lom
(12) stated that the uniformity of the buccal apparatus of
peritrichs is strong among closely related species, it is apparent
from the present study that the 6-row terminal peniculus (PI
and P3) of L. nassa, as described by Lom, differs from the
4-row terminal portion of the peniculus of L. callinectes. How-
ever, the 4-row terminal peniculus of L. callinectes appears
almost identical to the 4-row terminal peniculus of Carchesium
polypinum as described by Zagon & Small (24). Lom (12)
further described up to 9 rows of kinetosomes in the terminal
peniculus (PI, P2, P3) of Telotrochidium sp. This suggests
that protargol comparisons of other species in the large genus
Lagenophrys [52 species reported (21)] would be useful in
further species identification and characterization.
The lack of distinct scopularized regions in L. callinectes
trophonts probably reflects the high degree of specialization of
members of the genus Lagenophrys, including their existence
within loricae, and attachment to their loricae by adoral cyto-
plasm rather than by aboral poles of the trophonts.
REFERENCES
1. Bradbury, P. C. 1965. The infraciliature and argyrome of
Opisthonecta henneguyi Faure-Fremiet. /. Protozool. 12, 345-63.
2. Corliss, JT. 1959. An illustrated key to the higher groups of
the ciliated Protozoa, with definition of terms. /. Protozool. 6,
265-84.
3. & Brough, I. M..1965. A new species of Lagenophrys
(Ciliata:Peritrichida) from the Jamaican crab Metopaulias de-
pressus. Trans. Am. Micros. Soc. 84, 73-80.
4. Couch, J. A. 1967. A new species of Lagenophrys (Ciliatea:
Peritrichida: Lagenophryidae) from a marine crab, Callinectes
sapidus. Trans. Am. Micros. Soc. 86, 204-11.
5. 1971. Aspects of morphogenesis in Lagenophrys
callinectes (Ciliatea: Peritrichida). /. Protozool. 18 (Suppl.), 22.
6. Davidson, A. L. & Finley, T. E. 1972. A comparative study
of argentophilic structures in three peritrich ciliates. Trans. Am.
Micros. Soc. 91, 8-23.
7. Faure-Fremiet, E., Rouiller, C. & Gauchery, M. 1965. Les
structures myoides chez les cilies. Etude au microscope electronique.
Arch. Anat. Micros. 45, 139-61.
8. , Favard, P. & Carasso, N. 1962. Etude au microscope
electronique des ultrastructures d'Epistylis anastatica (Cilie,
Peritriche). /. Micros. 1, 287-312.
9. Finley, H. E. & Bacon, A. L. 1965. The morphology and
biology of Pyxicola nolandi n. sp. (Ciliata, Peritrichida, Vagi-
nicolidae). /. Protozool. 12, 123-31.
10. Honigberg, B. M. 1947. The characteristics of the flagellate
Monocercomonas verrens sp. n. from Tapirus' rnalayanus. Univ.
Calif. Publ. Zool. 53, 227-36.
11. Kane, J. R. 1965. The- genus Lagenophrys Stein, 1852
(Ciliata, Peritricha) on Australasian Parastacidae. /. Protozool.
12, 109-22.
12. Lom, J. 1964. The morphology and morphogenesis of the
buccal ciliary organelles in some peritrichous ciliates. Arch.
Protistenk. 107, 131-62.
13. & Corliss, J. O. 1968. Observations on the fine
structure of two species of the peritrich ciliate Genus Scyphidia
and on their mode of attachment to their host. Trans. Am. Micros.
Soc. 87, 493-509.
14. Noirot-Timothee, C. & Lom, J. 1965. L'ultrastructure de
1'haplocinetie des cilies peritriches. Comparaison avec la membrane
ondulante des hymenostomes. Protistologica 1, 33-40.
15. Pitelka, D. R. 1969. Fibrillar systems in flagellates and
ciliates, in Chen. T. T., ed., Research in Protozoology. Pergamon
Press, London and New York 3, 279-388.
16. Randall, J. & Hopkins, J. M. 1962. On the stalks of
certain peritrichs. Phil. Trans. Roy. Soc. Land. 245, 59-79.
17. Reynolds, E. S. 1963. The use of lead citrate at high
pH as an electron-opaque stain in electron microscopy. /. Cell
Biol. 17, 208-17.
18. Rouiller, C. & Faure-Fremiet, E. 1957. Ultrastructure
reticulee d'une fibre squelettique chez un cilie. /. Ultrastruct
Res. 1, 1-13.
19. Schneider, L. 1960. Elektronenmikroskopische Untersuchun-
gen iiber das Nephridialsystem von Paramecium. J Protozool 1
75-90. ' '
20. Shaw, B. L. & Battle, H. I. 1957. The gross and microscopic
anatomy of the digestive tract of the oyster. Crassostrea virginica
(Gmelin). Can. J. Zool. 35, 325-47.
21. Sprague, V. & Couch, J. A. 1971. An annotated list of
protozoan parasites, hyperparasites, and commensals of decapod
crustaceans. /. Protozool. 18, 526-37.
22. Williams, A. B. 1965. Marine decapod crustaceans of the
Carolinas. U. S. Fishery Bull. 65, 1-298.
23. Zagon, I. S. 1970. Carchesium polypinum: cytostructure after
protargol silver deposition. Trans. Am. Micros. Soc. 89, 450-68.
24. & Small, E. B. 1970. Carchesium polypinum:
somatic and buccal structure analysis after protargol staining
Trans. Am. Micros. Soc. 89, 443-9.
-------
CONTRIBUTION NO. 238
-------
A Clinical Centrifuge Tube for Small Blood Samples-
Occasionally, large amounts of whole -blood
are difficult to obtain for physiological studies.
Little blood is available when the animal is
small (e.g. some fish and crustaceans) or when
blood samples are taken periodically without
sacrificing the animal. It is often difficult to ob-
tain clear serum or plasma from small blood
samples. In many microanalytical procedures
(e.g. electrophoresis) only a few microliters of
serum are needed, but the serum must be free
of other blood components. Because of this, a
simple and inexpensive device for separating
components of small amounts of blood in a clin-
ical-type centrifuge is described.
To obtain separation of cells from serum in a
small sample, (e.g. one drop of blood) a long
centrifuge tube of small diameter is needed.
.Usually a microcentrifuge or hematocrit centri-
fuge is necessary to achieve separation. How-
ever, the cost of these instruments makes them
impractical for most students and some re-
searchers.
The device illustrated permits routine cen-
trif ugation of small blood samples in a clinical-
type centrifuge. Materials used to modify the
plastic centrifuge tube are inexpensive and
readily available.
The centrifuge tube l serves as a holder for
the sample tube made from a disposable Pas-
teur capillary pipet. The pipet is heat-sealed at
the tip, cut to an appropriate length, and the
end fire-polished. A rubber medicine dropper
bulb, inverted in the bottom, cushions the tip of
the sample tube. The top of the sample tube is
' international Autoclear ® Plastic Centrifuge Tube I. E, No.
1649 and corresponding polypropylene stopper, International Equip-
ment Co., Needham Heights, Massachusetts 02194.
CUT AND HREPOLISH
CUT 7mm HOLE
S> IN STOPPER
« GLASS PASTEUR PIPET
PLASTIC CENTRIFUGE TUBE -
SEAL TIP OVER BURNER
-* MEDICINE
DROPPER BULB
BLOOD
SAMPLE
ASSEMBLE
Clinical centrifuge tube adapted for small blood
samples.
retained by a polypropylene centrifuge tube
stopper l with a 7-millimeter hole at the center.
Other materials could be utilized to construct
a similar apparatus (e.g. an adapter for hepa-
rinized hematocrit tubes).
Approximately 50 microliters of blood is in-
serted into the sample tube with a syringe or
pipet. After centrifugation for 3 minutes at
1,800 fir's one drop of blood is distinctly sepa-
rated into its components at the tip of the sam-
ple tube. Several microliters of serum can be
removed for analysis using a capillary pipet or
a microliter syringe.
—PATRICK W. BORTHWICK, Department of
Biology and Marine Sciences, The Univer-
sity of West Florida, Pensacola, Fla. 32504.
The author's present address is U.S. Environmental
Protection Agency, Gulf Breeze, Fla. 32561, an affili-
ated laboratory of the National Environmental Re-
search Center, Corvallis, Oreg. 97330.
GB 238
184
THE PROGRESSIVE FISH-CULTURIST
36(3): 184. July 1974
-------
Bottle Rack for Primary Productivity Studies
Limnological studies on large midwestern
reservoirs have required primary productivity
measurements utilizing the method originally
developed by Steemann-Nielson in 1952 (The
Use of Radioactive Carbon for Measurements
of Organic Productivity in the Sea. Journal du
Conseil, vol. 28, p. 117-140). For these studies
we needed a compact bottle rack sturdy enough
to withstand rough water and inexpensive to
construct. The rack was designed to hold 300-
milliliter BOD bottles by the neck.
A single rack consisted of a 12- by 2- by %-
inch (30.5- by 5.1- by 1.9-cm) block of clear
plexiglass with a %-inch (1.9-cm) diameter
hole drilled on center (see diagram). On either
end of the rack medium size tool holder clips
have been attached using No. 10, l^-inch
(3.18-cm) sheet metal screws. The ends of the
clips have been bored to 3/16-inch (0.5-cm) di-
ameter to accept 3-inch (7.6-cm) hairpin cotter
keys which secure the bottles in the clip. We
have found it desirable to sheath the clips with
short pieces of l^-inch (0.6-cm) ID Tygon tub-
ing when using aluminum-foil-wrapped dark
bottles to prevent accidental tearing of the foil.
Metal conduit i/^-inch (1.3-cm) ID which
slides through the center of the rack is the sup-
port. A series of holes have been bored through
the conduit so that the racks may be held at a
desired depth with the use of a 3-inch (7.6 cm)
hairpin cotter key beneath the rack. To increase
the depth capability beyond 10 feet (3 meters),
additional conduit may be joined using smaller
diameter metal tubing for a ferrule. The joint
is made by connecting these pieces with hair-
pin cotter keys placed in holes drilled through
the tubing and ferrule. The rack may be as-
sembled or disassembled in minutes which
eliminates having to leave the rack in the wa-
ter overnight and discourages vandalism. Flo-
,-
12" X 2" X %" clear plexiglass (3D.5 cm x 1.9 cm]
#10 I'/," sheet metal screw
(3.18 cm) II
diameter (1.9 cm)
3/16" diameter (0.5 cm|
" hairpin cotter key (7.6 cm]
medium size tool holder clip
'/2" thin wall conduit (1.3 cm)
Primary productivity bottle rack for 300-milli-
liter BOD bottles.
tation for the assembled rack can be accom-
plished by using buoys or styrofoam logs at-
tached to another permanently anchored float
or dock. We have found that a cylindrical mar-
ker buoy with a hole through the center makes
a satisfactory float and does not shade the racks
to any appreciable degree.
We have been using the racks all summer for
nearly 700 sets without a broken or lost bottle.
Lake conditions ranged from calm to extremely
rough during exposure periods. Use of a small
float and the length of conduit extending per-
pendicular in the water makes this device ex-
tremely stable regardless of weather and lake
conditions.
Total cost of four complete racks assembled
on a 10-foot conduit was under $8, excluding
labor.
—STEPHEN L. BUGBEE, THOMAS F. LORENZ,
and LEOTIS Mossy, Surveillance and Anal-
ysis Division, U.S. Environmental Protec-
tion Agency, 25 Funston Road, Kansas
City, Kans. 66115.
VOL. 36, NO. 3, JULY 1974
183
-------
CONTRIBUTION NO. 242
-------
Cytopathology, Ultrastructure, and
Virus Infection in Pink Shrimp
Exposed to the PCB, Aroclor® 1254
by
JOHN A. COUCH and DELWAYNE R. NIMMO
U.S. Environmental Protection Agency
Gulf Breeze Environmental Research Laboratory
Sabine Island, Gulf Breeze, Florida 32561
(Associate Laboratory of the National Environmental
Research Center, Corvallis, Oregon)
Little information is available concerning the effects of pollutant chemicals
on the fine structure of tissues in aquatic invertebrates. Even less is known
concerning possible interactions of pollutant chemicals and natural pathogens
in commercially valuable invertebrate species. In experiments at the Gulf
Breeze EPA Laboratory we have exposed pink shrimp (Penaeus duorarmn) to 3 yg/£
Aroclor 1254 in flowing seawater from 30 to 52 days. During these exposures,
up to 50% or more of the animals died. Both living and dead shrimp were
analyzed for Aroclor residues and, after 30 days exposure, were found to
accumulate from 33 mg/kg to 40 mg/kg in their hepatopancreatic tissues.
Hepatopancreatic tissues from experimental shrimp (surviving the exposures)
and control and feral shrimp were prepared for histology and electron micro-
scopy. Light microscopic examination revealed that approximately 30% of the
exposed shrimp possessed intranuclear crystalloid inclusions in hepato-
pancreatic epithelial cells. Electron microscopy revealed that shrimp
possessing the crystalloid inclusions were infected by an intranuclear, rod-
shaped, free, and occluded virus similar morphologically to the nuclear
polyhedrosis viruses (Baculovirus group) of insects. To date, control and
feral shrimp have not shown this infection.
Cytopathologic changes in hepatopancreatic cells of exposed shrimp consisted
of: (1) proliferation and hypertrophy of rough and smooth endoplasmic re-
ticulum in large numbers of cells; (2) loss of cytoplasmic density and struct-
ural integrity; and (3) formation of small vesicles in the nucleoplasm of
degenerating nuclei of cells showing the above cytoplasmic changes. These
changes were not directly associated with virus infection because infected
cells demonstrated several alterations apparently directly attributable to the
influence of the virus. These changes were: (1) hypertrophy of the infected
nucleus; (2) loss of chromatin; (3) proliferation of nuclear membranes, and
(4) production of crystalline inclusion bodies containing virus rods. Further
work concerning the possible interactions between organochlorines and the
shrimp virus is presently underway.
Gulf Breeze Contribution No. 242.
(Abstract). Program of the Joint Meeting of
The Society for Invertebrate Pathology and
International Colloquium on Insect Pathology
and Microbial Control, p. 105. Oxford
University, England. (1973)
-------
CONTRIBUTION NO. 245
-------
In: Guidelines on sampling and statistical methodologies for
ambient pesticide monitoring. Federal Working Group on Pest
Management: Washington, D. C., Oct. (1974). Chapter V, pp: V1-V5.
CHAPTER V - ESTUARIES
Philip A. Butler, Ph.D.*
The decision to monitor an estuary for pesticides may derive from
any one or several specific needs. These needs or objectives will
largely determine the character and modus operandi of the program.
Obviously, two pesticide monitoring programs in the same estuary
might be entirely different because of the kinds of information
sought. Estuarine monitoring objectives may be for the purpose of
determining:
1. Background levels of an array of persistent waterborne
pesticides by randomized sampling of estuaries in a
particular geographical area.
2. The escapement of pesticides in surface run-off from
specific use areas in the drainage basin by sampling
deltaic sediments.
3. The cause of increased faunal mortalities or lack of
species diversity in an otherwise normal appearing
estuary.
4. Tissue residue levels of persistent pesticides to ensure
that they are within legal tolerance levels for edible
fish and shell fish or their products.
5. Pesticide residues in food chain organisms to alert resource
management agencies of possible mortalities resulting from
trophic magnification.
6. Pesticide residues in pre-spawning gonads of commercially
valuable species to identify causes of change in
productivity.
The choice of which physical or biological elements are to be
monitored in an estuary will be determined by specific program
objectives.
Water samples taken at infrequent intervals or at limited points in
an estuarine system will usually be of limited value. If the monitoring
program objective requires specific knowledge of pesticide residues
in the water, the guidelines enumerated in Chapter IV should be
followed for such samples as well as for sediment samples.
Sediment samples are useful in detecting persistent pesticides
in the estuary. However, interpretation of their analyses requires
knowledge of particle size, organic/inorganic composition, station
location with reference to current flow and similar data that frequently
are not readily available. Sediments in shallow estuaries can be
*Contibutiorv No. 245, Gulf Breeze Environmental Research Laboratory, EPA
V - 1
-------
disturbed by storm conditions and their pollution burden may change
drastically in time of flood or drought without reference to pesticide
usage in the area. Analysis of stratified estuarine sediments may
reveal unusual patterns of pesticide residue accumulation, but our
lack of information on aerobic and anaerobic degradation of persistent
pesticides complicates the interpretation of such sediment samples.
Carefully collected samples at the sediment-water interface along
the geographic axis of an estuary may be useful, however, in pinpoint-
ing up-stream sources of pesticide pollution.
The choice of a biological sample for monitoring the estuary is
determined largely by two factors: is the form to be sampled migratory,
and what is its position in the trophic web? Sessile or non-migratory
species, representing entire communities present obvious advantages
since they reflect pollution levels at specific locations. Their
life style usually facilitates age determination and permits some
degree of bracketing of the occurrence of the pesticide pollution.
Many non-mobile species, e.g., molluscs and barnacles, are detritus
and filter-feeders. Pesticide residues may be biologically retained
and magnified in their tissues and reflect the introduction of
pollutants into the lowest levels of the food web. Such residues
suggest direct contact with pollution sources. Residues in vertebrate
carnivores, on the other hand, are more likely to reflect trophic
magnification of persistent pesticides. Fish-eating birds or mammals
not only concentrate but store for long periods compounds that
contaminate their diets.
Plankton offers several advantages as a tool for estimating levels of
pesticide pollution. Its small particle size presents a relatively
large sorptive surface. The cells are usually high in lipid content
and readily take up organochlorine compounds. However, the rapid cell
division and growth rates favor dilution of tissue residues. Also
the interpretation of data on pesticide residues in plankton may be
confused by the facts that plankton is a composite of living and dead
materials that contains varying amounts of silt and other inorganic
materials, and its moisture content" varies widely depending on the
components. Plankton samples containing pesticide residues are
probably indicative of fairly recent contamination. When samples
are taken at frequent intervals, they may be especially useful in
identifying pollution sources.
Crustacea, such as commercial shrimp, are generally one of the least
satisfactory groups to monitor because of their extreme sensitivity to
insecticides. Both organophosphorus and organochlorine insecticides
-------
will kill crustaceans at concentrations in the parts-per-trillion
range. Carbamates are somewhat less toxic to them and herbicides
generally are not toxic at concentrations likely to be encountered •
The net result is that pesticide effects on crustaceans
are likely to be an all-or-none affair and crustacean samples may
reveal little about relative levels of pesticide pollution in the
estuarine environment.
Molluscs (oysters, clams, and mussels) have special merit as bioassay
tools because of their sensitivity to synthetic organochlorine
pollutants present in the ambient water. They detect and accumulate
these persistent pesticides to an astounding extent without being
themselves markedly affected by the pollution levels generally
encountered in estuaries. Not all molluscs are equally sensitive and,
as with other bioassay animals, care must be exercised in comparing
pesticide residue levels between individuals and species. Salt-water
mussels are especially useful as monitor species because of their
wide geographic distribution and their ubiquity over a broad range
of salinity regimes.
The chief objection to the use of molluscs lies in their rapid
metabolism of pesticide residues. Although they can concentrate
pollutants in their tissues by a factor of 50,000 or more, these
residues are lost in a matter of days when the ambient water becomes
free of contamination. Thus, oysters and mussels are monitor
animals of 'choice when sampling can be done on a monthly or more frequent
basis. They are relatively useless in reflecting trends in environment
pollution when sampling can be done only once a year.
Fish are often the most convenient group to monitor because of their
availability from the commercial catch. They are sometimes sensitive
to high pesticide residues in their environment as evidenced by
massive fish-kills, but may accumulate relatively large residues
and become pesticide-resistent when the concentration of a pollutant
is low. Experimental work has shown that they accumulate pesticides
directly from the ambient water as well as from their food supply.
The interpretation of pesticide residue data in fish is difficult
unless their life history is known.
Fish are highly selective in their diet and may accumulate small or
large pesticide residues in polluted estuaries, depending on their
food supply. Plank ton-feeding or herbivorous fish tend to have
significantly lower pesticide residues, for example, than carnivorous
species that feed on small fish in the same estuary. Populations
of even the same species of fish may have quite different diets in
two different estuaries. Comparison of pesticide residues in these
two populations could be very misleading if their food habits are not
known.
V - 3
-------
Fish store organochlorine residues primarily in tissues having a
high lipid content. Such residues accumulate as the fish age but
may diminish sharply at spawning or in starvation periods when
stored body fats are mobilized. Wide variations in pesticide
residues may occur also in fish having presumably similar back-
grounds. In one series of analyses of 15 "similar" specimens
collected simultaneously from a school of estuarine fish, for example,
DDT residues varied by two orders of magnitude.
Fish are most useful as monitors when they are small enough to permit
whole body analysis of at least 15 specimens, thus averaging individual
variations. If the fish are about 1 year in age or sampled prior to
their first spawning, the residues reflect pollution exposure during
a known time period.
Sample Preservation - The handling of estuarine samples for pesticide
monitoring poses the same types of problems as samples from any other
medium. Increased knowledge of the dangers of sample contamination
through contact with various kinds of synthetic wraps and containers
has demonstrated the necessity for glass and perhaps aluminum foil
containers to preserve the integrity of wet samples. Immediate
freezing and maintenance of frozen sample until analysis is still the
best way to protect samples and prevent degradation or loss of pesticide
residues. Freezing, because of its simplicity, is also perhaps the
best method for use by unskilled personnel.
The preservation of tissue samples for pesticide analysis at room v
temperature by the use of desiccants has been used with marked success
in estuarine monitoring. Although this method requires a modest amount
of personnel training as well as special chemicals, it avoids^ the loss
of frozen samples because of missed airmail schedules and power failure.
It has proven especially useful for samples collected long distances
from the analytical laboratory. In practice, field samples of plants
or animals are chilled, homogenized, and blended with a combination of
two desiccants sodium sulfate and powered silica. The resulting
mixture is a dry, free-flowing powder in which pesticide residues
remain intact for 15 days or more at room temperature. This desiccant
mixture is compatible with chromatographic procedures for organochlorine,
phosphorus, and phenoxy pesticide compounds.
Representative Samples - The collection of representative field samples
requries careful planning and pre-supposes a good understanding of
estuarine ecology. The introduction of stratification into the
selection of sample type, collection frequency, and station location
is advisable if the most knowledge is to be gained from samples that
are necessarily limited in number and areal coverage.
V
-------
It has been found in past monitoring programs, that apparent
pesticide pollution levels have been grossly altered or misinterpreted
because of drought, windstorms, substitution of one species of clam
for another, collection of samples on different tidal stages,
and by assuming that the diet of a particular species of fish
being monitored was the same in two different estuaries. Ultimately,
the understanding of field sample analyses is determined by knowledge
of the response of similar samples, either organic or inorganic, to
pesticide pollution under controlled conditions. Too often, money
and effort in environmental monitoring have been largely wasted
because of the failure to select samples whose analyses could be
meaningfully related to environmental conditions.
V - 5
-------
CONTRIBUTION NO. 247
-------
EPA-660/3-74-024
DECEMBER 1974
A CONCEPTUAL MODEL FOR THE MOVEMENT
OF PESTICIDES THROUGH THE ENVIRONMENT;
A contribution of the EPA
Alternative Chemicals Prooram
By
James W. Gillett
James Hill IV
Alfred W. Jarvinen
W. Peter Schoor
National Ecological Research Laboratory
aulf Breeze Environmental Research Laboratory
National Water Quality Laboratory
Southeast Environmental Research Laboratory
National Environmental Research Center
Corvallis, Oregon 97330
Project Element 1EA487
ROAP 21BCL, Task 03
NATIONAL ENVIRONMENTAL RESEARCH CENTER
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS, OREGON 97330
-------
ABSTRACT
This report presents a conceptual model of the movement and disposition
of pesticides in the environment. A multi-media model is built up from
simple modules representing basic processes and components of air, soil,
and water. More specific models are exposited for the atmospheric/
terrestrial, freshwater aquatic, and estuarine/marine environments.
Through iterative operations of expansion and systematic reduction of
the components and processes these models of segments of the environment
can be joined to provide a holistic view of the disposition of a
chemical and its attendant effects. Ultimately systems analysis and
mathematical simulation techniques can be employed to evaluate the
fate of a specific chemical in a particular environment. The conceptual
model is thus a first step in organizing facts, assumptions, and
hypotheses into a graphic and logical array capable of exploitation in
further experimentation of pesticide disposition and effects.
While rejecting formulation of a model with global validity, the authors
emphasize the commonalities of the basic processes and components in
the various environments. Thus, a multi-media approach to disposition
studies is made explicit even in the absence of a single, all-media
global model.
This report was submitted in fulfillment of Project Element 1EA487,
ROAP 21BCL, Task No. 10 by the National Ecological Research Laboratory
under the sponsorship of the Environmental Protection Agency. Work
was completed as of September 1974.
-------
CONTENTS
Page Page
ABSTRACT ii
LIST OF FIGURES iv
ACKNOWLEDGEMENTS vi
FOREWORD vii
SECTIONS
I CONCLUSIONS 1
II RECOMMENDATIONS 3
III INTRODUCTION 4
IV OVERALL CONCEPT OF THE MODEL 11
V THE ATMOSPHERIC/TERRESTRIAL MODEL 27
VI THE FRESHWATER AQUATIC MODEL 40
VII THE ESTl"\RINE/MARINE MODEL 55
VIII REFERENCES 59
IX KEY LITERATURE SOURCES FOR PESTICIDE
EFFECTS RESEARCH 63
APPENDIX 73
-------
FIGURES
No. Page
1 Variable-Form Module: Chemical 5
2 Global Array of Environmental Regions 7
3 Food Web Module 9
4 Diagram of the Atmospheric/Terrestrial Model 30
4A ATMOSPHERE 31
4B FAUNA 34
4C FLORA 37
4D SOIL and WATER 38
5 Diagram of Faunal Subsystem Model 33
6 Vertical Representation of a Stratified Lake 43
7 Horizontal Representation of a Lake 44
8 Horizontal Array of Vertical Columns for
Representation of Lotic Systems 45
9 So.ne of the Storages, Processes, and Subsystems
Associated with the Surface Layer Storage Compartment 47
10 An Expansion of the Hydrologic Input 48
11 A Skeletal Abstraction of a Food Web 49
12 Food Chain Model of DDT in a Freshwater Marsh 50
13 A Minimal Representation for a Pesticide
in a Dimictic Lake 52-53
14 Simple Model of Transport in Estuaries 56
15 Expanded, Iterated Basic Chemical Module for
Transport of Chemicals in Estuaries 57
Bi ota-nediated Flux (overlay) 57
"1 V
-------
FIGURES (Cont'd)
No. Page
A-l Relationship Ariong Graphical Representations 74
A-2 Streeter-Phelps Oxygen-Deficient Model
for a Stream 76
A-3 Vollenweider Lake Eutrophication Model 76
A-4 Nutrient Model for Lake with Biotic
and Abiotic Storage 78
A-5 Possible Coupling of Biomass (B) Subsystems
with Nutrient Concentration (Mb) Subsystems 79
-------
ACKNOWLEDGEMENTS
The authors graterully and most humbly acknowledge the contributions
of their colleagues Ray R. Lassiter and Edward J. Rykiel , Jr. (SERL);
Patrick W. Borthwick, Marl in E. Tagatz, and Gerald J. Halsh (GBERL);
and Eugene Elzy, F. T. Lindstrom, Marvin L. Montgomery, and Rizanul
Haque (Environmental Health Sciences Center, Oregon State University,
Corvallis, Oregon). Helpful and constructive comment was received
from N. R. Glass and A. S. Lefohn (NERL), J. Eaton (NWQL), T. W. Duke
(GBERL), and D. W. Duttweiler, 11. M. Sanders, and G. L. Baughman (SERL)
Timely preparation would not have been possible without the dedicated
assistance of Program Support Center (NERC-Corvallis).
-------
FOREWORD
This report is a pror.uct of the Environmental Protection Agency's
Substitute Chemicals Research Program, which seeks chemical
alternatives to certain pesticides. The report provides an overall
view of these chemicals regarding their pathways through and possible
effects on the environment. Since the substitute chemicals to be
investigated may exhibit properties similar to conventional pesticides,
such as bio-concentration and bio-degradation, this program was
initiated to study the environmental routes and rates of transport,
metabolic fate, and sinks for a variety of these substances.
Many chemicals, including the substitute chemicals, move throughout
all of the environment, and their total impact cannot be evaluated
by a research program dealing with only one part of the environment.
Experiments designed to provide data for regulatory function must
include as many parts of the environment as possible. For this
reason, the whole ecosystem approach has been adopted in this program.
We have thus presented an overall conceptual scheme from which
scientists, administrators, management executives, and other
interested persons with a concern for pesticide-related problems
can obtain an overview.
vn
-------
SECTION I
CONCLUSIONS
Pesticides are applied to the ecosystem of the pest and not to the
pest alone. An ecosystem by definition is a causally closed system
in which each process is influenced by overall system structure. The
concept of the ecosystem represented simply in thought or language is
of little operational use until translated into more functional diagrams,
Each of the many forms of system diagrams has strengths and weaknesses
depending upon their application. An iterative process of expansion
and systematic reduction of components to achieve an optimal balance
between resolution and effort can be employed to join various segments
of the environment.
Placing the pesticide problem in the control diagram format forces the
investigator explicitly to define and delimit a complex hypothesis.
Further, systems analysis and simulation techniques may be applied to
mathematical approximation of the hypothesis stated in the control
diagram. When applied to a preliminary system diagram, these analyses
allow systematic reduction to a less complex form. As a preliminary to
an experimental study, these techniques can provide answers to many
questions concerning the variables to be measured, the accuracy required
of the measurement, and the frequency of sampling. Thus, these methods
of modeling and techniques of analysis enable investigators to develop
models for the behavior of a specific pesticide in a specific ecosystem
yielding an approach to optimum information re resource expenditure.
Ultimately, mathematical modeling and analysis could precede
introduction of chemical which might be potentially hazardous in the
environment. By identifying those properties of the agent and the
-------
systems and by quantifying interactions of components, mathematical
simulation can direct critical experiments to verify hypotheses of
disposition and effect. The conceptual model is the first step in a
rigorous scientific treatment of the fate and effects of agents and
their alternatives in pest control.
-------
SECTION II
RECOMMENDATIONS
The conceptual model necessarily will benefit from criticism,
experimentation, and utilization in research. The process of
improving and updating the conceptual relationships should be
a continuing function of this program.
Analysis of the disposition of pesticides in particular segments of
the environment and of the effects accompanying their distribution and
fate should employ the conceptual models in developing more explicit
hypotheses and as an operational framework. Research in laboratory
microcosm and in field validation of laboratory studies of processes,
effects, etc., should be correlated through appropriate models derived
from this conceptual base.
In relation to the Substitute Pesticides Program, this conceptual
model should be employed in referencing the probable disposition of an
alternative chemical to that of the de-registered or suspect agent that
the substitute might replace.
-------
SECTION III
INTRODUCTION
Literally millions of chemicals and combinations of chemicals are now
manufactured and isolated, formulated, used, and ultimately disposed
of in the environment. Management of the resources of regulatory
agencies, supporting scientific institutions and manufacturers of the
chemicals demands effective and reliable shortcuts in evaluating the
potential hazard involved in such chemicals. The purpose of this
conceptual model is to elucidate the disposition of an agent in the
environment to permit judicious collection and evaluation of data
that indicate the critical points in that disposition. From the
conceptual model one could develop a more explicit model for the
behavior and disposition of a specific chemical in a particular
environment—a model that includes realistic parameters and by
computer simulation provides realistic estimates of the concentration
of that chemical in space and time.
A number of models have been proposed for the movement of specific
agents or classes of chemicals in various environments. Some attempt
to represent the global distribution of agents; others relate to
smaller portions of the whole environment or to generalized segments
(e.g., within man). Highly significant contributions to this effort
are the works of BISCHOFF AND BROUN (1966), WOODWELL et al., (1967,
1971), HARRISON et al., (1970), NISBET AND SAROFIM (1972), KENAGA
(1972), LINDSTROM et al ., (1974), and ELZY et al., (1974).
In setting forth this particular set of models encompassing the
atmospheric/terrestrial, freshwater aquatic, and estuarine/marine
-------
environments, this report has established limits of validity and
relevance focused locally rather than globally. The utility of the
conceptual model rests in its conversion and evolution into an explicit
mathematical statement capable of evaluation as a hypothesis. Current
and near-future capabilities for extrinsic control of environments will
limit such testing to laboratory microcosms, such as those of METCALF
et al., (1971), and to small external sites, both characterizable as
limited within the concepts of the model. Extension of the model
conceptually in space and time can be made to the extent that the
elements of the models can be grouped, subsected or interconnected.
Figure 1: Variable-form Module: chemical.
A chemical may exist "free" or "bound" in one of the states
shown, all of which can interact within a region (inside box)
or interact with adjacent modules of other environments
(indicated by arrows).
