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-------
EPA FOREWORD
This report has been prepared as one of the inputs to the
Environmental Protection Agency's Kepone Mitigation Feasibility Project.
The flanded participants included: The Corps of Engineers, Norfolk
District; the Department of Energy; Battelle Pacific Northwest
Laboratories; EPA's Gulf Breeze Environmental Research Laboratory; and
the Virginia Institute of Marine Science. The separate reports of the
participants appear as Appendices to the Kepone Mitigation Feasibility
Project Report.
While conclusions and recorTrendations are included in each
participant's report, they are conditional within the scope of each
project participant's tasks. Project conclusions and recommendations
are the responsibility of EPA and are included in the Kepone Mitigation
Feasibility Project Report.
The Executive Summary addresses the concept of Kepone "indices"
development for water, for sediment, and for food consumed by organisms.
At this time no formal criteria have been approved by EPA for Kepone.
Thus, the "indices" discussed here should be considered only as
"guides" for use in the Kepone Mitigation Feasibility Project.
U.S. Environmental Protection Agency
Office of Water and Hazardous Materials
Criteria and Standards Division
Washington, D.C. 20460
-------
TABLE OF CONTENTS
EXECUTIVE SUMMARY
/ BIONOMICS - EG&G, INC. 1976.
Acute toxicity of Kepone to embryos of the eastern oyster (Crassostrea
virginica). Toxicity Test Report submitted to U.S. Environmental Protection
Agency, Gulf Breeze Laboratory, Sabine Island, Gulf Breeze, Florida. March.
£, HANSEN, DAVID J., ALFRED J. WILSON, JR., DEL WAYNE R. NIMMO, STEVEN C. SCHIM>
AND LOWELL H. BAHNER. 1976.
Kepon^: Hazard to aquatic organisms. Science 193 (4253): 528.
3. NIMMO, DEL WAYNE R., LOWELL H. BAHNER, REBECCA A. RIGBY, JAMES M. SHEPPARD,
AND ALFRED J. WILSON, JR. 1977.
Mysidopsis bahia: An estuarine species suitable for life-cycle toxicity
tests to determine the effects of a pollutant. In: Aquatic Toxicology and
Hazard Evaluation. ASTM STP 634, F.L. Mayer and J.L. Hamelink, Eds. American
Society for Testing and Materials, pp. 109-116.
H. WALSH, GERALD E., KAREN AINSWORTH, AND-ALFRED J. WILSON, JR. June 1977.
Toxicity and uptake of Kepone in marine unicellular algae. Chesapeake
Science 18 (2)-.222-223.
JT. SCHIMMEL, STEVEN C. AND ALFRED J. WILSON, JR. June 1977.
Acute toxicity of KeponeR to four estuarine animals. Chesapeake Science
18 (2):224-227.
C,, HANSEN, DAVID J., LARRY R. GOODMAN, AND ALFRED J. WILSON, JR. June 1977.
KeponeR chronic ef|.ects on embryo, fry, juvenile, and adult sheepshead
minnows (Cyprinodon variegatus) . Chesapeake Science 18 (2) -.227-232.
7, HANSEN, DAVID J., DEL WAYNE R. NIMMO, STEVEN C. SCHIMMEL, GERALD E. WALSH,
AND ALFRED J. WILSON, JR. July 1977.
Effects of Kepone on estuarine organisms. In: Recent Advances in Fish
Toxicology: A Symposium. EPA publication 600/3-77-085. pp. 20-30.
%< COUCH, JOHN A., JAMES T. WINSTEAD, AND LARRY R. GOODMAN. August 1977.
Kepone induced scoliosis and its histological consequences in fish.
Science 197:585-587.
BAHNER, LOWELL H., ALFRED J. WILSON, JR., JAMES M. SHEPPARD, LARRY R. GOODMA1
GERALD E. WALSH, AND JAMES M. PATRICK, JR. September 1977.
Kepone bioconcentration, accumulation, loss, and transfer through estua]
food chains. Chesapeake Science 18 (3):299-308.
/6, BOURQUIN, AL. W., PARMELY H. PRITCHARD, AND WILLIAM R. MAHAFFEY. 1977.
Effects of Kepone on estuarine microorganisms. Developments in Industria.
Microbiology Volume 19 (in press).
//, BAHNER, LOWELL H. AND JERRY L. OGLESBY. October, 1977.
Test of model for predicting Kepone accumulation in selected estuarine
species. In: Proceedings of the ASTM Second Symposium on Aquatic Toxicology.
October 31-November 1, 1977. Cleveland, Ohio, (in press).
/£, GARNAS, RICHARD L., AL W. BOURQUIN, AND PARMELY H. PRITCHARD. 1978.
The fate of 14-C Kepone in Estuarine Microcosms. Presented at 175th
National Meeting of the American Chemical Society, Anaheim, California, Marc'
Pesticide Chemistry, paper 59.
-------
/S".
J4.
I 'I
/'%.
/?.
At.
RUBINSTEIN, NORMAN I. 3977.
A benthic bioassay usinp time-lapse photography to measure the effect
of toxicants on the feeding behavior of lugworms (Polychaeta: Arenicolidae) .
In: Symposium on Pollution and Physiology of Marine Organisms"! (Eds:
W.S. and J.F. Vernberg) Academic Press (in press).
SCHIMMEL, STEVEN C., JAMES M. PATRICK, JR., LINDA F. FAAS, JERRY L. OGLESBY,
AND ALFRED J. WILSON, JR.
Kepone: Toxicity to and bioaccumulation by blue crabs. Estuaries
(in press) .
COSTLPV, JOHN D. ?977.
Effects of insect growth renulatnry end .iuvenilp hormone mimics on
crustacean develonment. Quarterly Renort to the Environmental Protection
Agency, Environmental Research Laboratory, Gulf Breeze, Florida.
NICHOLS, MAYMARr/ M., Ar'!D RICHARD C. TROTM/w. 1977.
Kepone in Janes River Sediment. Annual Report to
Protection Agency, Environmental Research Laboratory,
the Environmental
Gulf Breeze, Florida
HUGGETT, ROBERT J. 1977.
The role of sediments in the storage, movement, and biological uptake
of Kepone in estuarine environments. Annual Report to the Environmental
Protection Agency, Environmental Research Laboratory, Gulf Breeze, Florida.
HAVEN, DEXTER S. AND REINALDO MORALES-ALAMO. 1977.
Uptake of Kepone from suspended sediments by oysters, Rangia and Macoma.
Annual Report to the Environmental Protection Agency, Environmental
Research Laboratory, Gulf Breeze, Florida.
O'CONNOR, DONALD J. AND KEVIN J. FARLEY. 1977.
Preliminary analysis of Kepone distribution in the James River.
Report to the Environmental Protection Agency, Environmental Research
Laboratory, Gulf Breeze, Florida.
PROVENZANO, ANTHONY J., KATHLEEN B. SCHMITZ, AND MARK A. BOSTON. 1977.
Survival, duration of larval stapes, and size of postlarvae of grass
shrimp, Palaemonetes pugio, reared from Keoone contaminated and uncon-
tamin?ted populations in Chesap°ake Bay. Final Report to the Environmental
Protection Agency, Environmental Research Laboratory, Gulf Breeze, Florida.
Accepted for publication in Chesaneat'e Science.
BOIWIN, AL
Fate and
W. PARMFLY H. ?D!TrH;"nO, Af1'^ HFDBFRT L. F^EH0TCKSO!1'. ]97,°..
effects of Kepone in artificial estuarine ecosystems.
Abstract for American Society
Las Vegas, Nevada.
of Microbiolopy National Meptino, May 197R,
GARNAS, RICHARD L., AL W. BOURQUIN, AND PARMFLY H. PRITCHARD. 1978.
The fate of 14C-Kepone in estuarine microcosms. Abstract for
American Chemical Society National Meetina, March 1978, Anaheim, California,
-------
EXECUTIVE SUMMARY
Prepared By
U.S. Env ronmental Protection Agency
Envirc mental Research Laboratory
.If Breeze, Florida
May 9, 1978
-------
I. Conclusions
Laboratory studies indicate that Kepone does not degrade either bio-
logically or chemically in simulated estuarine systems and this informatic
suggests that degradation processes will not significantly alter the level
of Kepone now found in the water and sediments of the James River.
Clean-up indices for water quality were devised for Kepone in estuari
waters following the "Guidelines for Developing Water Quality Criteria for
Aquatic Organisms for Consent Decree Chemicals." The final index for
24-hour average concentrations of Kepone in estuarine waters should never
exceed 0.008 ug Kepone/1 of water.
Concentrations of Kepone in food organisms should be less than 0.015
Kepone/kg in tissue to minimize undesirable impacts on consumer species.
Examination of partition coefficients between sediment and water, bioconce
tion factors for benthic organisms, and food chain effects indicate that c
centrations should be less than 0.015 mg Kepone/kg in sediment.
Conventional methods of analysis for Kepone in estuarine water, biota
and sediments have acceptable limits of detection of 0.020 ug Kepone/1 of
0.020 mg Kepone/kg in tissue and 0.020 mg Kepone/kg in sediment, respecti\
The pro2osed criteria are below acceptable limits of detection by conventi
methods of analysis and lead to the final conclusion that a hazard exists
Kepone can be detected in the James River Estuary by these methods of anal
-------
II. Introduction
The Gulf Breeze Laboratory was initially involved with the Kepone
problem in the James River Estuary late in 1975 with the initiation of
studies to determine the toxicity of Kepone to estuarine animals, bioaccumu-
lation of Kepone in estuarine aninals, and studies of Kepone residues in
animals living in the James River Estuary. These studies were gradually
increased in scope during 1976 and some of the information on Kepone toxicity
was presented in the federal criminal action brought against Allied Chemical
Corporation and Life Science Products in October 197_6.
During 1976 ongoing studies at ERL, Gulf Breeze, and research studies
supported by ERL, Gulf Breeze, provided information on the ecosystem effects
of Kepone and processes that xcere allowing crabs, oysters, and fish to
concentrate Kepone in their tissues to a level making them unsafe for human
consumption, i.e., reaching concentrations above the FDA designated action
levels. This required a complete study of the effects of Kepone on repre-
sentative species amenable for study under laboratory conditions, and to
correlate "these studies with all the information that could be gathered from
monitoring and field experiments. The results of this information were used
to develop saltwater indices1 for Kepone. The index for water was
developed using the methods outlined in the "Guidelines for Developing Water
Quality Criteria for Aquatic Organisms for Consent Decree Chemicals" prepared
by the Ecology Laboratories of the Office of Research and Development. These
guidelines are still evolving and the procedures used were current in April 24
1977. The criterion should include consideration of production, use, chemical,
and physical properties, occurrence, and human health implications. However,
our indices only considered protection of aquatic life and uses of aquatic life.
Also, the unique situation in the Janes River Estuary where production and use
have ceased and the major source of Kepone is recycled Kepone from the estuarine
3
-------
sediments requires a modified approach, the development of separate indie
for aquatic food organisms and for sediment.
Significant concentrations of Kepone are present in various phases oJ
the estuarine system of the James River—in solution, Huggett (1977) in tl
sediment, Nichols and Trotman (1977) and in the food chain, Bender et al.
(1977), particularly in fishes. The interrelationships, or more specific*
the transport, uptake, and release of Kepone, are thus affected by both pi
chemical mechanisms, Huggett (1977), Garnas et al. (1977), as well as bio-
gical phenomena, Bahner et al. (1977), Bahner and Oglesby (1977). The ph\
chemical mechanisms include the hydrodynamic transport through the estuari
system, adsorption to and desorption from the suspended and bed solids, an
settling and resuspension of these solids, Huggett (1977), Nichols and Tro
(1977). The latter incorporates the assimilation and excretion routes thr
the various components of the food chain, Bahner and Oglesby (1977), Schim
et al. (in press). The potentially significant transport and kinetic proce
transfer to the atmosphere, photochemical oxidation, and biological degrad
are not significant for Kepone, Garnas et al. (1978).
The basic information required as a major contribution to the mitigat
study was the development of a quantitative framework to evaluate the
dynamics of Kepone movement within the James River Estuary system. This
could lead to an evaluation of the time required to reduce the Kepone con-
centrations to an acceptable level. The major role was taken by Battelle
in providing this evaluation to the mitigation study and data from the
experimental program and the field studies mentioned above have been used
in the Battelle reports. The progress made by O'Connor and Farley (1977) j
a parallel modeling effort is reported in this volume. The GBERL program
has included a series of sponsored research projects at the Virginia Ins tit
-------
of Marine Science, principal investigator—Dr. Robert Huggett; Manhatten
College, principal investigator—Dr. Donald O'Connor; Duke University,
principal investigator—Dr. John Costlow; and Old Dominion University,
principal investigator—Dr. Anthony Provenzano.
III. Water Quality Clean-up Index
Table I. Derivation of Saltwater Clean-up Index for Kepone
The reader is referred to the "Guidelines for Developing Water Quality
Criteria for Aquatic Organisms for Consent Decree Chemicals" prepared by th
\
Ecological Effects Laboratories of the Office of Research and Development i
order to better understand the following summary and recommendation. The i;
of these "Guidelines" results in the following Tables and the calculations
contained in them. These "Guidelines" were developed exclusively for the
"65-21 Consent Decree Chemicals." Our use of the "Guidelines" for the
establishment of a Kepone index is tentative, and should not be con-
sidered final or precise. Additionally, the Kepone index has been re-
viewed only at ERL, Gulf Breeze and has not received proper review by all
ORD Ecological Research Laboratories and the Office of Water and Hazardous
Materials as required for criterion generation.
The. estuarine fish, spot (Leiostomus xanthurus) is particularly sensit
to Kepone; the Final Fish Acute Value calculated from this species is 6.6
ug Kepone/1 of water. The Final Invertebrate Acute Value is 0.60 ug Kepone
of water. Consequently the lower of the two, 0.60 ug Kepone/1 of water,
becomes the Final Acute Value.
Chronic studies have been conducted on sheepshead minnows and marine
mysids. The Final Fish Chronic Value is <0.01 ug Kepone/1 of water; the Fd
Invertebrate Chronic Value is 0.008 ug Kepone/1 of water. Therefore, the
Final Chronic Value is _<0.008 ug Kepone/1 of water.