WATER
ORGANIC
PftRTICULATE
INORGANIC
PARTICIPATE
-------
The regional models can be considered as amplified aspects of a basic
variable-form module (Figure 1) within which a chemical may exist in
a "free" or "bound" form. Since any chemical may be used as a pesticide,
a term describing its function, the fate and movement of any agent (and
effects consequent to that disposition) can be described and displayed
without regard to that extrinsic function. Thus, the model should
serve not only for pesticidal chemicals, but also for other natural
and man-made agents that are being evaluated. The subcompartments of
modules may exist in varying proportions and with diverse relationships
in different environments. Specification and elaboration of this basic
chemical module are employed to relate it more specifically to a region
or zone within the environment, and interrelating and interfacing such
subsystems generates models of broader relevance. Subsequently,
iteration of models can occur longitudinally (to represent stream flow,
geographical or climatic regions, or atmospheric processes), vertically
(to represent water depths, soil horizons, or meteorologic events), or
horizontally (to represent distances from interfaces) to develop
multi-media models.
At the interfaces of the regions explicit representation becomes most
difficult. Although the models exposited cannot be viewed as globally
valid, the iteration and conjunction of subsystems generate a global
array (Figure 2) that serves conceptually as an overall model. As
shown in Figure 2, some elements are "shared" in a more or less
regular manner through seasonal, circadian, or shorter cycles and in
an irregular manner through meteorologic and geologic changes. The
tidelands, flood plains, and marshes are not fully represented by
either aquatic or terrestrial models exposited herein, but both
provide sufficient elements for subsequent elaboration as knowledge
of the physical structure, physicochemical relationships, and
alteration rates of these interfaces is improved. These interactions
cannot be ignored simply because the mean flux appears to be zero,
-------
because the rates of change are so slow or so fast as to lie outside
apparent rate-limiting processes, or because events do not appear to
affect disposition or effects of pesticides directly.
GROUND
AND SURFACE
WATERS
Figure 2: Global array of environmental regions.
Modules can be arrayed as representing environmental regions
interacting by flow (open arrows) or other transport and
transfer phenomena (solid arrows) so as to represent global
disposition.
-------
A second major unifying thread of shared elements are the plants and
animals designated as "Biota" in Figure 1. The interfaces of the
physical environmental regions provide for considerable crossover of
an agent via the biota, yet explicit representation is difficult. The
phenomena of predation, migration, and vectorial transport (associated
with compartmental flows) are indicated in Figure 3. Similar to the
variable-form module of an environment, iteration of biological transfer
and storage modules provides extension and expansion of these routes
of disposition. Unlike interactions with the physical components of
the environment, however, the biocidal and physiologic activities of
pesticides can have pronounced direct and indirect effects on the
disposition of a given agent. Determination of such effects within
ecosystems would be vital to development of realistic simulation
models.
Chemicals are altered by both physical and biological systems in the
environment, so that site and rate of such change are highly significant
aspects of the disposition. Representing these changes in a single
model is difficult, especially when the agent (or its products) may
alter the rate of biotransformation. Where an agent is altered
chemically, we are assuming that the disposition of the product can
be considered to be into a model parallel to that of the parent
compound. The particulars of interaction may be describable for a
given relationship, so that defined systems can be set forth for a
specific chemical. In tracing the movements of an agent through this
conceptual model, the products of photolysis, chemical alteration, and
biotransformation can be visualized as leaving the global array (Figure
2) and entering a similar point on a model for each product. There
might be many points interacting between the model of the parent agent
and models of products.
-------
TRANSPORT
FLUX
PRIMARY
PRODUCERS
PRIMARY
CONSUMERS
HIGHER
CARNIVORES
SECONDARY
CONSUMERS
DECOMPOSERS
OMNIVORES
SCAVENGERS
Figure 3: Food web module.
Solid arrows indicate intra-web flux by predation and
feeding; open arrows indicate other transport and transfer
within the food web or between food webs of different regions
or zones.
-------
Another major concept utilized in these models is that components
can be represented as compartments equivalent to a well-stirred
chemical reactor in a processing plant. Definition of what
constitutes a compartment or component is part and parcel of the
process of bringing the conceptual model into specific focus with a
particular agent in a given segment or region of the environment. The
extent of correspondence between (a) the definition of a "compartment"
of the model, and (b) the characteristics of an environmental component
determines how well a given variable-form module represents reality.
Redefinition of compartments serves to make the model more sophisticated
or less complicated, as knowledge is gained about the component and its
functions.
The conceptual model for the transport of pesticides in the environment
has been devised from three units: atmospheric/terrestrial, freshwater
aquatic, and estuarine/marine. The nature of the presentations differ
somewhat as expected for diverse points of reference, but the basic
components and chemico-physical and biological flows are compellingly
similar. This report will attempt to synthesize these components and
processes further into an overall concept, then consider representations
for the three major areas.
10
-------
SECTION IV
OVERALL CONCEPT OF THE MODEL
An explicit, overall conceptual model derived from the principles of
chemistry, physics, and biology and valid for all pesticides and
environments, would over-reach the bounds of current knowledge. For
practical translation into a quantitative model, the common threads
of these principles and of the constituents of the environmental
regions must be woven into a fabric or matrix of systems capable of
analysis. Practically, we are forced to examine experimentally
relatively-small regions which can be characterized and/or controlled,
or we must generalize these models by summation (see HARRISON et al.,
1970; WOODWELL et al., 1971). Iterative simulation of the models over
all environmental regions would require an unachievable data base, but
much can be learned about the whole even from the parts. These will
tell.us where sampling and monitoring will be valid and helpful.
Attention could thus be focused on the processes and mechanisms
affording (and on these factors affecting) disposition.
PRELIMINARY SYSTEMS ANALYSIS FOR REDUCTION AND EXPERIMENTAL DESIGN
A diagrammatic representation of a system is usually of value to a
scientific investigation even if the potential applications of the
system representation are not realized. The trial-and-error expansion
and reduction of compartments forces the investigators explicitly to
acknowledge the boundaries and the level of definition of the system.
Deciding upon alternative representations of flow and control paths
promotes consideration of even the most remote possibilities. Finally,
many of the assumptions necessary to represent the system are explicit
in the diagram.
11
-------
The system diagram is a complex, qualitative hypothesis which must be
tested by experiment. The hypothesis cannot be realistically tested
in the graphical form of the conceptual models given so far (Figures
1 and 2). A more exact representation of the relationship between
storages and flow rates is needed. The many possible mathematical
forms for these relationships may be classified as linear or nonlinear
and as recipient-controlled, donor-controlled, or mixed.
The linear, donor-controlled form (PATTEN, 1971) is probably the most
elementary (CHILD and SHUGART, 1972). It can be represented mathe-
matically as
^| = AX + BZ (1)
in which X is a storage level vector, dX/dt is a flow rate vector, Z
is an input vector, and A and B are coefficient matrices.
Donor-control implies that flow rate depends only upon the storage
level from which the flow originates. Although this assumption may be
unrealistic, the use of a linear, donor-control approximation of the
system representation appears to be justified for these preliminary
analyses. Often, linear approximations are less sensitive to parameter
estimation errors than nonlinear representations. Also several
expedient techniques of analysis may be applied to the linear,
donor-control approximations. The following analysis techniques can
yield alternative statements of the system diagram hypothesis that
can be interpreted in terms of reduction and experimental design.
1. Topological analysis is currently being developed by
a group of Dr. B. C. Patten's graduate students at the
University of Georgia (PATTEN et al., In Press). This
technique is intended to allow determination of the
12
-------
influence of the topological structure on system
behavior. Such information is useful in evaluating
alternative system structures and particularly in
determining the effects of reduction or aggregation
of components.
2. Flow analysis (HANNON, 1973) or input-output analysis
(LEONTIEF, 1966) is based upon the manipulation of the
A coefficent matrix in the linear, donor-control
approximation. Briefly, a matrix & is generated by
G ^ A1' (2)
in which each element (G. .) is a relative measure of the
' 0
fraction of flow out of storage j_ that appears as input
to storage i_ under steady state conditions. This
information may be used to identify important processes
or flow paths in the system.
3. Sensitivity analysis (TOMOVIC and VUKOBRATOVIC, 1972;
PATTEN, 1973), may be used to evaluate the effect of a
perturbation, v(t) , upon the storage levels in the system.
The measure of sensitivity, S^, is useful in determining
which parameters have a prominent effect upon system
behavior. A linear approximation of S(t) is determined
from
dX dX.
S(t) + ["dT V(t) (3)
where the terms in brackets are Jacob i an matrices. With
a unit perturbation of each parameter, A. ., the steady-
state values of S^ for each storage variable may be used
as a relative measure of system sensitivity to each
parameter.
13
-------
4. Frequency response analysis (CHILD and SHUGART, 1972;
WEBSTER et al., In Press) provides frequency-related
measures of system behavior. Both the referenced
papers and current studies indicate that the sampling
ratio (g) and the undamped natural frequency (oo ) are
well described by a second-order control system
approximation of the system (KUO, 1962; DERUSSO et al.,
1965). When the system is overdamped (most ecosystems
appear to be so), then the undamped natural frequency
becomes a measure of the maximum required sampling
rate for system variables.
5. Component analysis (HILL, 1973) allows numerical
determination of a limited number of coefficient
values from the A matrix of the linear, donor-
controlled representation and the system transfer
function as determined from experimental input-
output data.
Topological analysis can be used as an aid in evaluating the influence
of connectivity upon process rates in the system. Flow analysis can
provide a measure of steady-state distribution of flow through the
process pathways. A preliminary sensitivity analysis can determine
the effect of an error in parameter estimation upon storage levels
and hence upon flows. These three evaluations of process-system
interaction provide criteria for elimination of components that have
the least effect on system behavior, thus systematically reducing the
graphic representation. This results in information that may be used
as a first approximation in choosing measurement methods and sampling
rates for evaluation of system hypotheses,
MECHANISMS OF DISPOSITION
Much of the movement and fate of a given agent is dependent on the
rate and nature of certain mechanisms or processes which do not differ
14
-------
in character or principle between the various compartments. Explicit
in this dependency are (a) the physical, chemical, and biological
principles of behavior of the chemical and environmental component and
(b) the organization of the constituents as described in the diagram.
It is convenient to divide these processes into two major groups:
transport processes, where the agent is moved vectorially in
association with an environmental component or by mass flow and
diffusion; and kinetic processes, where the movement can be described
by kinetic rate constants related more specifically and pointedly to
the agent. When considering distributions of a chemical with respect
to time, these diverse processes may play significant roles in
determining whether a given disposition is flow-limited (by transport
processes), compartmentalized (by kinetic processes), or some
combination of both. The reference time frame, not specified for
the conceptual model, is a highly significant parameter vital in
translating the conceptual model to realistic simulations. Similarly,
the spacial reference point (volume, location) has purposely been left
vague to permit the general case to be stated with the understanding
that specification of spacial and geographic localization will be
carried out in translation and elaboration of the modules (Figures 1
and 3) into models.
Examples of transport processes can be seen in dispositions primarily
dependent on stream flow, surface-to-ground water flow (leaching),
blood circulation, xylem transport, and precipitation from air.
Kinetic process-dependent dispositions may involve high or practically
irreversible sorption or binding, differential rates of sorption or
desorption between compartments of a major subsystem, or differential
chemical alteration. The following are offered as the principle
processes limiting or affording disposition of an agent in the
environment. More than one process may be occurring simultaneously
along the same route, so that the factors controlling the process
determine the proportion going by a particular pathway, which in turn
may alter that of another route.
15
-------
Transport Processes
Convective Mass Transport ("leaching," "drift") -
This physical process operates in all environments, in both the
gaseous and liquid phases, usually along the direction of mass flux.
In SOILS it would depend strongly on the degree of soil saturation,
in the ATMOSPHERE on the micrometeorological air flows, and in the
AQUATIC environment on hydrodynamics.
Inter-particle Diffusion (linear, eddy, etc.) -
This process operates where chemical gradients or local turbulence
exists Viscous solvent drag effects (included in the commonly used
term "dispersion coefficient") also operate.
Intra-particle Diffusion (absorption/de-absorption) -
Fickian chemical gradients act as driving forces causing chemical
mass to enter and diffuse into or out of particulate matter itself.
The structure of the particle, its degree of internal saturation with
water, the size and diffusivity of the chemical, and the chemical's
structure are important factors. Included in this category would be
"exclusion-type" processes, where pore size of inorganic particulates
may be large under one set of environmental conditions (pH, degree of
saturation), permitting entry of chemical to sites unexposed under
other conditions, and the subsequent trapping or binding of the agent
therein when the conditions change.
Co-distillation -
Volatilization in association with water evaporation takes place at
the soil/air and water/air interfaces and is highly dependent on the
temperature, degree of soil moisture or amount of water surface exposed,
and the chemical vapor pressure.
16
-------
Sublimation from a Surface -
This might be regarded as a compartment with the barrier consisting of
the heat of vaporization of the component and is significant for the
outer portions of multi-layered chemical adsorbed or held on a
surface exposed to the atmosphere.
Ingestion (includes feeding, drinking, imbibing.
inhalation, pinocytosis, etc.) -
The mass of the compartment ingested moves into biota at rates highly
dependent on age, physiological and nutritional status, species,
season, temperature, availability of alternative foods or sources of
water, etc. Several physicochemical and biological processes may be
involved with the intimate uptake (absorption, facilitated or active
transport, etc.).
Kinetic Processes
Adsorption-Desorption Phenomena (phase-surface interactions) -
The principle parameters of this movement are the enthalpy of sorption
of the chemical and the activation energy of the surface. Hence the
structure and properties of the agent and the total surface chemistry
of the interface are critical. The nature and type of surface
(composition of soil, tissue of animal, type of particle) and the
surface area presented to the phase containing the "free" agent are
important. This process is regarded as being represented by a pair
of kinetic equations, the ratio of which rate constants is the measure
of the equilibrium attainable between the surface and the medium. The
residence time of the medium (rate of change in compartment contact),
if small in relation to the rates of these reactions, may limit
disposition. Where the rate of binding exceeds very greatly the rate
17
-------
of desorption, the material may appear to be irreversibly bound.
Where these rates are both substantially slower than the rate of
media movement, the surface interaction will characterize the
disposition. Moisture level, pH, and temperature, as they affect
the chemical and the surface, will play major roles in this
phenomenon.
Chemical Transformation -
The non-biological alteration of a chemical introduced into any part
of the environment is dependent on the moisture, pH, and temperature
of that environment, on the nature of reactive groups on the agent,
and on the-presence of catalytic sites (on particles, etc.). The
nature and intensity of illumination additionally determines
photochemical reactions. At very high temperatures (pyrolysis) both
physical and chemical structure may be broken down to yield material
in the vapor state. In biota, soils, and water, and to a much less
extent in air, cation and anion exchange capacity coupled with
eletrolyte levels determines ionic interactions which may alter the
structure or availability of a chemical, such as by the formation of
insoluble complexes. In some instances the chemical reaction phenomena
are closely associated with adsorption-desorption processes, related
nonlinearly to the extent of coverage by, say, soil or air moisture
of the catalytic binding site where the reaction might be hydrolysis.
To the extent that the media are suitable for reaction or provide a
necessary reactant (e.g., ozone) these processes can appear to be
compartmentalized in rate of disposition.
Biological Alteration (includes activation,
degradation and conjugation) -
These processes are assumed to be catalyzed by enzymes, although
similar or identical chemical or photochemical reactions may be taking
18
-------
place at the same time (at reduced rates) in the same compartment or
others. The great increase in rate of the enzyme-catalyzed reaction
provides opportunity for compartmental differentiation of disposition.
These reactions are highly dependent on species, status (physiological,
nutritional, and previous chemical history), and route of exposure.
They may: provide for an agent becoming more or less biologically
active; for binding or conjugation in a form more or less available
to other organisms, compartments, etc., without altering the potential
biological activity; or for the covalent interaction of the agent with
an enzyme, thus altering the capacity of the system subsequently to
carry out alterations at the same rate (inhibition).
The biologtcal effects of an agent are difficult to separate from
disposition, inasmuch as one potential effect is to alter disposition
routes and/or rates. Known pesticide-induced enzymatic reactions in
both vertebrates and invertebrates include oxidation-reduction,
hydrolysis, conjugation, and carbon-carbon bond cleavage. The enzyme
activities induced may represent de novo synthesis of theretofore
unexpressed genomes (microbial) or amplification of the rate of genome
expression (higher animals). Biochemical alteration of environmental
contaminants and agents can be viewed as a function of the expression
of genetic material in coordination with the ability of the environment
and the biological species to provide for synthesis of enzyme and
cofactors to support the reactions. Changes in the course of this
expression may be one of the biological effects interacting strongly
on the disposition of a particular chemical.
Factors Affecting Disposition
As noted in the foregoing discussion of mechanisms, the disposition of
chemicals in the environment is governed by physicochemical, physical,
and biologic processes which can be related to properties of the chemical
19
-------
Table I. FACTORS AFFECTING DISPOSITION OF CHEMICALS IN THE ENVIRONMENT
Mechanism, Pathway,
or Process
Convecti ve mass
transport; Inter-
particle diffusion
Co-distillation;
volatilization/
Condensation
Intra-particle
diffusion
Ingestion
Adsorption/desorption
Properties of Anent
General-association with
compartmental component
Size, diffusion coeffi-
cient in media; vapor
pressure, latent heat of
vaporization; interaction
with media; intra-
molecular interactions
Size, diffusion coefficient
in particle, chemical
gradient
General-association with
compartmental component
Structure, enthalpy of
sorption (mono-layered);
enthalpy of fusion
(multi-layered)
Properties of Environmental
Component
Vectorial flux; degree
of saturation of immobile
matrix by movement
Water evaporation rate,
surface area, interaction
with aaent, degree of soil
moisture, extent of satura-
tion of air
Structure (micro), degree
of water saturation,
alterations of structure
by temperature, pH, ionic
strength
Nutritional value to
feeder, attractiveness
(chemical or physical),
availability of alternative
foods, dearee of competi-
tion with other feeders,
nutrietional and physiologic
status of feeder
Macro- and microstructure,
surface area, activation
energy of surface
Environmental Control
Physical (water or air
flow, soil movement);
temperature and gross
energy distribution
Temperature, energy flux
Temperature, pH, humid-
ity or soi1 moisture
General-biological
structure of ecosystem,
physical conditions
affecting rate or choice
of foods (temperature,
season, light)
Temoerature, humidity or
soil moisture, pH
-------
Table I (cont)
Mechanism, Pathway,
or Process
Chemical reaction
phenomena
Biological alteration
(activation, degrada-
tion, conjugation)
Properties of Agent
Structure (reactive
groups); energy of
activation, free energy of
reaction, nature of
mechanism
Structure (reactive
groups), energy of
activation, free energy
of reaction, nature of
reaction mechanism,
binding constant to
enzymes acting on it
Properties of Environmental
Component
Structure (catalytic
sites), energy of
activation, reactive
sites, dearee of coupling
to other systems providing
reactants or removing
products
Genetic capacity for
eliciting appropriate
enzyme, nature of enzyme;,
species status (physio-
logical, nutritional,
psychological), sensi-
tivity to aaent (inhibi-
tion, synergism, toxi-
city), degree of coupling
to other systems providing
reactants or removing pro-
ducts, presence or absence
of cofactors
Environmental Control
Temperature, humidity or
soil moisture, pH nature
and quantity of light
Temperature, pH, humidity
or soil moisture, biologi-
cal structure of eco-
system, previous chemical
history
-------
and environmental components. Table 1 summarizes these to indicate
those factors which should be known or determined in making judgments
as to the probable disposition of the chemical. Obviously, all
properties play some role in that disposition in the complex, real
world. As modeling proceeds from the conceptual level to mathematical
simulation, these values become the critical inputs, especially as the
disposition is related over time.
SOURCES OF CHEMICALS RELEASED INTO THE ENVIRONMENT
Each of the major compartments of the model can receive direct input
of certain chemicals as a result of the action of man. These inputs
are derived from "sources," which can be defined as the places and
activities leading to the release of a particular agent. A source
may result in a variety of inputs into major compartments and
subcompartments, and more than one source may have very similar input
into a model of pesticide behavior. For example, if methoxychlor were
sprayed on a forest in a diesel oil medium, this application ("source")
would have inputs into the atmosphere (both gases and aerosols), on to
the cuticular or dermal surfaces of biota, and on to the surfaces of
soil and water. A source may be deliberate, accidental, or
adventitous, but the inputs have been handled uniformly in the models.
The sources can be grouped generally according to the major compartments
to which inputs are directed and according to the time frame in the
history of a particular agent that it may enter a model from a source.
The latter might be divided into preconsumption (synthesis and
manufacture), distribution (transport, storage, consumption, application,
or use), and disposal (dumping, release). A chemical plant might serve
as a source of atmospheric release of a pesticide during manufacture,
a site of accidental spills during storage and transportation, and
then have to dispose of waste materials containing the agent in
22
-------
sanitary landfill, so that it would be a source having several inputs.
Other typical sources are shown in Table 2. For the purposes of models,
we then should consider the specific nature of sources providing inputs
into the environment.
Atmosphere
Considerable atmospheric input occurs upon application of the large
class of organic chemicals used as pesticides; i.e., insecticides,
fungicides, herbicides, and rodenticides. For example, DDT is
commonly applied by spraying a liquid suspension or solution by
aircraft or mobile ground equipment. UOODWELL et al., (1971) report
that in aerial applications of DDT to forests in the northeastern
United States 50 percent or less of the amount applied was deposited
in the forest. The rest was dispersed in the air either in the
gaseous form or as small droplets. While much of the airborne liquid
droplet fraction settles to the ground nearby, a significant amount
remains aloft, to become associated with other particles and distributed
in the environment at distances far from the point of initial application.
Table 2. SOURCES OF CHEMICALS FOR THE TERRESTRIAL ENVIRONMENT
Phase of
History of Chemical Examples
Preconsumption Manufacture, food processing, mining,
refining
Distribution Application of chemical in pest control,
agriculture, or for public health purposes;
unintentional release resulting from the
use of products containing or made of
chemicals which are not totally confined
or immobilized; accidental spills in
transport or storage
Disposal Release of wastes in air or industrial and
domestic waste water; landfill operations;
incineration; dumping and discarding
23
-------
The chemical input may be in the gaseous state or adsorbed onto
particulates released into the atmosphere. Accidental discharge
resulting from explosions, containment vessel failure, human error,
or other accidents involving vehicles or devices for transporting
chemicals can cause major problems in a local geographic area, but
are probably minor when considered on a global scale.
Chemical input into the atmosphere through routine use of products
made of chemicals not totally immobilized, either intentially or
unintentionally, is of major concern. For their model, NISBET and
SAROFIM (1972) had to estimate the amount of PCBs lost to the
atmosphere by evaporation of hydraulic fluids, lubricants,
dielectric-fluids used in transformers, and various plastics which
are manufactured using PCBs.
Flora
Except for the direct application of plant growth regulators and
chemicals used in pest control and for other agricultural purposes,
sources are generally separated from flora by atmosphere, soil, and
water of the environment. With direct application, input may occur
on the foliage and/or fruiting body; alternatively, soil or water
applications are sources of indirect inputs.
Fauna
As with flora, few sources directly input into these compartments.
Medical and veterinary application of drugs and medicines, cosmetic
and hygienic dermal applications, and consumption of food and non-
food items constitute typical types of deliberate exposure from
sources. In occupational use and, to a lesser extent, the general
public, exposure can occur by direct inhalation of vapors or
absorption through the skin. Hence, concern has been evidenced for
24
-------
workers breathing or otherwise coining into contact with chemicals
present at relatively low concentrations for long periods of time
or at relatively high concentrations intermittently for short periods
of time. Direct or indirect application of chemicals to flora or
fauna can constitute a significant input for animals higher in the
food chain if residues of the chemical or its alteration products
are retained in the food.
Inadvertant and accidental release or even purposeful misuse or
abuse of various chemicals and chemical products can also be a
serious and significant direct source of agents to fauna. Hhile some
such sources are moderated through the atmosphere, soil, water, or
flora, opportunities arise for direct inputs to fauna under some such
circumstances.
Soil and Water
Many of the direct introductions of pesticides into the environment
are sources closely connected to the soil and water regions.
Application of pesticides and fertilizers by spraying a solution,
liquid suspension, or granular formulation are important inputs to
the soil surface, subsurface and the aquatic surfaces on both a local
and global scale. In addition to adventitious contamination by
accidental spills, other usages, and leakage from sources, a local
region becomes a source by dumping or discarding material or by
creation of sanitary landfills. Since the latter are generally of
greater scope, the subsequent infiltration by rainfall and movement
of surface or ground water can be major inputs throughout the soil,
as detailed by ELZY et al., (1974).
Once a chemical is introduced into the soil and/or water environments,
those compartments may continue to act as a reservoir for long periods
of time, leading to transfer of an agent to flora and fauna. Depending
2.5
-------
on the time rate of change of the concentration of a chemical at a
site of localization, the compartment may act as a "sink" (where an
agent is effectively withheld from participation in the system) or
as a "reservoir" (where flows and transfer permit participation).
From the point of reference of a given species, a compartment may be
either a reservoir (and thus a "source" of an input) or a sink. A
breakdown product (such as DDE from DDT or methy!mercury from mercury)
may arise in the soil and biota and subsequently appear broadly in the
environment, even though it was not manufactured or synthesized as
such. Thus, as the ultimate repository of waste, unwanted materials,
and the products for which the chemicals were manufactured or prepared,
the SOIL and WATER have pervasive major inputs into other segments of
the environment.
26
-------
SECTION V
THE ATMOSPHERIC/TERRESTRIAL MODEL
The model consists of a set of assumptions derived from experiment,
experience, and physical law that are set forth graphically to
illustrate the principal components ("compartments") of the system,
the means by which the chemical itself or the components bearing the
chemical being modeled move or change in the environment, and the
relationships between compartments vis-a-vis this movement. Also
enumerated and elucidated are the factors affecting these routes,
such as the characteristics of the chemical and compartment.
ASSUMPTIONS
1. Elements or components of the terrestrial environment considered
are confined in a geographic and geophysical sense to a local
environment consisting of "ATMOSPHERE," "SOIL and WATER." "FLORA,"
and "FAUNA."
2. These elements and their constituent aspects can be set forth
as compartments, representing chemical reactors.
3. The model is directly applicable only to the agent; its breakdown
products or metabolites are representable as parallel models following
identical conceptual functions of disposition.
4. The interrelationships of compartments and the movement of
chemicals can be represented by a chemical process flow sheet.
DEFINITIONS
1. ATMOSPHERE. The gaseous phase containing suspended aerosols and
particulates above the earth and its biota.
a. Troposphere. The portion of the atmosphere in direct contact
with soil, water, and the biota.
27
-------
(1) Suspended participates. Solid matter, including
certain microscopic biota, suspended in the atmosphere.
Each particle has a surface subcompartment.
(2) Aerosols. Microscopic matter (solid or liquid)
dispersed in the atmosphere, each with a surface
subcompartment.
(3) Gases. The gases and vapor-phase components of
the atmosphere.
b. Stratosphere. A compartment above the troposphere and
beluw the mesosphere having components as in (1-a), but not
interacting directly with the earth and its biota.
c. Mesosphere. A compartment below the mesopause and
ionosphere and above the stratosphere having compartments
as in (1-a and b), but not interacting directly with the
earth except through the troposphere and stratosphere.
2. FAUNA. Biota excluding plants and microorganisms (except
protozoans) and including not only the terrestrial surface species
but also those living predominantly in the atmosphere and in the soil.
a. Man. Human beings representable by a subsystem of several
compartments based on anatomical and physiological characteristics.
b. Higher carnivores. Those creatures feeding on primary
carnivores (and perhaps in some instances on forms lower in
the food web).
c. Primary carniyores. Those species feeding predominantly
on herbivores and (to a lesser extent) primary producers.
d. Herbivores. Those species feeding on primary producers,
usually plants and related microorganisms.
e. Soi1-organisms. Those primary producers dwelling
predominantly in soil and the scavengers of the plant and
animal matter constituting the organic matter of soils.
3. FLORA. The biota including largely the photosynthetic primary
producers consumed by man, herbivores, and soil organisms. Generally
the plant is represented as having a subsurface portion (consisting of
subcompartments for the root tissues and potential storage or fruiting
bodies) and a surface portion (consisting of foliage and fruiting body),
all surrounded by a cuticular compartment.
4. SOILS AND WATER. In addition to soil organisms (2-e), this
compartment is separated into two regions containing the same components
-------
a. Surface. The top portion of the soil, capable of interacting
with the air directly.
(1) Surface water. The result of precipitation, ground
water springs, etc., but distinct from streams, ponds,
lakes, etc. (parts of the AQUATIC model); includes "free"
water associated with soils and all solutes.
(2) Organic particulates. Colloidal materials in suspension
including organic matter and decaying material derived from
biota.
(3) Inorganic particulates. Inorganic soil structural
materials (clay, silicates, minerals, etc.) and those
insoluble materials of non-biological origin.
b. Subsurface. Similar to (4-a) but containing the ground
water and associated soil water. Actually there exists a
series of parallel plates or zones through the soil profile
which will differ in composition, environmental condition, etc.
The subsurface region indicated in this model is considered to
be all that below the immediate surface in contact with the
atmosphere.
PATHWAYS
The foregoing compartments are displayed in Figure 4. A compound
introduced into the ATMOSPHERE (Figure 4A) may be in the vapor phase,
as an aerosol, or in the form of a large particle. Chemicals in the
vapor phase would-be expected to adsorb reversibly to the surface of
aerosols and other particulates, where the potential for alteration by
cnemical or photolytic means (due to catalytic sites thereon) is much
greater than in the vapor phase. The particles might condense or break
down, and chemicals would be redistributed. A chemical on the surface
of a particle or aerosol could absorb reversibly into the particle,
where photolysis would be very much less likely. All of these
interactions would be taking place in the Mesosphere and Stratosphere
as well as the Troposphere, which are mixed by diffusive and
meterological conditions and events. Photolysis would be expected to
29
-------
CO
CD
LEGEND:
Mass compartmsntal transft
Cfitmical movomtnt
R«
-------
MESOSPHERE
n
STRATOSPHERE
n
ii
6
n
TROPOSPHERE
GASES
'?%
O
AEROSOLS
surface
SUSPENDED
F*RTICULATE$
jrface
Precipitation washout
Fcllou*
Wash-ouU
/x
Erosion
Co-distillation
Figure 4A: ATMOSPHERE
31
-------
play a progressively great role in the upper atmospheric compartments
and, conversely, chemical reaction (except for ozonolysis) would be
expected to be of less importance in those upper compartments.
Iteration of the basic Troposphere model through the upper atmospheric
compartments is easily accomplished.
Materials can enter or leave the atmospheric compartments by
reversible sorption, interacting especially with SOIL and FLORA
surfaces, or by volatilization/condensation from these surfaces.
Particulate matter would settle out onto surfaces or be washed out
by precipitation. Winds, mechanical action (such as abrasion),
various modes of direct introduction (application, emission sources),
and meteorologic aerosol formation in association with codisti1lation
would result in particulate aerosol introductions into the ATMOSPHERE.
FAUNA, and to a lesser extent FLORA, would be subject to ingestion of
portions of the tropospheric compartment by respiration, while sorption
would provide dermal exposure. Inhaled particles not trapped in lungs,
spiracles, etc., and particles or aerosols trapped on skin, hair, etc.,
and thereby subjected to grooming (e.g., fur licking), may be ingested
with mucous. Air is also present in soils, in equilibrium with the
soil surfaces re_ any component chemical; it likely plays little
role per se in exposure of Soil Organisms and is therefore ignored.
Depending on atmospheric mixing and soil movement, the exposure of
Soil Organisms may be qualitatively and quantitatively different than
the exposure of surface FAUNA via the air.
A schematic fauna! subsystem (Figure 5) illustrates the probable
inputs and outputs of the several compartments in Figure 4B (compare
to Figure 3). Depending upon the food source, material from one or
more of the other major compartments may be ingested, exposing the
lumen tissue to the chemical in the food, air, or water. It may be
sorbed, broken down within the lumen, and/or passed out with the
32
-------
FOOD (From PLANTS,
FAUNA, SOILS, and
SOIL ORGANISMS)
WATER
(SOILS;
Death A-
Decay \._
To other^FAUNA
~\ <^
n
* . Blood (Hemolymph;'*
m
I V
"bound"
Lumen
I v
Gut
Digestive organs
(Liver, Kidney)
•iT Hair
| Feathers
Dermus
Endoderm
x»m
A | Respiratory organs
"X,nnX
Other tissues and organs
(muscle, bone, nervous system,
adipose)
e
Reproductive tissues
Offspring
(fetus, eggs)
-*
ATMOS
Inhalation
Figure 5: Diagram of Fauna! Subsystem Model.
(Based on BISCHOFF and BROWN, 1966). Arrows indicate
flows or transfers of agent and/or compartment mass.
33
-------
fecal matter, which then passes on to the SOIL or as food for other
FAUNA. The portion absorbed may be distributed throughout the
organisms to other tissues, which may alter the agent's structure
to more easily excreted products, store the agent (for later release
or for ingestion by a predator species), or provide for the agent's
excretion.
::::::: Respiration iiii ':'•':
Ingestion
t
MAN
M,X«-N
(see FAUNAL subsystem] \J
\\
HIGHER
CARNIVORES ._
M,X*"*\
(see FAUNAL subsystem) J
n
PRIMARY
CARNIVORES .__
MX*^
(see FAUNAL subsystem) 9
n
HFRBIVORFS ^.
M.X^y
(see FAUNAL. subsystem^
fr
'•:•. : | ! Decay
—
/-
V
<•
c
c
c
c
^-
From
AQUATIC
Figure 4B: FAUNA
34
-------
In the higher vertebrates this process is complicated by functions
such as the enterohepatic cycle (gut -»- liver -> bile •> intestinal
lumen). In higher animals material may be lost through the skin,
hair, or feathers. As noted earlier, these tissues also receive
exposure from the ATMOSPHERE (and some instances SOIL). Some agents
may be altered externally and some may not penetrate the dermal
barrier. Unabsorbed material could volatilize or be adsorbed by
atmospheric particles. Except for exhalation of unadsorbed material,
pulmonary losses of chemicals taken into animals by other routes
appear negligible.
Agents are also distributed to reproductive tissues, which can
constitute- a major outlet of chemical for the exposed animal. In
female mammals this release can continue on through parturition into
lactation. The route to offspring may be of great significance, since
the young of many species serve as food for higher trophic levels.
Another major loss route, in addition to excretion, is the death and
decay of tissues and organisms, leading to the entry of the material
into the SOIL and WATER compartments (Figure 4D). Initially on the
Soi1 Surface subcompartment, these materials become part of the
organic particulates and later free water of that compartment, but
are transferred by mechanical, geophysical, and biological action
into the Subsurface compartment. Soil Organisms then ingest these
particles, and one could propose an elementary version of the scheme
in Figure 5 for disposition of the chemical in those organisms.
Additionally some Soil Organisms may be purged of some chemicals by
reversible sorption of materials in the gut lumen onto the out-going
soil particles.
The other major biological compartment is that of FLORA (Figure 4C),
represented as a generalized model with both Subsurface and Aerial
35
-------
portions surrounded by a waxy cuticle. Materials can be deposited
on this latter surface by fallout or precipitation, by condensation,
or by reversible sorption. Some agents can pass on through into
plant tissue or may be broken down chemically or photolytically on
the surface. A portion may be washed off the leaves and added to the
SOILS compartment. Material bound to the foliage will subsequently
enter litter as decay occurs. An agent on the foliage may be
volatilized off or sorb onto air particulates.
In the Subsurface zone, material may be brought into the plant by
uptake of water or by sorbtion onto the root surface and subsequent
penetration of the cuticle. Some may "leak" out or be released to
the SOIL from the cuticle. Both the Aerial and Subsurface portions
of FLORA are subject to herbivorous feeding, moving material into
the Soil Organisms and other FAUNA, and an agent in either compartment
is subject to chemical or biochemical alteration. Once an agent is in
FLORA, it may be translocated to other tissues, including fruiting
bodies associated with either portion. Similar to animals, a given
species of herbivore may select only a limited tissue on which to
feed; all portions of a plant are seldom ingested by a single
creature at one time. Distribution within the plant of a given agent
would therefore have a very marked effect on the subsequent nature
and extent of movement of a chemical from FLORA to other major
compartments. All of these movements would be less complex in
photosynthetic microorganisms.
"Bound" agents, including material strongly sorbed (seemingly
irreversibly) and material covalently reacted (but bearing the active
groups intact) are difficult to define and determine. Some of the
"bound" residues may be released by extraction, when sorption is
reversed, or by chemical or enzymatic treatment, where the conjugating
bonds are cleaved. In both FLORA and FAUNA (including Sojl Organisms)
materials considered metabolized or altered so as to leave the scheme
may re-enter a compartment as a result of such action.
36
-------
Co-distillation
Figure 4C: FLORA
The most complex and probably most significant compartment in the
disposition of an agent entering the terrestrial environment is that
of SOILS and WATER (Figure 4D). Material can enter this compartment
directly at the Surface by sorption from the ATMOSPHERE, condensation,
37
-------
Wind, abrasion, mechanical)
T
Respiration
Volatilization
SOIL SURFACE
SOIL
ORGANISMS
;•:- Decay X-:-:'
;•>:•;;•;•;•;•>•; •
Tillage, geophysical, biological
SUBSURFACE
To
AQUATIC
To
AQUATIC
Figure 4D. SOIL and WATER
-------
settling and fallout, and precipitation (including material washed
off of plant surfaces). Excretion, exfoliation, and decay of animal
tissues and defoliation, withering, litterfall, and subsequent decay
of plant materials add to the routes of entry. Material can leave
the Soil Surface by erosion (wind, water, or mechanical), by
volatilization, by photochemical and chemical alteration, and by
ingestion by Soil Organisms and other FAUNA. Some material is lost
from the Surface by tillage, mechanical mixing (geophysical or
biological), and "leaching." The movement of Surface water into the
Ground water takes with it solubilized and reversibly sorbed materials.
Within the Surface compartment, much as in the case of the Troposphere,
materials can be bound to the surfaces of particulates. Surface
waters can become contaminated by a Ground water-source containing an
agent, which would then be distributed in ttie Soil Surface.
In the Subsurface zone, material can enter from the Soil Surface, be
brought into the zone by Soil Organisms or FLORA (through translocation,
leakage, and root decay), and can leave by routes noted earlier--
sorption into FLORA and Soil Organisms (and to a lesser extent, other
FAUNA), ingestion by Soil Organisms and FAUNA, and through the Ground
water into FLORA and out into other waters (streams, lakes, estuaries--
labeled AQUATIC). In actual cases, it would be necessary to
characterize each soil horizon by iteration of interconnected
Subsurface models.
39
-------
SECTION VI
THE FRESHWATER AQUATIC MODEL
This section develops a systematic approach to an optimal
representation of the behavior of pesticides in aquatic environments.
A quantitative discussion of processes and parameters important to
the fate and transport of pesticides-in-general is futile because of
the diverse chemical and physical properties of pesticides. This is
further complicated by the need to specify chemical, biological, and
physical characteristics of the aquatic ecosystem. Therefore, a
qualitative approach to studying and modeling the fate and transport
of pesticides in aquatic ecosystems is discussed.
There has been a shift in many areas of science toward studies of
wider scope. This has been brought about partly by increased
emphasis on "the environment" and partly by wider knowledge of the
techniques of system studies. According to MOORE (1967) the emphasis
in pesticide studies has shifted from
Pesticide -+• Pest
to
Pesticide -> Ecosystem
Past pesticide research resulted in few system studies and fewer
mathematical analyses of such studies.
One area of system studies is that of microcosm or partial system
studies (METCALF et al., 1971). These studies emphasize a particular
short food chain largely as an index for comparison of various studies,
Their quantitative applicability to real-world ecosystem is therefore
limited. Nevertheless, they provide the basis of a large portion of
our comprehension of the behavior of pesticides in the environment.
40
-------
Global model studies are important in setting an overall framework
within which smaller system studies may be placed. RANDERS and
MEADOWS (1971) studied the movement of DDT in the environment, and
WOODWELL et al., (1971) made a similar study. An important
conclusion of both these papers was that the DDT concentration in
food chain organisms would continue to increase long after the rate
of application was decreased or terminated. This conclusion was
based on computer simulation studies and comparative analyses.
Smaller system studies of greater detail bring us closer to
interactions at the ecological level. Analyses of pesticide
transformations and transports at the ecological level may make use
of both ecological theory and various applications of systems theory.
For example, EBERHARDT et al., (1971) applied system simulation to a
field study as an aid in interpreting the data.
The above examples deal with specific pesticides in relatively
defined ecosystems and are not generally applicable to a description
of fate and transport. The presentation that follows is applicable
to pesticides and aquatic ecosystems in general but can also be used
as a starting point for any specific pesticide and system.
SKELETAL DIAGRAMS FOR A PESTICIDE IN THE AQUATIC ENVIRONMENT*
The most effective aggregation of storage components and rate
processes varies as attention is turned from one aquatic regime to
another. Even within the range of lentic systems, the diagrammatic
representation for a deep dimictic lake would be inappropriate when
used for a freshwater marsh. For this reason, several basic frameworks
or skeletal models without detailed process embellishment are presented
for different aquatic environments.
*See Appendix for detailed background.
41
-------
The first skeletal diagram in Figure 6 is intended for a dimictic
lake in which process dynamics are affected by the presence of a
strong thermocline. The division between epilimnion and hypolimnion
may allow for long-term storage and release from the sediments of
the reduced forms of some chemical species (HUTCHISON, 1957; O'MELIA,
1972). The surface layer is isolated as a storage component in this
vertical model because of the possibility of enrichment in heavy
metals and pesticides (DUCE et a]., 1972) and the neuston food web.
The sediments are treated as a separate storage unit because of
possible long-term storage (AHR, 1973), sorption-desorption process
rates (HUANG, 1971) and the benthic food web.
A similar vertical skeletal structure without the hypolimnion may be
used for a holomictic lake or a freshwater marsh. However, a shallow
lake or a marsh may be better represented by a horizontal structure
(SCHINDLER, 1974) as presented in Figure 7. Here the storage is
divided among aquatic communities, which have varying response times
and process rates.
The independent variable implied in both of these lake models is time-.
However, either one may be used as a two-dimensional stream or river
model by choosing longitudinal distance (i.e., downstream) as the
independent variable and including hydraulic and morphologic effects
on settling and mixing.
Finally, the lotic system may be represented by a horizontal array
of vertical column structures (similar to that of Figure 6) with
longitudinal distance as the independent variable. The transfers
between columns represent the transverse mixing in the system
(HOLLEY and ABRAHAM, 1973). This concept is presented in the
diagram of Figure 8.
42
-------
Atmospheric
Parameters
'X
Atmospheric Inpi
t *
>' /-"
'
'*J >
1
Surface Layer
<
x^V,
i
Output to
Atmosphere
^ v
___ / \
Atmospheric
Parameters
*
[^
-------
Hydrologlc
Parameters
x
ff\
f— v_ ~\J
COo
t
Hydraulic
Input-Output
XII*
/
Atmospheric
Input-Output
x
X
*
Littoral Zone
Amospheric
Input-Output
fV/j Hydraulic
|/\| Input-Output
Pelagic Zone
Suspension
*
*
[X] Settling |
| Settling [XI [X] Suspension |
/
V
v
1
1 /-^ TL
I lf "V\
* t^
Hydraulic V/l
Input-Output /\|
Benthlc Zone
Figure 7: Horizontal Representation of a Lake.
-------
Morpholigic >v_ __ ---^.
Parameters J— ~~
I
Hydraulic
~~ — ~ V Parameters
Figure 8: Horizontal Array of Vertical Columns for Representation of Lotic Systems.
-------
In all of the preceding skeletal representations each storage
component may be divided into discrete physical, chemical, and
biological storage components with their associated transfers,
rate coefficients, and coupled subsystems (e.g., food web biological
uptake and storage with its associated growth, respiration, and
trophic dynamics). A typical expansion of the surface layer storage
component from the vertical representation of a lake in Figure 6 is
shown in Figure 9.
Inputs and outputs may be expanded in a manner similar to the
storage based upon their physical and chemical characteristics
(dissolved, particulate, or absorbed). Expansion of the storage
compartments may be necessitated by expansion of the hydraulic
input-output to the epilimnion (Figure 10) into three separate
inputs and outputs.
The storage labeled "Food Heb" in Figure 9 is at the heart of most
problems concerning pesticides in the environment (MOORE, 1967). A
skeletal abstraction of a food web structure is shown in Figure 11
(compare to Figure 3). One method of allowing for the influence of
food web dynamics upon the pesticide storages and fluxes in the model
is to assume that the biomass levels in the aggregates representing
the food web are at a steady state. Under this assumption the
influence of the food web may be included in rate coefficients that
affect the flux of pesticide between storage components representing
pesticide concentration in the food web compartments. This is
exemplified in the compartment diagram for the linear, donor-
controlled model of DDT in the freshwater marsh of Figure 12.
Another method of allowing for the effects of the food web on
pesticide behavior is to couple the explicit representation of the
food web to the related pesticide storages. Thus, there will be two
storage components coupled together in a manner similar to that of
46
-------
Inorganic Pa rticulates
/MeteorologicalN
1 Atmospheric J
V Coefficient ./ x~v
^~~~~~-^i~y ~Q^
* r
Solar Input Rate ty]
Available Solar Energy i
i
, i
i I
^ * *
/'"£' -^ IVl Transmission
r ~
1
1
1 1
* *
1 Adsorption p
H
Concentration on
Pa rticulates
Photo
Oeg
Cneffiripnt L»-
Concentration in Surface Layer
f* of Water
<
'
i
chemical V
radation /
J
//
tO:
1
t>
^J
f
3)
i
i
*
jSorbtionand lnjestion[)
f
^] Decomposition | (
!
i i .
i i 1
(f\
f~ —• ^J
COo
' f
[X] Biodegradation |
Surf ace Food Web
Organic ^articulates
1
t
i
_i
t^JJ-
Figure 9: Some of the Storages, Processes, and Subsystems
Associated with the Surface Layer Storage Compartment.
-------
co
Solubilization/
Precipitation
Pesticide Particles
Hydrologic Input of
Dissolved Pesticide
Dired Chemical Oxidation
and Microbial Degradation
Hydrologic Input of
Adsorbed Pesticide
N
Adsorbed on
Suspended Particles
Figure 10: An Expansion of the Hydrologic Input.
-------
.v>j-
D
Respiration
t
r s°n"ion |X1
£3>
.1
Respiration £X]
t '
p; %
>- ,^_. ^j
t
[XT 'ngestion ]
Respiration JX] |
[Xjnecrth and Excretion
I
L
Decomposers
[XI Sorption [
J__J
Figure 11: A Skeletal Abstraction of a Food Ueb.
49
-------
Figure A-5 (see Appendix) for each storage of pesticide in a biotic
component. The mathematical representation of this type of interaction
is presented by HARRISON et al., (1971).
r
r
r
Surface Concentration
of DDT
,
Concentration in Tadpole
1
Concentration in
Suspended Matter
r
r
Concentration in Sunfish
i
Concentration in
Bloodworm
\
/
Concentration in Water
rf
V
Concentration in Carp
1 '
Concentration in
Narrow-leaf Pondweed
DDT Granules (Input)
Figure 12; Food Chain Model of DDT in a Freshwater
Marsh (from EBERHARDT et al., 1971).
Remembering that there is no best or correct representation, a
plausible general model for a pesticide in the aquatic environment
based upon the vertical skeletal structure is presented in Figure 13,
an example of the result of the process of expanding the diagrams
to include the system storages or processes that may be considered
important. The system processes included in Figure 13 are intended
to constitute a minimal set of parameters to be considered when
investigating the movement and impact of a pesticide in the aquatic
envi ronment.
50
-------
In a typical mathematical representation, which may be derived from
the system diagram, each of the storage blocks accounts for one of a
set of simultaneous differential equations. Also, each of the valve
symbols accounts for a rate term in the set of equations. In addition,
the coefficients or parameters in circles (many of which are omitted
from Figure 13 for the sake of visual simplicity, e.g., pH, temperature,
and Eh) appear as rate modifying coefficients in the equations. Each
of these coefficients must be estimated from the literature or
determined by a set of measurements on the system. Thus even the
minimal set of variables of Figure 13 results in a complicated set of
mathematical equations and requires a large data base for evaluation.
This complex representation can be reduced by aggregation of the
least important variables for a particular pesticide and ecosystem.
The inclination to eliminate the least important variables is usually
intuitively focused either on very rapid processes, "which cannot be
rate limiting," or conversely on very slow processes, "which cannot
transport or transform much matter or energy," depending upon the
investigator's objectives. The possible dangers in using these bases
for eliminating variables lies in the synergistic behavior of causally
closed environmental systems.*
The effect of an individual process on system behavior is dependent
upon four levels of system interactions. These are
a. the rate coefficients and parameters for the process itself;
b. the hypothesized topology for the system interactions as
presented in the system diagram;
*An ecosystem is a completely connected system (COMMONER, 1971) that
is closed in the control sense (HUTCHISON, 1948; PATTEN, 1973). A
closed control system or feedback system may exhibit "emergent
properties" (CANNON, 1967) or "synergistic effects" (ODUM, 1971)
that are dependent upon system structure or total system properties
(BERTALANFFY, 1968). These properties can affect the influence of
a specific process upon system behavior.
51
-------
en
ro
-
coMSunuK*
-------
en
CO
^^~ - 1 C.OMCBMT"«*''''OKI T
^ATi 1 '?OM"^I ] ~
Figure 13: A Minimal Representation for a Pesticide in a Dir^ctic Lake.
-------
c. the hypothesized system structure, which includes the
influence of other process rates acting through the causal
topology; and
d. the time series of inputs to the system.
Analytical techniques for estimating the effects of individual processes
on the system behavior are summarized in Section I. The results provide
an analytical basis for reduction of complex system diagrams.
54
-------
SECTION VII
THE ESTUARINE/MARIME MODEL
The considerations incorporated in the freshwater aquatic model
continue in relevance and validity into the estuarine/marine system,
which can be viewed as specialized iterations of the general model
(Figures 1 and 3). The relationship to the terrestrial and
freshwater systems have been alluded to earlier (Figure 2). Thus,
it is sufficient here to outline the significant differences and
inter-relationships applicable to these regions of the environment.
The physical state of a compound in a system depends on its relation
with the other components of the system, a behavior which can ideally
be described by distribution constants when at equilibrium. For
instance, the pharmaco-dynamic action of many drugs depends on their
relative ability to bind to different sites. In such a fashion, the
bloodstream may act as a reservoir permitting slow release of a drug
to assure its long-term action. This ability to bind substances can
occur anywhere. The toxicity of a pollutant must thus be evaluated
in terms of the physical state(s) in which it shows toxicity and not
merely by its observed concentration. With regard to availability to
a carnivore, a pollutant adsorbed to detritus may be as unavailable
as that adsorbed to a grain of sand.
Figure 14 is a schematic diagram of flow of a chemical through an
estuary. It should be pointed out strongly that the estuarine system
is exceedingly complex and any simulation will require time and caution,
Figure 15 is a representation of functional interactions at the
interfaces between the estuary and other indicated ecosystems. The
arrows indicate possible flux of chemicals without regard to form and
origin. Interactions in the estuary are treated in a more precise
conceptual fashion in Figure 14.
55
-------
SOURCE
PHYSICAL FORM
RESERVOIR
LOSS
cn
01
Figure 14: Simple Model of Transport in Estuaries.
-------
The following definitions apply to Figure 15 only:
Run-off: Any transport from land adjacent to an estuary,
including drainage not covered by river flow, such as non-
specific drainage from swamps.
Tidal action: Any transport mediated by tidal flushing and
tidal currents.
Biota-mediated flux: Any transport of organisms from one
domain (sea coast, ocean, and fresh water) to another, such
as in the case where a predator leaves its domain to feed in
another domain, possibly itself becoming prey, or contamination
through excretions (feces, urine, and regurgitated pellets).
Emigration and immigration are also included.
River flow: Any transport mediated by a river or rivulet.
This includes adsorbed as well as non-adsorbed materials.
Atmospheric disturbances: 1) Any transport caused by
unusually high tides due to strong winds. 2) Any transport
caused by agitation of the sediment or shore/bank by
abnormally strong wave action or currents due to strong winds.
Turbulence: Any transport due to abnormal mixing caused by
eddies (underwater storms).
Tides and Currents: Any transport due to normal tides and
currents.
Fal1-out: Any transport via the atmosphere.
57
-------
en
00
ATMOSPHERE
INORGANIC
PARTIC
ULATES
ORGANIC
PARTJC
ULATES
AEROSOLS
T^
^
Figure 15: Expanded, Iterated Basic Chemical Module for
Transport of Chemicals in Estuaries.
Open arrows indicate transport between modules via Run-off, River flow, Tidal
action. Turbulence, Fallout, and Atmospheric disturbances.
Biota-mediated Flux (overlay).
Solid arrows indicate unspecified migration, predation, life cycle-related
changes, and transport-dependent movement between food webs associated with
chemical modules representing environmental regions.
-------
SECTION VIII
REFERENCES
AHR, W. M. (1973). Long lived pollutants in sediments from the
Laguna Atacosa National Wildlife Refuge, Texas. Geol. Soc.
Amer. Bull. 84, 2511.
BERTALANFFY, L. VON. (1963). "General Systems Theory." George
Braziller, Inc., New York. p. 289.
BISCHOFF, K. B. and R. G. BROWN. (1966). Drug distribution in
mammals. Chem. Eng. Prog. Symp. Ser. A 6_6, 32.
CANNON, R. H. (1967). "Dynamics of Physical Streams." McGraw-Hill,
New York. p. 1093.
CHILD, G. I. and H. H. SHUGART, JR. (1972). Frequency response
analysis of magnesium cycling in a tropical forest ecosystem,
J_n_ "Systems Analysis and Simulation in Ecology," Vol. II,
B. C. Patten (ed). Academic Press, New York. p. 592.
CLOSE, C. M. (1963). "Notes on the Analysis of Linear Circuits."
Rensselaer Polytechnic Institute, Troy, N. Y. p. 123.
COMMONER, B. (1971). "The Closing Circle." Alfred A. Knopf, Inc.,
New York. p. 326.
DeRUSSO, R. M., R. ROY and C. CLOSE. (1965). "State Variables for
Engineers." John Hi ley and Sons, Inc., [Jew York.
DUCE, R. A., J. G. QUINN, C. E. OLNEY, S. R. PIOTROWICZ, B. J. RAY
and T. L. WADE. (1972). Enrichment of heavy metals and organic
compounds in the surface microlayer of Narragansett Bay, Rhode
Island. Science 176, 161.
EBERHARDT, L. L., R. L. MEEKS and T. J. PETERLE. (1971). Food chain
model for DDT kinetics in a freshwater marsh. Nature 230, 60.
ELZY, E., F. T. LINDSTROM, L. BOERSMA, R. SWEET and P. WICKS. (1974).
Analysis of the movement of hazardous chemicals in and from a
landfill site via a simple vertical-horizontal routing model.
Oregon State Agricultural Experiment Station Special Report No.
414, Oregon State University, Corvallis, OR 97331. 110 pp.
59
-------
FORRESTER, J. W. (1971). "World Dynamics." Wright-Allen Press,
Cambridge, Mass. 142 pp.
HANNON, B. (1973). The structure of ecosystems. J. Theor. Biol.
41.
HARRISON, H. L., 0. L. LOUCKS, J. W. MITCHELL, D. F. PARKHURST,
C. R. TRACY, D. G. WATTS and V. J. YANNACONE, JR. (1970).
Systems studies of DDT transport. Science 170, 503.
HILL, J., IV (1973). Component Description and Analysis of
Environmental Systems. Masters Thesis. Utah State Univ.,
Logan, Utah. p. 94.
HOLLEY, E. R. and G. ABRAHAM. (1973). Field tests on transverse
mixing in rivers. J. Hydraulics Div. ASCE. HY12, 2313.
HUANG, J. (1971). Organic pesticides in the aquatic environment.
Water and Sewage Works. May, 139.
HUTCHISON, G. E. (1948). Circular causal systems in ecology.
Ann. N. Y. Acad. Sci . 50, 221.
HUTCHISON, G. E. (1957). "A Treatise on Limnology." John Wiley
and Sons, Inc., New York. p. 1015.
KENAGA, E. E. (1972). Guidelines for environmental study of
pesticides: determination of bioconcentration potential.
Res. Rev. 44, 73.
KARNOPP, D. and R. C. ROSENBERG. (1968). "Analysis and Simulation
of Multiport Systems." Massachusetts Institute of Technology.
Cambridge, Mass. p. 221.
KUO, B. C. (1962). "Automatic Control Systems." Prentice-Hall.
Englewood Cliffs. N.J. p. 504.
LEONTIEF, W. W. (1966). "Input-Output Economics." Oxford
University Press, New York.
LINDSTROM, F. T., J. W. GILLETT and S. C. RODECAP. (1974).
Distribution of HEOD (dieldrin) in mammals: I. Preliminary
model. Arch. Environ. Contam. Toxicol. 2_, 9.
MEADOWS, D. H., D. L. MEADOWS, J. RANDERS and W. H. BEHRENS, III.
(1972). "The Limits to Growth" Universe Books, New York.
p. 205.
60
-------
METCALF, R. L., G. K. SANGHA and I. P. KAPOOR. (1971). Model
ecosystem for the evaluation of pesticide biodegradability
and ecological magnification. Environ. Sci. Technol. 5_, 709.
MOORE, N. W. (1967). A synopsis of the pesticide problem, j_n_
"Advances in Ecological Research," Volume 4, J. B. Cragg,
(ed). Academic Press, p. 75-128.
NISBET, I. C. T. and A. F. SAROFIM. (1972). Rates and routes
of transport of PCBs in the environment. Environ. Health
Perspect. ]_, 21.
ODUM, E. P. (1971). "Fundamentals of Ecology." Saunders,
Philadelphia, p. 574.
ODUM, H. T. (1972). An energy circuit language for ecological
and social systems: Its physical basis, I.n_ "Systems Analysis
and Simulation in Ecology," Vol. II, B. C. Patten, (ed).
Academic Press. New York. p. 591.
O'MELIA, C. R. (1972). An approach to the modeling of lakes.
Hydro! ogie 34, 1.
PATTEN, B. C. (1971). A primer for ecological modeling and
simulation with analog and digital computers, J.n_ "Systems
Analysis and Simulation in Ecology," Vol.. I. B. C. Patten
(ed). Academic Press, New York.
PATTEN, B. C. (1973). Need for an ecosystem perspective in
eutrophication modeling, J.n_ "Modeling the Eutrophication
Process," E. J. Middlebrooks, D. H. Falkenborg, and T. E.
Maloney (eds). Utah Water Research Laboratory, Logan Utah.
p. 227.
PATTEN, B. C., W. G. CALE, J. FINN AND R. BOSSERMAN. (In Press).
Propagation of cause in ecosystems, lr\_ "Systems Analysis and
Simulation in Ecology," Vol. IV, B. C. Patten (ed.).
Academic Press, New York.
QUINLAN, A. (1974). Personal Communication.
RANDERS, J. and D. L. MEADOWS. (1971). "System Simulation to
Test Environmental Policy: A Sample Study of DDT Movement
in the Environment." System Dynamics Group, Alfred P. Sloan
School of Management, Massachusetts Institute of Technology.
Cambridge, Mass. 52 pp.
61
-------
SCHINDLER, J. (1974). Personal Communication.
TOMOVIC, R. and M. VAKOBRATOVIC. (1972). "General Sensitivity
Theory." Elsevier, New York.
ULANOWICZ, R. E. (1972). Mass and energy flow in closed ecosystems.
J. Theor. Biol. 34, 239.
WEBSTER, J. R., J. B. WAIDE and B. C. PATTEN. (In Press). Nutrient
cycling and ecosystem stability, In, "Mineral Cycling in
Southeast Ecosystems," F. Howell Ted). AEC Symposium Series.
WOODWELL, G. M., C. F. WURSTER, JR. and P. A. ISSACSON. (1967).
DDT residues in an East Coast estuary: A case of biological
concentration of persistent insecticide. Science 156, 821.
WOODWELL, G. M., P. P. CRAIG and H. A. JOHNSON. (1971). DDT in the
biosphere: Where does it go? Science 174, 1101.
62
-------
SECTION IX
KEY LITERATURE SOURCES FOR PESTICIDE EFFECTS RESEARCH
GENERAL TEXTS - CHEMISTRY, MODELING AND BIOLOGY
ASHTON, F. M. and A. S. CROFTS. (1973). "Mode of Action of
Herbicides." John Wiley and Co., New York. 504 pp.
AUDUS, L. J. (1964). "The Physiology and Biochemistry of
Herbicides." Academic Press, London. 555 pp.
DeRUSSO, R. M., R. ROY and C. CLOSE. (1965). "State Variables for
Engineers." John Wiley & Sons, Inc., New York.
FEST, C. and K. J. SCHMIDT. (1973). "The Chemistry of
Organophosphorus Insecticides; Reactivity, Synthesis, Mode of
Action, Toxicology." Springer Verlag, Berlin; New York. 339 pp.
JACQUEZ, 0. A. (1972). "Compartmental Analysis in Biology and
Medicine." Elsevier, New York. 237 pp.
KARNOPP, D. and R. C. ROSENBERG. (1968). "Analysis and Simulation
of Multiport Systems." Massachusetts Institute of Technology,
Cambridge, Mass. 221 pp.
KEARNY, P. C. and D. D. KAUFMAN. (1969). "Degradation of
Herbicides." M. Dekker, New York. 394 pp.
LUKENS, R. J. (1971). "Chemistry of Fungicidal Action." Springer
Verlag, New York 130 pp.
MEYER, J. H. (1971). "Aquatic Herbicides and Algaecides." Noyes
Data Corp., Park Ridge, N. J. 176 pp.
O'BRIEN, R. (1967). "Insecticides: Action and Metabolism."
Academic Press, New York. 332 pp.
SONDHEIMER, E. and J. B. SIMEONE. (1970). "Chemical Ecology."
Academic Press, New York. 336 pp.
63
-------
REPORTS OF FEDERAL AGENCIES OF BROAD PROBLEMS
BALDWIN, I. L. (1962). Pest Control and Wildlife Relationships.
National Acad. Sci., National Res. Council.
JENSEN, J. H. (1965). Report of the Pesticide Residues Committee.
National Acad. Sci., National Res. Council.
_. (1969). Report of the Committee on Persistent
Pesticides. Div. of Biology and Agriculture, National Res.
Council to USDA.
MacLEOD, C. M. (1963). Use of Pesticides. President's Science
Advisory Comm.
MRAK, E. M. (1969). Report on the Secretary's Commission on
Pesticides and Their Relation to Environmental Health. To
U.S.D.H.E.W.
TECHNICAL DATA
ANON. (1972). Ecological Research Series. Office of Research and
Monitoring. U.S. Environmental Protection Agency, Washington,
D.C. Example: An Evaluation of DDT and Dieldrin in Lake
Michigan. EPA-R3-72-003, August.
. (1969). Effects of Pesticides in Water. A Report of the
States. U.S. Environmental Protection Agency. Office of
Research and Development.
. (1969). "Fish and Chemicals." A Symposium on Registration
and Clearance of Chemicals for Fish Culture and Fishery
Management. 99th Annual Meeting of the American Fisheries
Society, New Orleans, Louisiana. September 12, 1969.
. (1958-59). Handbook of Toxicology. National Acad. Sci.,
National Res. Council, Saunders, Philadelphia.
Vol. I. Acute Toxicities of Solids, Liquids, and Gases to
Laboratory Animals. W. S. Spector, ed.
Vol. III. Insecticides. W. 0. Negherbon, ed.
Vol. V. Fungicides. D. S. Dittmer, ed.
. (1972). Pesticide Study Series 2, 3, 5, 6, 7, 8, 9, and 10.
Environmental Protection Agency, Office of Water Programs.
64
-------
. U.S. Department of the Interior. Office of Water Resources
Research. Bibliography Series. Water Resources Scientific
Information Center, Washington, D. C. Examples: DDT in Water -
VJRSIC 71-211; Dialdrin in Hater - WRSIC 72-202; Aldrin and
Endrin in Water - WRSIC 72-203.
. (1972). Water Quality Criteria. A Report of the Committee
on Water Quality Criteria. Environmental Studies Board.
National Academy of Sciences. National Academy of Engineering,
Washington, D.C.
. (1971). Water Quality Criteria Data Book. Volume 3. Effects
of Chemicals on Aquatic Life. Water Pollution Control Research
Series 18050 GWV 05/71.
DYRSSEN, D. and D. JAGNER. (1972). "The Changing Chemistry of the
Oceans." Proceedings of the 20th Nobel Symposium, August 16-20,
1971, Goteborg, Sweden. Wiley-Interscience, New York. 365 pp.
EISLER, R. (1970a). Factors affecting pesticide-induced toxicity
in an estuarine fish. U.S. Bureau of Sport Fisheries and
Wildlife Technical Paper No. 45.
. (1970b). Acute toxicities of organochlorine and
organophosphorus insecticides to estuarine fishes. U.S. Bureau
of Sport Fisheries and Wildlife Technical Paper No. 46.
EPSTEIN, S. S. and M. S. LEGATOR. (1971). "The Mutagencity of
Pesticides." MIT Press, Cambridge, Mass.
GILLETT, J. W. (ed.). (1970). "The Biological Impact of Pesticides
in the Environment." Environmental Health Sciences Series No. 1,
Oregon State University, Corvallis, Oregon.
HEATH, R. 6., J. W. SPAWN, E. F. HILL and J. F. KREITZER. (1972).