-------
The marine alga, Chlorococcum sp., is the most sensitive plant species
to Kepone; the Final Plant Value is <350 ug Kepone/1 of water.
The Residue Limited Toxicant Concentration (RLTC) is based on (1) a
study in which blue crab survival or molting was adversely affected after
being fed a diet of oysters which contained 0.15 mg Kepone/kg in tissue, and
(2) an average bioconcentration factor of 7688. This RLTC is <0.019 ug Kepone/1
of water.
CLEAN-UP INDEX: The 24-hour average concentration should never exceed
0.008 ug of Kepone/liter of water.
It is important to emphasize that the data on the chronic effects of
Kepone on fish, and the feeding studies on blue crabs provide "less than"
values. Results of laboratory tests with crabs, shrimp, fish, and shellfish
exposed only to Kepone in seawater underestimate the residues of Kepone
measured in similar animals exposed to similar measured concentrations in the
James River Estuary. Therefore, we consider that the index is conservative.
-------
'. .I*'
.'i...
Table II. Fish Acute Values for Kepone
Corrected
Organism
Sheepshead minnow,
Cyprinocion variegatus
Longnose killifish,
Funaulus similis
Spot,
Leiostomus xanthuruti
White mullet,
Mugil curema
Bioaseay
Method >v
FT
FT
FT
FT
Test
Cone. **
M
U
M
U
Time
(hrs.)
96
48
96
48
LC50
(Uq/1)
69.5
84.
6.6
55.
LC50
(uq/1)
69.5
52.4
6.6
34.3
Deference
Schimmel
Butler,
Schimmel
Butler,
and Wilson,
1963
ana Wilson,
1963
1977
1977
*S = static; FT = flow-through
**M = measured; U = unmeasured ••,
Geometric mean of corrected values = 30.1 pg/1
. 8 14
3.7
Lowest value from a flow-through test with measured concentration = 6.6 pg/1
-------
Table III. Fish Chronic Values for Kepone
Organism
Shcepshead minnow,
Cyprinodon variegatus
Test
Limits
(uq/i)
Embryo/ <0.0«
larval
Chronic
Value
(uq/1)
<0.08
Reference
Hansen, et al., 1977
Lowest chronic value = <0.08 pg/1 <0-08 = <0.01 ng/1
-------
Tdbie IV. Invertebrate Acute Values for Kepone
Ot iiiinigni
Eastern oyster,
Crassostrea virginica
Eastern oyster,
Crassostrea virginica
Mysid shrimp,
Mysidopsis bahia »*
Grass shrimp,
Palaemonctes pugio
Brown shrimp,
Penaeus aztecus
BiOcissay Test
M c 1 1 ion _*_ Cone .
FT U
FT
FT
FT
FT
M
M
Time
(HIS.)
96
96
96
96
. 48
LC50
57.
15.
10.1
120.
85.
Corrected
LCbO
120.
Ketetence
43.9 Butler, 1963
11.6 Butler, 1963
10.1 Niirano, et al.. 1977
Schimmel and Wilson, 1977
28.1 Butler. 1963
*S = static; FT = flow-through
'"'•'M = measured; U = unmeasured
Geometric mean of corrected value = 29.6 |ig/l "• ° = 0.60 pg/1
49
/l-i <
>*-f'6
- .-/V.V'
Lowest value from flow-through test based on measured concentrations = 10.1 pg/1
-------
O
Table v. Invertebrate Chronic Values tor Kepone
Organism
Mysid shrimp,
Hysidopsis bahia
Test
LC
Limits
(ug/l>
.026 to-'
0.072
Chronic
Value
(uq/1)
0.043
Reference
Nimmo, 1978
Pg/1
'
5.1
0.008 Mg/1
-------
Table VI. Plant Eifects tor Kepone
organism
Alga.
Chlorococcum sp.
Ettect
Growth
inhibition
(EC50)
Alga, Growth
Dunaliella tertiolecta inhibition
(EC50)
Alga,
Nitzschia sp.
Growth
inhibition
(EC50)
Alga, Growth
Thalassiosira pseudonena inhibition
(EC50)
Concentration
(uq/11
350
580
600
600
Natural phytoplankton 94.77. decrease 1,000
communities in productivity;
1,000 l"C in a
4-hr exposure
Reterfence
Walsh, et al., 1977
Walsh, et al., 1977
Walsh, et al., 1977
Walsh, et al., 1977
Butler, 1953
Lowest Plant Value = <350 pg/1.
-------
Table VII. Residues tor Kepone
Or q.mism
EnsLern oyster.
Grasses trea virginica
Ec-istern oyster,
Crassostrea virginica
Marine mysid.
Mysidopsis bahia
Grass shrimp, ^
Palaemonetes pugio
Gi ass shrimp ,
Palaemonetes pugio
Sheepshead minnow,
Cyprinodon variegatus
Bioconcentration Factor
9,354
t"
9,278
5,962
5,127
11,425
7,200
TIME
(days)
19
21
21
28
28
36
weterence
Bahner, et al. , 1977
Bahner, et al. , 1977
Bahner, et al . , 1977
Bahner, et al. , 1977
Bahner, et al. , 1977
Hansen, et al . , 1977
Maximum Permissable Tissue Concentration
Organism
Man
Man
Blue crabs
Average bioconcentration factor =
Lowest residue concentration = <0.
Action Level or Effect
Shellfish
Fish
Food
7,688
,15 <0- 15 =
-------
Table VIII.other Data tor Kepone
Oj
Organism
Alga.
Chlorococcum sp.
Alga,
Dunaliella tertiolecta
Alga,
Nitzschia sp.
Test
Duration
24 hrs
24 hrs
24 hrs
24 hrs
Alga,
Thalassiosira Pseudonena
Eastern oyster (larvae),48 hrs
Crassostrea virginica
Blue crab, 4 days
Gallinectes sapidus
Grass shrimp, 4 days
Palaemonetes pugio
Grass shrimp (larvae), £21.7
Palaemonetes pugio ~
Sheepshead minnow, 4 days
Cyprinodon variegatus
Spot, 4 days
Leiostomus xanthurus
Blue crab, 96 hrs
Callinectes'sapidus
Blue crab, 96 hrs
Callinectes sapidus
Blue crab, 56 days
Callinectes sapidus
Result
luq/11
Ettect
Bioconcentration
factor - 800X
Bioconcentration
factor - 230X
Bioconcentration
factor = 410X
Bioconcentration
factor - 520X
EC50 66.
Bioconcentration
factor =8.1
Bioconcentration
factor = 698
,No effect on survival,
size or duration of
larval stages for larval
with residues of <0.63
mg/kg Kepone
Bioconcentration
factor = 1548
Bioconcentration
factor = 1221
207. mortality 1,000.
07. mortality' 210.
Survival or molting
was reduced in crabs
fed oysters containing
0.15 mg/kg Kepone
Reference
Walsh, et al., 1977
Walsh, et al., 1977
Walsh, et al.. 1977
Walsh, et al., 1977
Bionomics Report, 1976
Schlmmel and Wilson, 1977
Schimmel and Wilson, 1977
Provenzano, et al., 1977
Schimmel and Wilson, 1977
Schimmel and Wilson, 1977
Butler. 1963
Schimmel and Wilson, 1977
Schimmel, et al., In Press
-------
IV. Food Clean-up Index
Acute exposure of blue crabs to Kepone in sea water in the laboratory
indicated relatively low toxicity and bioconcentration, Schimmel et al.
(1977). In contrast, monitoring data indicated that blue crabs from the James
River Estuary accumulated significant concentrations of Kepone, Bender et al.
(1977). Schimmel et al. (in press) found that the major route of Kepone entry
is through contaminated food and not water. Therefore, estimates of an index
for contaminated food were developed as shown in Tables 1 and VII of the water
\
index. Guidelines have not been developed by OR&D laboratories for these
types of data to estimate safe concentrations for untested species and to provide
adequate protection when data do not support a no-effect concentration. We
support a Kepone food index of 0.015 mg Kepone/kg of tissue obtained in the
following manner.
Toxic Effects of Kepone in Food
Effects of Kepone on growth and survival of blue crabs fed oysters
contaminated with Kepone, Schimmel et al. (in press), are the only laboratory
data demonstrating adverse effects of. this''pesticide in food on an aquatic organism.
Concentrations of 0.15 mg Kepone/kg of oyster meat fed to blue crabs diminished
survival, or molting. However, because the data do not provide us with a no-effect
concentration, we applied a safety factor of 0.1 to this concentration to provide,
in our judgement, an index (0.015 mg Kepone/kg of tissue) that should be
protective of consumer species.
Food_ciean-up Index Relative to Kepone in James River Food Organisms
A criterion of 0.015 mg Kepone/kg in food organisms is far less than monitoring
data revealed in animals from the James River Estuary. An analysis of the data
of Bender et al. (1977) indicated that the average concentration of Kepone in
fishes and invertebrates from the James River which could be eaten ranged from
-------
0.090 to 2.0 mg Kepone/kg of tissue. Our data on effects of Kepone in oysters
fed to blue crabs support the hypothesis that undesirable impacts on survival
and molting of blue crabs are occurring in the James River.
v. Sediment Clean-up Index
The monitoring studies by Huggett (1977), the State of Virginia (1977), and
Battelle (1977) clearly demonstrate that most of the discharged Kepone is
now resident in the sediments of the James River Estuary. The main sink is
in the bed sediment of the estuary, in the zone of the turbidity maximum, where
suspended sediments'are trapped and deposited. The hypothesis used to explain
this distribution, and the processes used in modeling Kepone transport (and long-
term persistence and bioavailability in the estuary), is that Kepone is bound
to sediment and sediment transport processes control the distribution of Kepone,
Huggett (1977), O'Connor (1977).
The concentrations of Kepone are orders of magnitude greater in the bed
sediments than dissolved in estuary water. Garnas et al. (1978), Huggett (1977),
and Battelle (1977) have shown that partition equilibria for Kepone between
sediment and water are directly affected by the sediment quality. Therefore,
mitigation must address Kepone in sediments.
It _is important to use all the available information to estimate the sediment
concentration that must be achieved to eliminate the hazard to aquatic life and
its consumers. Additionally, it is essential to consider: 1) the direct toxic
effects and the bioavailability of Kepone contained within the bed sediments,
Bahner (1977), Rubinstein (1977), and adsorbed to suspended sediment, Haven and
Morales-Alamo (1977); 2) bioconcentration in the food chain directly from sediment;
and 3) the dissolved Kepone in the estuary water which is controlled by the
sediment/water equilibrium, Garnas et al. (1978).
Indices for acceptable concentrations of Kepone in sediments can be
derived by examining how Kepone partitions among water, sediments, and
-------
benthic biota. Experiments have shown that benthic organisms (lugworms,
Arenicola cristata, and fiddler crabs, Uca pugilator) that injested James River
sediments with 0.250 ng Kepone/kg of sediment attained whole-body residues of
0.250 to 0.300 mg Kepone/kg of tissue within 21 days, Banner et al. (in prepara-
tion). Lugworms did not survive exposure to these sediments after 21 days.
Concentrations as low as 2.8 ug Kepone/1 seawater caused a reduction in the
normal substrate reworking activity of the lugworm and 29.5 ug Kepone/1 seawater
was acutely toxic within 144 hours to lugworms burrowing in sediments, Rubinstein
(1978). Kepone did not depurate from lugworms and fiddler crabs over a period of
a few weeks in clean water, Banner et al. (in preparation).
C]ean-up Indices Formulation for Kepone in Sediments
Three methods for determining indicies for Kepone-laden sediments are
feasible:
1. a safety factor can be applied to sediment concentrations shown to be
toxic to animals;
2. food quality index f°r estuarine food chain based on accumulation
of Kepone by animals from sediments to concentrations that exceed the food
quality index;
3. index • based on desorption of Kepone from James River sediment into
water at concentrations that exceed the water quality index;
Sediment Clean-up Index Based on Sediment Toxicity and Safety Factors
Insufficient data are available to determine if reducing Kepone concentra-
tions in sediment by any factor will be protective to benthic organisms.
Sediment Clean-up Index Obtained from the Food Quality Index
Benthic organisms attained Kepone concentrations similar to the amount
in sediments. The food quality index is 0.015 mg Kepone/kg of tissue.
Therefore, Kepone concentrations in sediment should not exceed 0.015 mg Kepone/kg
of sediment to insure that Kepone concentrations are less than the food quality
index.
-------
Sediment Clean-up Index Obtained from the Water Quality Index and Sediment/
Water Partition Coeffients
Establishment of an acceptable concentration of Kepone in sediments
may be based upon the premise that an equilibrium exists for Kepone between the
sediment and water [Kp == (ug/kg sediment)/(ug/1 water)]. An examination of
laboratory Kp-values indicates numbers ranging from 2.5 to 1700, Garnas et al.
(1978), Huggett (1977), Dawson (1977). If pure reference clays and sand are
ignored (Kp = 2.5-50), the range is between 100 to 1700 and is related to the
quality and quantity of organic material in the sediment. Using these values
to derive acceptable sediment concentrations, a Kepone water index not
greater than 0.008 ug Kepone/1 of water at equilibrium is related to a range
from 0.0008 to 0.014 mg Kepone/kg in sediment. The average concentration of
Kepone in James River sediments from December 1976 through July 1977 was 0.150
mg Kepone/kg of sediment, Huggett (1977), with the limit of Kepone analysis in
sediments at 0.020 mg Kepone/kg of sediment.
Derivation of a Final Kepone Sediment Clean-up Index
If a Kepone partition between water and James River sediment of Kp=lOOO is
, ' if
utilized, concentrations of 0.008 mg Kepone/kg of sediment would result in
equilibrium concentrations equal to the Water Quality Index of 0.008 ug
Kepone/1 of water. Because the food quality index is 0.015 mg Kepone/kg
of tissue, the concentration of Kepone in sediment must not exceed 0.015 mg
Kepone/kg of sediment. Since the lower limit of analytical detection of Kepone
in sediments is usually 0.020 mg Kepone/kg of sediment, both derived concen-
trations are below analytical detection. Therefore if Kepone is present in
measurable quantities, it is hazardous to aquatic life.