Comparative Dietary Toxicities of Pesticides to Birds. U.S.D.I.,
Fish and Wildlife Service, Bureau of Sport Fisheries and Wildlife.
Special Sci. Report-Wildlife No. 152. Washington, D.C.
KRAYBILL, H. G. (ed). (1969). Biological Effects of Pesticides
in Mammalian Systems. Ann. M. Y. Acad. Sci. 160.
PIMENTAL D. (1971). "Ecological Effects of Pesticides on Non-Target
Species." Executive Office of the President, Office of Science
and Technology. Washington, D.C.
ROSEN, A. A. and H. F. KRAYBILL (eds).\ (1966). "Organic Pesticides
in the Environment." Adv. in Chem. Series 60_. American
Chemical Society, Washington, D.C.
65
-------
STICKEL, L. F. (1968). "Organochlorine Pesticides in the
Environment." U.S.D.I., Fisheries and Wildlife Service, Bureau
of Sport Fisheries and Wildlife. Special Sci. Report-Wildlife
No. 119.
TUCKER, R. K. and D. C. CRABTREE. (1970). Handbook of Toxicity
of Pesticides to Wildlife. U.S.D.I., Fish and Wildlife
Service, Bureau of Sport Fisheries and Wildlife. Research
Publication No. 84, Washington, D.C.
WILKINSON, B. K., L. S. CORRILL and E. D. COPENHAVER. (1974).
"Environmental Transport of Chemicals Bibliography."
Oakridge National Laboratory (ORNL-E1S-74-68). 185 pp.
TECHNICAL JOURNALS AND PERIODICALS
Archives of Environmental Contamination and Toxicology. Springer
Verlag (Quarterly).
Bulletin of Environmental Contamination and Toxicology. Springer
Verlag (Monthly).
Comparative Biochemistry and Physiology. Pergammon (Monthly).
Environmental Science and Technology. American Chem. Soc. (Monthly).
Journal of Agricultural and Food Chemistry. American Chem. Soc.
(Bimonthly).
Journal of Fisn Biology (Quarterly).
Journal of the Fisheries Board of Canada (Monthly).
Journal of the Water Pollution Control Federation (Quarterly).
Journal of Wildlife Management (Quarterly).
Limnology and Oceanography (Bimonthly).
Marine Pollution Bulletin (Monthly).
Nature (Weekly).
Pesticide Abstracts. EPA - Office of Pesticide Programs,
Washington D.C. (Monthly).
66
-------
Pesticide Biochemistry and Physiology. Academic Press.
New York (Monthly).
Pesticide Monitoring Journal. EPA, Washington, D.C. (Quarterly).
Residue Reviews. Springer Verlag (Irregular - several volumes
per year).
Science. Amer. Assoc. Adv. Sci. (Weekly).
Soil Science. Amer. Soc. Soil Science (Monthly).
Toxicology and Applied Pharmacology. Society of lexicologists
(Monthly).
Transactions of the American Fisheries Society. Allen Press, Inc.
(Quarterly).
Water Pollution Control Federation Journal (Monthly).
Water Research (Monthly).
Weed Science. Amer. Weed Soc. (Bimonthly).
ABSTRACT SOURCES
Biological Abstracts (Semi-monthly).
Chemical Abstracts (Weekly).
Pesticide Abstracts (Monthly).
Sport Fishery Abstracts (Quarterly)..
Water Pollution Abstracts (Monthly).
COMPUTER LITERATURE SEARCH DATA BASES
Medline (Biomedical) National Library of Medicine.
Toxline (Toxicology) National Library of Medicine.
WRSIC Water Resources Scientific Information Center
67
-------
SIE Science Information Exchange. Smithsonian Institution,
Washington, D.C.
NTI Search U.S. Department of Commerce.
ISI Institute for Scientific Information.
FEDERAL AND STATE RESEARCH LABORATORIES WHERE
BOTH DATA AND INTERPRETATION OF DATA IS AVAILABLE
National Water Quality Laboratory. Pesticide Research Team.
Mr. John G. Eaton, Coordinator. Duluth, Minnesota 55804.
Fish Control Laboratory, U.S. Bureau of Sport Fisheries and Wildlife,
P.O. Box 862, LaCrosse, Wisconsin 54601.
Fish-Pesticide Research Laboratory, Bureau of Sport Fisheries and
Wildlife, Route 1, Columbia, Missouri 65201.
Radiation and Metabolism Laboratory, U.S. Department of Agriculture,
Fargo, North Dakota 58102.
Gulf Breeze Environmental Research Laboratory, Sabine Island, Gulf
Breeze, Florida 32561.
Newtown Fish Toxicology Station, U.S. Environmental Protection Agency,
3411 Church Street, Cincinnati, Ohio 45244.
Southeast Environmental Research Laboratory, U.S. Environmental
Protection Agency, College Station Road, Athens, Georgia 30601.
Perrine Primate Laboratory, Wenatchee Research Section, U.S.
Environmental Protection Agency, P.O. Box 73, Wenatchee,
Washington 98801.
U.S. Environmental Protection Agency Laboratory, Region 10, 15345 N.E.
36th Street, Redmond, Washington 98052.
Office of Pesticide Programs, Criteria and Evaluation Division, U.S.
Environmental Protection Agency, Washington, D.C. 20250.
Gulf Coast Water Supply Laboratory, U.S. Environmental Protection
Agency, P.O. Box 158, Dauphin Island, Alabama 36528.
Idaho Fish and Game Department, P.O. Box 25, Boise, Idaho 83707.
68
-------
Fish Control Laboratory, U.S. Bureau of Sport Fisheries and Wildlife,
Route 1, Box 9, Warm Springs, Georgia 31830.
Southeastern Fish Cultural Research Laboratory, U.S. Bureau of Sport
Fisheries and Wildlife, Marion, Alabama 36756.
U.S. Environmental Protection Agency, Pesticide Monitoring Laboratory,
Bay St. Louis, Mississippi 29520.
Great Lakes Fishery Laboratory, Bureau of Commercial Fisheries, Fish
and Wildlife Service, U.S. Department of the Interior, Ann Arbor,
Michigan 48107.
Wisconsin Department of Natural Resources, P.O. Box 450, Madison,
Wisconsin 53701.
Agricultural Research Service Laboratories (U.S. Department of
Agriculture) Regional.
(Studies on nuisance aquatic insecticides, herbicides, etc.)
Department of Defense, Naval Ship Research and Development, Center,
Annapolis, Maryland 21402.
(Anti-Fouling Agents)
National Agricultural Library, U.S. Department of Agriculture,
Beltsville, Maryland 20705.
Alaska Department of Environmental Conservation, Pouch 0, Juneau,
Alaska 99801.
Conservation Library Center, Denver Public Library, 1357 Broadway,
Denver, Colorado 80283.
Division of Pesticide Community Studies, Office of Pesticide Programs,
Environmental Protection Agency, 4770 Buford Highway, Chamblee,
Georgia 30341.
Gulf South Research Institute, P.O. Box 1177,.New Lberia, Louisiana
70560.
Fish Control Laboratory, U.S. Bureau of Sport Fisheries and Wildlife,
Route 1, Box 9, Warm Springs, Georgia 31830.
Fish Farming Experimental Station, U.S. Bureau of Sport Fisheries and
Wildlife, Box 860, Stuttgart, Arkansas 72160.
69
-------
National Agricultural Chemicals Association, 1155 15th St. NW,
Washington, D.C. 20005.
Division of Biology and Agriculture, National Research Council, 2101
Constitution Ave. NW, Washington, D.C. 20418.
New Hampshire Pesticides Control Board, State House Annex, Room 201,
Concord, New Hampshire 03301.
New York State Department of Environmental Conservation, 50 Wolf Rd. ,
Albany, New York 12201.
Patuxent Wildlife Research Center. Laurel, Maryland 20810.
lexicological Research Laboratory. Veterinary Sciences Research
Division. Agricultural Research Service, USDA, P.O. Box 311,
Kerrville, Texas 78028.
Community Study Pesticide Project. Idaho Department of Health,
Statehouse, Boise, Idaho 83707.
Division of Wildlife Services. Bureau of Sport Fisheries and
Wildlife. U.S. Department of the Interior, 1717 H Street NW,
Washington, D.C. 20240.
Denver Wildlife Research Center. U.S. Bureau of Sport Fisheries and
Wildlife, Building 16, Federal Center, Denver, Colorado 80225.
COLLEGES AND UNIVERSITIES ASSOCIATED WITH
PESTICIDE RESEARCH OR PESTICIDE INFORMATION
Water Resources Research Institute, 314 Nuclear Science Center,
Auburn University, Auburn, Alabama 36830.
Lake Ontario Environmental Laboratory, College at Oswego, State
University of New York, Oswego, New York 13126.
Colorado State University, Fort Collins, Colorado 80521.
Department of Zoology, Mississippi State University, Mississippi
State, Mississippi 39762.
Department of Fisheries and Wildlife, Michigan State University, East
Lansing, Michigan 48823.
70
-------
Department of Entomology, School of Life Sciences, University of
Illinois, Urbana-Champaign, Illinois 61801.
Oregon State University, Corvallis, .Oregon 97331.
Department of Entomology, Fisheries, and Wildlife, University of
Minnesota, St. Paul, Minnesota 55101.
Cornell Pesticide Residue Laboratory, Cornell University, Ithaca,
New York 14850.
Trace Level Research Institute, Purdue University, Lafayette,
Indiana 47907.
Department of Environmental Health, University of Cincinnati College
of Medicine, Cincinnati, Ohio 45219.
Biological Sciences Library, University of New Hampshire, Kendall
Hall, Durham, New Hampshire 03824.
College of Agriculture and Environmental Science, Rutgers—the State
University, New Brunswick, New Jersey 08903.
Institute of Biological Sciences, School of Agriculture and Life
Sciences, North Carolina State University, Box 5306, Raleigh,
North Carolina 27607-
Rhode Island Agricultural Experiment Station, University of Rhode
Island, 113 Woodward Hall, Kingston, Rhode Island 02881.
University of California, Berkeley, Department of Entomology and
Parasitology, Berkeley, California 94720.
University of California, Davis, Department of Environmental
Toxicology, Davis, California 95616.
University of California, Riverside, Department of Entomology,
Riverside, California 92502.
Louisiana Cooperative Wildlife Research Unit. Louisiana State
University, Baton Rouge, Louisiana 70803.
Massachusetts Cooperative Wildlife Research Unit. University of
Massachusetts, Amherst, Massachusetts 01003.
South Carolina Community Pesticide Study. Medical University of
South Carolina, 80 Barre Street, Charleston, South Carolina
29401.
71
-------
College of Forest Resources. University of Washington, Seattle,
Washington 98105.
SOME PRIVATE CORPORATIONS HAVE PERFORMED
PESTICIDE RESEARCH AS RELATED TO AQUATIC LIFE
Bionomics, Inc., P.O. Box 135, Main Street, Wareham, Massachusetts
02571.
Industrial Bio-Test Laboratories, Inc., 1810 Frontage Road,
Northbrook, Illinois 60062.
Envirogenics Company, Division of Aerojet-General Corporation,
El Monte, California 91734.
Union Carbide Corporation, Tarrytown Technical Center, Tarrytown,
New York 10591.
Lakeside Laboratories, 1707 East North Ave., Milwaukee, Wisconsin
53201.
Syracuse University Research Corporation, Merrill Lane, University
Heights, Syracuse, New York 13210.
72
-------
APPENDIX
GRAPHIC REPRESENTATION OF PESTICIDES IN AQUATIC SYSTEMS
In approaching any problem, an investigator must first form a
mental image or conceptual model of the system. This conceptual
model usually is not well defined and varies considerably from one
investigator to another. With the complex problems associated with
environmental systems, solving and/or communicating the conceptual
model requires translation into the nonempirical language of
mathematics or symbolic logic.
Since direct translation of the conceptual model into mathematical
representation is awkward and difficult, the initial description is
best formulated into a graphic symbolism. The nature of the graphic
description is dependent upon the investigator's conceptualization of
the processes, the degree of resolution required, and data that are
available or that can be measured from experiments with the system.
The graphical representation is the heart of systematic experimental
design because the applicability of the ensuing analysis is limited
by the ability of the investigator to represent his conceptual model
of the system processes in graphic form. There is no best or correct
graphical representation of a system. They differ only in the degree
of realism and utility.
Graphical representations can be improved by iteration. After
application of analytical techniques, any unusual or unexpected
storage levels or flow rates may require modification of the
components or connectivity of the original graphical representation.
The nature of the iterative interactions among the graphical
representation, the mathematical model, and data acquisition is
presented in Figure A-l.
73
-------
Graphical Representation
Alternative Hypotheses
Qualitative
Mathematical Model
Analysis
Quantitative
Data Aquisition
Experiment
Constraints
Figure A-l:
Relationship Among Graphical Representations,
The Mathematical Model and Data Acquisition
(from QUINLAN, 1974).
Circuit diagrams (CLOSE, 1963) compartment diagrams (ODUM, 1971),
block diagrams (KUO, 1962) signal flow graphs (KUO, 1962), bond
graphs (KARNOPP and ROSENBERG, 1968), energy circuit language (ODUM,
1962), and Forrester diagrams (FORRESTER, 1971; MEADOWS et al., 1972)
are all examples of graphical representations of systems. Each has
advantages and disadvantages depending upon the nature of the system
to be described.
Bond graphs are excellent symbolic representations for
environmental systems in which energy flow is of primary concern
and in which complementary variables (a potential and a related flux)
may be defined. Compartment diagrams are useful representations of
74
-------
environmental systems when mass or energy storage and their rates
of exchange are of interest but complementary variables are not
explicitly defined (ULANOWITZ, 1972). Signal flow graphs and
associated control system analysis techniques are valuable when
feedback control properties of the system are of primary concern.
Forrester diagrams may be used in the general case to represent
interactions, transformations, and transports of mass or energy
without recourse to specific component equations or other constraints
upon the system variables.
A Forrester diagram can be used to present a conceptual model of the
transport and transformation of pesticides in the aquatic environment.
From this presentation a reduced or working model in compartment form
may be derived for a specific pesticide and specific ecosystem. The
compartment diagram should include the mathematical form of the
interactions and can provide a basis for preliminary system analysis
as an aid to experimental design.
Forrester Diagrams
In Forrester diagrams of dynamic systems, six symbols are commonly
used.
A solid line represents a directed pathway for transfer
of matter or energy.
A dashed line represents a directed pathway for control
or information transfer.
The cloud symbol represents a source or sink (input or
output) outside the defined system boundaries.
A rectangle indicates storage of matter or energy.
The valve symbol indicates rates along the associated
pathway.
Finally, the circle represents coefficients and
parameters that affect flow rates.
The degree of resolution or complexity of the Forrester diagram of a
75
-------
system may vary considerably depending upon application and resources
available for evaluating the hypothesis. While there appears to be
no upper limit to the resolution of a model, the lower limit (a single
storage component) is demonstrated for a stream and a lake in the
following examples (Figures A-2 and A-3) from O'MELIA (1972). The
low level of resolution in these examples does not necessarily imply
that there is a better representation for a particular application.
1
i
Input Rate
Output Rate
f
1
[
/
/
/
©
Figure A-2:
— =K,L-K?D where D = oxygen deficit, 1= BOD remaining,
KI = deoxygenation coefficient, and
Kj = reaeration coefficient.
Streeter-Phelps Oxygen Deficit Model for a Stream.
t
[X] '"put Rate
©
i
*
Output RKe
O
©
M,
Nutrient Concentration
-5^ = — -|o + q)M, where M. = concentration of nutrient,
* Z
j = flux of M to lake,
o = sedimentation coefficient,
q = flow coefficient, and Z= mean l*e depth.
Figure A-3: Vollenweider Lake Eutrophication Model
76
-------
One manner of increasing the resolution of a model is to divide a
single storage component into sub-units, which may have differing
rates of input or output for the stored variable. For example, the
nutrient concentration in the lake (Mw) from Figure A-3 may be divided
between abiotic storage (Ma) and biotic storage (Mb) with the result
shown in Figure A-4. If the output rate (from sedimentation and flow)
of the nutrient stored in the biotic component differs from that of
the nutrient stored in the abiotic component, then the mean residence
time is changed and the dynamic behavior of the nutrient output may
be changed considerably from the single storage representation.
Another means of increasing the resolution of the representation is
to include a time-varying parameter instead of the mean value of an
exogenous variable that controls a rate of flow for an endogenous
variable. Thus, instead of a mean lake depth (Z), a time varying
lake depth [Z(t)] could be incorporated in the model.
Finally, the rate controlling flow and storage may be explicitly
included in the representation and the two resulting subsystems can
be realistically coupled (HARRISON et al., 1970). This is
demonstrated in Figure A-5 for the biotic component of the lake
model in Figure A-4. These expansions of the diagrams may continue
until the point of diminishing returns is reached with respect to
either application or resources.
77
-------
*
[X] Input Rate
0©
i *
| Output Rate |)>
-------
Concentration in
Biotic Component
Rate
Coefficient
t
[X] Uptake Rate
Growth Rate
Rate
Coefficient
Rate
Coefficient.
t
•| Output Rate |Xl
Death Rate
Rate
Coefficient
i
B
Biomass
Coefficient "H R"Pira"°n 1X1
Figure A-5:
Possible Coupling of Biomass (B) Subsystems
with Nutrient Concentration (Mb) Subsystems,
79
-------
iJAlA
(I'lrase read Instructions on the reverse before completing)
4. TITLt AND SUBTITLE
A Conceptual Model for the Movrment of Pesticides
through the Environment: A Contribution of the EPA
Alternative Chemicals Program
I. HI PORT NO.
2.
3. RECIPIENT'S ACCESSION-NO.
5. REPORT DATE
November 1, 1974
6. PERFORMING ORGANIZATION CODE
. AUTHOR(S)
.
James W. Gillett, James Hill IV, Alfred W. Jarvinen
and W. Peter Schoor
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
National Ecological Research Laboratory
Environmental Protection Agency
Corvallis, Oregon 97330
10. PROGRAM ELEMENT NO.
1EA487
11. CONTRACT/GRANT NO.
17. SPONSORING AGENCY NAME AND ADDRESS
Same
13. TYPE OF REPORT AND PERIOD COVERED
.Final
14. SPONSORING AGENCY CODE
16. SUPPLEMENTARY NOTES
16. ABSTRACT xhis report presents a conceptual model of the movement and disposition of
pesticides in the environment. A multi-media model is built up from simple modules
representing basic processes and components of air, soil, and water. More specific
models are exposited for the atmospheric/terrestrial, freshwater aquatic, and
estuaring/marine environments. Through iterative operations of expansion and
systematic reduction of the components and processes these models of segments of the
environment can be joined to provide a holistic view of the disposition of a chemical
and its attendant effects. Ultimately systems analysis and mathematical simulation
techniques can be employed to evaluate the fate of a specific chemical in a par-
ticular environment. The conceptual model is thus a first step in organizing facts,
assumptions, and hypotheses into a graphic and logical arm capable of exploitation
in further experimentation of .pesticide disposition and effects. While rejecting
formulation of a model with global validity, the authors emphasize the commonalities
of the basic processes and components in the various environments. Thus, a multi-
media approach to disposition studies is made explicit even in the absence of a
single all-media global model.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Air Pollution
Conceptual Model
Ecology
Environmental Biology
Hazardous Materials
Mathematical Model
Pesticides
18. DISTRIBUTION STATEMENT
Systems Analysis
Water Pollution
Alternative Chemicals
Program
Laboratory Microcosms
Simulated Ecosystems
1201
0611
0606
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
20. SECURITY CLASS (Thispage)
22. PRICE
EPA Form 2220-1 (9-73)
U.S. GOVERNMENT PRINTING OFFICE: 1974-697-650/65 REGION 10
-------
CONTRIBUTION NO. 250
-------
Gulf Breeze Contribution 250
In: Marine Bioassays
Proceedings of workshop sponsored by
The Marine Technology Society, 1975.
(Entire proceedings may be obtained from
The Marine Technology Society, 1730 M Street, N.W.,
Washington, D. C. $18.00).
155
-------
HISTOIflGICAL AND PHYSIOLOGICAL EVALUATIONS IN SOME MARINE FAUNA
The development of pathology, as applied to aquatic toxicology, depends
heavily on the knowledge of normal histology and physiology if anomalies, due
to pollutants or disease, are to be accurately defined. However, at present,
knowledge of normal morphology or metabolic activities is either incomplete or
lacking for most marine or coastal organisms.
Obviously, well-coordinated efforts will be required to characterize nor-
mal ranges and interpret the morphological or physiological responses of aqua-
tic organisms to various factors including pollutants. Most laboratories can-
not justify well-defined pathological units, although the need often arises
through governmental enforcement organizations and other activities. The tech-
niques and examples thus presented are intended to offer some means of obtain-
ing evaluations of aquatic organisms' well-being or disorders.
I - Histological Techniques
A.- Fixation
Freezing is useful for preserving tissue for chemical analyses, isolation
of viruses or for sectioning on a cryostat if very small pieces of fresh tis-
sue are frozen very rapidly as by irrmersion in liquid nitrogen. Specimens
should never be frozen prior to routine histology because it destroys cells
and produces artifacts which make diagnoses difficult or impossible and pro-
hibits the taking of publishable photomicrographs. A common freezing arti-
fact is rows of vacuoles resembling fat cells. If a chemical fixative is not
readily available, refrigeration or wet ice, for up to 24 hours, and then fix-
ing, is preferable to freezing.
Chemical fixation, which is one of the most important steps in the prepa-
ration of specimens for histology, should be at or very close to the time of
death to insure that tissues are maintained as near to their natural state as
possible. The chemical fixatives most commonly utilized for vertebrate and
invertebrate marine organisms are Bouin's, Buffered Formalin, Davidson's, Die-
trich's, Zenker's and Kelly's.
1) Davidson's (Shaw S Battle, 1957) has proven to be an excellent fixa-
tive for marine organisms. This formula is close to that of Dietrich's, dif-
fering principally by using filtered seawater instead of distilled water, along
with the addition of glycerol. Results are particularly gratifying when the
seawater used is close to the salinity of-water where organisms were obtained.
For Davidson's, prepare a stock solution consisting of 1 part glycerin; 2 parts
formalin; 3 parts 95% ethyl alcohol; and 1 part seawater. Before use, add one
part glacial acetic acid to 9 parts stock solution. Fix in cold from 2t hours
to one week. Store in 60-70% ethyl alcohol.
2) Ten percent formalin (1 part 1*0% formalin; 9 parts H.O) is a good
general fixative with several practical advantages: a) it can be transported
as a concentrate and diluted with local water at the collecting site, greatly
reducing the weight and volume that must be carried. After a mininum of 18
hours' fixation, the excess formalin can be discarded to facilitate the return
of the specimens to the laboratory where they should be reformalized; (b) spe-
cimens can be stored for extended periods in neutral buffered formalin. If
initial fixation is unbuffered, it should be replaced as soon as practical
with buffered formalin; and (c) embedded formalin fixed tissue can be repro-
cessed for electron microscope studies with fair results. Tissues fixed with
the acid fixatives are unsuitable for subsequent electron microscope studies.
The major disadvantage of formalin is that it penetrates slowly, allowing
deep tissues to autolyze before being fixed. This disadvantage can be avoided
if all body cavities are completely opened up and large tumors and organs are
sliced part way through, every centimeter.
3) Bouin's (75 ml picric acid; 25 ml UO* formalin; and 5 ml glacial ace-
tic acid) is excellent for fish tissue; it penetrates well and decals scales
and small bones. Bouin's should be replaced with 70-80% ethanol between 18
and H8 hours and the alcohol changed regularly until the picric acid is mostly
156
-------
removed (routine histological processing removes some picric acid, but if the
initial burden is heavy, residual amounts will be carried through to the fini-
shed microscope slides and can be seen as crystals in the tissue).
4) Glutaraldehyde is recommended for tissues to be studied by electron
microscopy! Porter's "formula contains 3% glutaraldehyde; 0.1 M (2.14 gm/100
ml) sodium cacodylate buffer; 0.002 M (0.022 gm/100 ml) caCl,; and 10% glucose.
Adjust pH to 7.U with HCL or NaOH. Cut pieces of tissue witn one dimension
less than 1.0 mm with new alcohol-cleaned razor blades and immerse for a mini-
mum of two hours. Keep refrigerated. Tissues can be safely stored in the
fixative indefinitely.
5) Dietrich's FAA (10 ml 40% formalin; 2 ml glacial acetic acid; 30 ml
95% alcohol; 60 ml JO) is recommended for terrestrial and aquatic arthropods
and molluscs. The arthropods should be fixed initially in a vacuum chamber
to facilitate replacing the air in the trachea with the fixative. In the
field, Dietrich's fixative has proven to be a very satisfactory fixative of
marine organisms, based on the following criteria: (a) rapid tissue penetra-
tion, (b) tissue preparation does not require transfers to other liquids,
(c) tissues, when fixed, are firm but not brittle, (d) fixed tissues may re-
main in this preparation for extended periods (not to exceed 6 months, prefe-
rably), (e) decalcification occurs simultaneously with fixation (in small te-
leosts and shrimps, decalcification is completed within approximately 10 days),
and most important, (f) superior cellular detail is obtained with routine his-
tological processing. Tissue-fixative proportion should not be less than 100
grams of tissue per liter of fixative, or 10 times the volume of fixative.
In the laboratory, excellent fixation of tissues for light microscopy is
accomplished with Kelly's or Zenker's fixative, but time and management of
liquid changes can often be a deterrent to the use of these preparations.
Therefore, Davidson's or Dietrich's fixative is used routinely in many labora-
tories .
Teleost tissue fixation and preparation for light microscopy should be
done according to the following general guideline: teleosts having a total
length of t cm or less are routinely immersed directly into the fixative, and
require no further preparation prior to trimming for parafin embedding. Tele-
osts t to 12 cm in total length are prepared as follows: (1) immerse the whole
animal in fixative for 5 to 10 minutes, (2) remove the whole animal and tran-
sect the caudal peduncle immediately posterior to the anal opening, (3) open
the body cavity with a longitudinal, ventral incision, (4) remove the opercula,
and (5) complete the sagital section of the organism after approximately 30
additional minutes of fixation. All fishes greater than U cm in length and
all crustaceans should be decalcified prior to final tissues trimming. Com-
mercially available products (Decal*) are available for tissue decalcification.
Flat pieces from biopsies and autopsies, such as skin, should be backed by a
piece of thin cardboard before fixing to prevent shriveling.
One of every five specimens, at least, are prepared for examination by
transverse sectioning. Transections, at intervals of approximately 1 cm, are
made beginning with ventral incisions that penetrate the soft tissues to the
vertebral column. Following a preliminary fixation (approximately 30 minutes),
the transection is completed with the scalpel by a "sawing action". However,
some instances may require "crushing", or forced penetration of ossified and
cartilagenous tissues.
The following additional steps are normally required to prepare teleosts
having a total length greater than 12 cm: (1) excess dorsal musculature is
trimmed away, (2) a dorsal-median incision is made to open the cranium, (3)
lateral musculature of at least one flank is removed, and finally (1) the ver-
tebral column is severed at intervals of approximately 2 cm with scissors.
These tissues are returned to the fixative prior to triuming and embedding.
Fixation is accomplished by immersion of select tissues or whole animals
into the fixative. Prior to immersion, soft-bodied species (i.e., those
(*) Omega Chemical, Cold Spring in Hudson, New York, N.Y. Mention of this
product does not constitute endorsement.
157
-------
without firm skeletons such as roundworms, flatworms, and octopods) are relax-
ed in MgCl2, chlorobutanol, refrigeration, etc.), arthropods have their exo-
skeletons opened and trapped air evacuated from their trachea by vacuum cham-
ber, and bivalve and gastropod molluscs are shucked from their shells. _After
an initial fixation of 10-15 minutes in Dietrich's and somewhat longer in Zen-
ker's for large whole arthropods such as lobsters and certain crabs, one
should: (1) remove appendages, (2) make^a sagittal section through_the cara-
pace, and (3) remove visceral tissues and return them to the fixative. As a
further aid to penetration of the fixative,• whole large organisms such as the
lobster may be step-sectioned according to convenience, but the slices should
not exceed 2 cm in thickness. The larger molluscs should be sectioned at in-
tervals of approximately 1 cm after initial fixation and returned to new fixa-
tive. When selected tissues are fixed rather than the whole animal, they
should be suspended in the container by placing cheesecloth or absorbent paper
towels on the bottom. Buoyant tissues, on the other hand, should be held im-
mersed in the fixative by placing paper towels at the surface.
For final trimming, the tissue blocks are oriented so that the microtome
sections will come off in the plane desired for examination. In preliminary
studies of an organism (control-or exposed), the tissues taken for histologi-
cal examination should be as extensive and complete as possible.
Data accompanying organisms in shipment should be recorded on hard paper
tags or keyed to numbered plastic tabs placed in the fixative with the res-
pective tissue. Data accompanying a specimen's container is preferably recor-
ded with lead pencil since inks (ballpoint) usually dissolve when placed in
fixatives, or on package labels during freezing and thawing. Fixed tissues
can be shipped in compact containers such as small, plastic "whirl bags" with
excellent results. The volume of fixative in each tissue container need only
be sufficient to immerse the previously fixed tissue or organism. Cotton is
added to the container to insure that all the tissue remains moist in the
event of leakage during shipment. All tissue containers should be placed in
a shipment carton lined with a large, sealed, plastic bag to contain possible
leakage during transit.
The rate at which, postmortem changes or tissue autolysis occurs have been
determined for the mummichog by light microscopy (Gardner, unpublished). Fif-
ty-four adult mummichogs (Fundulus heteroclitus) were sacrificed in natural
seawater (20°C and 20°'oo salinity) containing 2 mg/1 or MS-222. Following
death, the.specimens were allowed to remain for preselected intervals of time
in the aquaria prior to removal and fixation. Two specimens were removed
every 2 minutes until 30 minutes and two every 15 minutes thereafter (or up
to 3.5 hours).
Histological examination of these Fundulus revealed obvious tissue auto-
lysis , first appearing apically in villus folds of the intestinal mucosa 10
minutes after death. Peripheral areas of the liver were atrophied at approxi-
mately 20 minutes. The changes proceeded to encompass the complete organ wi-
thin a period of 3.5 hours. Autolysis also occurred in tissues adjacent to
the gall bladder after 28 minutes, while changes in the intestinal track in-
cluded the submucosa after 1 hour-30 minutes. The first indication of auto-
lysis in the respiratory and oral epithelium occurred after 1 hour-15 minutes.
The pseudobranch, sensory, renal, and gonadal tissues were noticeably affec-
ted after 2 hours-45 minutes. It must be realized that these autolytic chan-
ges will conceivably be intensified by increased temperature, for instance.
Atrophy, or distortion of epithelial surfaces will also occur if the organism
is removed from the aquatic environment and exposed to prolonged air-drying.
Thus, it is imperative that tissue fixation be completed as quickly as possi-
ble. Msribund specimens are valuable for revealing the nature of a toxic res-
ponse, however, those collected dead usually have little value for histopatho-
ligical evaluation.
The importance of proper tissue fixation can be further illustrated by
the observations of intestinal lesions produced in the ileum and rectum of
adult Fundulus by 5500 mg/1 (LC-50, 168 hrs.) of NTA (Eisler et al.., 1972).
The induced lesions originated at the apice of the villus structure as indica-
ted by subacute NTA exposure (1 mg/1). It is easy to realize that inadequate
fixation would have produced similar changes. Cadmium-induced damage in the
158
-------
intestine of Fundulus also originates at the apice of the villus structure as
early as 2 homj-s after exposure to the product (Gardner 6 Yevich, 1970).
Therefore, delayed or improper fixation which promotes autolysis would un-
doubtedly conceal early development of lesions, as indicated above.
II Processing of Tissues for Microscopic Examinations
"Detailed technical procedures for fixation, tissue preparation, staining
and microscopy are to be found in a large number of publications. Among the
more valuable sources for methods in histopathology, histochemistry and elec-
tron microscopy are: Jones (1966); Luna (1968); Galigher S Kbzloff (1971);
Pease (1964); Lillie (1965); Hayat (1970); and Zugibe (1970).
These texts can also be complemented by more specialized studies (Gard-
ner 6 Yevich, 1970; Gardner 6 La Roche, 1973; Couch, 1974b; Couch £ Nimmo,
1974a).
Ill Morphological and Physiological Changes Resulting from Pollutant
Select organs of some common species are briefly discussed as normal
structures or morphological alterations resulting from toxicant exposures.
These limited examples serve to illustrate the use of histology as an essen-
tial complement to marine bioassays.
The following species are among thosB considered to be valuable marine
species for use in bioassays of toxic materials:
A.- American Oyster (Crassostrea virginica) an estuarine mollusc
1) Normal histological parameters:
As already indicated, an organism that would serve as an indicator of
biologically damaging pollutants in estuaries must possess a number of charac-
teristics. Wide geographic range is desirable to allow comparative studies
of control and exposed organisms under differing environmental conditions and
different locations. A species whose morphological and physiological proper-
ties are understood. The ability to rear the organism under controlled popu-
lations and specimens exposed to various changes of the natural environments.