-------
VI. Degradation of Kepone
Test Results
Studies by Garnas et al. (1978) have employed static and flowing water-
sediment systems to assess both biological and non-biological degradation of
Kepone. Sediments with and without Kepone contamination were taken from the
James River and used in these systems. The fate of Kepone was monitored using
radiolabelled (^C) material and total budget chemical analysis. Using a
variety of experimental conditions (oxygen concentration, nutrient additions,
Kepone levels, sediment sources, sunlight, temperature, and salinity), these
studies indicate that Kepone does not degrade (i.e., complete recovery of
Kepone after extended incubation periods) either biologically or chemically
in laboratory systems. These data suggest that degradation processes will not
significantly alter the levels of Kepone now found in the water and sediment
of the James River, and place further emphasis on the indices derived for
water, food, and sediment.
-------
Acute toxicity of KEPONE to
embryos of the eastern oyster
(Crassostrea vi rgi ni ca)
Toxicity Test Report
Submitted to
U.S. Environmental Protection Agency
,£ulf Breeze Laboratory
Sabi ne Island
Gulf Breeze, Florida
Bionomics - E G & G , Inc.
Marine Research Laboratory
Route 6, Box 1002
Pensacola, Florida 32507
March 1976
-------
A marine toxicity test was conducted to determine the acute
effect of KEPONE on embryos of the eastern oyster (Crassostrea
v i r g i n i c a). The criterion for effect was reduction in the num-
ber of normal embryos in test concentrations (those which developed
to the fully-shelled, straight-hinged veliger stage within 48
hours) as compared to the number of normal control embryos. Re-
sults of the test are expressed as a 48-hour EC50 (the concen-
tration of KEPONE estimated to be effective in preventing normal
development of 50% of the embryos).
MATERIALS AND METHODS
Test material
A 2.5 gram sample- of a white powder was received in a small glass
vial labeled "KEPONE, 88% pure, 2.5 g." Accompanying instruction
from U.S. Environmental Protection Agency Research Biologist Steven C.
Schimmel directed us to assume 100% purity for testing purposes.
Test concentrations are reported here as micrograms (yg) of KEPONE
per liter (a} of sea water or parts per billion (ppb).
Test animals
Oyster embryos were obtained by induced spawning of oysters
which had been conditioned in the laboratory for 6-8 v.'eeks in
flowing, unfiltered natural sea water at 25±2 degrees Celsius (°C).
T_est conditions
Methods for the acute (48-hour) -oyster embryo test were based
on those of Woe Ike (1972) and U.S. Environmental Protection Agency
(1975). Individual, sexually mature female oysters held in glass
chambers containing 1 £ of filtered (5 micrometers, ym) sea water
-------
were induced to spawn by increasing the water temperature in
the chamber from 25°C to 33°C over a 30-minute interval in the
presence of viable sperm excised from the gonad of a sexually
mature male oyster. Fertilization occurred upon release of the
\
eggs into the spawning chambers. Microscopic examination con-
firmed fertilization success to be >95%. Density of the embryos
was determined by averaging three 1-nu Sedgewick-Rafter counts of
1:99 dilutions (1 mi embryo suspension to 99 ml sea water) from
the spawning chamber.
Concentrations for the definitive 48-hour test were based
on a preliminary assay and were 56, 65, 75, 87, and 100 ppb. All
test concentrations and the control were triplicated. Test con-
tainers were 1-x. glas,s_ jars, each containing 900 nu of filtered
(5 ym), natural sea water. Test concentrations were prepared by
pipetting appropriate amounts of KEPONE, dissolved in reagent
grade acetone, into each test container. Control jars received
900 mi of sea water with no KEPONE. Salinity was 21 parts per
thousand (°/oo) and initial pH was 8±0.5 for all test concentra-
tions and the controls.
For the definitive test, each test container was inoculated
with an estimated 26,000±1,000 embryos and then maintained at
20±1°C in a controlled environmental chamber. After 48 hours
of exposure, the embryos from each test container were collected
by seive (37yrn)5 rinsed into a 100-nn- graduated cylinder from
which a 10-ma aliquot was transferred to a small glass bottle
and preserved with 0.3 mi. of neutralized formalin. The number
of normally developed 48-hour embryo present in 1 m;, of each
-------
preserved sample was determined by Sedwich-Rafter counts. The
percentage reduction of normal 48-hour embryos was determined
as fol 1 ovfs :
'; umber of normal 43-hour control embryos
•* minus the number of normal 48-hour embryos
Percentage _ in each test concentration
reduction ~ ^ 100
Number of normal 48-hour control embryos
The number of normal embryos was based on the average of two counts
of 1 m2. of preserved sample from triplicate test concentrations
and control..
Statistical Analysis
Based on results of the test, a 48-hour EC50 for eastern
oyster embryos was calculated. The test concentrations were con-
verted to 1ogarithms'and corresponding percentage reduction of
normally developed 48-hour embryos to probits. The 48-hour EC50
was then calculated by linear regression.
RESULTS AMD DISCUSSION
A preliminary test with oyster embryos showed the concentration
of KEPONE effective in preventing normal development to be >56<100
ppb (Table 1). It also' showed the volume of acetone used as a sol-
vent/carrier did not affect normal development of embryos (Table 2).
Further, it is interesting to note the increased number of normal
embryos in the lower test concentrations (10, 32, and 56 ppb) of the
preliminary test. We have observed this effect in tests with several
different compounds and do not know whether it is caused by the pre-
sence of the toxicant, a synergistic effect between toxicant and
carrier,or is random error.
-------
The definitive test confirmed the results of the prelimi-
nary test. After 48 hours exposure, percentage reduction of
normally developed exposed embryos as compared to the control was
from 0% in the 56 ppb test concentration to 100% in 87 and 100
\
ppb (Table 3). The calculated 48-hour EC50 for eastern oyster
embryos exposed to KEPONE in static, unaerated sea water was 66
ppb with 95% confidence limits of 60-74 ppb.
»
The average number of normal embryos counted per 1 nil of a
1:99 dilution of the embryo population collected from each test-
concentration ranged from 0 in 87 and 100 ppb to 152 in 56 ppb
(Table 4). The 48-hour embryos from 56 ppb test concentrations
appeared to be smaller and not as well formed as the control;
however, they were fully shelled and straight-hinged. In 65
ppb, although only an average of 44 normal embryos were counted,
there were numerous abnormal embryos present. The severity of
abnormal development increased in the higher test concentrations
of 75, 87, and 100 ppb.
-------
LITERATURE CITED
U.S. Environmental Protection Agency. 1975. Methods for Acute
Toxicity Tests with Fish, Macroinvertebrates, end Amphibians.
Ecological Research Series EPA-660/3-75-009: 61 pp.
Woelke, Charles E. 1972. Development of a Receiving Water Quality
Bioassay Criterion Based on the 48-Hour Pacific Oyster (Crassos-
trea gigas) Embryo. Technical Report 9: 93 pp.
-------
TABLE 1. The acute toxicity of KEPONE to embryos of the
eastern oyster (Grassestrea vi rg1n1ca) exposed
for 48 hours in static, unaerated sea water.
Salinity was 21 °/oo and temperature, 20±1°C.
The criterion for effect was the reduction in the
^number of normal embryos in test concentrations
as compared to the number of normal control
embryos.
Hominal concentratien Percentage reduction of
(yg/£;ppb) normal 48-hour embryos3
Sea water control 0
Acetone control 0
10 ' 0
32 . 0
56 0
TOO" • 100
180 100
Number of normal -48-hour control embryos
minus the number of normal 48-hour embryos
Percentage _ in each test concentration
reduction Number of normal 48-hour control embryos
-------
TABLE 2. Number of normal eastern oyster (Crassostrea v1rg1ni ca'
embryos per milliliter counted following 48 hours of
exposure to KEPONE in static, unaerated sea water.
If all embryos in the initial inoculum had developed
normally, the expected count would have been 314±16
''embryos per milliliter. Salinity was 21 °/oo and
temperature, 20±1°C.
Nominal concentration Number of normal embryos
{yg/£ ;ppb)
*•
Sea water control A
Sea water control B
Acetone control
10
32
56__
• 100
180
Rep A
198
179
160
206'
285
264
0
0
Rep B
179
175
212
253
280
236
0
0
Avg.
188
177
186
229
283
250
0
0
-------
TABLE 3. The acute toxicity of KEPONE to embryos of the
eastern oyster ( Cra_ssos trea v 1 r g 1 n 1 c a ) exposed
for 48 hours in static, unaerated sea water.
Salinity was 21 °/oo and temperature, 20±1°C.
The criterion for effect was the reduction in
x the number of normal embryos in test concen-
trations a^s compared to the number of normal
control embryosT"
Nominal concentration Percentage reduction of
(ug/£;ppb) normal 48-hour embryosa
Sea water control 0
56 0
65 68
75 97
87 100
100 100
Number of normal 48-hour control embryos
minus the number of normal 48-hour embryos
Percentage in each test concentration
reduction ~;"7:~X 100
Number of norms! 48-hour control embryos
-------
TABLE 4. Number of normal eastern oyster (Crassostrea v1rgi n1ca )
embryos per mill-Miter counted following 43 hours of
exposure to KEPONE in static, unaerated sea water.
If all embryos in the initial inoculum had developed
normally, the expected count would have been 247±12
embryos per milliliter. Salinity was 21 °/oo and
^temperature, 20±1°C.
Nominal concentration
( y g / 1 ; p p b )
»
Sea water control
56
65
75
87
100
Number
Rep A
128
151
42
2
0
0
of no
Rep B
138
147
43
8
0
0
rmal emb
Rep C
145
158
46
2
0
0
ryos
Avg
137
152
44
4
0
0
-------
10
SUBMITTED BY
Bionomics - EG&G, Inc.
Marine Research Laboratory
Route 6, Box 1002
Pensacola, Florida 32507
March 1976
PREPARED BY:
Tom Heitmuller
Biologist
APPROVED BY:
Rod Parrish
Director, Marine
Research Laboratory
-------
Table III. Fish Chronic Values for Kepone
Chronic
Limits Value
Organism Test (uq/H (ug/1) Reterencfe
Shcepshead minnow, Embryo/ <0.08 <0.08 Hansen, et al., 1977
Cyprinodon variegatus larval ""
Lowest chronic value = <0.08 pg/1 <2^§§. «=
-------
LETTERS
Kepone: Ha/ard to
Aquutic Organisms
Rudolph J. Jaeger (Letters, 9 July, p.
94) reports the chronology of mammalian
toxicity tests with Kcpone (chlordecone)
and the exposures of workers at the Life
Science Products Company of Hope-
well, Virginia, Initial concern has proper-
ly been focused on the results of toxicity
and carcmogenicity tests on rats, rab-
bits, dogs, and mice and on the disease
that Kcpone produced in exposed work-
ers. \Vc would like to document our con-
cern about the ha/ard of Kepone to
aquatic organisms in the James River and
the Chesapeake Bay.
On-site tests of oiganisms taken from
the James River showed significantly
high Kepone concentrations. These
tests, conducted at the Virginia Institute
of Marine Science and funded by the
Environmental Protection Agency
(EPA), revealed that concentrations in
edible tissues of most fresh and estuarine
fin- and shellfish commonly ranged from
0.1 to more than 1 microgram per gram.
These concentrations exceeded allowable
health limits for commercial and sport
fisheries and forced closure of the river to
some commercial and sport fishing. This
year Kepone concentrations have in-
creased m anadrofnous fishes as they
spent more time in the river.
Further, after laboraloiy exposures at
the EPA laboratory in Gulf Bree/e, Flor-
ida, we found that Kepone, like other
chlorinated insecticides, is highly
bioaccumuiative and persists in estuar-
ine organisms. Ovsters, grass shnmp, and
n\hes ha\e bioconcentrated Kepone
."ro." ^2:~ to 10.i .":0 t.mes the :cr,cer'.ra-
tion in the surrounding water. Therefore,
action lc\els for edible seafoods now in
force might be reached by as little as 5
parts of Kepone per tnllion parts of ua-
ler (nanogranis perlitei). In Kepone-free
\vatei. oysteis can depurate about 90
percent of the accumulated Kcpone in 4
days, but fish may require moie than 3
weeks to lose 30 to 50 percent. Five
weeks after fertili/ation of sheepshead
minnow eggs containing Kepone, the ju-
venile lish tetained as much as 46 per-
cent of the Kepone piesent in the eggs.
Kepone can be accumulated by fish to
concentrations that exceed those in their
food.
Kepone is acutely toxic to estuarine
oigamsms, but long-term bioassays reveal
that the ha/ard to these organisms is
greatly underestimated by the 96-hour
tests. The concentrations in miciograms
per liter, estimated to be lethal to 50
percent of the test animals m 96 hours
(LC-,n), were 6.6 for spot, 70 for sheeps-
head minnows, 10 for an estuarine mys-
)d, 121 for grass shrimp, and moie than
210 for blue crabs. Kepone was lethal lo
adult sheepshead minnows exposed to
O.X microgram per liter foi 2X days. A
significant number of embryos fiom
adults exposed to 1.8 nucrogiams per
liter weie abnormal and died. When em-
bryos were exposed to O.OS microgram
of Kepone per liter of watei, 36 days
l.ilei, resulting jusenile lish were shorter
than control fish and some exhibited sco-
liosis. Mysid shrimp exposed for 20.days
to about 0.2 microgram per liter pro-
duced fewer progeny: with gieater con-
centrations, their growth and survival
were reduced. We are concerned because
all concenlrations tested thus far in long-
term expostucs of sheepshead minnows
and mysids ha\ e reduced sur\ ival. icpro-
duction. or growth.
The threat of an even greater impact of
Kepone to aquatic organisms in the
James River and expansion of this im-
pact into the Chesapeake Hay, therefore.
is real and it may continue for some
years to come. It is essential that
we use knowledge now available to
attempt to make decisions that may mini-
mi/e the fuiuie impact of this insecticide
on the aquatic enviionment.
D/\\ H3 J. H \NSI N. A: i KtD J. Wn SUN
DLI.WWSC R. N'IMMO
Si r\ liN C. SCH I.MM EL
Low 1.1 t II. BAHNLK
Ein'irvHint'HUil I'ratcciion Agency.
Environmental Research Lahonilury,
Gut] Breere, Florida 32561
ROHbKT Hu'C.eiETT
\'irt;ini(i Institute of Munne Science,
Gloucester Point 23062
-**>.