The American oyster, Crassostrea virginica (Gmelin), fills these crite-
ria. Its geographic range extends frcm Prince Edward Island, Canada, along
the Atlantic coast to the Gulf coast of Texas. It has been claimed that the
oyster is the best known marine organism. It is now feasible to spawn adult
oysters, rear the larvae and maintain the spat and juvenile oysters under con-
trolled laboratory conditions. For these reasons, the American oyster should
make a valuable bioassay organism to evaluate estuarine conditions.
The voluminous literature on oyster biology is scattered through numerous
scientific publications. However, for individuals initiating studies on the
American oyster, biology and disease, several references may serve as entries
into the field (Galtsoff, 1964; Cheng, 1967; Johnson, 1968; Sinderman, 1970;
Sparks, 1972).
Histological study of oysters can be carried out using standard techni-
ques of tissue fixation and staining. As in all histological work, investiga-
tors have developed special procedures found to be especially useful when par-
ticular cellular features are to be studied. Because of this, i*ien a specific
problem arises, one must consult the original literature for variant techni-
ques. By utilizing the references in the literature, appropriate techniques
can be found and adopted readily. One should be aware that most histological
techniques developed for vertebrate tissues require variations to be useful
for invertebrate tissues.
There is no single publication containing a detailed description of oys-
ter histology, but Galtsoff's monograph (1964) contains much useful informa-
tion and numerous references. Histological descriptions of various oyster
tissue are scattered through the literature so that each tissue or organ may
159
-------
be described with particular emphasis. For example, Loosanoff (19142) descri-
bed the histology of gamete development in C_. virginica when the laboratory
was involved in development of techniques for artificial spawning. In 1957,
Shaw and Battle published the definitive work on digestive tract histology
while studying digestion in the oyster. Considerable information and specula-
tion about "normal" oyster tissues has been published by persons studying oys-
ter diseases; for example, Pauley S Sparks (1966) described acute inflammatory
reactions and necessarily described the comparable "normal" tissue of control
oysters. Thus, the best way to find a detailed histological description of
oyster tissues is to seek appropriate references to original research papers.
Assuming one knows basic oyster histology, it is still necessary to iden-
tify common pathological conditions unrelated to the specific insult under
consideration. Two examples of this problem are: (1) effects of non-specific
stress and (2) infection. Oysters in unfavorable environments have a tendency
to seal their valves and "ride out the storm". Thus, heavy rains, decreasing
salinity below a tolerable minimum, or silting, or blooms of non-food orga-
nism, may cause oysters to close their valves tightly. With time, this ces-
sation of feeding may lead to tissue and cell destruction, disruption of meta-
bolism and reduction of reproductive potential. In some areas, infection of
oysters by certain parasites is nearly universal (i.e. Nematopsis ostrearum
in Delaware Bay), but little tissue damage is noted in most infected indivi-
duals. These natural occurences have to be evaluated individually and with
some experience of local conditions, reasonable interpretations of tissue da-
mage are possible. With this baseline information mastered, one may then turn
to the effects of additional factors.
The oyster chiefly consists of parenchymal cells (leydig cells, vesicu-
lar connective tissue cells), abductor muscle, heart, kidney, gills, mantle,
gut, gonad, and hepatopancreas, all bounded by various epithelial layers.
The normal histology of the oyster is described in numerous publications, the
most extensive of which is the book by Galtsoff (196t).
2) Toxicant-induced histopathology:
The following conditions may indicate altered well-being in oysters chro-
nically exposed to toxicants when compared to oysters from control groups:
a) gross emaciation; watery, pale digestive gland (effects of post-
spawning stresses also produce this condition);
b) abcesses or pustules on mantle or inner shell;
c) production of large amounts of pseudofeces;
d) loss of vesicular pattern of parenchymal cells with breakdown
in supportive stromata; this may be associated with gross ema-
ciation (see a);
e) massive or heavy infiltrates of leukocytes into regions of pa-
renchymal cells and basement membrenes;
f) metaplasia in digestive diverticula, particularly involving
the non-ciliated epithelium of the distal tubules, and/or the
epithelium of the proximal tubular ciliated epithelium; atro-
phic changes in the distal epithelium of the digestive tubules
may indicate a critical irritant response (Couch, in Lowe et al,
1972), and be associated with gross emaciation (see a) resulting
from chronic interference with normal absorption of nutrients;
g) edema histological separation of parenchymal tissues from
basement membranes of digestive tubules, stomach, or intestine
due to fluid pressure;
h) hyperpLasia or generally, metaplasia, of gill and mantle epi-
thelia may produce abnormal epithelial tissues several cell
layers deep, instead of normal cuboidal or columnar epithelia.
Hyperplasia may indicate an irritant response of exposed epithe-
lial surfaces;
i) higher prevalences of the following oyster pathogens in exposed
versus control bioassay oysters:
Minchinia nelsoni (MSX) - Couch, 1966, 1967;
Minchinia costalis (SSO) Couch, 1967;
- Labyrinthbmyxa marina (Dermocystiduim marinum)- Ray, 1954;
160
-------
Hexamita (flagellate);
• Nematopsis (gregarine);
j) Finally, inhibition of gonadal development or gametogenesis as
compared to that in control or feral oysters of the same size or
age, during spring, summer and early fall seasons.
3) Physiological Indices:
a) Shell deposition
Under optimal conditions, yearling oysters usually grow rapidly and uni-
formly. A technique for determining suboptimal environmental conditions is
to measure the lack of shell deposition in young oysters (Butler et al., 1960).
Young oysters selected from a population for size, have the new shell filed
off. Half of these oysters are placed in uncontaminated flowing water with
an adequate food supply and the other half are placed in suspect water. Every
other day, the total length of each oyster is measured.
After 3 weeks, each oyster is refiled to the original length and the ex-
periment repeated. The presence of pollutants affecting the oyster will ge-
nerally affect adversely shell growth rates when compared to oysters maintai-
ned in uncontaminated water (Andrews, 1961). To determine the permanency of
the damage, oysters whose shell growth has been inhibited can be placed in un-
contaminated water and their growth rate compared to controls. In most cases,
the absence of toxic materials will allow a resumption of normal shell growth
rate. Growth inhibition is not a specific reaction and independent chemical
analysis of test waters are essential to identify toxic chemicals (Butler,
1966).
b) Water pumping
Lamellibranch molluscs are dependent on the flow of water over certain
tissues to supply oxygen and food and to remove wastes. Water transport is
accomplished by synchronized continuous beating of thousands of lateral cilia.
The adductor muscle, the mantle edge, the gill muscles and ostea all play a
part in regulation of the flow of water. Numerous techniques have been devi-
sed for determining rate of water flow and several are discussed in detail by
Galtsoff (Chap. IX). Several groups of substances or changes in seawater
will influence rate of pumping and, for this reason, care should be taken to
establish reproducible control conditions before toxic response studies are
undertaken. Considerable variations between individual oysters are also
known to occur normally and must be taken into account. Toxic materials, ex-
cessive particulate matter, drastically modified salinities will, for instance,
be sensed by oysters and will often result in decreased water pumping. This
technique would thus appear to be relatively sensitive, but nonspecific in
the establishment of water quality events which would affect this species.
Kymograph recordings of shell movements of a group of test oysters, compared
to comparable data from control animals, can yield valuable information about
water quality (Butler, et al., 1960).
In oysters, water pumping (or transport) and respiration are intimately
interdependent. Rate of oxygen uptake can be measured directly by microdeter-
mination of oxygen content of water taken from the inhalent and exhalent cur-
rents of individual oysters. These procedures are discussed in Galtsoff (Ch.
IX). Reproducible values are obtained with practice and toxic concentrations
of pollutants in water would generally cause significant changes in the abi-
lity of oysters to extract sufficient oxygen from the water, even though pum-
ping rates may remain unaffected for a period of time. Prolonged lowering of
oxygen fixation will inevitably reduce physiological activities, including
pumping rates.
c) Blood proteins
A traditional method for gauging the overall health of an organism is to
monitor its blood protein composition or concentration. In vertebrates, this
valuable information can assist in the diagnosis of certain anomalies; in oys-
ters and other invertebrates, the information obtained is less definitive but
may be useful in the establishment of disorders related to nutritional or me-
tabolic activities.
161
-------
Tbtal blood cell (amoebocyte) counts can be made vising simple enumerative
techniques (Feng, 1965). Total hemolymph protein concentrations can be deter-
mined by standard methods (Lowry, et'al., 1951). Electrophoresis of hemolymph
protein can also be done with minor modifications of standard procedures
(Feng and Canzonier, 1970). Lysozyme is found in oyster hemolymph and can be
quantified (Feng and Canzonier, 1970). This sort of analysis of oyster^hemo-
lymph, to relate concentrations and distributions of natural products with
overall health, can be a sensitive and valuable tool; however, this approach
has not been fully exploited thus far.
d) Other tissue biochemistry
If oysters recognize and react to changes in their environments, it_can
be assumed that measurable biochemical changes may accompany their adaptive
or toxic responses. For instance, gross observation of oysters subjected to
prolonged exposure to low salinity demonstrates that they become "thin" and
"watery" (i.e. the tissues have become translucent). This is often associated
with loss of tissue glycogen; the product concentrated for eventual energy
production under anaerobic conditions. Sophisticated studies show other ef-
fects of stress that are reflected in biochemical changes (Hammen, 1969). The
basic patterns of tissue metabolism are similar to those found in other spe-
cies. However, the development of selective analysis,identifying key enzyma-
tic reactions and quantitative measures of products formed, need to receive
further attention. A comparative approach, evaluating changes between control
and exposed organisms, would be a powerful tool in the identification of envi-
ronmental changes affecting the survival of oysters in a particular area.
e) Gonadal development
For the continued presence of the species to be assured, in a particular
location, gamete formation and spawning must occur in a significant number of
oysters each year. It is known that molluscan spawning follows irregular an-
nual cycles, but it is evident that prolonged low yield reproduction will ul-
timately accompany the disappearance of a population. Colonization by plank-
tonic oyster larvae from distant points via tidal movements is possible, but
not nearly as efficient or effective as the successful spawning of the local
population, particularly if local conditions limit the survival of setting or-
ganisms. Thus, any factor that decreases the ability of adult oysters to
form gametes and insure adequate fertilization is a threat to the population.
In adult oysters, gamete formation is dependent upon obtaining adequate
nutrients, active feeding and successful incorporation of elements essential
for gamete synthesis. Anything interfering with feeding, digestion and meta-
bolic activities for prolonged periods of time would adversely affect repro-
duction. Gamete production can be monitored by gross observation of indivi-
dual oysters during the normal spawning season (mid-July through early-Septem-
ber, in the mid-Atlantic region: significant variability is notable in other
areas). Semi-quantitative measurements of gonad development can be made
(Tripp, 1974) and related to environmental parameters. This is done by cut-
ting sections through the middle of the oyster so as to include the maximal
cross-sectional gonadal area. The tissues are fixed, stained and examined
microscopically. A semi-quantitative scale for measurement of gonad maturity
is established. A rough measure of the condition of the population can be ob-
tained by calculating the "gonad index" (the average degree of gonad develop-
ment for several individuals sampled). This, in turn, can be correlated with
environmental parameters and certain inferences can be drawn (Tripp, 1974).
As indicated earlier for several measurable parameters, this type of biologi-
cal reaction is not specific to any particular product or environmental chan-
ges. Probing environmental analyses are the only means of identifying the
single or multiple causative factors inducing anomalies.
f) Larval development
Perhaps the most developed of all bioassay techniques involving oysters
is the use of larvae as early indicators of water quality (Davis, 1961; Woelke
1962, 1967 and 1972). This procedure requires collection of adult oysters
from natural populations and maintenance under optimal developmental condi-
tions until gonads are fully mature. At this time, spawning of individuals
-------
may be stimulated by raising the temperature of running seawater to 25-30°C
for 2-3 hrs. Embryos may then be collected and held in large cultures (20,000-
30,000 fertilized eggs per liter) until 48 hrs. old. During this time, they
are exposed to test water and, at the end of the 48 hr. test period, they are
examined and compared to normal control larvae. The. effect of any variable
tested is described in terms of the per cent oyster larvae which develop ab-
normally (Woelte, 1967). Mortality can also be determined and LC_n calculated
(Davis, 1961). su
B.- Pink Shrimp (Penaeus duorarum) - an estuarine, marine decapod
The pink shrimp is found fron Brazil to Virginia, in estuaries, as juve-
niles, and in the ocean, as adults and larvae. It is one of three corcmercial-
ly valuable penaeid shrimp species in the South Atlantic, and Gulf of Mexico
(Williams, 1965). The species has been used successfully in many bioassays.
These bioassays have included LC50 and EC^Q determinations for several toxi-
cants as well as estimates of effects on several physiological parameters
(Nimnro et al., 1974). Couch (1974a,b) and Couch and Nimmo (1974a) have stu-
died the histology, ultrastructure and pathology of this species in relation
to toxicant exposures. The normal histology of select organs of the pink
shrimp has also received considerable attention.
The hepatopancreas of the pink shrimp appears to be a sensitive organ in
the histopathological detection of toxicant effects in Crustacea. Both light
microscopical and electron microscopical methods are proving useful in deter-
mining morphological changes (Couch, 1974b, Couch and Nimmo, 1974a).
1) Hepatopancreas normal appearance
This organ consists of acini or digestive tubules which extend from two
hepatopancreatic ducts that have their origins in the lateral walls of the py-
loric stomach. The functional and structural unit of the organ, the acinus,
consists of a tube lined with epithelial elements of varied structurally and
functionally defined cell-types. These cells are:(a) the embryonic cells,
found at the blind-pouch end of the acinus, distal to the hepatopancreatic
ducts; these cells are active mitotically and serve as generative or blas-
toid cells which give rise, through differentiation, to the cell types more
proximal (in tubule epithelium) to the hepatopancreatic ducts; (b) absorptive
cells, found distal-to-medial along the acinus, possess fine structural fea-
tures similar to absorptive cells in the vertebrates, (i.e. microvilli, api-
cal region of phagocytic vesicles, mitochondria and lysosomes, and a base nu-
cleus) ; these cells have been studied intensively by light and electron mi-
croscopy following exposure of shrimp to toxicants (Couch and Nimmo, I974a);
(c) secretory cells, found medial and proximal to hepatopancreatic ducts along
the acinus epithelium; these cells are characterized by the presence of large
granules and vacuoles and may be both holocrine or apocrine in secretion of
contents (presumably digestive and lubricative materials) into the lumen of
the acinus.
The epithelium making up the walls of the ducts and tubules of the center
of the hepatopancreas consists of cells usually in an atrophied condition.
Renewal and growth of the whole organ occurs at the distal ends of the acini
in the peripheral region of the organ where embryonic cells abound.
2) Hepatopancreas pathological alterations
Certain of the following histological and cytological alterations have
been found in hepatopancreas of pink shrimp exposed to polychlorinated biphe-
nyls (PCB's) in flowing seawater bioassays; most of these alterations are pro-
bably non-specific and, thus, may also be indicators of toxicity caused by
other pollutants:
a) histological changes (light microscopy): hepatopancreatic epithelial
cell lysis, nuclear pyknosis, vacuolization (larger vacuoles than
found in normal secretory cells); large pyramidal, tetrahedral in-
clusion bodies of Baculovirus penaei in nuclei (Couch, 1974a,b,d).
163
-------
b) histochemical abberrations: excessive lipid accumulation, glycogen
loss, reticulin stromatic changes from normal, and selected - enzyme
loss of activity.
c) ultrasrructural changes in the c;-toplasm, and nucleus of absorptive
cells; loss of normal microvillui. boarder at the apical end of cells;
swelling of mitochondria; and formation of myelin bodies in cytoplasm;
loss of normal composition of cytoplasm, i.e. abnormal increase in
free ribosomes or loss of all organelles; nuclear hypertrophy, chro-
matin abberrations, or diminution and formation of vesicles within
nucleoplasm (Couch and Nimmo, 1974a).
d) higher prevalence and intensities of viral hepatopancreatic infections
(Baculovirus penaei, Couch, 1974a,b,d,) in exposed compared to control
shrimp, i.e. possible augmentation of natural pathogens in the pre-
sence of pollutants (Couch and Nimmo, 1974b). Both light microscopy
and electron microscopy are needed to detail the diagnosis.
Most of the above lesions have been found in experimentally exposed
shrimp. However, more research is needed to clarify their significance in
relation to functional anomalies.
C.- Spot (Leiostomus xanthurus) - an estuarine, marine fish
The spot is found in abundance along the Gulf and Atlantic coasts, in es-
tuaries in the spring, summer, fall and even in the winter in the South. This
fish is found as far north as New York and throughout the Gulf of Mexico coas-
tal regions. It also may be obtained in several life-cycle stages, accord-
ing to season of the year. The spot may be easily obtained for bioassay ex-
periments in its range. For a biology of the spot, the reader is referred
to Dawson (1958).
1) Liver - normal histological parameters
The normal liver of spot is of the tubulosinusoidal type (Elias £ Ben-
glesdorf, 1952). The liver parenchymal cells are arranged in cords or muralia
which are usually two hepatic cells thick. There are no well-defined lobules,
although the hepatic artery, portal vein and bile duct are found close toge-
ther. The portal vein and bile duct are usually paralleled by disseminated
pancreatic exocrine tissue (Couch, 1974c).
In normal, well-fed fish, hepatic cells show moderate to heavy PAS posi-
tive reactions and diastase lability indicating the presence of glycogen.
Starved or stressed fish show less PAS affinity. Normal spot usually reveal
few, small lipid droplets or no lipid in hepatic cells (oil Red 0 method,
with frozen sections).
2) Liver - pathological Alterations
The liver of this fish has been histologically and histochemically exami-
ned following exposure to the following toxicants: PCB's (Aroclors 1254, 1016;
Couch 1974c), Dieldrin (Parrish, 1974), Endrin (Lowe, 1965) and Sevin (Couch,
1974c). The above listed chemicals may be found in various estuaries and ma-
rine environments. The pathological studies reported herein were performed on
fish exposed in laboratory flow-through bioassays to which controlled low le-
vels of the toxicant were administered continuously. The following changes in
livers of spot exposed to any given toxicant may be observed in the course of
such exposures:
a) alterations in the orientation of liver cord relationships and
hepatic cells;
b) lytic or pyknotic changes in hepatic cells and nuclei;
c) vacuolization, fibrosis, cirrhosis and necrosis of hepatic tissues'
d) morphological anomalies in intrahepatic pancreatic exocrine tissues
such as excessive vacuolization, loss of basophilia and nuclear de-
generation ;
e) glycogen loss or excessive accumulation; lipid increase, particu-
larly "fatty liver" syndrome, or fatty metamorphosis (histochemical
methods); researchers should be aware that these changes may also
164
-------
result from normal seasonal variations;
f) prehepatomatic signs, and hepatoma or other neoplastic lesions;
g) cholangiolar proliferation;
h) red blood cell occlusion or stasis in portal or central veins;
i) excessive pigment deposition in or between hepatic cells (i.e.
ceroid or hemosiderin);
j) ultrastructural changes (as observed with electron microscopy),"
such as abnormal proliferation of endoplasmic reticulum, lipid
accumulation in hepatocytes, myelin body formation, and nuclear
change.
The preceding list of possible morphological alterations is not meant to be
exhaustive, by any means, but it includes alterations noted in livers from
fishes exposed to known levels of toxicants (Couch, 197Mc). Alert observers
should note combinations of the above or other lesions not listed.
Certain dysfunctional states in the organism may be related to specific
lesions in the list above. Identification of functional disorders associated
with specific lesion or histopathological syndrome would have to be resolved
by appropriate physiological or biochemical investigations.
D.- Remarks
Guided by observations made under natural conditions, exposure experi-
ments of organisms to toxicants must be conducted under controlled laboratory
conditions. With acute exposure studies, the researcher is generally able to
estimate the concentrations of toxicants to be used in chronic bioassays. From
the histological standpoint, organisms are evaluated for presence and repro-
ductability of lesions in all instances. Whenever possible, the exposure and
histopathologic studies are followed by, or associated with observations of
field conditions to determine whether the reaction(s) of non-captive organisms
exposed under natural conditions compare with laboratory findings. These re-
sults , combined with those from other scientific approaches, can provide the
scientific basis for establishment of Water Quality Criteria (W.Q.C.). His-
tology provides an essential dimension which often confirms or explains the
damaging effects of certain products before they are introduced into the en-
vironment, such as would have been the case with NTA (a proposed substitute
to phosphate in laundry detergents) (Eisler et al., 1972).
Heavy metals, pesticides and petroleum products represent major concerns
in terms of environmental contamination. Minimata, or Itai-Itai disorders are
disturbing examples of what too much mercury in water can do to the human
body. Too much cadmium affects the kidney of some fishes in a fashion simi-
lar to that documented for humans (Gardner S Yevich, 1970). For instance, ac-
cumulation of cadmium in the kidney to certain levels will induce irreversible
damage in the renal tubules of Fundulus.
Copper is also known to promote lesions in the kidney of some fish and
of some invertebrates. In addition, copper has demonstrated neurotoxic pro-
perties . Preliminary observations have indicated that behavioral changes were
associated with exposures to sublethal copper concentrations in fish. Histo-
logical examinations of Fundulus and Menidia menidia (Atlantic silverside)
exposed to copper confirmed the presence of morphological alterations in vital
sensory organs (Gardner £ La Roche, 1973). These lesions are significant be-
cause they involve the lateral line (mechanoreception) and the olfactory or-
gans (chemoreception). Obviously, these organs are vital to perception and
normal behavior patterns such as those of feeding, schooling, reproduction
and migration. From the observations reported (Gardner 6 La Roche, 1972), it
is unlikely that affected fish could cope with either preys or predators in
a natural environment. Mercury and silver also damages some perception organs
of the sensory system, although the lesions differ in appearance. Damage to
sensory organs have also been demonstrated in menhaden (Brevoortia tyrannus)
obtained from three different sites in the environment (Gardner 6 La Roche,
1973; Gardner, unpublished). In these instances, circumstantial evidence as-.
sociates the lesions with high soluble copper concentrations in the water co-
lumn at a power plant discharge (70 ppb), in Narragansett Bay, Warwick, R.I.
(30-100 ppb) and in the Acushnet River, New Bedford, Mass. (230 ppb).
165
-------
The prospect of combined metal wastes can be disturbing, especially
when additive or synergistic responses are expected. The combination of cad-
mium arid copper represent an example of two metals that can act synergistical-
ly (Eisler £ Gardner, 1973). In Fundulus, low acute toxicity for a concentra-
tion of copper alone, added to a concentration of cadmium of undetectable to-
xicity by itself, becomes markedly toxic when both are added in short-term
laboratory bioassays. For these low metal concentrations, copper would only
induce sensory lesions, while cadmium alone would fail to elicit any renal le-
sion. However, in combination at these concentrations, the metals induced
both neurosensory and kidney lesions and increased acute toxicity by several
orders of magnitude.
Some physical factors are known to influence toxicity of some compounds,
such as cadmium. Nevertheless, changes in salinity, temperature, pH, or dis-
solved oxygen, do not alter the eventual nature of lesions (Gardner 6 Yevich,
1969).
Various chemical pesticides, including chlorinated hydrocarbons and or-
ganophosphates, have been found to cause histological alterations in livers
of fishes similar to those of mammals exposed to toxicants (Couch, 197tc).
Preliminary histological evaluations of methoxychlor,exposed Fundulus hetero-
clitus have indicated that the compound causes lesions in the lateral line
system (Gardner £ La Roche, 1974).
Chemical carcinogens occur among the polycyclic hydrocarbons, N-nitroso-
compounds, radioactive nuclides, mycotoxins, heavy metals, and other groups.
Field collected cold-blooded vertebrates and invertebrates from numerous fresh-
water to marine environments are regularly discovered with neoplastic diseases
suggesting that carcinogenic chemicals are present. The most likely sources
of such chemicals include domestic and industrial discharges; leaching of pes-
ticides, fertilizers, natural materials and residues from atmospheric fallout;
oil spills, and natural synthesis when the precursors are available.
Examples of some lower animal neoplasms discovered in such possibly con-
taminated aquatic habitats are: (1) Russell £ Kotin (1957) reported 10 out of
353 cases of oral non-invasive squamous-cell papillomas in white croakers col-
lected in the Pacific Ocean, within 2 miles of the Santa Monica, California
sewage outfall, while no similar lesions were found on 1,116 white croakers
collected 50 miles away in unpolluted water. Subsequently, white croakers
with oral papillomas were reported from Los Angeles Harbor (Young, 196H) and
numerous examples were also discovered in white croakers feeding near the San-
ta Ana, California sewage outfall (Harshbarger, 1972, 197t); (2) Lucke and
Schlumberger (1911) discovered 166 brown bullheads (Ictalurus nebulosus)with
transplantable onl epitheliomas, in the Delaware and Schuylkill Rivers in
southeastern Pennsylvania, presumably heavily polluted from effluents from
Philadelphia. Harshbarger (1972, 197t) discovered similar neoplasms in Icta-
lurus nebulosus in lakes in central Florida, where there is extensive use of
chemicals to control citrus pests and where water hyacinths in the lakes are
sprayed with herbicides; (3) Rose (in press) has discovered a population of
neotenic tiger salamanders (Ambystoma tigrinum) living in a treated, domestic
sewage settling pond in which at least 50% of the population has developed
conspicuous neoplasms of the skin - primarily epidermal papillomas, papillary
dermal fibromas, and melanomas; CO There have been numerous reports of inva-
sive neoplasms in the filter-feeding (bivalve) molluscs from widely distribu-
ted estuaries receiving effluent from many sources since the initial report
in 1968; previously, there were only occasional reports of benign neoplasms
in these animals, going back to 1887. Wolf (1969, 1971) reported 12 invasive
neoplasms of the mantle epithelium from Crassostrea commercialis from 2 rural
(agricultural) estuaries in southeastern Australia, although one estuary had
a pulp mill upstream. Pauley and Sayce (1972) described a single invasive
mantle epithelioma in C_. gigas from the lightly industralized estuary, Willapa
Bay, Washington, while a second £. gigas from Willapa Bay had a ganglioneuroma
(Pauley et al., 1968). Fourteen cases of gonadal neoplasms have been descri-
bed in both male and female quahogs, Mercenaria mercenaria, from the Narragan-
sett Bay area of Rhode Island, which is near a large metropolitan area (Yevich
S Barry, 1969; Barry 6 Yevich, 1972). In the same general area, 40% of the
soft-shelled clams (Mya arenaria) examined showed atypical gill and/or kidney
epithelial hyperplasia (Barry et al., 1971). In the Chesapeake Bay and its
166
-------
tributaries, hematopoietic neoplasms have been discovered in a large number
of C. virginica (Farley, 1969; Couch, 1969; Frierman 6 Harshbarger, 1974), a
retTculosarcoma-like lesion in a C_. virginica (Couch, 1970), and an epizootic
highly proliferative, anaplastic neoplasm arising from the gill epithelium of
Maeoma balthica (Christensen et al., 1974). An epizootic of an undifferentia-
ted mesenchymal neoplasm in Ifytilus eduli s and hematopoietic neoplasms in Os-
trea lurida have been found in Yaquina Bay, Oregon, downstream frcm.a pulp
mill (Farley 6 Sparks, 1970); and, (5) Following a spill of a combination of
jet fuel and number 2 fuel oil, Barry (in preparation) associated a 22% inci-
dence of neoplastic-like lesions in soft-shelled clams. Other lesions of- a pos-
sible preoancerous nature have also been demonstrated in animals exposed to
crude oil. Gardner et al (1974) found such lesions in the olfactory organs
of Menidia, apparently from the salt water soluble fraction of crude oil. In
addition, the pseudobranch of Menidia was especially vulnerable to crude and
waste oil and vascular lesions occurred in scallops, oysters, Henidia and
Fundulus.
The neoplasms and related diseases correlated with environmental pollu-
tants in the five examples just cited, all occurred in bottom-feeding fish and
filter-feeding molluscs, a trend also borne out by the specimens which have
been sent to the Registry of Tumors in lower Animals for diagnosis and regis-
tration (Harshbarger, 1974), and by the other published, but not cited, cases
of lower animal neoplasms (Dawe 6 Harshbarger, 1969). This is a logical ob-
servation since bottom-feeders and filter-feeders would be apt to encounter
larger amounts of suspended and sedimented chemicals than surface feeders and
it provides further circumstantial evidence of the harmful effects of water
pollution.
Neurosensory lesions have also been linked to pulp mill waste exposures,
since olfactory lesions have been demonstrated in the Atlantic salmon (Gard-
ner, 1972). These lesions were induced in an experimental system within the
natural waters of the St. Croix River. Thus, neurosensory lesions, detectable
by histology, represent an important aspect of incidious damage to marine fish
exposed to relatively low doses of varied chemicals likely to be introduced
in the marine environment.
As previously stated, normal histology, accounting for seasonal and all
other normal variations, forms the basis from which histological evaluations
of anomalies must be made. Since the morphological and physiological states
of marine organisms varies annually according to environmental conditions and
including those of captivity, results may be greatly influenced by them. For
this reason alone, it is imperative that researchers be convinced of the neces-
sity to maintain and compare exposed organisms with appropriate controls.
The copepods (Acartia clausi and A. tonsa), routinely cultured for labo-
ratory experimentation, serve to illustrate the -importance of routine histo-
logical examination of experimental stocks maintained in captivity. Histo-
logical evaluations with these organisms have shown that captivity will af-
fect the abundance of unicellular glands (Gonzalez et al., 1973). These
glands are usually numerous in natural populations and~~ih laboratory cultures
that are well fed. These glands diminish in abundance from both laboratory
and field populations when food densities are low. These glands can, further-
more, be used as an index of nutritional condition.
Investigations of the bay scallop, (Aquipectin irradians) have determined
that these organisms undergo significant annual variations which can be fol-
lowed by observing the content of acid-staining granules in its nervous sys-
tem (Blake 6 Yevich, 1972). The annual cycles are considered normal and can
be correlated with ambient water temperature. Therefore, it is essential that
these histophysiological variations be recognized when appraising the effects
of toxicants on aquatic species.
The above studies indicate that normal histology must be carried out on
feral organisms taken from natural environment to appreciate both the morpho-
logical and physiological states of the species which may be affected by pol-
lutant exposures. Although controls must be used in all experimental work,
both control and exposed organisms may undergo changes in a laboratory system
167
-------
which might not otherwise be recognized or evaluated in terms of significance.
Normal histological evaluations of marine organisms are essential to better
understand morphological anomalies which are due to pollutants. Weekly col-
lections and examinations of stocks of the bay scallop (Aquipectin irradians),
mussel (Mytilus edulis), soft-shelled clam (Mya arenaria), and Atlantic sil-
versides (Menidia menidia), has been demonstrated to be reasonable means of
establishing normal seasonal variations, especially in natural populations
likely to be used experimentally (Yevich, 1971*).
Because of a wide geographic distribution and commercial value, the oys-
ter has been studied extensively for its responses to changes in water quality.
In a few instances (i.e. to evaluate pulpmill effluents), it has been used
routinely for acute larval responses. Some information on its morphological
and physiological responses is also available. This is, generally speaking,
quite rudimentary and it is hoped that systematic efforts will be made to im-
prove techniques leading to an understanding of metabolic activities involved
with specific environmental changes. With the increased attention being paid
to pollution of estuarine waters, this important sedentary adult mollusc may
serve as a biological indicator of the quality of these vulnerable waters.
An entirely new approach is now feasible as well. This is the rearing
of oysters under controlled laboratory conditions and transferring them to
field situations to determine their reaction to particularly suspicious envi-
ronments. The advantage of this approach is to eliminate the possibility that
biological damage or impairment observed in oysters from the field may limit
the use of oysters as bio-monitoring elements.
Early histological evaluations must be as complete as possible to offer
a broad survey of tissues which may be affected through specific exposures.
In some instances, reported histological investigations are incomplete in that
attention is focused too rapidly on one or two major tissues, such as to res-
piratory and hepatic tissues. In other cases, only the short-term study of
one section of an organism may have been explored. Many lesions, reportedly
due to toxicants or diseases, may appear questionable since they may only re-
present normal variations. The need for normal histology, established over
extended periods of time, remains most important. In-depth studies on select
tissues and organs is always' indicated once they have been identified as tar-
gets of toxicants.
Respiratory tissues represent an area of prime histological interest in
aquatic organisms. These tissues require a great deal of histological studies
to properly assess normal and abnormal developments. In fish pathologist cir-
cles, it has been observed that "the degree of hyperplasia in respiratory epi-
thelium depends largely on the angle of the cut". The statement is a propos.