-------
Authorized Reprint from
Special Technical Publication 634
Copyright
American Society for Testing and Materials
1916 Race Street, Philadelphia, Pa. 19103
1977
D. R. Nimmo,' L. H. Bahner,' R. A. Rigby,'
/. M. Sheppard,' and A. J. Wilson, Jr.l
Mysidopsis bahia: An Estuarine
Species Suitable for Life-Cycle
Toxicity Tests to Determine the
Effects of a Pollutant
REFERENCE: Nimmo, D. R., Bahner, L. H., Rigby, R. A., Sheppard, J. M., and
Wilson, A. J., Jr., "Mysidopsis bahia: An Estuarine Species Suitable for Life-Cycle
Toxicity Tests to Determine the Effects of a Pollutant," Aquatic Toxicology and
Hazard Evaluation. ASTM STP 634, F. L. Mayer and J. L. Hamelink, Eds.. American
Society for Testing and Materials, 1977, pp. 109-116.
ABSTRACT: This study documents the successful use of a mysid, Mysidopsis bahia,
for life-cycle toxiciiy tests. These tests were conducted to determine acute and chronic
toxicities of metal (cadmium) and pesticide (Kepone). Delay in the lormauon of mysid
brood pouches and release of young were noted in low concentrations <6.4 Mg cad-
mium/litre. Fewer young produced per female and decreased growth were other indica-
tors of effects of Kepone.
KEY WORDS: water analysis, toxicology, toxicity, cadmium, Kepone, life cycles,
insecticides
Many freshwater but a few estuarine or marine animals are available
to the biologist for life-cycle toxicity tests. Few biologists have worked on
methods of culturing marine animals; therefore, less is known about their
nutritional, behavioral, or environmental requirements than those of fresh-
water species. Life cycles of marine species may be more complex, that is,
many require an estuarine existence as larvae or juveniles, followed by adult
migration to deeper waters offshore to reproduce. Culturing and maintaining
estuarine and marine species require elaborate and expensive facilities be-
cause equipment must include temperature or salinity controls, anticorro-
sion surfaces, and often special filtration systems. Sufficient quantities of
1 Research aquatic biologists, biological technician, biologist, and supervisory research
chemist, respectively, U.S. Environmental Protection Agency. Environmental Research
Laboratory, Gulf Breeze, Fla. 32561.
-------
110 AQUATIC TOXICOLOGY AND HAZARD EVALUATION
good quality saltwater may not be as available as freshwater and may be
more expensive to obtain.
A water quality criteria report by the Environmental Protection Agency
(EPA) [I]2 indicated that only 12 percent of 332 experiments with estuarine
animals were conducted in flow-through tests with organic materials and
that no tests were conducted on entire life cycles of any estuarine or marine
species. Since this report, an entire life-cycle bioassay using the sheepshead
minnow, Cyprinodon variegatus, has been accomplished [2].
We report progress in conducting life-cycle toxicity tests with small
crustaceans called mysids. Commonly called an "opossum shrimp" because
the female carries the young in a brood pouch during development,
Mysidopsis bahia Molenock [3] was first described from West Bay, Galves-
ton, Texas, and also occurs in southwest Florida [4]. Dr. T. E. Bowman3
of the Smithsonian Institution, Washington, B.C., identified the species.
We have completed tests on the effects of the metal, cadmium, and the
pesticide, Kepone, which has been detected in the water, sediments, and
biota of the James River and Chesapeake Bay [5]. The only other reported
use of mysids for flow-through toxicity tests was to assay the toxicity of
Kraft mill effluent [6].
Life History
Mysidopsis bahia is an estuarine species, but it has not been found at
salinities below 9 parts per thousand salinity [4]. In our first collection,
most animais were found in snallow ponds near the substratum, oriented
positively towards the current. However, they made vertical migrations in
early morning hours co feed at the surface. Scintiia de Almeida Prado [7]
found that six species of mysids closely related to M. bahia were primarily
coastal-water species: the young migrate to surface waters at night, while
adults remain close to the bottom. The vertical migration of the young
facilitates their dispersion towards the sea or into adjacent mangrove areas.
We have found that newly released M. bahia are planktonic for the first 24
h; thereafter, they orient to a current and actively pursue Anemia. Feeding
habits of M. bahia have not been described. However, a related estuarine
species, M. almyra, is omnivorous: 31 percent of its digestive tract contents
is vascular plant detritus and 11 percent, copepods and diatoms [4].
Ecological Significance
The various stages in mysid life cycles are important links in estuarine
and marine food chains, and many studies-emphasize the importance of
2 The italic numbers in brackets refer to the list of references appended to this paper.
3Bowman, T. E., Smithsonian Institution, Washington, D.C., personal communication.
12 Jan. 1976.
-------
NIMMO ET AL ON MYSIDOPS1S BAHIA 111
mysids as food for fishes [8-21]. Stomachs of young-of-the-year striped
bass, Morone saxatilis, captured from the York River, Va., contained as
much as 12.7 percent mysids by volume [11]. The composition of the
stomach contents by volume of tidewater silversides, Menidia beryllina, in-
cluded 65 percent mysids during night feeding [4]. Stickney et al [12]
found that three of four species of flounders captured mysids as a primary
staple in their diets. Percentage of total number of organisms in stomach
contents were: Etropus crossotus, 3.1 percent; Citharichthys spilopterus, 72
percent; Ancylopsetta quadrocellata, 81 percent; and Scopthalmus aquosus,
96.3 percent. It is obvious that, in some instances, the loss of mysids as a
food source would have serious impact on the next trophic level.
Materials and Methods
Mysids were collected from small shallow ponds that received a constant
supply of saltwater from Santa Rosa Sound near Pensacola, Florida.
Mysids were cultured in the laboratory in 40-litre glass aquaria supplied
with filtered (20 Mm) flowing water (10 to 27 parts per thousand salinity)
at 18 to 28°C and were fed daily 48-h-old Anemia salina larvae. Overflow
from each aquarium was through a standpipe to which ?. ring of screen
«,:r..ji£-T.;=n: .-'.' .":! '-"._ :". oi-'.:urc ~.r.z ..:}:.,^ , :...:./,ujasiy lor la months
without major fluctuations in population density.
All tests were conducted in intermittent flows from a diluter [13] or con-
tinuous flow with the toxicant added by an infusion pump [14]. As each
aquarium achieved maximum volume, a self-starting siphon drained the
water to a \olu:r.e 01 a^out one litre in aquarium. Fluctuating levels, oc-
curring at about 30 min each, ensured an exchange of water within each
aquarium and the small chambers devised to retain the mysids. The cham-
bers consisted of a standard glass petri dish to which a 15-cm-high cylinder
of Nitex screen (mesh Number 210) was attached. To begin each test,
twenty to thirty two 48-h-old juveniles, 4 or 5 per chamber, were exposed to
each concentration. Acclimation was not necessary since the culture and
test water were identical. A small stream of compressed air was delivered
into each chamber to safeguard against possible anoxic conditions and to
create a current that apparently aids orientation of the animals. In record-
ing daily changes in populations, each chamber was lifted gently from the
aquaria, water was drained through the Nitex cylinder to the level of the
petri dish, and the chamber was then placed on a lighted counter top.
Number of live animals by sex, number of females with and without brood
pouches, and number of young were recorded.
4Nitex is a registered trademark of Tobler, Ernst and Trabor, Inc., Murray St., New York.
N.Y. Reference to commercial products does not constitute endorsement by the Environmental
Protection Agency.
-------
112 AQUATIC TOXICOLOGY AND HAZARD EVALUATION
We conducted 96-h, flow-through tests beginning with 48-h-old juvenile
mysids, followed by life-cycle bioassays. The procedures were similar, ex-
cept that test concentrations in the life-cycle tests were lower than in the
96-h tests.
Water samples were analyzed for Kepone by extracting one litre of sea-
water twice with 100-ml volumes of methylene chloride in a two-litre sep-
aratory funnel. The combined extracts were concentrated to about 5 ml in
a Kuderna-Danish Concentrator on a steam table. Fifteen millilitres of
benzene was added and the extract reconcentrated to remove the methylene
chloride. Extracts were adjusted to appropriate volumes for analyses by
electron-capture gas chromatography. Determinations were performed on
Varian Aerograph Model 2100, and 1400 gas chromatographs equipped
with 182-cm by 2-mm inside diameter glass columns packed with 2 percent
SP 2100 and 0.75 percent SP2250: 0.97 percent SP2401 on 100/120 mesh
Supeloport.
The operating modes were: oven temperature, 185°C; injector tempera-
ture, 200°C; detector temperature, 210°C; nitrogen carrier-gas flow rate,
25 ml/min. Average recovery of Kepone from saltwater was 85 percent;
concentrations reported here were not corrected for efficiency of the ana-
lytical method. All samples were fortified with an internal standard (dichloro-
benzophenone) prior to analysis to evaluate the integrity of results.
Cadmium in water was measured by atomic absorption spectrophotometry,
by using the procedure of Nimmo et al [15],
The LC^s and 95 percent fiducial limits were calculated by linear re-
gression analysis after probit transformation. We employed two-sample t
tesi to mean brood size; we used Dunnett's test comparing multiple treat-
ments with control to mean adult length. Significant (a = 0.05) differences
were attributed to Kepone.
Results and Discussion
Life-cycle toxicity tests, using survival as the criterion for effects, were
first conducted with cadmium; the procedures for quantifying effects or the
reproduction and growth were developed while testing the pesticide, Kepone.
Seventeen days was chosen as the test requirement for a life cycle because
animals in the control aquarium released their brood in this time. In sub-
sequent experiments, we reduced the time required to complete a life cycle
by maintaining the temperature at 29 °C.
For cadmium, the 96-h calculated LC50 was 15.5 ^g/litre (95 percent
fiducial interval, 12.6 to 19.6) at 25 to 28°C and 10 to 17 parts per thou-
sand salinity. In a 17-day life-cycle test, the LQo was 11.3 ^g/litre (95 per-
cent fiducial interval, 4.2 to 12.9 at 20 to 28°C and 15 to 23 parts per
thousand salinity (Table 1)). In addition, we observed a 48-h delay in the
formation of brood pouches and a 24-h delay in the release of brood in
-------
NIMMO ET AL ON MYS/DOPSIS BAHIA 113
TABLE 1—Survival (numbers) o/ Mysidopsis bahia in measured concentrations oj cadmium
chloride m seuwaier. Temperature range, 20 to 28°C, salimt\, 15 to 23 pans per thousand.
Cadmium, Mg/litre
Days
0
11
13
17
18
20
23
Control
20
19"
19
19*
19
19
19
4.8
20
19"
19
19fc
19
19
19
6.4
21
16
16"
16
16*
16
16
10.6
20
16°
16
13
11
8*
2
28.0
20
1
0
0
0
0
0
" Formation of brood pouches noted in the chambers.
* Young released. Average number of young/female at 23 days was 7.0 in control, 8 in 4.8
Mg/litre, 3 in 6.4 Mg/litre, and 4 in 10.6 Mg/litre. This average was based on all females, not
producing females alone.
females exposed to 6.4 Mg/litre. Although we were unable to prove statisti-
cal significance of these observations, we believe that these delays, and a
72-h delay in release of brood by females in the 1C.6 ug-'irre, could have
a deleterious effect ecologically since production of maximum brood is out
of phase with food or predator cycles. Also, fewer young were produced by
females in the 6.4 and 10.6 Mg/litre aquaria than by females in the control
and 4.8 Mg/litre aquaria.
The 96-h calculated LCSO for Kepone toxicity to M. bahia exposed to
Kepone was 10.1 _^. iicrt1 v:*5 percent f.Jjviai interval. 8.1 to 12.4; ^t LL ;o
28°C and 10 to 16 parts per thousand salinity. The 19-day (life-cycle) LC50
was 1.4 Mg/Htre 195 percent fiducial interval, 1.1 to 1.8) at 25 to 28 °C and
10 to 20 parts per thousand salinity (Table 2). The duration of the test
allowed production of several broods. We discovered that a single female
could commence brood production as early as Day 12 at higher tempera-
tures, and could produce two additional broods by Day 20; some females
produced as many as 35 young. In the Kepone test, the average number of
young per female at 20 days was 15.3 in the controls, and 8.9 in 0.39 Mg/
litre. Differences between controls (15.3 juveniles/female) and the 0.39Mg/
litre concentration (8.9 juveniles/female) were significant (a = 0.05, 2-
sample t test).
In preliminary tests, growth of some mysid individuals in higher con-
centrations of Kepone appeared to be less than control mysids. To evaluate
this effect, we began two separate, 14-day tests that began by exposing
24-h-old juveniles to Kepone and concluded by- measuring their total
lengths (tip of carapace to end of uropod) (Table 3). We found that female
mysids exposed to 0.072 Mg/litre Kepone grew less than the control mysids.
This effect was consistent with apparent effects on reproductive success
-------
114 AQUATIC TOXICOLOGY AND HAZARD EVALUATION.
TABLE 2—Survival (numbers) of Mysidopsis bahia in measured concentrations of Kepone in
seawater. Temperature was 25 to 2S°C; salinity, 10 to 20 parts per thoutnnd.
Kepone, >ig/litre
Days
0
2
4
6
8
10
15
19
Control
32
32
32
32
32
31
30
29
0.39
32
32
32
29
29
29
27
27
1.55
32
26
22
21
21
19
17
16
4.4
32
19
9
6
2
2
1
1
8.7
32
5
0
0
0
0
0
0
NOTE—Average number of young per producing female at 19 days was 15.3 in control, 8.9 in
0.39 Mg/litre, and 0 in all higher concentrations tested. The difference between control (15,3)
and 0.39 Mg/litre (8.9) was significant (2-sample t test).
TABLE 3—Lengths of 14-day mysids, Mysidopsis bahia. maintained in suKlethal measured
concentrations of Kepone. Measurements were from tip of carapace to end of uropod. Tem-
perature, .4 to 29°C: :almir\: IS to 27parts per thousand.
Kepone,
Average Length,
mm
Males
Control
7.96
0.026
7.88
S.34
0.072
7.70
3.11-
0.11
7.93
i.lcr
0.23
7.67
a.33
0.41
7.71
8 06J
^S;;f-:i-"r*rri?.gf5fe.-*^:;5i'r"C-S^v"''-''
4
'Significant at 95% confidence level compared to control (Dunnett's test).