Very often, histological diagnosis will acquire much greater significance
by complementing routine histology with techniques available in histochemistry,
autoradiography, electron microscopy and electron scanning. The light micros-
cope and electron microscope are providing an increasing amount of knowledge
about the morphological responses of marine organisms to acute concentrations
of toxicants. The light microscope can, in many instances, indicate the sites
of initial tissue reactions to chronic toxicity. In this manner, light micros-
cope serves to guide the investigator in probes to define tissue damage with
more elaborate diagnostic procedures including electron microscopy. In chro-
nic toxicity evaluations, only a limited number of animals in any one popula-
tion nay became visibly affected in a specified time frame. However, these
evaluations will generally supplement findings established at more acutely
toxic levels. It is essential that diagnostics of other scientific endeavors
such as behavior and physiology, for instance, be applied to the overall ana-
lysis of normal or abnormal performance by exposed species. Histopathology
is essential as an element in these investigations, and by being applied broad-
ly, it can offer realistic visible evidence of pollutant damage.
Histological evaluations, therefore, should be considered as essential
elements in the scientific establishment of Water Quality Criteria. However,
a successful approach to the problem of defining these criteria depends on
well-developed interdisciplinary diagnostic strategies to identify the effects
of pollutants.
168
-------
REFERENCES
ANDREWS, J.D. 1961. Measurement of Shell Growth in Oysters by Weighing in
Water. Proc. Ntl. Shellfish. Assoc. 52: 1-11.
BARRY, M.M. 1974. Incidence of. Cancer in Soft-shelled Clam (Mya arenaria)
from an Oil Spill. In: Neoplasms in Aquatic Animals. Prog. Exp. Tumor
Res., F. Homburger (ed.), Vol. 21, S. Karger: Basel, MUnchen, Paris, New
York, (in press).
BARRY, M.M. & YEVICH, P.P., 1972. Incidence of Gonadal Cancer in the Quahaug,
Mercenaria mercenaria. Oncology 26: 87-96.
BARRY, M.M., YEVICH, P.P. and THAYER, N.H. 1971. Atypical Hyperplasia in the
Soft-shell Clam, Mya arenaria. J. Invert. Pathol. 17: 17-27.
BLAKE, N.H. and YEVICH, P.P., 1972. The Neurosecretory Cycle of the Bay Scal-
lop, Aquipectin irradians. Gen. Camp. Endocrinology, 18(3) abstr.
BUTLER, P.A. 1965. Reaction of Estuarine Mollusks to Some Environmental
Factors. In: Biological Problems in Water Pollution. C.M. Tarzwell,(ed).
U.S.D.H.E.W., P.H.S., Div. of Water Supply and Pollution Control, Cin-
cinnati, Ohio. 424 pp.
BUTLER, P.A. 1966. The Problem of Pesticides in Estuaries. Amer Fish. Soc.
Spec. Publication no. 3: 110-115.
BUTLER, P.A., WILSON, A.J. and RICK, A.J. 1960. Effect of Pesticides on Oys-
ter. Proc. Natl. Shellfish Assoc. 51: 23-32.
CHENG, T. 1967. Marine Molluscs as Hosts for Symbioses with a Review of Known
Parasites of Commercially Important Species. Advan. Marine Biol. 5: 1-
424.
CHRISTENSEN, D.J. FARLEY, C.A.and KERN, F.G. 1974. Epizootic Neoplasms in
the Clam Hacoma balthica (L.) From Chesapeake Bay. J. Natl. Cancer Ins-
titute, 52(6): 1739-1949.
COUCH, J. 1967. Concurrent Haplosporidian Infections of the Oyster, Crassos-
trea virgLnica. Joum. Parasitology, 53: 248-253.
COUCH, J. 1969. An Unusual Lesion in the Mantle of the American Oyster (Cras-
sostrea virginica. Nat. Cancer Inst. Monogr. 31: 557-562.
COUCH, J. 1970. Sarcoma-like Disease in a Single Specimen of the American
Oyster. In Bibl. haemat. no. 36, ed. R.M. Dutcher, p. 647, Kager, Basel/
Munchen/Paris/New York.
COUCH, J. 1974a. Free and Occluded Virus, Similar to Baculovirus, in Hepato-
pancreas of Pink Shrimp. Nature, 247: 229-231.
COUCH, J. 1974b. An Enzootic Nuclear Polyhedrosis Virus of Pink Shrimp. I.
Ultrastructure, prevalence and enhancement. J. of Invert. Path, (in
press).
COUCH, J. 1974c. Histopathologic Effects of Pesticides and Related Chemicals
on the Livers of Fishes. In: The Pathology of Fishes. University of
Wisconsin Press (in press).
COUCH, J. 1974d. Possible Activation or Enhancement of Latent Baculovirus
Infections in Penaeid Shrimp. In: Neoplasms in Aquatic Animals. Prog.
Exp. Tumor Res., F. Homburger (ed.), vol. '21, s. Rarger: Basel, MUnchen,
Paris, New York, (in press).
169
-------
COUCH, J., FARLEY, A. and RQSENFIELD, A. 1966. Sporulation in Minchinia nel-
soni (Haplosporida, Haplosporidiidae) in Crassostrea virginica. Science,
TS3T 1529-1531.
COUCH, J. and NIMMO, D. 1974a. Ultrastructural Studies of Shrimp Exposed to
the Pollutant Chemical, Polychlorinated Biphenyl (Aroclor 1254). Bull.
of Pharmaeol. and Environm. Pathologists, 11: 17-20.
COUCH, J. and NIMMO, D. 197tb. Detection of Interactions between Natural Pa-
thogens and Pollutants in Aquatic Animals. In: Proc. Symposium on Di-
seases of Aquatic Animals (Regional) LSU-Sea Grant Sponsored (in press).
DAVIS, B.C. 1961. Effects of Some Pesticides on Eggs and Larvae of Oysters
(Crassostrea virginica) and Coams (Venus mercenaria). Comm. Fish. Rev.
23: 8-23.
DAWE, C.J. and HARSHBARGER, J.C. (eds.) 1969. Neoplasms and Related Disorders
of Invertebrate and Lower Vertebrate Animals. Natl. Cancer Inst. Monogr.
31, 772 pp.
DAWSON, 1958. A Study of the Biology and Life History of the Spot (Leios-
tomus xanthurus) with Special Reference to South Carolina. Bears Bluff
Laboratory Contribution, no. 28, South Carolina, U.S.A.
EISLER, R. and GARDNER, G.R. 1973. Acute Toxicology to an Estuarine Teleost
of Mixtures of Cadmium, Copper and Zinc Salts. J. Fish Biol. 5: 131-142.
EISLER, R., GARDNER, G.R., HENNEKEY, R.J., LA ROCHE, G., WALSH, D.F. , YEVICH,
P.P. 1972. Acute Toxicology of Sodium Nitrilotriacetic Acid (NTA) and
NTA-Containing Detergents to Marine Organisms. Water Res. 6: 1009-1027.
ELIAS, H. and BENGELSDORF, H. 1952. The Structure of -&ie Liver of Vertebrates.
Acta Anatomica, It: 24.
FARLEY, C.A. and SPARKS, A.K. 1970. Proliferative Disease of Hemocytes, En-
dothelial Cells and Connective Tissue Cells in Molluscs. In: Comparative
Leukemia- Research 1969, Bibl. Haemat., No. 36, R.M. Dutcher (ed.), pp.
610-617, S. Karger: Basel, MUnchen, Paris, New York.
FARLEY, C.A. 1969. Probable Neoplastic Disease of the Hematopoietic System
in Oysters, Crassostrea virginica and Crassostrea gigas. Natl. Cancer
Inst. Monogr. 31: 541-555.
FENG, S.Y. 1965. Heart Rate and Leucocyte Circulation in Crassostrea virgini-
-ca (Gmelin). Biol. Bull. 128: 198-210.
FENG, S.Y. and CANZONIER, W.J. 1970. Humoral responses in the American Oys-
ter (Crassostrea virginica) Infected with Bucephalus sp. and Minchinia
nelsoni. In: Diseases of Fishes and Shellfishes, S.F. Snieszko, (ed.)
Amer. Fish. See., Washington, D.C. 526 pp.
GALIOER, A. and KOZLOFF, E. 1971. Essentials of Practical Microtechnique.
Second edition. Lea & Febiger, Philadelphia. 531 pp.
GALTSOFF, P.S. 1959. Environmental Requirements of Oysters in Relation to
Pollution. In: Biological Problems in Water Pollution. U.S.D.H.E.W.,
P.H.S., Div. of Water Supply and Pollution Control, Cincinnati, Ohio.
GALTSOFF, P. 1964. The American Oyster. Fishery Bull. 64: 1-480.
GARDNER, G.R. 1972. Appendix K, p. K1-K5. In: St. Croix River. A Study of
Water Quality and Benthic Conditions in the St.Croix River, Grand Falls
to Milltown, Maine and New Brunswick. U.S. Env. Protection Agency, Re-
gion I, Boston, Mass.
GARTNER, G.R..LAROCHE, G. 1974. Chemically Induced Lesions in Estuarine or
Marine Teleosts. In: W. Ribelin and G. Migaki (ed.) The Pathology of
Fishes. Univ. of Wisconsin Press, (in press).
170
-------
GARDNER, 6.R., LA ROCHE, G. 1973. Copper Induced Lesions in Estuarine Teleosts.
J. Fish. Res. Bd. Can. 30: 363-368.
GARTNER, G.R. and YEVICH, P.P. 1969. Toxicological Effects of Cadmium on Fun-
dulus heteroclitus Under Various Oxygen, pH, Salinity and Temperature
Regimes. Amer. Zool. 9(4): 1096.
GARDNER, G.R. and YEVICH, P.P. 1970. Histological and Hemtological Responses
of an Estuarine Teleost to Cadmium. J. Fish. Res. Bd. Can. 27: 2185-2196.
GARDNER, G.R., YEVICH, P.P. and ROGERSON, P.P. 1974. Morphological anomalies
in Adult Oyster, Scallop and Atlantic Silversides Exposed to Emulsified
Waste Motor Oil. (unpublished).
GONZALEZ, J., YEVICH, P.P., GENTILE, J. and LACKIE, N. 1973. Problems Asso-
ciated with Cultured Marine Copepods (in press).
HAMMEN, C.S. 1969. Metabolism of the Oyster, Crassostrea virginica. Am. Zool.
9: 309-318.
HARSHBARGER, J.C. 1972. Work of the Registry of Tumors in Lower Animals with
Emphasis on Fish Neoplasms. Symp. Zool. Soc. London, no. 30, pp. 285-303.
HARSHBARGER, J.C. 1974. Activities Report (of the) Registry of Tumors in
Lower Animals 1965-1973. Smithsonian Institution, Washington, D.C. 141 pp.
HAYAT, M. 1970. Principles and Techniques of Electron Microscopy. Biological
Applications. Vol. 1. Van Nostrand Reinhold Co., New York. 412 pp.
JOHNSON, P.T. 1968. An Annotated Bibliography of Pathology in Invertebrates
Other Than Insects. Burgess Publ. Co., Minneapolis, Minn. 322 pp.
JONES, R. 1966. Basic Microscopic Technics. The University of Chicago Press,
334 pp.
LA ROCHE, G., GARDNER, G.R., EISLER, R., JACKCM, E., YEVICH, P.P. and ZAROO-
GIAN, G. 1973. Analysis of Toxic Responses in Marine Poikilothenns. In:
Gary E. Glass (ed.) Bioassay Techniques and Environmental Chemistry. Ann
Arbor Science Publishers, Ann Arbor, Michigan.
LILLTE, R. 1965. Histopathologic Technic and Practical Histochemistry. Mc-
Graw-Hill, New York, 715 pp.
LOOSANOFF, V.L. 1942. Seasonal Gonadal Changes in the Adult Oysters, Ostrea
virginica, of Long Island Sound. Biol. Bull. 82: 195-206.
LOWE, J. 1965. Some Effects of Andrin on Estuarine Fishes. Proc. 19th Ann.
Conf. Southeastern Assoc. Game and Fish. Comm. Tulsa, Okla. p. 271.
LOWE, J., PARRISH, P., PATRICK, J. and FORESTER, J. 1972. Effects of the
pdychlorinated biphenyl Aroclor®1254 on the American Oyster Crassostrea
virginica. Mar. Biol. 17: 209-214.
LOWRY, O.K. , ROSENBROUGH, N.J., FARR, A.L. and RANDALL , R.J. 1951. Protein
Measurement with the Folin Phenol Reagent. J. Biol. Chem. 193: 265-275.
LUCKfi, B. and SCHLUMBERGER, H. 1941. Transplantable Epitheliomas of the Lip
Lower Animals 1965-1973. Smithsonian Institute, Washington, D.C. 141 pp.
LUNA, L.G. (ed.) 1968. Manual of Histological Staining Methods of the Armed
Forces Institute of Pathology. McGraw-Hill, New York, 258 pp.
, D., HANSEN, D., COUCH, J., COOLEY, N., PARRISH, P. and LOWE, J. 1974.
Toxicity of Aroclor©1254 and its Physiological Activity in Several Es-
tuarine Organisms. Archives of Envir. Contam. and Toxicol. (in press).
171
-------
PARKISH, R., COUCH, J., FORRESTER, J., PATRICK, J., and COOK, G. 1974. Diel-
drin: Effects on Several Estuarine Organisms. Proc. of the 27th Arm.
Conf. of the Southeastern Assoc. Game and Fish. Coimu (in press).
PAULEY, 6.B. and SPARKS, A.K. 1966. The Acute Inflammatory Reaction in Two
Different Tissues of the Pacific Oyster, Crassostrea gigas. J. Fish. Res.
Bd. of Can. 23: 1913-1921.
PAULEY, 6.B., SPARKS, A.K. and SAYCE, C.S. 1968. An Unusual Internal Growth
Associated with Multiple Watery Cysts in a Pacific Oyster (Crassostrea
gigas). J. Invert. Pathol. 11: 398-405.
PAULEY, G.n,. and SAYCEj G.S. 1972. An Invasive Epithelial Neoplasm in a Paci-
fic Oyster, Crassostrea gigas. J. Natl. Cancer Inst. 49(3): 897-902.
PALILEY, G.B. 1969. A Critical Review of Neoplasia and Tumor-like Lesions in
Molluscs. Natl. Cancer Inst. Monogr. 31: 509-540.
PEASE, D. 1964. Histological Technics for Electron Microsoopy. Second Edi-
tion. Academic Press, New York, 381 pp.
RAY, S. 1954. Biological Studies of Dermo-cystidium marinum a Fungus Parasite
of Oysters. Rice Inst. Pamphlet, Monogr. in Biology.
ROSE, F.L. 1974. Neoplasms and Related Lesions in a Population of Tiger Sala-
manders Living in Treated Sewage Effluent. In: Neoplasms in Aquatic Ani-
mals, Prog. Exp. Tumor Res., F. Homburger (ed.), vol. 21, S. Karger:
Basel, MUnchen, Paris, New York (in press).
RUSSELL, F.E. and KOTIN, P. 1957. Squamous Papilloma in the White Croaker.
J. Natl. Cancer Inst., 18(6): 857-861.
SHAW, B.L. and BATTLE, H.I. 1957. The Gross and Microscopic Anatomy of the
Digestive Tract of the Oyster, Crassostrea virginica (Gmelin). Can. J.
Zool. 35: 325-347.
SINDERMANN, C. 1970. Principal Diseases of Marine Fish and Shellfish. Aca-
demic Press, New York, 369 pp.
SPARKS, A.K. 1972. Invertebrate Pathology. Non-cormunicable Diseases. Aca-
demic Press, New York. 387 pp. General principles of invertebrate patho-
logy with numerous references to oyster diseases throughout. A second
volume on infectious diseases is in preparation, but probably will not be
available before 1976.
TRIPP, M.R. 1974. Effects of Organophosphate Pesticides on Adult Oysters
(Crassostrea virginica) In: Pollution and Physiology of Marine Organisms,
F.J. Vemberg, (ed.) (in press).
WILLIAMS, A. 1965. Marine Decapod Crustaceans of the Carolines. Fish. Bull.
65: 1-298.
WOELKE, C.E. 1962. Bioassays of Pulp Mill Wastes with Oysters. In: Biologi-
cal Problems in Water Pollution, D.M. Tarzwell (ed.), U.S.D.H.E.W., P.H.
S., Div. Water Supply and Pollution Control, Cincinnati, Ohio.
WOELKE, C.E. 1967. Measurement of Water Quality With the Pacific Oyster Em-
bryo Bioassay. Water Quality Criteria, ASTM STP 416, Am. Soc. Testing
Mats, 112-120.
WOELKE, C.E. 1972. Development of a Receiving Water Quality Bioassay Crite-
rion Based on the 48-hour Pacific Oyster (Crassostrea gigas) Embryo.
Washington, Dept. of Fisheries, Tech. Report no. 9, pp. 1-93.
WOLF, P.H. 1969. Neoplastic Growth in Two Sydney Rock Oysters, Crassostrea
coinnercialis (Iredale E Roughley), Nat. Cancer Inst. Monogr. 31: 563-573.
172
-------
WOLF, P.H. 1971. Unusually Large Tumor in a. Sydney Rock Oyster. J. Natl.
Cancer Inst., 46(5): 1079-1084.
YEVICH, P.P. and BARRY, M.M. 1969. Ovarian Tumors in the Quahog, Mercenaria
mercenaria. J. Invert. Pathol. 14: 266-267.
YEVICH, P.P. 1974. Unpublished.
YOUNG, P.H. 1964. Some Effects of Sewer Effluent on Marine Life. Calif. Fish
Same, 50(1): 33-41.
ZU6TBE, F. 1970. Diagnostic Histochemistry. C.V. Mosby Co., St. Louis, 366pp.
The presentation: "Histological and Physiological Evaluations in Some
[ferine Fauna" is a composite of information compiled by (in alphabeti-
cal order):
John Couch
Gulf Breeze Environmental Research Laboratory,
United States Environmental Protection Agency,
Gulf Breeze, Florida 32561
Tel. no. (904) 932-5326
George Gardner
National Marine Veter Quality laboratory,
United States Environmental Protection Agency,
South Ferry Road,
Narragansett, Rhode Island 02882
Tel. no. (401) 789-1427
John C. Harshbarger
Director,
Registry of Tumors in Lower Animals,
National Museum of Natural History,
Smithsonian Institution,
Washington, D.C. 20560
Tel. no. (202) 628-4422
M. R. Tripp
Department of Biological Sciences,
117 Wolf Hall,
University of Delaware,
Newark, Delaware 19711
Tel. no. (302) 738-2275 or 2281
Paul P. Yevich
National Marine Water Quality Laboratory,
United States Environmental Protection Agency,
South Ferry Road,
Narragansett, Rhode Island 02882
Tel. no. (401) 789-1427
173
-------
RESPIROHETRY
Although respirometry, applied to aquatic organisms, cannot be claimed as
a technique with any degree of specificity in the identification of toxic me-
chanisms, it is valuable in detecting signs of metabolic involvements. Well-
standardized respirometry may also be relatively accurate in measuring the de-
gree of metabolic changes associated with specific treatments (Voyer £ Morri-
son, 1971, Cheng S Sullivan, 1973).
A number of aquatic poikilotherms are suited for these measurements and
it would appear "that respirometry is among the simplest and earliest means of
establishing quantitative assessments of toxicant effects by comparing control
and test organisms. Test organisms used were aquatic gastropod molluscs:
Biomphalaria glabrata, Bulinus truncatus, and Nassarius obsoletus. Results on
the mudflat snail, N. obsoletus, are presented herein.
I - Materials and methods
The specimens of the estuarine snail N_. obsoletus used in this study were
collected at Wickford Harbor, North Kingston, Rhode Island, during November
1973. They were brought back to the laboratory and maintained in 170-gallon
aquaria at 22 C. Overcrowding was avoided by maintaining no more than 500
snails in each aquarium. The salinity of the artificial seawater (Instant
Ocean)* was 30-31°/oo and the snails were fed frozen turbot and fresh clams,
Mercenaria mercenaria, ad libitum.
All of the snails used measured between 18-22 mm in shell length and all
had been ascertained to be free of helminth parasites.
In preparation for respirometric determinations, the exterior of the shell
of each snail was cleaned and blotted dry prior to being placed in a reaction
vessel of a Model GRP 14 Gilson Differential Respirometer (Middletown, Wiscon-
sin). The vessel also contained 73 ml of the test solution, (i.e. seawater
plus an experimental additive).
One ml of potassium hydroxide was placed in the center well of each flask,
together with a filter paper fan (Whatman No. 2) for the absorption of the 002
evolved. One control snail was placed in a similar reaction vessel containing
seawater with each determination and respirometric data obtained for these
served as controls. By following this procedure, data obtained for the con-
trol snails can be compared with those of test snails with greater reliability
than if the determination of oxygen consumption of controls and experimentals
were made separately, at different times.
Once the molluscs are in place, reaction vessels are submerged in the
water bath maintained at 20°C and allowed to equilibrate for 50 minutes with
shaking at 80 oscillations per minute. After this period, respirometric read-
ings are recorded at 20-minute intervals for a 2-hour period.
At the end of the 2-hour period, snails were dissected from their shells,
rinsed in deionized water, dried overnight at 70°C, and weighed. Respirometric
data were converted to microliters of oxygen consumed per gram dry weight.
II - Test solutions
Simulated pollutants added to the seawater were 1 ppm of Zn as ZnSO^,
1 ppm Cu as CuSOn, 1 ppm. Cu as copper ethylenediamine tetraacetic acid (CuEOTA),
1 ppm Bayluscide, and 1 ppm Frescon. Copper sulfate was selected because it
is known to be a molluscicide (Cheng, 1974 for review) and CuEDTA was selected
because it is known not to be molluscicidal, at least to Biomphalaria glabrata
(Cheng £ Sullivan, 1973). Both Bayluscide and Frescon are commercial mollus-
cicides, and 2nSOi| was chosen because certain zinc-containing compounds, such
as zinc dimethyldithiocarbamate, have molluscicidal properties (Malek E Cheng,
1974).
* Aquarium Systems, Inc., Eastlake, Ohio.
174
-------
CONTRIBUTION NO. 251
-------
6ulf Breeze Contribution No. 251
BEHAVIORAL BIOASSAYS
In: Marine Bioassays
Proceedings of workshop sponsored by
The Marine Technology Society, 1974.
(Entire proceedings may be obtained from
The Marine Technology Society, 1730 M Street, N.W.,
Washington, D. C. $18.00).
-------
BEHAVIORAL MEASURES OF ENVIRONMENTAL STRESS
Bori L. Olla, Chairman and Editor
Jelle Atema James S. Kittredge
Charles C. Coutant John J. Magnuson
Patricia De Coursey Don Miller
David Hansen Mark J. Schneider
Winona Vernberg
-------
AFFILIATIONS
Jelle Atema
Woods Hole Oceanographlo Institution
Woods Hole, Massachusetts 02543
Charles C. Coutant
Ecological Sciences Division
Building 2001
Oak Ridge National Laboratory
P.O. BoxX
Oak Ridge, Tennessee 37830
Patricia DeCoursey
Belle W. Baruch Coastal Research Institute
University, of South Carolina
Columbia, South Carolina 29208
David Hansen
Environmental Protection Agency
Gulf Breeze, Florida 32561
James S. Kittredge
Marine Blomedical Institute
University of Texas
200 University Blvd.
Galveston, Texas 77550
John J. Magnuson
Laboratory of Limnology
Department of Zoology
University of Wisconsin
Madison, Wisconsin 53706
Don Miller
U. S. Environmental Protection Agency
National Marine Water Quality Laboratory
South Ferry Road
Narragansett, Rhode Island 02882
Bori L. Olla
U.S. Department of Commerce
National Oceanic and Atmospheric Administration
National Marine Fisheries Service
Middle Atlantic Coastal Fisheries Center
Sandy Hook Laboratory
Highlands, New Jersey 07732
Mark J. Schneider
Battelle Northwest
Ecosystems Department
Richland, Washington 99352
Winona B. Vernberg
Belle W. Baruch Coastal Research Institute
University of South Carolina
Columbia, South Carolina 29208
-------
INTRODUCTION
Until fairly recently, water quality bioassay techniques have been limited to obser-
vations of the lethal concentrations of a pollutant. Such measures as LDso, TLm5o,
and LCso were, and still are, commonly used to assess the acute effect of a pollutant,
usually based on the mortality of adult organisms. While this approach was probably
inevitable as a step in the evolution of both the philosophy and techniques of bioassaying,
it has become abundantly clear that this concept has serious limitations as a measure
of the effects of a pollutant on the environment.
The need for additional, more comprehensive measures of organismic response to
contaminants has stimulated the search for new testing techniques in a variety of disci-
plines, including behavior. The most important advantage to using behavior as a tool
to measure stress is that the results of behavioral tests often lend themselves to direct
interpretation regarding environmental quality as related to possible consequences at
the population and ecosystem levels. Also complex biochemical and physiological
responses of an organism may be reflected in rather easily observable acts. Although
behavioral measures may suffer in regard to quantification because of the high degree
of inherent variation, they are highly sensitive to stress.
The general aim of the Workshop was to explore various aspects of applying
behavioral measures to bioassay. Although in a few instances the use of behavioral
bioassays has reached the standard test stage, the state of the art is still very young.
Consequently, the scope of the discussion was intended as a beginning toward integrat-
ing a variety of basic research techniques into logical steps towards developing stand-
ard tests.
SPECIES TO STUDY (INDICATOR ORGANISMS)
Assuming that the objective of behavioral or any other bioassays is to protect the
structure and function of marine ecosystems from degradation by pollutant sources,
the organisms selected for study must be representative of the ecosystem that will
receive the impact. The exact selection will, by necessity, differ from ecosystem to
ecosystem. The kinds and number of species required to adequately represent various
ecosystems will differ according to the complexity of the ecosystem. * Rarely will
selected species transcend ecosystems in general. The examination of "standard" test
organisms, nationally designated or from commercially-produced laboratory stocks
will not be sufficient, except when there is an obvious commonality of species of simi-
lar ecosystems. In essence, the subject of bioassays should be determined on an eco-
system-by-ecosystem basis and often on a site-by-site basis where discrete locations
for waste disposal or discharge have been identified.
The following criteria are offered as guidelines for selection of species. There
are two principal categories: social (wherein the importance is predominantly humanis-
tic) and ecological.
2
No hard and fast number can be recommended, but will in most cases exceed 10.
Broad functional generalizations should be encouraged wherever possible. However,
even when tests are on the same species from widely-spaced locations the applica-
bility of numerical results may be questionable.
-------
Social
Species of economic Importance
Species which sustain economically important commercial or recreational harvests
should be included. These may not represent the most important species ecologically,
but economic concerns usually will justify their preservation or enhancement.
Rare and endangered species
National law protects species judged to be rare or endangered (see lists published
by the U.S. Dept. of the Interior). Wherever these species are found they must be
evaluated if at all practical. Of course, it is obvious that many of the endangered spe-
cies may be so limited in number as to preclude extensive assays.
Nuisance species
Some species may be judged as nuisances from both social and economic view-
points, and events which promote expansion of these species are thus deemed undesir-
able. For example, thermal modifications may allow northward proliferation of
undesirable southern fish species. These expansions may be possible through a variety
of means Including behavioral changes caused by elevated temperatures. It is also
possible that a particular environmental stress may favor less desirable species caus-
ing imbalance in population structure.
Ecological
Interaction of the organism with the pollutant
Bioassays should be designed on an ecologically realistic basis wherein there is a
significant potential for interaction of the organism with the pollutant. Some knowledge
of the engineering design, hydraulics or chemistry of the waste discharge will be
necessary to predict the components of the ecosystem that will be susceptible. Species
selected should be spatially representative of the zone affected by the pollutant. For
example, a shoreline discharge will probably first influence nearby littoral species and
not benthic organisms a mile or more offshore. Temporal considerations may be as
important as spatial in some instances, as for example, when seasonal discharges
such as food processing wastes are discharged into waters with transient, migratory
fishes.
Trophic level representation
Major trophic levels should be represented in the overall scope of bioassays.
While behavioral assays of primary producers may be a rarity, the several levels of
consumers (herbivores, carnivores, detritivores, etc.) should be represented. Of
particular importance will be those species which contribute a major portion of the
biomass or which functionally have a rapid material or energy transfer function.
These may be temporarily short, but none the less Important for the ecosystem (e.g.,
meroplankton, fish larvae).
Habitat structure
Some organisms should be considered for assays because they may be ecologically
important by virtue of their role in directly serving as habitats for other organisms
(e. g., corals and attaching bivalves) or secondarily affecting other species that serve
as substrates. For example, behavior or larval corals (especially settling) will deter-
mine the locations of coral reef structures. Feeding behavior of sea urchins affects
the kelp of southern California waters, which is a dominant "physical" habitat feature
there.
-------
Community structure
Certain species are particularly important because they strongly structure the
remainder of the biotic community. For example, starfish of the Pacific coast main-
tain a characteristic community of the rocky intertidal zone through selective preda-
tion (Paine, 1969). In the Great Lakes invasion of the marine lamprey has greatly
changed the indigenous community structure (Smith, 1973).
Consideration of life history phases
Particularly among invertebrates, there has been ecological diversification within
the life cycle. Different life phases are behaviorally diverse and exhibit different
levels of sensitivity to pollutants. Key phases should be examined independently.
Organisms with behavioral traits pertinent for pollutant effects
All ecologically significant species will not have behavior patterns that are sensi-
tive and ecologically pertinent in terms of having high probability of interacting with
the specific contaminant. Since the assays will be of behavior, the normal behavioral
patterns of the indigenous fauna must be known and pertinent behaviors selected for
study.
Coverage of sensory repertoire
Behavior patterns that depend upon diverse sensory mechanisms (e.g., olfaction,
vision, acoustic, electrical) should be evaluated depending upon the nature of the pollu-
tant. For example, a chemical discharge may interfere with chemoreceptors impor-
tant for feeding behavior while at the same time cause turbidity which would affect
visually-dependent behavior. The relative importance may not be readily apparent.
Species with behavior patterns that can be accommodated in the laboratory
Practicality dictates that behavioral traits of species to be studied must be amen-
able to laboratory (or controlled field) examination. This will involve (a) maintenance
of organisms in the laboratory, (b) ability of the organism to perform under controlled
conditions, and (c) capability of investigators to isolate relevant behaviors, especially
those that may be influenced by the pollutant in question. It seems clear that much
behavioral research must precede selection of suitable behavioral bioassays. (This
subject is covered more fully below.)
SOURCE OF TEST ANIMALS
Preferably, local test animals should be used in assessing possible pollution
effects. Few areas in the world are completely free today of man-induced pollutants;
since each pollutant can significantly alter physiological and behavioral responses to
environmental factors, effects of an added stress should be assessed on these organ-
isms. If species are not available from a local area, then representative species
from nearby localities offer an alternate source. As a last resort, commercially
available species can be utilized for "first-cut" tests.
Variables to be Considered
One of the prime reasons local organisms should be used is that even within the
same species there may be significant differences in the way an organism responds
physiologically between separate populations. Some of these differences have been
recognized for many years. For example, Mayer (1914) found that populations of the
horseshoe crab, Limulus, from Woods Hole died at 38. 5°C, but specimens from
Florida survived up to46. 2°C. In 1936, Sparck, Fox, and Thorson demonstrated
independently that the physiological responses of species from northern waters were
not the same as those from some more southern latitudes when measured at the same
-------
temperature. Since these earlier works, there have been many studies documenting
the many physiological parameters that differ between populations of the same species
(for review see Vernberg and Vernberg, 1972a).
Thermal history must also be considered. For example, phototactic response of
larvae of the fiddler crab. Pea pugilator. reared at 20. 0°C is not the same as larvae
reared at 25.0°C (Vernberg, DeCoursey, and Padgett, 1973). Temperature may also
interact with pollutants to modify behavioral responses. For example in goldfish,
increase in water temperature from 21. l°Cto 21.5°C in the presence of CuCl2 can
increase the attractiveness of the water (Kleerekoper et al., 1973), while exposure to
DDT can modify the thermal preference of salmon fry (Ogilvie and Anderson, 1965).
Stage in the life history is another important variable. Larvae of U. pugilator are
several magnitudes more sensitive to Hg poisoning than are adults or the eggs (DeCour-
sey and Vernberg, 1972; Vemberg, DeCoursey, and O'Hara, 1974) or the salinity pre-
ference of mosquitofish, Gambusia afftnis (Hansen, 1972).
The use of animals cultivated in the laboratory Is debatable. Preliminary evidence
indicated that animals reared in the laboratory under optimum conditions often do not
respond physiologically in the same manner as do field animals. There may, however,
be some utility in testing limited numbers of cultivated animals for obtaining reference
data (e. g., for comparing techniques among different laboratories).
Many abiotic and biotic factors must be considered before making valid assess-
ments of the effects of a particular pollutant. In the estuarine environment, which is
potentially the most likely to have the greatest pollution, salinity, dissolved oxygen,
and photoperiod all fluctuate daily and seasonally. In the temperate zone, temperature
is also an important consideration. Thus, multiple factors must be taken into account.