(fewer juveniles/female). Tests with 13 species of malacostracans (including
5 species of mysids) have established that the number of eggs produced was
a direct function of body length [16].
Our toxicity data for mysids exposed to cadmium were similar to those
reported for other species. For example, after 96 h, the estimated LCX for
sand shrimp, Crangon septemspinosa, and hermit crab.s, Pagurus longi-
carpus. to cadmium was 320 Mg/litre [17]. By comparison, the 30-day
LC50 value for pink shrimp, Penaeus duorarum, was 720 ng/litre, and a 29-
day LC5() for grass shrimp, Palaemonetes vulgaris, was 120 ng/litre U5].
Thus, in either 96-h or life-cycle bioassays, the sensitivity of M. bahia to
cadmium was an order of magnitude greater than that of the four species
of decapod crustaceans just reported. In addition, crustaceans were found
-------
NIMMO ET AL ON MYSIDOPSIS BAHIA 115
to be more sensitive to cadmium than were echinoderms, annelids, mol-
lusks, or fishes [17].
gfc^-rc-r,s.-0;,w^<:.,.:fev4 Mysids were sensitive to Kepone in 96 h (10.1 jug/litre) [5]. The 96-hour
'„."-.-?•*•_•• v~--.. : •:"'*----^l LC50 for several other estuarine organisms were: 6.6 ^g/Htre for spot
_ > (Leiostomus xanthurus),10 Mg/litre for sheepshead minnows (Cyprinodon
variegatus), 121 ^g/litre for grass shrimp (Palaemonetes pugio), and more
than 210 pg/litre for blue crabs (Callinectes sapidus).
Our data for cadmium and Kepone provide bases for calculation of
application factors. This factor is the numerical ratio of the "safe" concen-
tration (conducted in a long-term toxicity test) to the acutely lethal con-
centration (usually a 96-h toxicity test) [18]. Although application factors
have been used primarily for fishes, sufficient data are lacking to allow
such estimates for invertebrates or for extrapolating between phylogenetic
lines, especially for marine species for which few data exist. However, by
using the procedure given by Eaton [18], the application factor for cadmium
can be calculated by dividing the concentration that did not delay produc-
tion of young mysids nor decrease number of young per female (4.8 pg/
litre) by the 96-h LC50 (15.5 fig/litre). The resulting ratio is 0.31. The
application factor for Kepone can be calculated by dividing the concentra-
tion that had no apparent effect on growth of female mysids (0.026 ^g/litre)
by the 96-h LC^ (10.1 /ug/litre). This ratio is O.C125. Since both cadmium
and Kepone are considered persistent and cumulative, the water quality
criteria report [1] has recommended that concentrations of these chemicals
should not exceed 0.05 of the 96-h LQo at any time or place. In addition,
:ti^h:i'7^r-<:-.^t.^-^j^--;^---^ the 24-h average concentration should not exceed 0.01 -of the 96-h LC-^.
' . . . • .i Our research with mysids suggests that extreme caution should be taken
when estimating application factors (for example. 0.011 without data from
laboratory studies, because the laboratory-derived application factor is
"overprotective" for cadmium (0.31) and "underprotective" for Kepone
(0.0025).
General Conclusions
Ease of culture and maintenance in flow-through bioassays, apparent
sensitivity to toxicants, and shortness of life cycle are advantages for using
the crustacean Mysidopsis bahia in life-cycle toxicity tests. Some sublethal
effects observed after prolonged exposure to cadmium and Kepone singly
were: (a) delay in the formation of brood pouches, (b) delay in the release
of young, (c) fewer young produced per female, and (d) reduced growth. In
nature, the loss of mysids due to the direct toxic effects of pollutants or the
indirect effects on their growth or population size could affect the food
supply of many fishes.
-------
116 AQUATIC TOXICOLOGY AND HAZARD EVALUATION
References
[/] "Water Quality Criteria 1972," EPA-R-73-033, Environmental Protection Agency, U.S.
Government Printing Office, Washingtion. D.C., 1973.
' [2] Hansen, D. J.. EPA-600-3-76-079, Environmental Protection Agency, U.S. Government
'- " Printing Office. Washington, D.C., 1976, pp. 63-76.
[.?] Molenock, J.. Tulane Studies in Zoology and Botanv, Vol. 15, No. 3, 1969, pp. 113-
116.
[4] Odum, W. E. and Heald, E. J., Bulletin of Marine Science, Vol. 22, No. 10, 1972,
pp. 671-738.
[5] Hansen, D. J., Wilson. A. J., Nimmo, D. R., Schimmel, S. C., and Bahner, L. H.,
Science, Vol. 193. 1976, p. 528.
\6] Jacobs. F. and Grant, G. C., Water Research. Vol. 8, 1974, pp. 439-445.
[7] Scmtila de Almeida Prado, M., Boleiim de Zoologia e Biologia Marinha, Brazil. Vol.
30, 1973, pp. 395-417.
\8} McLane. W. M., unpublished Ph.D. Dissertation, University of Florida, 1955.
[9] Darnell, R. M., Publications of the Institute of Marine Science. Vol. 5, 1958, pp.
353-416.
[10] Carr. W. E. S. and Adams, C. A.. Transactions, American Fisheries Society, Vol. 102.
No. 3, 1973. pp. 511-540.
U/l Markle, D. F. and Grant, G. C., Chesapeake Science, Vol. 11, No. 1. 1970. pp. 50-54.
112} Stickney. R. 'R., Taylor, G. L., and Heard, R. W., Ill, U.S. Fish and Wildlife Service
Fishery Bulletin. Vol. 72, No. 2, 1974. pp. 515-525.
\13] Mount. D. 1. and Brungs, W. A., Water Research. Vol. 1, 1967, pp. 21-29.
,{14} Bahner. L. H., Craft. C. D., and Nimmo, D. R., Progressive Fish-Culturist. Vol. 37,
No. 3, 1975. pp. 126-129.
[/5] Nimmo, D. R., Lightner, D. V., and Bahner, L. H., Physiological Responses of Marine
Biota to Pollutants, F. J. Vernberg. A. Calabrese, F. P. Thurberg, and W. B. Vernberg,
Eds., Academic Press, 1977," pp. 131-183.
{10} Jensen. J. P.. Mcddclelser Fra Danmarks Fiskeri-og Havundersogehtr. Vol. 2, No. 19,
1958. pp. 1-25.
.«v~S \17] Eisler. R., Journal of the Fisheries Research Board of Canada. Vol. 28, No. 9 1971
"^ pp. 1225-1234.
[18} Eaton. J. G.. Bioassay Techniques and Environmental Chemistry. C. E. Glass. Ed.,
Ann Arbor Publishers. Inc., 1973, pp. 107-115.
-------
Short Papers and Notes
Toxicity and Uptake of Kepone in Marine
Unicellular Algae1
ABSTRACT: Four species of marine unicellular al-
gae were exposed to Kepone in laboratory bioassays.
EC50 values after seven days' growth, in mg/Iiter
(ppm), were: Chlorococcum sp., 0.35; Dunaliella ler-
tiolecta, 0.58; \it:schia sp., 0.60; Thalassiosirapseu-
donana, 0.60. When exposed to 100 jig/liter (ppb)
Kepone for 24 hr. residues associated with the algae, in
mg/kg (ppm) wet weight, were: Chlorococcum sp., 80;
D. (erliolecta, Z3; i\itzschia sp., 41; T. pseudonana,
52.
Kepone® (decachlorooctahydro-1,3.4-metheno-2H-
cycloSuta jcdj pemalene-2-one) is used for control of
banana and potato pests and as an ant and roach
killer. The insecticide has been reported in water and
biota of the Appomatox and James rivers. Virginia
(Environmental Protection Agency 1975). Kepone is
structurally similar to the insecticide mirex (dodecach-
lorooctahvdro-l.3,4-metheno-2H-cyclobuta [cd] pen-
talene) and may be a product of its photodegradation
(Ivie etal. 1974).
We exposed four species of marine unicellular algae
to Kepone to determine its effects on growth and its
uptake by phytoplankton. Since algae are food for
animals, oxygenate svater. and regulate nutrients, an
effect on them could affect physical, chemical, and
Materials and Methods
Algae tested were Chlorococcum sp. (Milford "C").
Dunaliella icrnolecta (chlorophues). Nnzscliia sp. (LB
684). and Thalassiosira pseudonana (bacillanophytes)
All were obtained from the Culture Collection ot Al-
gae. Indiana University.
in growth studies, algae were grown either in' (1) 25
ml of medium m optically matched test tubes shaken at
approximately 60 excursions per minute on a New
Brunswick Model G shaker fitted with an ErlAngle®
(New Brunswick Scientific Co.. New Brunswick. Con-
necticut) clamp, or (2) 50 ml of medium in 125-ml
Erienmeyer flasks shaken at approximately 60 excur-
sions per minute. In the (ormer. optical density at 525
nm on a Fisher electrophotometer was determined each
day for seven days alter inoculation; in the latter.
optical density was determined after seven days of
growth.
Growth medium was artificial seawater of 30 parts
per thousand salinity with full and half-nutrient
' Contribution No. 292 from the Environmental Re-
search Laboratory - Gulf Breeze
® Registered trademark. Allied Chemical Corp.,
New York. Mention of trade names in this publication
does not constitute endorsement by the U.S. Environ-
mental Protection Agency.
strengths (Hollister et al. 1975). Temperature was 20°
± 0.5°C under 5.000 lux illumination from cool, white
fluorscent tubes with alternate 12-hr periods of light
and darkness. Technical grade Kepone was added in
0.1 ml acetone and 0.1 ml acetone was added to all
control cultures. This concentration of acetone did not
affect growth. During seven growth tests, each expo-
sure was performed three times Concentrations that
reduced growth by 50 per cent (EC50) after seven days
were calculated (American Public Health Association
1971).
In uptake studies. 12 cultures of each alga were
grown in 100 ml of full nutrient medium for six days.
All that time, cell numbers were between 2 x 105and 3
x 105 cells/ml, and Kepone, in 0.1 ml acetone, was
then added at the nominal concentration of 100 /xg/
liter. This was well below its solubility limit of 2-4 mg/
liter in seawater (May. unpublished). On the seventh
day. the algae were harvested by centnfugation,
washed four times with uncontaminated medium, and
analyzed for kepone by gas chromatography. Each
value in Table 1 is the average of 12 replications.
For residue analysis, algae samples were weighed in
125 mm x 15 mm (O D.) screw-top test tubes and
extracted with two 5-ml portions of acetomtrile for 30
sec with a Model PT 10-ST \Villems Polvtron (Bnnk-
— - '".^rurnents. '•V.-^:l:ury . Ne\v Yi -\ T; _• • • • • -
;:..-- extract was transferred to a 250-ml separators
tunnel after centnfugation, and the algae again ex-
tracted, first with 5 ml and then with 10 ml ot j;ctonc.
After each extraction, the tube was centrifuged. and
the extract placed in the separatory funnel. Then, 100
ml of 2.0% aqueous sodium sulfate and 10 ml of 1:1
diethyl ether-petroleum ether was added, and the mix-
ture sliaken for 1 minute. Alter the solvent phases
separated, the lower aqueous phase was drained into a
250-ml beaker and the upper layer collected in a 25-ml
Kuderna-Damsh concentrator tube. The ether extrac-
tion was repeated three times with 5 ml of 1:1 diethyl
ether-petroleum ether. The combined extracts were
concentrated to dryness by placing the tube in a water
bath at 45°C and evaporating the solvent with a gentle
stream of nitrogen. The residue was transferred to a
200mm x 9 mm (l.D.) Chromatlex® column (Kontes
Glass Co. Vmeland. N.J.) containing 3.0 gm of
Flonsil® overlain by 2 0 gm of anhydrous sodium sul-
fate. The column was first washed with 10 ml of hexane
and the residue transferred with four 0.5-ml volumes of
5% diethyl ether in hexane to remove PCB and pesti-
cides. Kepone was removed by a second elution with 40
ml of 1 % methanol in benzene Extracts concentrated
or diluted to appropriate volume were analyzed b\
electron-capture gas chromatography on Vanan Aero-
graph Models 2100 and .1400 Gas Chromatographs
equipped with 182 cm x 2 mm (l.D.) glass columns
packed with 2% SP2100 and 0.75% SP2250:0.97%
222
Chesapeake Science Vol 18, No. 2, June, 1977
-------
Short Papers and Notes
223
TABLE 1. Depression of growth of marine unicellular alga by Kepone and concentrations of Kepone in
algae after exposure to 100 /ng/'l for 24 hours.
Algae
Chlorococcum sp.
D. lernolecia
Nitzschia sp.
T. pseudonana
EC50° /xg/1
0.35 (0.27-0.40)
0.58 (0.51-0.64)
0.60 (0.53-0.66)
0.60 (0 50-0.70)
Cell Residue ^.g/g
80
23
41
52
Concentration Factor
800
230
410
520
" Range in parentheses.
SP2401 on 100/120 mesh Supelcoport®. The opera-
tion conditions were: oven temperature, 185°C: injec-
tor temperature, 200°C; detector temperature, 215°C;
nitrogen carrier gas flow rate, 25 ml/mm. Al! samples
were fortified with an internal standard (dichioroben-
zophenone) prior to analysis to evaluate the integrity of
results. The average recovery rate of Kepone from
fortified algae was 85%. Residue concentrations were
calculated on a wet weight basis without correction
factor for percentage recovery.
Results
Effects and uptake of Kepone are given in Table 1.
Because results from bioassays with full and half-
strength nutrient media were identical, the data were
combined. Seventh-day EC50 values are representative
of effect because the degree of growth inhibition was
constant from day to day. Chlorococcum sp. was the
most sensitive species: its EC50 values never over-
lapped those of the others. The EC50 values of the
other species were nearly identical.
Chlorococcum sp. also accumulated the greatest
sure, and all differences, except between Niizschia sp.
and T. pseudonana, are highly significant statistically
(t-test for two means, Brownlee 1965).
Discussion
Butler (1963) stated that 1 mg/liter of Kepone re-
duced carbon fixation by estuanne phytoplankton by
^5 per cent aitcr exposure lor 4 hr. Our data show that
toxicity occurred at much lower concentrations and that
algae can accumulate the chemical from water. Since
EC50 concentrations were higher than those reported
from the Appomatox and James rivers (Environmen-
tal Protection Agency 1975). Kepone may have little
effect on phytoplankton in the field. However, uptake
by algae could result in accumulation at higher trophic
levels.