The studies of Haefner (1970) on Crangon septemsplnosa illustrate the Interaction of
some of these factors. These shrimp are well adapated to the normal temperature-
salinity fluctuations of their estuarine environment, and calculations of surface
response survival curves of adults indicate that they can tolerate a wide range thermal-
salinity regime. However, when the dissolved oxygen levels are reduced to 2 ppm in
contrast to 6 ppm, survival is markedly narrowed. Lowered dissolved oxygen levels
often are indicative of polluted areas.
Often the effects of a pollutant cannot be properly assessed under optimum envi-
ronmental conditions. When U. pugilator adults were exposed to 0.18 ppm mercury
under optimum conditions, the crabs could survive indefinitely. However, under
thermal-salinity stress (33.0°C, 5 °/oo) female crabs died after 26 days; male crabs
after an 18-day exposure (Vernberg and Vernberg, 1972b). Phototactic responses are
not modified in II. pugilator larvae reared under optimum conditions, but marked
modification of response to mercury is noted when larvae are reared under suboptimal
regimes (Vernberg, DeCoursey, and Padgett, 1973).
FIELD OBSERVATIONS: PREREQUISITE FOR THE DESIGN
OF LABORATORY BEHAVIORAL BIOASSAY
Behavior is best suited for pollution bioassay when the particular behavioral modi-
fications may be related to possible consequences at the population and ecosystem level.
Thus, it is important that the choice of a behavioral bioassay arise from extensive
knowledge of natural behaviors in the environment which, if altered, may intefere sig-
nificantly with certain defined vital life processes. Key among these are ones closely
related to reproductive success, survival to maturity and body growth. Experiments
conducted under controlled laboratory situations have much greater significance when
they can be compared and verified. Further observations in nature are essential to
choices of realistic laboratory conditions, proper choice of experimental organism,
and life history stage.
-------
Direct and Indirect Field Observations
Many advances have been made in techniques of both direct and indirect observa-
tion of behavior of aquatic organisms. Table I provides a number of pertinent exam-
ples of how problems involved with to situ observations have been solved by a variety
of approaches and techniques. Animals can be observed from above, on and below the
sea surface. Limitations of direct observation center around the tolerance and fatigue
of the observer, technological limits to access to particular habitats, and the responses
of organisms to the observer and his facilities. The deeper or more distant the obser-
vation, the more difficult and costly it is.
Technological advances in the use of indirect methods such as closed-circuit tele-
vision, acoustic and radio telemetry, and active sonar have greatly increased our
abilities to observe in the aquatic environment. While these techniques do not have the
resolution of direct visual observation, they nevertheless have extended the capabili-
ties of observing aquatic organisms. Limitations of these techniques primarily relate
to species identification and a variety of technological limitations.
Sampling as a Behavioral Tool
Insights into an organism's behavior in nature can often be gained by not observing
behavior at all, but by sampling changes in the spatial distribution of the animals over
time with conventional gear such as nets, traps and dredges. Knowledge of habits such
as on and offshore movements, migration into estuaries and rivers and vertical migra-
tions may be gained. Variations in number caught per unit of sampling effort help
determine whether animals are distributed in uniform, random, or clumped spatial
arrays. To some extent, even changes in the relative locomotory activity can be fol-
lowed over time by changes in catch per unit effort with passive or stationary sampling
gear.
Likewise feeding behavior can be inferred by stomach content. Locomotory
behavior, feeding behavior, and sensory ability can also be inferred to some extent
from anatomy w!th elusive marine species. These methods are often used and in some
cases, the only available.
The Bridge Between the Laboratory and the Field
Relating what we observe in the field to what we observe under controlled labora-
tory conditions is the crucial link before the behavioral bioassay may be employed to
assess and predict pollution effects. The following hypothetical example illustrates the
interrelation between the field and laboratory.
To study the effects of an industrial waste we select an animal important in the
trophic ecology from the proposed site. This animal is observed to feed on worms
and to mate once a year. After its feeding behavior is studied in the laboratory using
a similar substrate and food, it is established that feeding is not modified in the labora-
tory. Mating behavior is too infrequent to be useful for bioassay or, alternatively, may
not withstand the transition to the laboratory negating the use of reproduction as a bio-
assay. After testing the effects of the effluent on feeding, we .find that feeding behavior
is altered. The animal takes a significantly longer time to locate a certain amount of
food. Its efficiency has decreased. From this simple observation we conclude that the
animal will grow more slowly, an effect which will be felt at the population and ecosys-
tem level. Fewer individuals will reach maturity and populations of the next generation
will be smaller (Atema and Stein, 1974).
In some cases, the use of a particular behavioral bioassay may be promising for
development even though direct links of this behavior with survival of the species have
not been demonstrated. An example is the effect of pollutants on learning .ability of
aquatic organisms. It would seem counter-productive to discard this bioassay at the
present, as it seems evident that learning affects many aspects of an animal's daily life.
-------
Table I. Techniques used in field observations of behavior.
Category
Technique
Reference
Direct Observation
Surface
Underwater
Boat
Snorkel (free diving)
SCUBA
Submersibles
Underwater habitats
Boat/rafts with viewing portals
Periscope
Field enclosure
Newman (1956); Wisby and Nelson (1964); Jenkins (1969); Nelson et al.
(1969); Keenleyside (1971)
Keenleyside (1962); Brown et al. (1973); Ogden and Buckman (1973); Reinboth (1973)
Hobson (1965), (1968), (1971), (1972); Starck and Davis (1966); Myrberg et al. (1967);
Olla et al. (1969); Green and Farwell (1971); Sale (1971); Reese (1973); Olla et al.
(1974)
Ballard and Emery (1970)
Clarke et al. (1967); Collette and Earle (1972); Sartori and Bright (1973)
Strasburgand Yuen (1960); Hobson (1963); Gooding and Magnuson (1967); Nakamura
(1972)
Magnuson and Karlen (1970)
Jenkins (1969); Magnuson and Karlen (1970); Popper et al. (1973)
Remote Sensing
Photography
Photography-Sonar
Sonar
Underwater television-Video tape
Sound (acoustic methods)
Yuen (1961), (1963), (1966); Nakamura (1972); Reinboth (1973)
Groot and Wiley (1965)
Gushing (1973)
Tyler (1971); Myrberg (1972); Stevenson (1972)
Winn (1964); Tavolga (1964), (1967); Morris (1966); Myrberg et al. (1971); Bright
(1972); Sartori and Bright (1973)
-------
Table I. (cont'd)
Category
Technique
Reference
Telemetry
Acoustic tags
Radio tag
Johnson (1960); Henderson et al. (1966); Poddubnyi et al. (1966); Haler et al. (1969);
Yuen (1970); Stasko (1971); Dodson et al. (1972); McCleave and LaBar (1972);
Young et al. (1972); Carey and Lawson (1973); Scholz et al. (1973); Stasko et al.
(1973); Olla et al. (1974); Rochelle and Coutant (1974)
Lonsdale and Baxter (1968)
-------
BEHAVIORAL PATTERNS IN LABORATORY BIOASSAYS
The modification of a normal behavioral response or impairment of any physiologi-
cal function by a toxicant may in many cases so diminish the changes of survival of an
organism that it will be eliminated. These impairments may affect a spectrum of acti-
vities from the rate of photosynthesis by algae, to the feeding efficiency of herbivores,
to the detection and avoidance of predators or success in prey capture (DeCoursey and
Vernberg, 1972; Vernberg and Vernberg, 1972a; Sprague, 1971). The survival of a
species may also be seriously threatened if the sublethal levels of a pollutant impair
the location of sexual partners or capacity to mate, though not affecting the survival
of the adult organism (Takahashi and Kittredge, 1973). Furthermore, the life cycles
of many marine invertebrates involve a series of developmental stages occupying
different niches, utilizing diverse food sources and being susceptible to different pollu-
tants. An example might be the numerous larval forms (e. g., gastropod veligers) that
feed in the natural surface film of neretic waters and thus are most susceptible to oil
pollution at this stage. Larval forms may also be less tolerant to a given toxicant, for
example, the adults of the fiddler crab, Uca pugilator, can tolerate 0.18 ppm Mercury
for two months while this concentration kills the stage I larvae in one to three days and
the stage V megalops in six hours (DeCoursey and Vernberg, 1972).
The question then becomes the detection of the sublethal effects of pollutants on the
various life stages of crucial representative species of the marine ecosystem under
study. The initial decision, as considered in an earlier section, is the optimum choice
of species and developmental stage. The behavioral criteria utilized for detecting and
measuring effects of contaminants in the laboratory is the primary subject of this sec-
tion.
Experimental Design
Behavioral analysis.
Most neretic marine organisms have evolved adaptive behavior to overcome the
stresses imposed on them by the environment. Quantitative studies of these altered
behaviors can provide the most sensitive bioassays of pollution stress. Behavioral
studies are also necessary to define the limits at which the behavioral adaptations
cannot compensate for the imposed stress and the organism's response is impaired.
The design of a behavioral bioassay should be based on both field and laboratory
observations of the behavioral repertoire of the species. These observations will
suggest both the scope and the limits of the behavioral characteristics of the organism
that may be amenable to analysis. While a complete analysis, documented to form a
behavioral ethogram, is desirable, that is seldom feasible. The analysis should, how-
ever, be sufficiently complete to allow the investigator to define the major behavioral
sequences and to recognize those components that are relatively stable and those that
are variable.
The field studies should initially establish the prime characteristics of the behavior
of the species. Although covered in an earlier section it would be illuminating to reit-
erate certain points. These include:
a. The spatial distribution of the species in the water column, or, for benthic
species, the type of substrate occupied or niche preference;
b. Spontaneous activities, e.g., ventilation or pumping rate, locomotor activities;
c. Feeding behavior, e.g., detection, capture, satiation;
d. Sexual behavior, e. g., attraction, courtship, copulation, spawning;
e. Parental care, e.g., nest preparation, guarding;
-------
f. Social behavior, e. g., aggregation, aggression, territorial instincts, social
structure;
g. Defensive behavior, e.g., warning displays, chemical secretions, withdrawal;
h. Evidence of phototactlc or thigmotactic behavior or other taxes;
i. Behavioral response to changes in light intensity or sound;
]. The presence or absence of diel or tidal rhythms in their behavior.
Laboratory studies based on the information derived from adequate field studies
will maximize the opportunities for true measures of the effects of the experimental
stress on the "normal" behavior of the animal. This does not imply that the laboratory
situation must mimic the field conditions in every detail. One must, however, under-
stand the behavior in the field in order to recognize which features of the laboratory
design must match the natural environment. Often one can succeed with a relatively
simple experimental approach if it satisfies a few basic criteria. Most investigators
recognize that proper substrate and water quality are essential. Light levels and dis-
turbing vibrations are the two physical parameters often given insufficient attention,
Both the appropriate light intensity and light/dark cycle must be provided for most
organisms. One can often reverse the normal light/dark cycle in studying the behavior
of nocturnal organisms. Random fluctuations in light intensity should be avoided. Due
to the incompressibility of water, most fish and invertebrates will respond to sources
of vibration that the observer may be unaware of; circulation pumps and refrigeration
compressors are prime sources of these vibrations.
When the appropriate laboratory situation has been established, a more detailed
study of the behavior observed in the field can follow with some confidence that the
results observed resemble the natural behavior. The criteria for the selection of the
behavioral traits best suited for the development of a bioassay should include:
a. Is the behavior obviously Important in the survival of the species?
b. Can the behavior be analyzed as a sequence of components?
c. Which components of the behavioral sequence are stable and reproducible?
d. Does the behavior have a well-defined endpoint?
e. Is the stimulus that triggers the behavior easily presented?
f. Can the strength of the stimulus be quantified ?
g. Is there a potential for quantifying the response or is it "all or none"?
It is also desirable that the behavioral characteristics selected for study yield data
that can be evaluated in the field.
Statistics
At this stage in the design of the bioassay, it is imperative that the statistical
methods to be utilized in the analysis be considered before the fact rather than after
data has been collected. It has been our experience that parametric statistics are
often inappropriate for the data generated by behavioral bioassays. One seldom has
any assurance that the spread of the observed criteria in the population has a normal
distribution. Nonparametric techniques must be applied in analyzing data when the
distribution of the measurement in the population is unknown. It is, however, a com-
mon misconception that only nonparametric statistics are useful with small samples.
A number of shortcut parametric tests have been developed that are applicable to small
samples. The price one pays for the use of nonparametrics is usually a loss of power
in testing hypotheses and some loss of accuracy in estimating confidence intervals.
Thus, if it can be established that the data are drawn from a normal population, the
power of the short cut parametric tests can be utilized. If the sample size is approxi-
mately fifty or greater, one may apply the ehi-square test for the goodness of fit to
-------
determine whether the frequencies in the classes of a sample distribution differ signi-
ficantly from the theoretical normal frequencies. Alternately, one may utilize the
Kolmogorov-Smirnov test to examine whether cumulative percentage distribution of the
sample differs significantly from a normal distribution. This test is easier to apply
than the chi-square test and it can be used for both grouped and ungrouped data. In
addition, the data can be plotted on normal distribution paper.together with the parallel
straight lines delimiting the confidence bands. An excellent example of small sample
parametric tests is the shortcut t-test (Lord's test). When it can be assumed that the
two independent samples are randomly drawn from a normal population of equal dis-
persion, this teat, utilizing only the two means and the two ranges, provides an esti-
mate of confidence limits that is nearly as good as the standard t-test in small samples.
Data from behavioral tests may be continuous or discrete, but often the data may
provide only ranking or only indicate the presence or absence of a criterion. We will
suggest only a few of the nonparametric tests that are both powerful and quick in evalu-
ating this data.
The rationale of both parametric and nonparametric inference rests on the random-
ness of the sampling. In behavioral bioassays, randomneaa in the distribution of the
results from a long sequence of tests is a measure of lack of bias in the results that
might have been imposed, for example, by gradual changes in the stock of experimental
animals. When the results are in the form of a simple two-sided alternative, a Run
Test will indicate the presence or absence of randomness in the sequence.
Many times an inference must be derived from a single set of samples. The Bank-
Difference Correlation test can be applied to bivariate data available only in ranks or
to ordered or continuous data to test the correlation between two variables in a set of
data. It provides a ready measure of the independence (or conversely, the degree of
relationship) or two parameters, as, for example, the size of an animal and its
response time. Another test of independence, the Contingency Test, can also be
applied when the variables are continuous, diacrete, or qualitative. It ia simple and
extremely general. Its application in a 2 x 2 contingency table is one of the most com-
mon statistical applications; however, it should be limited to those cases in which the
expected frequence in any cell is greater than four.
Often in behavioral bioassays one wishes to draw inferences from two samples. If
the two samples are independent and capable of being combined into a single ordered
series, the Rank Test, or Wilcoxon T test, is frequently used. This test is the non-
parametric test most nearly analogous to the standard parametric _t test and it is
approximately ninety percent as powerful. It is ideally suited for calculating, for
example, whether the response times of two different species of animals exposed to
the same stress are significantly different. If the two samples are related, as occurs,
for example, when each test organism is measured before and after exposure to a
stress, or the individuals in the experimental group are matched with individuals In
the control group, the tests of significance designed for independent samples may give
over-conservative results (these procedures reduce sampling error). The most useful
nonparametric tests for two related samples are the proportions, the sign and the
signed-rank tests.
Variability and noise
The final stage of the development of a behavioral bioassay is a study of the "noise"
in the system. Initially there is usually considerable scatter in the reaults which can
be reduced by conaidering separately each of the abiotic and biological components of
the assay. This refinement is largely a matter of trial and error, determination of the
optimum temperature and the effects of a temperature fluctuation, unexpected variations
in the water supply, proper aging of the experimental tanks, etc. A major part of the
variability is usually due to variations in the physiological condition of the experimental
organisms. It is usual to provide for an acclimation period for the experimental ani-
mals before they are used in an experimental situation. Paired experiments will reveal
-------
the importance of acclimation and statistical analysis will provide a measure of the
contribution of this factor to the variability of the measurements. It is perhaps
unnecessary to point out that large numbers of replicate measurements are often not
the optimum solution to overcoming the variability. Studies which statistically define
the contribution of acclimation, or feeding cycle, or age of the organisms, etc., to the
variability provide both for the selection of the optimum conditions and often allow one
to reduce the work required in the bioassay by permitting one to discard some of the
initial precautions without increasing the variability.
It is often desirable to raise the experimental organisms under controlled condi-
tions. As an example, in refining a study of the locomotor activity of the fiddler crab,
Uea pugilator, it was found necessary to select the test animals from hatches which
exhibited 90% or greater viability in order to reduce the variability of the assay
(DeCoursey and Vernberg, 1972).
It is sometimes suggested that there is a real dichotomy between those bioassays
that are used as research tools and the bioassays suitable for water quality monitoring.
Primarily this apparent dichotomy is an expression of the extent of the debugging that
has been applied to the bioassay. There are some research bioassays that are inordi-
nately time consuming or require, in their present stage of development, elaborate
instrumentation making them unsuitable for routine use. Often a research worker will
tolerate undesirable variability because the course of his study permits him to make
inferences from a minimum number of replications and it would thus be uneconomical
for him to refine the technique further. Many behavioral bioassays, however, can be
developed into routine assays.
Presentation of pollutant
A wide variety of pollutants are currently being added to both the marine and
freshwater ecosystems. The class of the pollutant(s), mode of action and where and
how it may be potentially added to the environment will determine the choice of species
and the specific behavioral assay. Conversely, chemical contaminants may be intro-
duced into an ecosystem in high concentrations as in a "slug" dose, e. g., vast dumping
from a barge or effluent from an outfall. These high concentrations will decrease with
time depending on certain environmental parameters (e. g., turbulent diffusion, floccu-
lation, biodegradation, etc.). It is also possible that the concentration of a chemical
pollutant may be relatively constant over prolonged periods as in the leaching of a
toxicant from dyked dredge sediments (DeCoursey and Vernberg, 1974: submitted for
publication).
No matted how added, the effect of exposing a test organism to an environmental
stress will generally be some form of an inverse relationship between the intensity of
the stress and the length of exposure for a given biological effect. The design of the
bioassay should include provisions for the exposure of the organisms to a range of con-
centrations of the pollutant and to several lengths of exposure. Generally, it is most
economical to explore short-term exposures to a series of three or more concentra-
tions differing by an order of magnitude. Realistic concentrations of a chemical pollu-
tant for this exploratory phase may range from 10"4 to 10~8 wt. /vol. The results of
these short-term exposures can then guide the selection of concentrations for long-
term exposures. It has been our experience that, for any long-term exposure experi-
ment, one must provide for maintaining the concentration of the pollutant, either by
infusion into an intermittent or continuously flowing system or by dialysis against an
immiscible solvent. Both the concentrations and the time intervals selected should be
realistic when compared to the field considerations under study.
Selection and application of the bioassay
In selecting and refining a behavioral bioassay for routine application in water
quality studies, there are additional criteria beyond those considered initially in estab-
lishing the bioassay that will contribute to its potential application:
-------
a. Is the species readily available throughout the year?
b. Is the species hardy?
c. Are there undesirable changes in its behavior during its spawning season?
d. Are larvae readily available?
e. Is there a potential for automating the measurement of the behavior?
Unfortunately, undue consideration of the above criteria is a major factor in often
selecting inappropriate organisms in water quality bioassays. The species selected
for study should be part of the normal biota and susceptible, at a behavioral level, to
the range of pollutant concentrations that are dangerous to the other species in the
ecosystem under study.
A major advancement in water quality bioassays has been the trend toward auto-
mation. Several simple behaviors such as locomotion, phototaxis and filtration rate,
are readily automated. Others, such as the pumping rate of oysters, can be automated
but require technical preparation of the oyster. The sensitivity of this assay and the
minimum time required per assay, offset the set-up time. Many other behaviors of
marine invertebrates, such as the defensive withdrawal time of many species of Poly-
chaeta, Mollusca and Tunicata, the feeding response of Anoumura and Brachyura, the
feeding movements of the Cirripedia, should be amenable to automation. The loco-
motor activities of certain marine fish species are also amenable to automation (for
example, Schuyf and de Groot, 1971; Gibson, 1973). Automation, however, of a poorly
designed experiment may obscure the results. We are reminded of feeding response
studies which were automated and analyzed in a computer to produce histograms that
were almost uninterpretable. In most instances, there is no substitute for simple
observation.
Another factor to consider in selecting a behavior for water quality studies is the
advantage of studying behaviors that are responses to sensory stimuli as compared to
spontaneous behaviors. These responses necessarily reflect the integrity of sensory
organs, the central nervous system and the musculature involved. One may measure
quantitatively the threshold of the receptors for response or the time before a response
is elicited. A sequence of many measurements can be completed in a short period of
time. Although too few examples exist to permit a comparison, elicited behaviors
probably provide the most sensitive types of bioassay available.
Behavioral bioassay literature
A large number of behavioral responses have been used at the research level to
measure the effects of sublethal concentrations of pollutants on aquatic organisms.
They are shown in tabular form, grouped for convenience into two major groups (Table
n). The performance of individual organisms has been studied at the level of the sen-
sory organs, an endogenous time sense, motor activities, motivation and learning
phenomena, as well as physiological responses closely related to the measurement of
particular behavioral patterns. A second group of responses involving the interactions
of animals has been classified as inter-individual responses. The examples of each
behavioral response are annotated with the speoies studied, the stress administered
and the literature citation.
INTEGRATION OF BEHAVIOR WITH STANDARD BIOASSAY TECHNIQUES
Behavioral observations used in conjunction with standard acute and chronic bio-
assays can significantly increase the value of tests. Bioassays which use functional
death as the measure of effect are ignoring the fact that alterations in behavior may be
occurring well before death. In this section we will present procedures that can be
used by an observer in the course of other testing which will serve to improve the sen-
sitivity of bioassays.
-------
Table II. Summary of Sublethal Behavioral Bioassays Used Primarily as Research Tools.
A. Performance of Individuals
Category Specific behavior Species
Sensory capacity Phototaxis Uca pugilator larvae
Calonodia americana
Acartia lillejeborgi
Paracalanug crassirostris
Salinity preference Gambusia affinis
Visual-optomotor Lepomis macrochirus
(flicker frequency)
Temperature preference Salmo salar
Poecilia reticulata
Caraesius auratus
Cyprinus carpio
Lepomis macrochirus
Micropterus salmoides
Pomoxis nigromaculatus
Ambloplltes repestris
Perca flavescens
Sensor inhibition Ictalurus natalis
Chemical attraction Nassarius obsoletus
(chemotaxis)
Pollutant or stress
Mercury
Xanthine dyes
DDT and malathion
Parathion
DDT
Sodium pentobarbitol
Copper
Various
Detergents
Kerosene fraction
Reference
Vernberg et al. (1973)
BJornberg and Wilbur (1968)
Hansen (1972)
Scheier and Cairns (1968)
Ogilvie and Anderson (1965)
Ogilvie and Fryer (1971)
Kleerekoper et al. (1973)
Neill et al. (1972)
Bardach et al. (1965)
Jacobsen and Baylou (1973)
-------
Table II. (cont'd)
Category
Sensor capacity (cont'd)
Time sense
Motor activity
Specific behavior
Chemoreception
Rheotaxis
Lateral line sensitivity
Endogenous timing of
daily activity
Avoidance of contami-
nants
Attraction to contami-
nants
Shelter seeking
Equilibrium
Swimming performance
and spontaneous loco-
motor activity
Species
Homarus americanus
Pachygrapsus crassipes
juvenile salmon
Salvelinus fontinalis
Uca pugilator adult
Salmo gairdneri
Cyprinodon variegatus
Gambusia affinis
Homarus americanus
Lepomis macrochirus
Oncorhynehus tshawtscha
Salmo gairdneri
Oncorhynehus nerka
Pomatomus saltatrix
Uca pugilator (larvae)
Carassius auratus
Pollutant or stress
Crude oil
Crude oil fractions and
aromatic hydrocarbons
Temperature
DDT
Mercuric chloride
Zinc sulphate
Pesticides
Pesticides
Kerosene fraction
Zinc
Temperature
Temperature
Temperature
Mercury
DDT
Reference
Atema and Stein (1974)
Takahashi and Kittredge (1973)
Keenleyside and Hoar (1954)
Anderson (1968)
Vernberg et al. (1974)
Sprague (1968)
Hansen (1969)
Hansen et al. (1972)
Atema et al. (1973)
Sparks et al. (1972)
Coutant and Dean (1972)
Brett (1967)
Olla and Studholme (1971)
DeCoursey and Vernberg (1972)
Davy et al. (1972)
-------
Table II. (cont'd)
Category
Motor activity (cont'd)
Motivation and learning
phenomena
Physiological responses
Specific behavior Species
, Swimming performance Lepomis macrochirus
and spontaneous loco-
Salvelinus fontinalis
Uca pugilator (adult)
Paleomonetes pugio
Carassius auratus
Carassius auratus
Feeding motivation Brachydanio rerio
Pomatomus saltatrix
Homarus americanus
Uca (sp. )
Learning Salvelinus fontinalis
Salvelinus fontinalis
Carassius auratus
Salmo salar
Ventilation rate Crassostrea virginica
Breathing rate Lepomis macrochirus
Pollutant or stress
Zinc
Copper
Mercury
Cadmium and low dis-
solved oxygen
Copper
Copper
Detergent
Temperature
Crude oil
Mercury
DDT
DDT
Various metals
Four insecticides
Hydrocarbons
Zinc
Reference
Cairns et al. (1973)
Drummond et al. (1973)
Vernberg et al. (1974)
White (1974)
Kleerekoper et al. (1972)
Kleerekoper (1973)
Cairns and Loos (1967)
Olla and Studholme (1971)
Atema and Stein (1974)
Klein and Linoer (1974)
Anderson and Peterson (1969)
Anderson and Prins (1970)
Weir and Hine (1970)
Hatfield and Johansen (1972)
Galtsoff et al. (1947)
Cairns et al. (1973)
-------
Table n. (cont'd)
B. Inter-Individual Responses
Category
Social motivation
Intraspecific visual
attraction
Aggregation and schooling
Aggression
Predation vulnerability
Species
Ictalurus natalis
Mugil cephalus
Pomatomus saltatrix
Lepomis cyanellus
Lepomis macrochirus
Iptalurus natalis
Predator
Gulls
Amia calva
Oncorhynchus klsutch
Micropterus salmoides
Salmo gairdneri (adult)
Micropterus salmoides
Micropterus salmoides
Prey
Fish
Fish (varied species)
Oncorhynchus nerka
Gambusia affinis
Salmo gairdneri (Juvenile)
Oncorhynchus tshawatscha
Gambusia affinis
Micropterus salmoides
Ictalurus punctatus
Pollutant or stress
Thermal
Thermal
Thermal
Turbidity
Zinc
Electric shock
Thermal
Poor condition
Thermal
Mercury
Thermal
Radiation
Thermal
Reference
Todd et al. (1972)
Olla (1974)
Olla and Studholme (1971)
Heimstra et al. (1969)
Sparks et al. (1972)
Todd et al. (1967)
Prentice (1969)
Herting and Witt (1967)
Sylvester (1972)
Kania and O'Hara (1974)
Coutant (1973)
Goodyear (1972)
Coutant etal. (1974)
-------
Acclimation
Standard Methods for the Examination of Water and Waste Water (1971) suggests
that before routine bioassays are performed, test animals should be acclimated for a
week or longer to laboratory conditions that are similar to test conditions. During a
period of four days immediately preceding a test, incidence of death or disease must
be less than ten percent. Also, specimens must show no abnormalities in appearance
or behavior at the time of their transfer to the test containers. It is at this point that
knowledge of the normal habits of the animal in both the field and laboratory can aid in
determining if the experimental subjects are in fact behaviorally acclimated. The
results of these observations may be considered a reflection of the relative health and
state of the test organisms.
Knowledge of test organisms' normal habits and environmental requirements as
learned from field observations are essential in determining such factors as the size
and shape of holding and testing aquaria, water quality, feeding schedules, and the
various physical parameters such as temperature, salinity, pH, etc. The aim is to
provide facilities which will enable the organism to express certain critical behaviors
which, if inhibited, may significantly alter their physiological condition. For example,
elliptical shaped aquaria should be provided for fast swimming pelagic forms; suitable
substrate for burying and burrowing forms; cover for reef residents; enough space as
defined by normal observations for animals which tend to be territorial; light levels
appropriate for the species.
Determining the fitness of a stock of animals for bioassays may utilize observable
behavioral activity in addition to more conventional criteria. The use of behavioral
characteristics in a diagnostic manner assumes that the behavior of the species is
already well understood by the investigator. Diagnostic behavior characteristics that
one might use in determining fitness might include feeding activity with the organisms
expected to feed during acclimation, particularly if they are offered food that comprises
their normal diet (as determined by field observations and stomach analysis). Altera-
tions in photo-responsiveness, increases in excitability, changes in motor activity,
schooling configuration, agonistic displays, and opercular movements are all observa-
tions which may indicate that organisms are not yet acclimated or suitable as test sub-
jects.
Acute Eioassays
Standard Methods (1971) suggests that physiological death should be the prime
measure of effect for routine bioassays. It also suggests that bioassayists keep
records of the number of animals which are alive but show pronounced symptoms of
intoxication and distress, such as loss of equilibrium and other markedly abnormal
behavior. Because death can be predicted if certain behavioral activities are altered,
the usefulness of bioassays can be significantly improved by accurate and consistent
reporting of the number of animals with impaired behavior, a description of the symp-
toms of the impairment, and a discussion of its probable ecological significance. Bio-
assays will also be improved If the habitat and behavioral requirements of the animals
are considered when designing the bioassay.
Most behavioral observations that were applicable in improving acclimation are
also applicable for bioassays. Animals should be provided adequate physical, chemi-
cal and biological surroundings and the animals should orient normally to them. For
example, phototropic animals, such as crab larvae, should react to light, burrowing
animals should burrow, gregarious animals should interact and animals should exhibit
normal rhythms of activity. An inventive researcher can improve observations by
devising procedures, such as timing ventilation rates, to quantify changes in behavior
without disturbing animals or interrupting the progress of the bioassay.
-------
Long-term Bioassays
In chronic bioassays, i.e., testa that are conducted over extended time periods
and/or over entire life cycles it is particularly important to provide a suitable environ-
ment for the test organisms to grow, mature, and reproduce. The more closely the
environmental requirements of the species can be met in the laboratory the more repre-
sentative will be the behavior of the animals. In bioassays that include the total life
cycle of the animals this may be particularly important since such behaviorally involved
phases as courtship and reproduction may be altered, and in turn the significance of the
results. Since the end point in chronic bioassays is not necessarily death, the signifi-
cance of behavior to the interpretation of the data is much more critical. Detailed and
careful observations of behavior during the course of an experiment may also provide
explanations for trends in the data.
PROCEDURES AND FACILITIES FOR CAPTURING AND HOLDING ORGANISMS
Great care and expense may be committed to the major aspects of the actual
experimentation, but often little attention is given to the critical aspects of animal
acquisition and care. Questions that should be considered by the experimenter work-
ing with marine organisms include:
a. How may organisms be captured and transported from the field to the labora-
tory with minimal mechanical damage and physiological stress?
b. What physical and biological conditions must be provided in laboratory holding
facilities for keeping aquatic organisms in the best possible shape ?
c. How is disease to be monitored and controlled?
d. How can the build-up of metabolites be controlled in closed systems?
e. Which animals might be raised in the laboratory?
To reiterate what was mentioned in an earlier section of this chapter, there is no sub-
stitute for defining the normal requirements of study organisms by a variety of techni-
ques and methods of study in the natural environment.
What we present in this section may be considered an introduction to some of the
more important aspects of acquisition, maintenance and culture of marine bioassay
organisms, with reference to some of the most significant publications on the subject.
Field Collection and Transport
It is obvious that the capture and transport of organisms should be done in a man-
ner which minimizes mechanical injury and physiological trauma. Fishing gear used
in commercial endeavors or in normal scientific collecting, when taxonomy or biologi-
cal surveys are the aim, are not often adaptable to collection of live animals for
laboratory use.
The individual characteristics of the organism, including their hardiness, will
determine how best to capture and transport them. For example, delicate types of
fishes such as Atlantic silversides, Menidia menidia, or any of the herrings and ancho-
vies are easily captured by shore seine. However, rather than beaching the seine, it
is far less deleterious to the fish to scoop them with a bucket or soft net from the water
and into a transport container while they are still swimming within the enclosed net in
shallow water. Another excellent method of catching animals with little injury is with
barbless hook and line. A variety of adult species including tuna (Nakamura, 1972)
bluefish, Pomatomus saltatrlx and Atlantic mackerel, Scomber scombrus, (Olla,
unpublished) have been collected successfully in this way. Juvenile bluefish, spot,
Leiostomus xanthurus, and winter flounder, Pseudopleuronectes americanua, have
also been successfully collected in this way. Collection of slow-moving benthic ani-
mals by hand, with the aid of SCUBA, is less damaging to the animal than traps or
-------
dredges. This method has been successfully employed in capturing tautog, Tautoga
onitis. while the fish were in a quiescent sleep phase during the night (Olla et al.,
1974). Collecting positively photo-responsive invertebrate larvae and adults and a
variety of other juvenile organisms can be accomplished with a night light. Innovative
modifications in plankton nets, which avoid mechanical injury, are also employed.