ACKNOWLEDGEMENT
We thank Shelley Alexander, Sharon Edmisten. Ka-
thy Nolan and Jerrold Forester for their technical as-
sistance.
REFERENCES
AMERICAN PUBLIC HEALTH ASSOCIATION. 1971. Stan-
dard Methods for the Examination of Water and
Wastewater. Thirteenth ed. APHA, New York. 874
P-
BROWNLEE, K. A. 1965. Statistical Theory and Meth-
odology in Science and Engineering. John Wiley and
Sons, New York. 590 p.
BUTLER, P. A. 1963. Pesticide-Wildlife Studies. U.S.
Department of the Interior, Circular 167, Washing-
ton, D.C. pp.11-25.
ENVIRONMENTAL PROTECTION AGENCY. 1975. Fact
sheet on Kepone levels found in environmental sam-
ples from the Hopewell, Va. area. Health Effects
Research Laboratory. EPA, Research Triangle
Park. NC, unpublished, 15 p.
HOLLISTF.R. T. A.. G. E. WALSH. AND J. FORESTER.
1975. Aiuex jnu marine uniccilu.^r.. _..;:. J..VL.JT...HI-
tion, population growth, and oxygen evolution. Bull.
Environ. Comam. Toxicol. 14:753-759.
IVIE. G W., H W. DOROUGH, AND E. G. ALLEY.
1974. Photodecomposition of mirex on silica gel
chromatoplates exposed to natural and artificial
light. J. Agric. Food Chem. 22:933-936.
MAY, J. R. Pesticide Reference Standards Manual
Nat. Commun. Disease Cen.. U.S. Pub. Health Ser..
Pest. Repository, Pest. Res. Lab.. Pernne. FL. un-
published
GERALD E. WALSH, KAREN AINSWORTH AND ALFRED
J. WILSON
Environmental Protection Agency
Environmental Research Laboratory —Gulf Breeze
Gulf Breeze, Florida 32561
-------
224
Short Papers and Notes
Acute Toxicity of Kepone® to
Four Estuarine Animals1
ABSTRACT: Recent contamination of the James
River estuan. Virginia, with Kepone prompted acute
flow-through bioassays to determine the 96-hour toxic-
it} of the insecticide to four estuarine species native to
that ecosystem. The species and their 96-hour LC50
values were: grass shrimp (Palaemonetes pugio), 121
fig/liter; blue crab (Callinectes sapidus}, >210 /xg/
liter; sheepshead minnow (Cyprinodon variegaius),
69.5 fig/liter; and spot (Leiostomus xanthurus) 6.6 p.g/
liter. Surviving animals were anahzed for Kepone.
Average bioconcentration factors (the concentration of
Kepone in tissues divided by the concentration of Ke-
pone measured in seawater) were: grass shrimp, 698;
blue crab 8.1; sheepshead minnow, 1.548; and spot,
1,221.
Introduction
Few published data are available on Kepone toxicity
to estuarme animals. Butler (1963) reported EC50
values (based on mortality or loss of equilibrium in 48
hours for shrimp and on inhibition of shell deposition in
96 hours for oysters) of 85 ^tg/liter for brown shrimp
(Penaeus aziecus): 57 /^g/Iiter and 15 fig/liter for east-
ern oysters (Crassostrea nrginica) exposed at seawater
temperatures of 14°C and 31°C. respectively. Twenty
percent of the blue crabs (Calhnecies sapidus) exposed
to 1,000 jug/liter Kepone died in 48 hours. Butler's
data 'vere derived from !n-«'.-t!-rr".^h b'oassnys ^nd
based on nominal, not measured, concentrations of
Kepone in seawater
Recent discharge of the insecticide. KeponeK. into
the James Riser estuary. Virginia has resulted in con-
tamination of that system's water, sediment, and biota.
This contamination raised questions about the acute
and chronic effects of Kepone on the aquatic life m the
t:stuar\ and the potential uunger tu humans tn eating
contaminated animals.
In Januap, l'17'i. we initialed flow-thr"'1*:!'. bioas-
says to determine bioconcentration and acute toxic
effects of Kepone on representative species found in
the James River estuary These were grass shrimp (Pa-
laemoneles pugio), blue crab (Callmecles sapidus),
sheepshead minnow (Cyprinodon variegatus), and spot
(Leiosiomus xanthurus).
Methods and Materials
Acute toxicity was determined by exposing 20 ani-
mals per aquarium to different concentrations of Ke-
® Registered Trademark for decachlorooctahydro-
1.3.3-metheno-2H-c\clobuta (cd) pentalen-2-one. Al-
lied Chemical Company. 40 Rector Street, New York.
New York 10006 Mention of commercial products
does not constitute endorsement b\ the Environmental
Protection Agency.
1 Contribution No 293, Environmental Research
Laboratory, Gulf Breeze.
pone for 96 hours in flow-through bioassays similar to
those described by Lowe et al. (1972). All test animals
were acclimated to laboratory conditions for at least ten
days prior to testing. The temperature and salinity of
seawater in which they were held were allowed to vary
with those of Santa Rosa Sound. Florida, during accli-
mation and testing Our acclimation and testing proce-
dures were compatible with those of Standard Methods
(A.P.H.A. 1971). Ail Vest animals were captured in the
vicinity of the Gulf Breeze Laboratory and samples
contained no detectable Kepone (<0.02 Mg/g)- During
acclimation, test animals were fed frozen brine shrimp
daily. Animals were not fed during the tests, but could
obtain food (plankton and other paniculate matter)
from the unfiltered seawater. Seawater was pumped
from Santa Rosa Sound into a constant-head trough in
the laboratory and delivered to each 18 liter aquarium
by a calibrated siphon that delivered approximately 68
luer/hr. One control and five experimental aquaria
were used in each test. Stock solutions of Kepone
(88% pure), in reagent-grade acetone, were metered
into experimental aquaria at the rate of 60 ml/hr.
The 96-hour LC50 values were determined for both
nominal and measured concentrations of Kepone in
seawater. Nominal concentrations were those calcu-
lated to be in seawater. based on the concentration of
the stock solution, plus the stock solution and seawater
flow rates The LC50 values were based on measured
Kepone concentrations de'crri!"ed K chemical analy-
sis of the exposure water. Mortality data were sub-
jected to probit anaUs;" to determine LC50 values and
their 45*7 confidence limits (Finnev 19~M).
At the end of each 96-mnir test, surviving animals
from each concentration were sacrificed, rinsed with
acetone, and pooled as a single sample for residue
analysis.
\\aier samples were aruhzed o; - .
of seawater twice with 100 ml of rrothvlene cnionJe.
The combined extracts wore concer.ira'.eu to about 5 mi
in a Kuderna-Danish Concentrator on a steam table.
Fifteen milliliters of benzene were added and the ex-
tract reconcentrated to remove the methylene chloride
The extract was cleaned up on a Flonsil Column as
described below.
Tissues of shrimp, crabs, or fish were weighed in 150
mm x 25 mm (O.D.) screw-top test tubes and ex-
tracted twice with 5 ml volumes of acetomtnle for 30
seconds with a model PT 10-ST Willems Polytron
(Bnnkman Instruments, \\estbury. New York). The
mixture was centifuged and the acetomtrile transferred
to a 250-ml separatory funnel. After the second extrac-
tion, the tissue was extracted with one 5-ml and one 10-
mi volume of acetone. After each acetone extraction
the tube was centnfuged and the acetone added to the
250-ml separatory funnel. To the combined extracts,
100 ml of 2.0% aqueous sodium sulfate and 10 ml of
1:1 diethyl ether-petroleum ether were added. The
separatory funnel was shaken for one minute. After the
solvent phases had separated, the lower aqueous phase
Chesapeake Science Vol 16, No 2, June, 1977
-------
Short Papers and Notes
225
TABLE 1. Toxicity of Kepone to and uptake by four estuarine organisms after 96 hours exposure.
Water Concentration (^g/liter)
Mortality
'
Species
Nominal
Measured
Whole-Body
Residue (#ig/g
wet weight)
Bioconcentration
Factor
Grass shrimp (Palaemoneles
pugio)
Blue crab (Callinectes sapidus)
Sheepshead minnow (Cyprino-
don variegatus)
Control
13.5
24.
42.
75.
135.
Control
42.
75.
135.
187.
240.
Control
ND°
12.
15.
39.
69.
121
ND
no.
164.
210.
ND
0
0
0
5
50
0
0
10
5
0
0
ND"
5.1
14.
29.
42.
94.
ND
0.85
1.7
1.3
ND
425
933
744
609
111
x = 698
7.7
10.4
6.2
x = 8.1
10.
18.
32.
56.
100.
Spot (Leioslomus xanthurus) Control
2.4
4.2
7.5
13.5
24,
7.1
14.
23.
51.5
78.5
ND
1.5
3.4
4.4
7.8
15.9
0
0
5
20
65
0
5
10
45
40
95
11.2
20.9
44.4
63.6
118.4
ND
1.7
3.2
7.0
10.8
16.8
1577
1493
1930
1235
1506
x = 1548
-
1133
941
1591
1385
1057
x = 1221
° ND —non detectable: <0 02 .us/liter in water. <0 02 ,uc/g in t'
TABLE 2 96-hour toxictty of Kepone to several estuarine animals in flowing seawater bioassays. The 95^7
confidence intervals are in parentheses. Animal sizes are rostrum-telson length for shrimps, carapace width
for crabs, and standard length for fishes.
Species
Grass shrimp (Palaemoneles
pugio )
Blue crab (Callinectes sapi-
dus)
Sheepshead minnow (Cypri-
nodon variegatus)
Spot (Leiosiomus xanthurus)
Size
(x. mm)
27.8
34.3
20.0
33.9
Nominal 96-hour Measured 96-hour
LC50 in ng/lner (,9fZLC50 in pig/hter (95 Tr
Confidence Limit) Confidence Limit)
134.8
(114.1-193.9)
>240
83.0
(67.9-115)
10.5
(8.3-14.0)
120.9
(103.0-171.6)
>210
69.5
(56.3-99.5)
6.6
(5.3-8.8)
Temperature
(x. °C)
20.0
19.0
18.0
25.0
Salinity
(x. "/„,')
16.0
20.0
15.0
18.0
was drained into a 250-ml beaker and the upper ether
layer was collected in a 25-ml Kuderna-Danish concen-
trator tube. The ether extraction was repeated three
times with 5 ml of 1:1 diethyl ether petroleum ether
The combined extracts were concentrated just to dry-
ness b\ placing the concentrator tube in a water bath at
45°C and blowing off the solvent with a gentle stream
of nitrogen. The residue was transferred to a 200 mm x
9 mm (l.D.) Chromaflex column (Kontes Glass Co.)
containing 2.3 gm of Flonsii topped with 2.0 gm of
anhydrous sodium sulfate. The column initially was
washed with 10 ml of hexane and the residue trans-
-------
226
Short Papers and Notes
ferred with four 0.5 ml volumes of 5% diethyl ether in
hexane The column was eluied with 20 ml of 5%
diethyl ether in hexane to remove PCB and pesticides.
Kepone was eiuted in a second elution of 40 ml of 1 %
methanol in benzene. Extracts were concentrated or
diluted to appropriate volumes for analyses by electron
capture gas chromatography.
Determinations were obtained by Vanan Aerograph
Model 2100 and 1400 Gas Chromatographs equipped
with 182 cm x 2 mm (I.D.). glass columns packed with
2% SP2100 and 9.75% SP2250: 9.97% SP2401 on
100/120 mesh Supelcoport. The operating parameters
were: oven temperature 185°C, injector temperature
200°C. detector temperature 216°C, and nitrogen car-
rier gas flow rate 25 ml/min
The average recovery rate of Kepone from fortified
tissue was 87%; from water. 85%. Residue concentra-
tions were calculated on a wet-weight basis without a
correction factor for percentage recovery. All samples
were fortified with an internal standard (dichloroben-
zophenone) prior to analysis to evaluate the integrity of
the results.
Results and Discussion
Kepone, at concentrations tested, was acutely toxic
to shrimp and fishes but not to blue crabs. The LC50
values varied wideh among species. Spot were the most
sensitive with a 96-hour LC50 of 6.6 ^.g/liter; the
sheepshead minnow LC50 was over ten times higher
(69.5 /xg/liter). The two crustaceans were less sensi-
tive. Grass shrimp LC50 was 120.9 /^.g/liter, and no
significant mortality was observed in blue crabs at mea-
sured concentrations as high as 210 /ig/liter (Tables 1
differed, the s\ mptoms of Kepone poisoning were simi-
lar. An early symptom was lethargic behavior followed
by loss of equilibrium. These symptoms occurred in
sheepshead minnows at 48 hours in 56 and 1 00 /j.g/liter
concentrations and 96 hours in 18 and 32 ^.g/liter
concentrations. Spot exhibited the symptoms in 48
hours when exposed to 7.5, 135 and 24 ^g/liter Ke-
pone. An advanced stage of poisoning was evident in
dark coloration of portions of the fish's body. This
color change was striking in that some fish had normal
coloration on one side ot their bodies, while the other
side was nearly black with a sharp line of demarcation.
Some spot and sheepshead minnows were darkened in
only one quadrant of the body; for example, the lett
side posterior to the pectoral fins. These color changes
were always more marked and appeared earlier in the
higher Kepone concentrations. Hansen et al. (1977)
also noted color changes in sheepshead minnows ex-
posed to a lower Kepone concentration (0.8 ^.g/lner)
over a longer duration (11 days). The same authors
also noted that growth, reproduction and survival of
sheepshead minnows were affected in 36 days by Ke-
pone concentrations as low as 0 08 /xg/liter, which is
0.001 of our sheepshead minnow LC50 (69.5 pig/liter).
If we assume that the same ratio exists for spot as
Hansen et al. reported for sheepshead minnows, then
the no-effect level for spot would be less than 0.007 ^.g/
liter based on our spot LC50 (6.6
No color changes were observed in the two crusta-
ceans although they also were lethargic in Kepone
concentrations greater than 75 /j.g/liter.