When beam or otter trawls must be used, slow tows which are very short in duration
are suggested.
Transport containers, especially for highly motile species, should be elliptical in
shape to prevent animals from crowding in corners or damaging themselves by striking
the walls (Nakamura, 1972). Verheijen (1956) suggested holding sardines and anchovies
in plastic bags, a method which has proved satisfactory (Tardant, 1962). Plastic bags
or a plastic curtain suspended in a way which forms a cushion from the container wall
will greatly aid in preventing physical damage.
Maximum density in a container will vary with the requirements of the species, as
well as with the water quality and the duration of holding. Aggressive animals should
be isolated in separate containers.
When these temporary holding and transporting measures must be sustained for
much more than 30 minutes, adequate safeguards must be taken to shield the animals
from sunlight and from extreme temperature and dissolved oxygen changes. Styrofoam
coolers and addition of ice around the outside of uninsulated containers provide tempo-
rary temperature maintenance. The use of anesthetics should be avoided, if at all
possible, during transport and handling due to potential after effects on organism
behavior (Goddard et al., 1974).
For many intertidal organisms, especially sessile types, large volumes of water
are not needed. Items such as wet seaweed may be an ideal transport medium for
these animals. If containers with appreciable volumes of water must be transported
by motor vehicle, an elliptical tank with a "conning tower" will prevent sloshing of the
water and organisms (Nakamura, 1966).
Laboratory Holding Requirements
Basic specifications for designs and materials for marine aquarium facilities have
been detailed by a number of authors: Clark and Clark (1964), Hagen (1970), Congres
International d'Aquariologie (1962-63), Olla et al. (1967), Spotte (1970, 1973). The
chapter by Atz in Clark and Clark (1964) is particularly useful for such basic principles
and practices as avoidance of toxic materials and maintenance of water condition. Still
one of the most useful papers on marine closed system maintenance is by Saeki (1958)
(also consult Davis, 1953 and Lewis, 1963). The suitability of various construction
materials in culture systems is further discussed in papers by Dyer and Richardson
(1962) and Bernhard et al. (1966). While the problem of disease is discussed by a
number of workers, Sindermann (1970) presents a useful and definitive treatment of
marine fish and shellfish diseases.
When organisms are to be used in behavioral bioassays, the basic requirements
iterated above must be supplemented. Ideally, conditions in the laboratory should
approximate the organism's normal requirements. For example, natural cycles of
light, temperature and salinity must be maintained, as well as habitat needs and food
which quantitatively and qualitatively approach natural requirements. The quality of
the light should also be considered. Acute illumination changes are to be avoided with
the most desirable method of light introduction as part of a daily cycle employing a
system which slowly increases and decreases light levels approximating sunrise and
sunset. Other physical conditions such as current, pressure, turbulence and natural
diurnal and tidal ranges of temperature may also be required.
Suitable animal density depends partly on natural conditions (see Section II).
Benthic and cover-dependent organisms should be provided with suitable substrate
and/or habitat materials.
22
-------
Type of food and mode and time of feeding should approximate natural conditions
as closely as possible as determined from field observations. Although it is often not
possible to duplicate the exact kinds of food the animal eats, it is often possible to
approximate their nutritional level and quality. At the most basic level, carnivores
should be supplied with a diet that suits their specific requirements, which are obvi-
ously different from those of herbivores. At present, unfortunately, knowledge of the
nutritional requirements of marine organisms is at best deficient in many aspects.
Failure to eat is common in animals recently brought into the laboratory. This
initial period of non-feeding may be shortened by social facilitation, i. e., adding ani-
mals which are already feeding to the holding tank. When laboratory animals do not
begin to eat or stop eating, once having begun feeding in the laboratory, this may be
indicative of an unhealthy condition in the holding facility, rendering the animals use-
less for experimentation.
Laboratory Culture of Organisms
Laboratory culture techniques are now sufficiently developed for certain species
to consider culture as a feasible method for obtaining experimental larval material,
when the much more desirable method of using animals from the particular locale that
will receive the impact is not possible (see earlier section of this chapter). Cultiva-
tion of marine organisms was the topic of the International Helgoland Symposium in
1969 (Kinne and Bulnheim, 1970). Methods of bivalve rearing are reported by
Loosanoff and Davis (1963). Facilities for bivalve larval bioassay are discussed by
Woelke (1972). For Crustacea culture, numerous references are cited by Costlow and
Bookhout (1968) and others in a symposium volume on decapod larval development. A
method for polychaete culture has been developed by Reish and Barnard (1960). For
two very useful general works on invertebrate culture, see Galtsoff et al. (1937) and
Costello et al. (1957). A bibliography of marine fish culture efforts has been compiled
by May (1971).
The prime requirement for laboratory reared organisms is that they be physio-
logically compatible with natural populations. Physiological compatibility should be
tested by comparing the results of bioassays on both laboratory animals and on natural
populations. Other criteria must be developed for larval forms. Histological compari-
son, which will indicate tissue normality and nutritional condition, can also be used.
-------
LITERATURE CITED
Anderson, J.M. 1968. Effect of sublethal DDT on the lateral line of brook trout,
Salvellnus tontlnalls. J. Fish. Res. Board Can. 25:2677-2682.
Anderson, J.M., and M.R. Peterson. 1969. DDT: Sublethal effects on brook trout
nervous system. Science 164:440-44!
Anderson, J.M., and H.B. Prlns. 1970. Effects on sublethal DDT on a simple re-
flex in brook trout. J. Fish. Res. Board Can. 27:331-334.
Atema, J., S. Jacobson, J. Todd, and D. Boylan. 1973. The Importance of chemical
signals in stimulating behavior of marine organisms: effects of altered environ-
mental chemistry on animal communication, p. 177-197. JnG. E. Glass (ed.)
Bloassay Techniques and Environmental Chemistry. Ann Arbor Science Pub-
lishers Inc., Ann Arbor, Michigan.
Atema, J., and L. Stein. 1974. Effects of crude oil on the feeding behavior of the
lobster Homarus americanus. Environ. Pollut. 6:77-86.
Atz, J.W. 1964. Some principles and practices of water management for marine
aquariums, p. 3-16. In_J.R. Clark and H. L. Clark (ed.) Sea-water systems
for experimental aquariums. U.S. Fish Wlldl. Serv. Res. Rep. 63.
Ballard, R.D., and K. O. Emery. 1970. Research Submerslbles in Oceanography.
Marine Technological Society, Washington, D. C., 70 p.
Bardach, J.K., M. Fujlya, and A. Holl. 1965. Detergents: Effects on chemical
sense of the fish Ictalurus natal is (Le Sueur). Science 148:1605-1607.
Bernhard, M., A. Zattera, and P. Filesl. 1966. Suitability of various substances
for use In the culture of marine organisms. Pubbl. staz. zool. Napoll 35:80-104.
BJornberg, T.K. S., and K. M. Wilbur. 1968. Copepod phototaxis and vertical
migration influenced by xanthine dyes. Blol. Bull. 134:398-410.
Brett, J.R. 1967. Swimming performance of sockeye salmon (Oncorhynchus nerka)
In relation to fatigue time and temperature. J. Fish. Res. Board Can.
24:1731-1741.
Bright, T.J. 1972. Blo-acoustlc studies on reef organisms. Nat. His. Museum,
Los Angeles County Sol. Bull. 14:45-69.
Brown, J.H., M. A. Cantrell, andS.M. Evans. 1973. Observations on the behavior
and coloration of some coral reef fish (Family: Pomacentrldae). Mar. Behav.
Physlol. 2:63-71.
Cairns, J., Jr., andJ.J. Loos. 1967. Changed feeding rate of Brachvdanio rerlo
(Hamilton-Buchanan) resulting from exposure to sublethal concentrations of
zinc, potassium dichromate, alkyl benzene sulfonate detergent. Proc. Penn.
Aca. Sci. 40:47-52.
Cairns, J., Jr., R.E. Sparks, and W.T. Waller. 1973. The relationship between
continuous biological monitoring and water quality standards for chronic exposure,
p. 383-402. In G.E. Glass (ed.) Bioassay Techniques and Environmental Chem-
istry. Ann Arbor Science Publishers Inc., Ann Arbor, Michigan.
Carey, F.G., and K.D. Lawson. 1973. Temperature regulation In free-swimming
bluefln tuna. Comp. Blochem. Physlol. 44A375-392.
24
-------
Clark, J.R., andR.L. Clark (ed.) 1964. Sea-water systems for experimental
aquariums. U.S. Fish Wildl. Serv. Res. Rep. 63:1-92.
Clarke, T.A., A.O. FlechsLg, andR.W. Grigg. 1967. Ecological studies during
Project Sealab II. Science 157:1381-1389.
Collette, B. B., and S. A. Earle. 1972. Results of the Tektite program; ecology of
coral reef fishes. Nat. Hist. Museum, Los Angeles County Sci. Bull. 14:1-179.
Congres international d'aquariologie, I6r Monaco 1960. 1962-1963. Bull. Inst.
Oceanogr. Monaco numero Special IA-ID.
Costello, D.P., M.E. Davlson, A. Eggers, M. H. Fox, and C. Henley. 1957.
Methods for obtaining and handling marine eggs and embryos. Marine Biol.
Lab., Woods Hole, Mass. 247 p.
Costlow, J.D., Jr., andC.G. Bookhout. 1968. The complete larval development of
the land-crab Cardlsoma guanhuml Latreille in the laboratory (Brachyura,
Gecarclnldae). Crustaceana, Supplement 2:259-270.
Coutant, C. C. 1973. Effect of thermal shock on vulnerability of juvenile salmonlds
to predation. J, Fish. Res. Board Can. 30:965-973.
Coutant, C.C., andJ.M Dean. 1972. Relationships between equilibrium loss and
death as responses of juvenile chlnook salmon and rainbow trout to acute thermal
shock. U.S. At. Energy Comm. Res. Devel. Rep. BNWL 1520- 19 p.
Coutant, C.C., H.M. Ducharme, Jr., and J.R. Fisher. 1974. Effects of cold
shock on vulnerability of juvenile channel catfish (Ictalurus punctatus) and large-
mouth bass (Mlcropterua salmoldes) to predation. J. Fish. Res. Board Can.
31:351-354.
Cushlng, D. 1973. The Detection of Fish. Pergamon Press, Oxford. 200 p.
Davis, H.S. 1953. Culture and Diseases of Game Fishes. Univ. Calif. Press,
Berkeley. 332 p.
Davy, F.B., H. Kleerekoper, and P. Gensler. 1972. Effects of exposure to sub-
lethal DDT on the locomotor behavior of the goldfish (Carassius auratus).
J. Fish. Res. Board Can. 29:1333-1336.
DeCoursey, P., and W. B. Vernberg. 1972. Effect of mercury on survival, metabo-
lism and behavior of larval Uca pugllator (Brachyurs). Olkos 23:241-247.
1974. The effect of dredging in a polluted estuary on the physiology of
larval zooplankton. (submitted to Water Research).
Dodson, J.J., W. C. Leggett, and R. Jones. 1972. The behavior of adult American
shad (Alosa sapidlsslma) during migration from salt to freshwater observed by
ultrasonic tracking techniques. J. Fish. Res. Board Can. 29:1445-1449.
Drummond, R.A., W.A. Spoor, and G. F. Olson. 1973. Some short-term indicators
of sublethal effects of copper on brook trout, Salvellnus fontlnalls. J. Fish. Res.
Board Can. 30:698-701.
Dyer, D. L., and D. E. Richardson. 1962. Materials of construction in algal culture.
Appl. Microblol. 10:129-131.
Fox, H.M. 1936. The activity and metabolism of poikilothermal animals in different
latitudes. I. Proc. Zool. Soc. London 1936:945-955.
Galtsoff, P.S., W.A. Chlpman, Jr., J.B. Engle, and H. N. Calderwood. 1947.
Ecological and physiological studies of the effect of sulphate pulpmill wastes on
oysters In the York River, Va. U.S. Fish Wildl. Serv. Fish. Bull. 43:58-186.
Galtsoff, P.S., F.E. Lutz, P.S. Welch, andJ.G. Needham (ed.) 1937. Culture
Methods for Invertebrate Animals. Comstock Publ. Co., Ithaca, New York 590 p.
25
-------
Gibson, R. N. 1973. Tidal and clrcadlan activity rhythms In juvenile plaice, Pleuro-
nectes platessa. Mar. Blol. 22:379-386.
Goddard, C.I., J.W. Lllley, andJ.S. Talt. 1974. Effects of MS-222 anesthetization
on temperature selection In lake trout, Salvellnus namaycush. J. Fish. Res.
Board Can. 31:100-103.
Goodlng, R.M., and J. J. Magnuaon. 1967. Ecological significance of a drifting
object to pelagic fishes. Pae. Sol. 21:486-497.
Goodyear, C. P. 1972. A simple technique for detecting effects of toxicants or other
stresses and predator-prey interaction. Trans. Amer. Fish. Soc. 101:367-370.
Green, J.M., and M. Farewell. 1971. Winter habits of the cunner, Tautogolabrus
adapersus (Walbaum 1792), In Newfoundland. Can. J. Zool. 49:1497-1499.
Groot, C., and W. L. Wiley. 1965. Time-lapse photography of an ASDIC Echo-
Sounder PPI-Scope as a technique for recording fish movements during migration.
J. Fish. Res. Board Can. 22:1025-1034.
Haefner, P. A., Jr. 1970. The effect of low dissolved oxygen concentrations on
temperature-sallnty tolerance of the sand shrimp, Crangon septemsplnosa
Say. Physlol. Zool. 43:30-37.
Hagen, W. 1970. Aquarium design criteria. Drum and Croaker, Special Edition.
Nat. Fisheries Center and Aquarium, U.S. Dept. Interior Washington, D.C.,
107 p.
Hansen, D.J. 1969. Avoidance of pesticides by untrained sheepshead minnows.
Trans. Amer. Fish. Soc. 98:426-429.
1972. DDT and malathlon: Effect on salinity selection by mosquito
fish. Trans. Amer. Fish. Soc. 101-346-350.
Hansen, D.J., E. Matthews, S.H. Nail, and D. P. Dumas. 1972. Avoidance of
pesticides by untrained mosquito fish, Gambusla afflnls. Bull. Environ. Contain.
Tox 8:46-51.
Hasler, A. D., E.S. Gardella, R.M. Horral, andH.F. Henderson. 1969. Open
water orientation of white bass, Roeeus chrysops, as determined by ultrasonic
tracking methods. J. Fish. Res. Board Can. 26:2173-2192
Hatfleld, C.T., andP.H. Johansen. 1972. Effects of four Insecticides on the ability
of Atlantic salmon parr (Salmo salar) to learn and retain a simple conditioned
reflex. J. Fish. Res. Board Can. 29:315-321
Heimstra, N. W., D.K. Damkot, andN.G. Benson. 1969. Some effects of silt
turbidity on behavior of juvenile largemouth bass and green sunfish. U. S. Fish
Wlldl. Serv. Tech. Paper 20:1-9.
Henderson, H. F., A.D. Hasler, and G. C. Chipman. 1966. An ultrasonic trans-
mitter for use In studies of movements of fishes. Trans. Amer. Fish. Soc.
95:350-356.
Herting, G.E., and W. Witt, Jr. 1967. The role of physical fitness of forage
fishes in relation to their vulnerability to predation by the bowfln (Amla Calva).
Trans. Amer. Fish. Soc. 96:427-430.
Hobson, E.S. 1963. Feeding behavior in three species of sharks. Pac. Scl.
17:179-194.
1965. Diurnal-nocturnal activity of some Inshore fishes in the Gulf
of California. Copeia 1965:291-302.
1968. Predatory behavior of some shore fishes in the Gulf of Callfor
nia. U.S. Fish Wlldl. Serv. Res. Rep. 73:1-92.
-------
1971. Cleaning symbiosis among California Inshore fishes. Fish.
Bull., U.S. 69:491-535.
1972. Activity of Hawaiian reef fishes during the evening and morning
transitions between daylight and darkness. Fish. Bull., U.S. 70:715-740.
Jaoobson, S.M. andD.B. Boylan. 1973. Effects of sea water soluble fraction of
kerosene on chemotaxis In a marine snail, Nassarhis obsoletue. Nature London
241:213-215.
Jenkins, T.M., Jr. 1969. Social structure, position choice and miorodlstrlbutlon
of two trout species (Salmo trutta and Salmo gairdnerl) resident in mountain
streams. Anlro. Behav. Monogr. 2:1-123.
Johnson, J.H. 1960. Sonic tracking of adult salmon at Bonneville Dam, 1957.
U.S. Wildl. Serv. Fish. Bull. 60:471-485.
Kanla, H. J., and J. O'Hara. 1974. Behavioral alterations In a simple predator-
prey system due to sublethal exposure to mercury. Trans. Amer. Fish. Soc.
103:134-136.
Keenleyslde, M.H.A. 1962. Skin-diving observations of Atlantic salmon and brook
trout In the Mlramlchl Elver, New Brunswick. J. Fish. Res. Board Can.
19:625-634.
1971. Aggressive behaviour of male longear sunflsh (Lepomls
megalotls). Z. Tierpsychol. 28:227-240.
Keenleyslde, M.H.A., and W. S. Hoar. 1954. Effects of temperature on the res-
ponses of young salmon to water currents. Behavior 7:77-87.
Klnne, O., and H.P. Bulnhelm (ed.) 1970. Cultivation of marine organisms and its
Importance for marine biology. Intl. Helgoland Symp. 1969. Helgolander wlss.
Meeresunters. 20:1-721.
Kleerekoper, H. 1973. Effects of copper on the locomotor orientation of fish.
Office of Research and Monitoring, Res. Rep. EPA-R3-73-045.
Kleerekoper, H., J. B. Waxman, and J. Matls. 1973. Interaction of temperature
and copper Ions as orienting stimuli In the locomotor behavior of the goldfish
(Carasslus auratur). J. Fish. Res. Board Can. 30:725-728.
Kleerekoper, H., G. F. Westlake, J.H. Matls, andP.J. Gensler. 1972. Orienta-
tion of goldfish (Carasslus auratus) In response to a shallow gradient of sub-
lethal concentration of copper in an open field. J. Fish. Board Can. 29:45-54.
Klein, M. L., andJ.L. Lincer. 1974. Behavioral effects of dleldrin upon the
fiddler crab, Uca, pugllator. (In press).
Lewis, W.M. 1963. Maintaining Fishes for Experimental and Instructional Pur-
poses. So. 111. Univ. Press, Carbondale. 100 p.
Lonsdale, E.M., and G. T. Baxter. 1968. Design and field tests of a radio-wave
transmitter for fish tagging. Prog. Fish-Cult. 30:47-52.
Loosanoff, V.L., and H. C. Davis. 1963. Rearing of bivalve molluscs, p. 1-136.
In_F.S. Russell (ed.) Advances In Marine Biology, Vol. 1. Academic Press,
London.
Magnuson, J. J., and D. J. Karlen. 1970. Visual observation of fish beneath the
ice in a winterkill lake. J. Fish. Res. Board Can. 27:1059-1068.
May, R. C. 1971. An annotated bibliography of attempts to rear the larvae of
marine fishes in the laboratory. NOAA Tech Rep. NMFS-SSRF-632, 24 p.
Mayer, A. G. 1914. The effects of temperature upon tropical animals. Pap. Tortuga
Lab. 6:3-24. Carnegie Institution, Washington, D. C.
27
-------
McCleave, J.D., and G. W. LaBar. 1972. Further ultrasonic tracking and tagging
studies of homing cutthroat trout (Salmo clarkl) In Yellowstone Lake. Trans.
Amer. Fish. Soc. 101:4-45.
Myroerg, A. A. 1972. Using sound to Influence the behavior of free-ranging marine
animals, p. 435-468. In H. E. Wlnn and B. L. Olla (ed.) Behavior of Marine
Animals, Vol. 2 Plenum Press, NewYork.
Myrberg, A. A., B.D. Brohy, and A.R. Emery. 1967. Field observations on
reproduction of the damselfish, Chromls multlllneata (Pomacentridae) with
additional notes on general behavior. Copeia 1967:819-826.
Myrberg, A.A., Jr., S.J. Ha, J. Banbury, and S. Walewskl. 1971. Effectiveness
of acoustic signals in attracting "blue-water" sharks to the Immediate vicinity of
an underwater sound source. Office of Naval Research Tech. Rep., Dept. of
the Navy.
Nakamura, E.L. 1966. Fiberglass tanks for transfer of pelagic fishes. Progr.
Fish-Cult. 28:60-62.
1972. Development and use of facilities for studying tuna behavior,
p. 245-277. Jn H. E. Wlnn and B.L. Olla (ed.) Behavior of Marine Animals,
Vol. 2. Plenum Press, NewYork.
Netll, W.H., J.J. Magnuson and G. G. Chlpman. 1972. Behavioral thermoregu-
latlon by fish: A new experimental approach. Science 176:1443-1445.
Nelson, D.R., R.H. Johnson, andL.G. Waldrop. 1969. Responses In Bahamian
sharks and groupers to low-frequency, pulsed sounds. Bull. So. Calif. Acad.
Sol. 68:131-137.
Newman, M.A. 1956. Social behavior and interspecific competition in two trout
species. Physiol. Zool. 29:64-81.
Norrls, K.S. (ed.). 1966. Whales, Dolphins, and Porpoises. University of Calif-
ornia Press, Berkeley. 789 p.
Ogden, J.C. , and N S. Buckman. 1973. Movements, foraging groups, and diurnal
migrations of the striped parrotflsh Scarus crolcensis Bloch (Scaridae). Ecology
54:589-596.
Ogllvie, D.M., andJ.M. Anderson. 1965. Effect of DDT on temperature selection
by young Atlantic salmon, Salmo salar. J. Fish Res. Board Can. 22-503-512.
Ogilvie, D.M., and J.N. Fryer. 1971. Effect of sodium pentobarbital on the tem-
perature selection response of guppies (Poecllla retlculata). Can. J. Zool.
49:949-951.
Olla, B. L. 1974. The effect of temperature of fish-to-fish attraction in an Inshore
schooling species, striped mullet, Mugil cephalus. In U. S. At. Energy Comm.
Prog. Rep. AT (49-7) 3045. Unpublished manuscript.
Olla, B. L., and A. L. Studholme. 1971. The effect of temperature on the activity
of blueflsh, Pomatomus saltatrix L. Biol. Bull. 141:337-349.
Olla, B.L., A. J. Bejda, andA.D. Martin. 1974. Dally activity, movements,
feeding and seasonal occurrence in the tautog, Tautoga onitis, Fish. Bull. ,
U.S. 72:27-35.
Olla, B.L., W.W. Marchloni, andH.M. Katz. 1967. A large experimental aquarium
system for marine pelagic fishes. Trans. Amer Fish. Soc. 96-143-150.
Olla, B.L., R. Wicklund, and S. Wllk. 1969. Behavior of winter flounder in a
natural habitat. Trans. Amer. Fish. Soc. 98:717-720.
28
-------
Paine, R. T. 1969. The Plsaster-Tegula Interaction; prey patches, predator food
preference, and Intertidal community structure. Ecology. 50:950-961.
Poddubnyl, A.C., Y. I. Spektor, and S. M. Kldun. 1966. The results of preliminary
experiments In tracking sturgeon carrying electronic tracer tags. Vop. Bchtlol.
6:725-734.
Popper, A.N., M. Salmon, and A. Parvulescu. 1973. Sound localization by the
Hawaiian squirrel fishes, Myrlprlstls berndtl and M. argyromus. Anlm. Behav.
21:86-97.
Prentice, E.F. 1969. Gull predatlon In a reactor discharge plume. U.S. At.
Energy Comm. Develop. Rep. BNWL 1050, Pt. 2:2.33-2.34.
Reese, E. S. 1973. Duration of residence by coral reef fishes on "home" reefs.
Copela 1973:145-149.
Relnboth, R. 1973. Duallstlc reproductive behavior In the protogynous wrasse
Thalassoma blfaslcatum and some observations on Its day-night changeover.
Helgolander wlss. Meersunters. 24:174-191.
Relsh, D.J., andJ.L. Barnard. 1960. I ield toxlclty In marine waters utilizing
the polychaetous annelid Capltella capltata (Fabrlclus). Pac. Nat. 1:1-8.
Rochelle, J.M., andC.C. Coutant. 1974. Temperature sensitive ultrasonic fish
tag, Q-5099. Oak Ridge Nat. Lab. ORNL-TM-4438.
Saekl, A. 1958. Studies on fish culture in filtered closed-circulation aquaria. Bull.
Jap. Soc. Scl. Fisheries 23:684-695. Trans. byE.R. Hope. 1974. Dlv. Scl.
Info. Services, DRB, Canada.
Sale, P. F. 1971. Extremely limited home range In a coral reef fish Dascyllus
aruanus (Plsces:Pomacentrldae). Copeia 1971:324-32,
Sartorl, J.D., andT.J. Bright. 1973. Hydrophonlc study of feeding activity of
certain Bahamian parrotflshes, Family Scarldae. Hydrolab J. 2:25-56.
Scheler, A., and J. Cairns, Jr. 1968. An apparatus for estimating the effects of
toxicants on the critical flicker frequency response of the blueglll sunflsh.
Purdue Univ. Eng. Ext. Series No. 132, Part 2:849-855.
Scholz, A. T., J. C. Cooper, D.M. Madison, R.M. Horrall, A.D. Hasler, A. E.
Dizon, andR.J. Poff. 1973. Olfactory Imprinting In coho salmon: behavioral
and electrophysiologlcal evidence. Proc. 16th Conf. Great Lakes Res. p.
143-153.
Schuyf, A., and S. J. deGroot. 1971. An Inductive locomotion detector for use In
diurnal activity experiments in fish. J. Cons. int. Explor. Mer 34:126-131.
Sindermann, C.J. 1970. Principle Diseases of Marine Fish and Shellfish. Academic
Press, New York. 369 p.
Smith, S. H. 1973. Application of theory and research In fishery management of the
Laurentlan Great Lakes. Trans. Amer. Fish. Soc. 102:156-163.
Sparck, R. 1936. On the relation between metabolism and temperature In some
marine lamelllbranchs, and its zoogeographlcal significance. Blol. Medd.
13:No. 5.
Sparks, R. E., W. T. Waller, and J. Cairns, Jr. 1972. Effect of shelters on the
resistance of dominant and submissive bluegllls (Lepomls macrochlrus) to lethal
concentrations of zinc. J. Fish. Res. Board Can. 29:1356-1358.
Spotte, S.H. 1970. Fish and Invertebrate Culture. Wiley-Intersclence, New York.
145 p.
-------
1973. Marine Aquarium Keeping. Wiley-Interscience, New York.
171 p.
Sprague, J.G. 1968. Avoidance reactions of rainbow trout to zinc sulphate. Water
Research 2;367-372.
1971. Measurement of pollutant toxicity to fish - III. Sublethal effects
and "safe" concentrations. Water Research 5:245-266.
Standard Methods for the Examination of Water and Wastewater. 1971. Amer. Pub.
Health Assoc., Inc., New York. 874 p.
Starck, W.A., II, andW.P. Davis. 1966. Night habits of fishes of Alligator Reef,
Florida. Ichthyologica 38:313-356.
Stasko, A. B. 1971. Bibliography of underwater telemetry in biological applications.
Underwater Telemetry Newsletter 1(1). Fish. Res. Board Can., St. Andrews,
New Brunswick.
Stasko, A.B., R.M. Horrall, A.D. Hasler, and D. Stasko. 1973. Coastal move-
ments of mature Fraser River pink salmon (Oneorhynchus gorbuscha) as
revealed by ultrasonic tracking. J. Fish Res. Board Can. 30:1309-1316.
Stevenson, R.A., Jr. 1972. Feeding behavior of the bicolor damsel-fish (Eupoma-
centrus partitas Poey) by environmental factors, p. 278-302. In_H.W. Winn and
B. L. Olla (ed.) Behavior of Marine Animals, Vol. 2. Plenum Press, New York.
Strasburg, D.W. , and H. S. H. Yuen. 1960. Progress in observing tuna underwater
at sea. J. Cons. int. Explor. Mer. 26:89-93.
Sylvester, J. R. 1972. Effect of thermal stress on predator avoidance in sockeye
salmon. J. Fish. Res. Board Can. 29:601-603.
Takahashi, J.T., andJ.S. Kittredge. 1973. Sublethal effects of the water soluble
component of oil: Chemical communication in the marine environment, p. 259-264.
In_D.G. Ahearn and S. P. Meyers (ed.) The Microbial Degradation of Oil Pollu-
tants. Louisiana State Univ. Press, Pub. No. LSU-SG-73-01.
Tardent, P. 1962. Keeping Clupeidae, Scombridae, and Scomberesocidae in the
Naples aquarium. Bull. Inst. Oceanog. Monaco No. special 1A: 29-34.
Tavolga, W. N. (ed.). 1964. Marine Bio-Acoustics. MacMillan Co., New York.
413 p.
1967. Marine Bio-Acoustics, Volume 2. Pergamon Press, New York.
353 p.
Thorson, G. 1936. The larval development, growth and metabolism of arctic marine
bottom invertebrates compared with those of other seas. Medd. Gronland 100:
No. 6.
Todd, J.H., J. Atema, and J. Bardach. 1967. Chemical communication in social
behavior of a fish, the yellow bullhead (Ictalurus natalls). Science 158:672-673.
Todd, J.H., D. Engstrom, S. Jacobson, and W. O. McLarney. 1972. An introduction
to environmental ethology: A preliminary comparison of Sublethal thermal and
oil stresses on the social behavior of lobsters, and fishes from a freshwater and
a marine ecosystem. U.S. At. Energy Comm. Prog. Rep. AT (11-1) 3567.
Unpublished manuscript.
Tyler, A. V. 1971. Surges of winter flounder Pseudopleuronectes americanus into
the tntertidal zone. J. Fish. Res. Board Can. 28:1727-1732.
Verheijen, F.J. 1956. On a method for collecting and keeping Clupeids for experi-
mental purposes, together with some remarks on fishery with light-sources and
a short description of free cupulae of the lateral line organ on the trunk of the
sardine, Clupea pilchardus Walb. Pubbl. Staz. zool. Napoli 28:225-240.
30
-------
Vernberg, W. B., and F. J. Vernberg. 1972a. The synerglstlc effects of temperature,
salinity, and mercury on survival and metabolism of the adult fiddler crab, Uca
pugllator. U.S. Fish Wlldl. Serv. Fish. Bull. 70:415^420.
1972b. Environmental Physiology of Marine Animals. Sprlnger-
Verlag, New York. 246 p.
Vernberg, W.B., P.J. DeCoursey, and J.O. O'Hara. 1974. Multiple environmental
factor effects on physiology and behavior of the fiddler crab, Uca pugllator.
In_F. J. Vernberg and W. B. Vernberg (ed.) Pollution and the Physiology of
Marine Organisms. Academic Press, New York (In press).
Vernberg, W. B., P. DeCoursey, and W. J. Padgett. 1973. Synerglstlc effects of
environmental variables on larvae of Uca pugllator (Bosc). Mar. Biol. 22:307-312.
Weir, P. A., and C. H. Hlne. 1970. Effects of various metals on behavior of con-
ditioned goldfish. Arch. Environ. Health. 20:45-51.
White, A.Q. 1974. Effect of sublethal doses of cadmium and low dissolved oxygen
levels on locomotion of adult Paleomonetes puglo. (In preparation).
Wlnn, H.E. 1964. Biological problems In acoustic systems (Behavior of fishes with
special reference to their sensory capacities). Office of Naval Research. Final
Report: 104-489.
Wlsby, W. J., and D. R. Nelson. 1964. Airplane observations of acoustic orientation
In sharks (abst.). Amer. Fish. Soc. Conf., Session on Fish Behavior and
Sensory Physiology.
Woelke, C. E. 1972. Development of a receiving water quality bloassay criterion
based on the 48-hour Pacific oyster (Crassostrea glgas)embryo. Wash. Dept.
of Fisheries, Tech. Rep. 9, 93 p.
Young, A. H., P. Tyler, F.G. T. Holllday, and A. MacFarlane. 1972. A small sonic
tag for measurement of locomotion behavior in fish. J. Fish. Biol. 4:57-65.
Yuen, H. S.H. 1961. Bow wave riding of dolphins. Science 134:1011-1012.
1963. Schooling behavior within aggregations composed of yellowfin
and skipjack tuna. Proc. World Scientific Meeting on the Biology of Tunas and
Related Species, July 2-14, 1962, FAO Fish. Rep. 3:1419-1429.
1966. Swimming speeds of yellowfin and skipjack tuna. Trans. Amer.
Fish. Soc. 95:203-209.
1970. Behavior of skipjack tuna, Katsuwonus pelamts as determined
by tracking with ultrasonic devices. J. Fish. Res. Board Can. 27:2071-2079.
------- |