Kepone was bioconcentrated by all test animals in
96 hours, although bioconcentration factors (concen-
tration of Kepone in tissue divided by measured Ke-
pone in water) varied between species (Table 1). (The
bioconcentration factors for fish were similar, x =
1200-1500). The two fishes bioconcentrated Kepone
an average of 1.7 to 2.2 times the bioconcentration
factor for the grass shrimp and 150 to 190 times that
measured in the blue crab. This difference in biocon-
centration has been noted in similar bioassays with
other organochlonne insecticides. Schimmel et al.
(1976) reported that sheepshead minnows, spot, and
pmfish (Lagodon riiomboides) bioconcentrated 4 to 70
times more heptachlor than grass shrimp or pink
shrimp (Penaeus duorarum). A similar relationship oc-
curred when some of the same species were exposed to
toxaphene (Schirnmel et al.. in press) and dieldrin (Par-
nsh et al. 1974) in 96-hour bioassays. The reason for
an extremely low bioconcentration factor in the blue
crab compared to grass shrimp is not known.
Further studies over a longer period of time are
required to better understand the more subtle effects of
Kepone on estuanne animals. One reason for this as-
sessment is that most deaths occurred after 48 hours in
our tests. These studies should include. (1) long-term
bioconcentration studies; (2) bioassays which include
the hatching and early development of an estuanne
animal such as spot; and (3) studies to determine move-
ment of Kepone through an estuanne food web.
ACKNOWLEDG" t^*-?
The aui.iors ^ ..•>;: c- .XvO^r...^ ne s.ipificant contri-
butions of Jerrold Forester and Johnnie Knight in the
chemical analyses of water and tissue samples and of
James M Patrick. Jr., for help in the bioassays.
LITERATURE CITED
AMERICAN PUBLIC HEALTH ASSOCIATION. 1971. Stan-
dard Methods for the Examination of Water and
Wastewater. American Public Health Association.
14th ed. Washington, D.C. 1193 p.
BUTLER. P. A. 1963. Pesticide-Wildlife Studies-A
Review of Fish and Wildlife Service Investiaations
During 1961 and 1962. U.S. Fish and Wildhfe Cir-
cular. 167 pp. 11-25.
FINNEV, D. J. 1971. Probit Analysis. Cambridge Uni-
versity Press. Great Britain. 333 p.
LOWE, J. I,, P. R. PARRISH, J. M. PATRICK. JR., AND J.
FORESTER. 1972. Effects of the polychlonnated bi-
phenyl Aroclor® 1254 on the American oyster,
Crassosirea Mrgmica. Mar. Biol. (Berlin) 17:209-
214
HANSEN. D. J., L. R GOODMAN AND A. J. WILSON.
JR. 1977. Kepone: Chronic effects on embryo. fr\,
juvenile and aduks sheepshead minnows. Cypnno-
don variegarus. Chesapeake Sci. 18 (2):227-232.
PARRISH, P. R., J A. COUCH, J. FORESTER, J, M.
-------
Short Papers and Notes 227
PATRICK. JR.. AND G. H. COOK. 1974 Dieldnn. and toxicity of Toxaphene in several estuarme orga-
Etfects on several estuarme organisms. Proc. 27th nisms. Arch. Environ. Contam. Toxicol. (in press).
Annu. Conf. Southeastern Assoc. Game Fish.
Comm. 427-434. STEVEN C SCHIMMEL
SCHIMMEL, S. C.. J. M. PATRICK. JR., AND J. FOR- AND
ESTER. 1976. Heptachior: Toxicity to and uptake by ALFRED J. WILSON. JR.
several estuarme organisms. J. Toxicol Environ. U.S. Environmental Protection Agency
Health. 1:955-965. Environmental Research Laboratory
. J. M. PATRICK. JR., AND J. FORESTER Uptake Gulf Breeze, Florida 32561
-------
Kepone®: Chronic Effects on Embryo, Fry,
Juvenile, and Adult Sheepshead Minnows
(Cyprinodon variegatus)l
ABSTRACT: \Ve investigated the Joxicity of Kepone
to, and uptake by embryo, fry, juvenile, and adult
sheepshead minnows (Cyprinodon variegatus) using
intermittent-flow toxicity tests. Concentrations of Ke-
pone and percentage of adult fish surviving in a 28-day
exposure were: Control, 95%; 0.05 fig/liter, 95%;
0.16 Mg/Hter. 100%; 0.80 /ig/liter, 78%; 1.9 Mg/"'»er,
20%; and 7.8 ftg/liter and 24 fig/liter, 0%. Concentra-
tion factors (concentrations in fish divided by concen-
trations measured in water) for adult fish averaged
5,200 (range 3,100 to 7,000). Symptoms of poisoning
included scoliosis. darkening of the posterior one-third
of the boch. hemorrhaging near the brain and on the
body, edema, fin-rot, uncoordinated swimming, and
cessation of feeding. Vdults surviMns the first exposure
in1;, jnti -:irvi.ai and growth "i ir\ ; jnd died e\en when incubated in Kepone-free
water. Kepone in water was not as lethal to progeny as
to adults: 36-da> LC50 for juveniles was 6.7 /ig/liter;
Ic5-dav I.t'50 for adult1.. 1.3 /i'i'liicr. However, the
average standard lenglh of juvenile fish was signifi-
cant!) reduced b> exposure to 0.08 ^ig of Kepone/liter
of water; some fish developed scoliosis. Concentration
factors in juvenile sheepshead minnows averaged 7,200
and increased from 3.600 to 20,000 as exposure con-
centrations decreased.
introduction
Kepone. decachlorooctahydro-1,3,4-metheno-2H-
cyclobuta cd pentalene-2-one. has been found recently
® Registered trademark. Allied Chemical Corpora-
tion. 40""Rector Street. New York. New York 10006.
Kepone used was purchased from Chem Service. West
Chester. PA as 99% pure. Our analyses indicated 88%
purity Mention of trade names or commercial products
does not constitute endorsement by the Environmental
Protection Agency.
1 Contribution No. 295, Environmental Research
Laboratory, Gulf Breeze.
in estuarine organisms in the James River, Virginia
(Hansen et al. 1976). The acute toxicity of this insecti-
cide to estuarine fishes and invertebrates has been
investigated (Butler 1963; Schimmel and Wilson
1977), but the chronic effects of Kepone on estuarine
fish have not.
Our study was conducted to determine: 1) the ef-
fects of Kepone exposure on survival and reproduction
of adult sheepshead minnows, 2) the effect on survival
and growth of embryos, fry, and juvenile fish spawned
from exposed adults, and 3) the extent of bioconcentra-
tion in adults and progeny.
Materials and Methods
TEST AVIMM.S
Environmental Research Ljooniiur.. Ou.. _>r-^..v.
Florida and acclimated in test aquaria in 30°C water tor
15 days before exposure. Mortality during acclimation
was less than 1 percent, and the fish fed, spawned, and
otherwise boh^ed ^ortia'!-. Tcved fi-ih a\ erjced 40
mm standard liij:, • and 2.3 g Adult tisn and tneir
food. Biorell® and Irozen adult brine shrimp (Anemia
so/mo). COT.- • __ •• . Jotectav-'c (-- 0 0: ^ y- Kj-
pone, other chlorinated insecticides or PCB's.
ADULT EXPOSURE
We exposed 32 female and 32 male fish in aquaria
containing zero, 0.16, 0.80, 1.9. 7.8, and 24 /ig/liter of
Kepone. and 108 fish to 0.05 /j.g/hter for four weeks in
an intermittent-flow toxicity test. Our apparatus was a
modified model of that used by Schimmel et al. (1974).
We delivered Kepone, 0.0088 ml of the solvent tnethy-
lene gtycol, and 1.5 liter of filtered 30°C (± 1°C)
seawater averaging 15"/(1(, salimt (range 8-26 "/,»,) to
each 70 liter aquarium during each cycle of the appara-
tus. Water and solvent without Kepone were delivered
to the control aquarium Number of cycles per day for
the adult exposure averaged 440.
EMBRYO. FRY, AND JUVENILE EXPOSURE
To determine the effect of Kepone on sheepshead
minnow embryo, fry, and juvenile survival, we en-
Chesapeake Science Vol. 18, No 2, June, 1977
-------
228
Short Papers and Notes
hanced egg production b\ injecting hormones in ex-
posed adult fish, fertilizing the eggs artifically, and
monitoring their development in Kepone for 36 days.
Twenty female sheepshead minnows exposed to none.
0.05. 6.16. or 0.80 fig/liter of Kepone and 8 females
exposed to 1.9 ^tg/hter were injected with 50 1. U. of
human chonomc gonadotrophic hormone on exposure
days 25 and 27 (Hansen et al. 1974). On the 28th day
of exposure eggs were stripped manually from injected
fish and were fertilized in control water with sperm
from excised macerated testes from ten or more males
exposed to 0.8 /^g/liter or less of Kepone and from five
fish exposed to 1.9 ^.g/hter. Some spawned adult fish
from each concentration were frozen for chemical anal-
yses ot Kepone content.
Embryos were placed in appropriate aquaria previ-
ously used for adult fish to determine the effects of
Kepone 1) in water: 2) within the egg; and 3) in both
eggs and water on embryos, fry. and juvenile fish.
Twenty embryos from control fish were placed in each
of four egg cups (Petn dishes to which a nine-cm high
collar of 450p. nylon mesh was attached) in the control
aquarium and in each aquarium receiving Kepone.
Twenty embryos from fish in each aquarium receiving
Kepone were placed in each of four egg cups in that
aquarium and into four egg cups in the control aquar-
ium. Thus a total of 80 embryos w-as observed in each
of 15 separate treatments. Remaining eggs were frozen
for chemical analysis of Kepone content. The dosing
apparatus cycled about 350 times per day delivering
water to each aquarium containing egg cups. The action
of a self-starting siphon in each aquarium caused water
le\els to fluctuate approximately 5 cm about 40 times
per day insuring that water in the egg cups was ex-
changed. Fry were fed live brine shrimp nauplii that
contained no detectable (<0.02 Mg/g) Kepone, other
. ,:o--...t.. •^:.i.^:^ > : r'CB'- At ;hc .iJ < :u,e
36-day emDryo-lr\-juveniie exposure, juveniles were
photographed for length measurements, weighed, and
some were frozen for chemical analyses.
CHEMICAL ANALYSES
Water samples were analyzed by extracting one liter
of seawater twice with 100 ml of methylene chloride.
The combined extracts were concentrated to approxi-
mately 5 m) in a Kuderna-Danish Concentrator on a
steam table, and 15 ml of benzene was added. The
extract was reconcentrated to remove the methylene
chloride and was cleaned on a Flonsil Column (de-
scribed below).
Tissues of fish and fish eggs were weighed in 150
mm x 25 mm (O.D.) screw-top test tubes and were
extracted twice with 5-ml volumes of acetomtrile for 30
seconds with a model PT 10-ST Willems Polytron
(Bnnkman Instruments. Westbury, New York). The
test tube was centrifuged and the acetomtrile was trans-
ferred to a 250-ml separatory funnel. After the second
extraction, the tissue was extracted with one 5-ml and
one 10-ml \olume ol acetone. After each acetone ex-
traction, the tube was centrifuged and the acetone was
added to the 250-ml separatory tunnel. To the com-
bined extracts were added 100 m! of 2.0% aqueous
sodium sulfate and 10 ml of 1:1 diethyl ether-petro-
leum ether The separatory tunnel was shaken for one
minute. After the solvent phases separated, the lower
aqueous phase was drained into a 250-ml beaker and
the upper ether layer was collected m a 25-ml Kuderna-
Danish concentrator tube. The ether extraction was
repeated three times with 5 ml of 1:1 diethyl ether-
petroleum ether. The combined extracts were concen-
trated to just dryness by placing the concentrator tube
in a water bath at 45°C and blowing off the solvent \Mth
a gentle stream of nitrogen. The residue was trans-
ferred to a 200 mm x 9 mm (l.D.) Chromaflex column
(Kontes Glass Co.) containing 3.0 gm of Flonsil topped
with 2.0 gm of anhydrous sodium sulfate, The column
was washed initially with 10 ml of hexane and the
residue was transferred with tour 0.5 ml volumes of 5%
diethyl ether in hexane. PCB's and pesticides were
eluted from the column with 20 ml of 5% diethyl ether
in hexane. Then, Kepone was eluted in 40 mi of 1 %
methanol in benzene. Individual extracts were concen-
trated or diluted to appropriate volumes tor analyses by
electron-capture gas chromatography.
Determinations were performed on Vanan Aero-
graph Model 2100 and 1400 Gas Chromatographs
equipped with 182 cm x 2 mm (l.D.) elass columns
packed with 2% SP2100 and 0.75% SP2250: 0.97%
SP2401 on 100/120 mesh Supelcoport. The operating
conditions were: oven temperature. 185°C; injector
temperature. 200°C: detector temperature, 210°C; ni-
trogen carrier-gas flow rate. 25 ml/minute.
The average recovery rate of Kepone from fortified
tissue was 87 % and from water. 85 %. Residue concen-
trations were calculated on a wet-weight basis without a
correction factor for percentage recovery. All samples
were fortified with an internal standard (dichloroben-
zophenone) prior to analysis to evaluate the integrity of
the results.
7- PSTICAL ANALYSES
Probit analyses of mortality data were used to deter-
mine LC50's. Chi-square tests (* = 0.05) were used to
determine statistical significance of mortality data.
Analysis of covariance and the N'ewman-Kuels (SNK1
test was used to test Uilierences in grow in ol ir\ in
Kepone at a - 0.01.
Results
ADULT EXPOSURE
Kepone was toxic to and was accumulated by adult
sheepshead minnows exposed for four weeks (Table 1).
Mortality increased in relation to the increase in con-
centration and duration of exposure (Figs. 1 & 2). All
fish exposed to 7.8 and 24 fie. of Kepone/lner of salt-
water died by day 15 Twenty-two percent of fish in
0.8 Mg/'iter and 80% of the fish in 1.9 ^g/liter died;
most of the surviving fish exhibited symptoms of Ke-
pone poisoning.
Symptoms of poisoning were related to concentra-
tion and duration of exposure, but were not typical of
poisoning by other organochlorme pesticides (Hender-
son et al. 1959). Symptoms of poisoning progressed
from scoliosis. darkening of the posterior one-third of
the body, hemorrhaging near the brain and at the
anterior point ot darkening, to increased hemorrhaging
posteriorly, swelling of the darkened area, fin rot. un-
-------
Short Papers and Notes
229
TABLE 1. Toxiciu and uptake of Kepone b\ adult sheepshead minnows (Cyprmodon variegatus) exposed
for 28 days in an intermittent flow toxicny test. A total of 108 fish were exposed to 0.05 /us/liter Kepone and
32 males and 32 females were exposed to other concentrations. Samples from 1.9 /ug/liter consisted of eight
females and their eggs and five males; other samples consisted of a minimum of 16 females and their eggs
and ten male fish.
Exposure Concentration (^g/hler)
Concentration in Fish (/ug/g, Wet Weight)
Desired
Control
0.1
0.32
1.0
3.2
10.0
32.0
Measured
ND*
0.05
0.16
0 80
1.9
7.8
24.
Mortality %
5
5
0
22
80
100
100
Females
ND*
0.35
0 90
3.6
12.
_
-
Males
ND
0.25
0.65
2.5
11.
_
-
Eggs
ND
0.26
1.0
4.7
11.
_
-
ND = Kepone not detected: <0 02 ^g/liter, <0.02 /ig/g.
12 16 20 24 28
TIME loo^i)
FIG. 1. Mortality of adult sheepshead minnows (Cv-
pnnodon vanegatus) exposed continuously to Kepone
for 28 days.
20
15
10
I
P
I
i
\ EMBRYO, FRY AND
JUVENILE
•
ADULT
3 \Z 16 20 24 28 32 36
EXPOSURE (doyt)
Fig. 2. Concentration of Kepone in water lethal to
50% (LC50) of embryo, try. and juvenile sheepshead
minnows continuously exposed for 36 days and adults
exposed for 28 days.
coordinated swimming, and cessation of feeding These
symptoms were observed on the first day in 24 /xg/liter,
the second in 7.8 /u.g/liter, the third in 1.9 p.g/liter, and
the eleventh day in 0.8 /xg Kepone/liter of water. These
symptoms increased in severity and frequency before
death from five to eight days later (Fig. 1).
Chemical analyses of adult sheepshead minnows ex-
posed to Kepone for four weeks showed that Kepone
was bioconcentrated in proportion to the concentration
in the exposure water (Table 1). Concentration factors,
concentration in tissue divided by concentration in wa-
ter, averaged 5,200 (range 3.100 to 7,000). Concen-
trations of Kepone in females were greater than in
males. Concentrations in eggs were generally similar to
amounts in females. The concentration of Kepone in
dead fish was similar to that found in fish surviving the
exposure (Table 1). Dead fish in 7.8 /ug of Kepone/liter
of water contained 17 ^g/g, in 1-9 jig/liter, 10 fig/g; in
0.8 Mg/Iiter- 3.4 /ig/g.
EMBRYO, FRY, AND JUVENILE EXPOSURE
Monalny. The effect of Kepone in water on mortal-
ity of embryos, fry, and juvenile sheepshead minnows,
tested by exposing progeny of adults from the control
aquarium to zero,"o.08rO.IS, 0 72, 2.0. 6.6, and 33 ptg
of Kepone/hter of water, was less than that observed
with aduit tish exposed to similar concentrations (Table
2, Fig. 2). The 36-day LC50 to juveniles exposed to
Kepone in water was 6.7 /ig/hter (95% confidence
interval: 4.7-13.4 ^ig/liter) and the 28-day LC50 to
adults was 1.3 ne./\'ner (95% confidence interval: 1.1
to 1.5 ^tg/liter). Kepone in water affected mortality of
embryos prior to hatching. Chi-square tests revealed
that mortality of embryos, in each Kepone concentra-
tion did not differ from that of controls; however, com-
parisons between embryo mortality in control, 0.08.
and 0.18 ng/liter (average 9%) with concentrations
greater than 0.18 ng/liter (average 17%) indicate that
embryo mortality was significantly increased by
Kepone. Fry from the embryos exposed to 6.6 and 33
/xg/liter were visibl\ affected within 24 hours of hatch-
ing. Juvenile fish exposed to 2.0 or 0.72 /xg/liter
beginning as embryos did not appear visibly affected
until 10 and 16 days after hatching, respectively.
Symptoms of poisoning in fry less than one week old in-
cluded diminished activity .'loss of equilibrium, cessa-
tion of feeding, and emaciation Symptoms in fry older
than one week were identical to those observed in adult
-------
230
Short Papers and Notes
TABLE 2. Mortality of embryo, fry, and juvenile sheepshead minnows and bioconcentration of Kepone in 19
to 63 juvenile fish (average 50) in a 36-day exposure In some instances parental fish were exposed to Kepone
for 28 days thus their eggs contained Kepone
Exposure Concentration
Embrvo, Fry, & Juveniles
Control (ND)°
Control (ND)°
Control (ND)°
Control (ND)"
Control (ND)"
0.08
008
0.18
0.18
0.72
0.72
2.0
2.0
6.6
33.
(jig/liter, measured)
Parental Fish
Control (ND)
0.05
0.16
0.80
1.9
Control (ND)
0.05
Control (ND)
0.16
Control (ND)
0.80
Control (ND)
1.9
Control (ND)
Control (ND)
Embryos
6.2
8.8
5.0
11.5
25.0
11.1
8.8
8.8
6.2
21.5
13.9
15.8
26.2
17.5
13.8
Mortality (%)
Fry and Juve-
nile Fish
3.8
3.8
12.5
0
3.8
11.1
0
3.8
11.2
6.3
3.8
24.4
36.2
22.5
86.2
Total
10.0
12.6
17.5
11.5
28.8
22.2
8.8
12.5
17.5
27.8
17.7
40.2
62.5
40.0
100
— Concentration in Ju-
veniles (Mg/g. Wet
Weight)
ND"
ND
ND
ND
0.13
1.1
1.6
1.4
1.0
2.6
1.9
7.8
8.4
? *>
-
" ND = not detectable, <0.02 /ig/1. <0.02
fish except for lack of hemorrhaging and the presence
of edema in fry exposed to 0.72, 2.0 or 6.6 /ig/liter.
Exposure of adult sheepshead minnows to Kepone
affected mortality of their embryos in Kepone-free wa-
ter (Table 2). Sixteen percent of the embryos spawned
by adult fish exposed to 1.9 /ng/liter failed to develop
normally and died, embryos from adults exposed to
lesser concentrations developed normally Fertiliza-
tion, cleavage, gastrulation. and early differentiation of
circulatory system, appeared normal for about 48
hours. Thereafter, development ceased and although
some embryos survived for nine days, all eventually
died. Teratogenicity of Kepone might be related to us
effect on the gametes or to its presence within the
In some instances, the presence oi Kepone in eggs
and water aitected tr. and juveniles to a greater extent
than in water or eggs alone, eftect on embryos was not
increased (Table 21. Fourteen percent of the embryos
spawned by adults exposed to 1.9 /ig.-liter developed
abnormally and died Mortality of embryos to hatching
averaged 7.1% in 0 18 /ig/liter or less ot Kepone and
20.1% in concentrations greater than 0.18 pig/liter.
Fry from adults exposed to 1.9 ptg/hter showed symp-
toms ot poisoning one day after hatching in 2.0 /^g/liter
and as juveniles began to die 10 days later; symptoms
were more pronounced and deaths occurred ten days
earlier than those for juveniles of unexposed parents.
The combined effect ot Kepone in eggs and water was
negligible at lower concentrations
Although juveniles that survived 36 days of expo-
sure to 0.08 /ug/hter of Kepone showed no symptoms
of Kepone poisoning, three oi five juvenile fish re-
covered at termination of the test had scohosis and
blackened tails. These five fish, spawned naturally by
adult fish previously exposed to 0.05 /ig/liter. were
exposed to Kepone longer than were juv eniles from the
36-day duration embryo, frv. and juvenile exposure. In
Kepone-iree water scoliosis persisted for more than 10
days. We believe, therefore, that long-term effects of
Kepone on juvenile fish were underestimated in the 36-
day test.
Growth. Kepone affected growth of sheepshead
minnows (Fig. 3). The average standard length of juve-
niles exposed to 0.08 to 6.6 jigof Kepone/hter of water
was less than that of unexposed juveniles (P
<0.000000). Juvenile length decreased in direct pro-
portion to increased concentration of Kepone and was
generally Limnilj.T.ced by a hSMry of previous expo-
sure of their parents. Lengths of juveniles in Kepone-
free water did not differ even when parents were ex-
posed to Kepor.e concentrations ot 0.05 to 0.8 fig/liter.
However, juveniles from parents exposed to 1.9 /ig/1
were shorter than unexposed juveniles (p <0.01).
Bioconccntranon, Chemical analyses of juvenile
sheepshead minnows exposed to Kcro'-.o ••! <.,>.:!.- ••<;
embrvos and fr\ for 3f> days showe^. :.;„; .Cv\ -c •, as
bioconccntrated and that prior exposure of parental
fish had little effect on quantities concentrated (Table
2). Concentrations of Kepone in juvenile^ increased
with increased concentrations of Kepone in water.
Concentration factors increased from 3.600 to 20.000
(average 7,200) as exposure concentration decreased
(Fig. 4). This relationship was not observed with adult
sheepshead minnows exposed to Kepone. Juvenile
progeny of adult fish exposed to 1.9 ^.g of Kepone/
liter —though hatched and grown in Kepone-tree wa-
ter—contained 0.13 fjig Kepone/g or about 46% of the
0.011 micrograms of Kepone originally in an ege re-
mained in the juveniles even after 36 days in Kepone-
free saltwater.
Discussion
The hazard of Kepone to fish is greatly underesti-
mated by acute toxicity tests and is incompletely re-
vealed by the results of our tests. Although the 96-hour
LC50 of Kepone to sheepshead minnows is 70 p.g/hter
(Schimmel and Wilson 1977). adult fish in this study
-------
Short Papers and Notes
231
14
1.2
§10
OO5'
016
CONTROL
'08
19
z
1-
l/>
^ O "
• PARENTS UNEXPOSED
• PARENTS EXPOSED
20
0 01 10 10.0
JUVENILE EXPOSURE CONCENTRATION (jjg/l)
Fig. 3. Average standard length of juvenile sheeps-
head minnows exposed as embryos and fry for 36 days
to zero, 0.08, 0.18, 0.72, 2.0. or 6.6 /ig of Kepone/
liter of water. Concentrations to which parent fish were
exposed were: zero, 0.05. 0.16, 0.80, or 1.9 /iig/hter.
* Concentration of Kepone in water, /^g/liter, for par-
ent fish exposed prior to placement of their embryos in
Kepone-free water.
died when exposed to concentrations as low as 0.8 /ug/
liter for 2S days In exposures that began with embryos.
juvenile fish exposed to 0.08 /ig/hter were smaller than
unexposed control fish Other effects, such as scoliosis.
were also observed in these tish. Growth, reproduc-
tion, or survival was affected by all concentrations
tested. It is generally accepted that 0.01 oi the acute
toxicity of a persistent organic chemical to an organism
should be protective of a species (NAS-NAE.1973).
O'.T c:.;a. however, indicaic that a Kepone cen.vntra-
tion of 0.001 of the 96-hour LC50 for sheepsh-.- l min-
nows aliected this species uc;r.nicniaii\ in cnromc to.v
icit\ test.
Reproduction of adult sheepshead minnows was af-
fected by exposure to Kepone in water, but the effect
appears to be less pronounced than in birds and mam-
mals fed this insecticide. Some sheepshead minnow
embryos from exposed parents developed abnormally.
although the majority developed, hatched, and sur-
vived as successfully as embryos from unexposed par-
ents. Effects on quail, pheasants, and pigeons have
ranged from complete inhibition of reproduction to
effects which include cellular, physiological, and endo-
cnnological alterations (De Witt et al. 1961; Elder
1964. McFarland and Lacy 1969; and Eroschenko and
Wilson 1 975) Kepone in food fed to cows was bioaccu-
mulated with no apparent ill effect (Smith and Arant
1967). whereas reproduction in mice was reduced as
ingested concentrations increased (Good et al. 1965).
Our use of a hormone to enhance egg production and
artificial spawning procedures may have masked possi-
ble additional reproductive effects of Kepone on
sheepshead minnows.
15
cc
o
1 10
005
01 05 10 50
KEPONE CONCENTRATION IN WATER (ug/l)
100
Fig. 4. Concentration factor of Kepone in juvenile
sheepshead minnows after 36 days exposure that began
with embryos as a function of concentration of expo-
sure. (Concentration factor = concentration measured
in juvenile fish divided by concentration measured in
water). Dashed lines indicate 95% confidence limits.
Kepone is a highly bioconcentrated pesticide. Bio-
concentration factors from 3.600 to 20.000 observed in
juvenile sheepshead minnows exposed to Kepone were
similar to those observed in juveniles similar!) exposed
to the •T>ecticide<; ep'Jr'i. 3.300-4.800 (Schimmel et
al. i .-75): chloraane, 6.51/0-12.300 (Parrish et al.
1976); toxaphene. 6,100-14.400 and heptachlor.
2,400-4.600 (Goodman et al. In press) and to the
polychlorinated biphenyls. Aroclor 1016. 2.500-8.000
(Hansen et al. 1975)' and Aroclor 1254, 16.000-
32,000 (Schimmel et al 1974). However, the concen-
tration factor tor Kepone in juveniles increased with
decreased Concentration:, in water. \W would expect
that concentration factors would have been unaffected
by changing concentrations of exposure as obser\ed
with juvenile sheepshead minnows exposed to the four
insecticides and both PCB's. Concentration factors in
adult sheepshead minnows exposed to Kepone. 3,100-
7,000: Aroclor 1016, 4.700-14.000 (Hansen et al.
1975). and Aroclor 1254. 15,000-30.000 (Hansen et
al. 1974) also were unaffected by change in concentra-
tion of exposure.
In these laboratory tests, Kepone was toxic to and
accumulated by adult sheepshead minnows and their
progeny. This data may be helpful in evaluating the
impact of Kepone in estuaries.
ACKNOWLEDGMENTS
The significant contributions of Jerrold Forester and
Johnnie Knight in chemical analyses of water and fish
samples and Walter Burgess. Jr. and Charles S. Man-
ning for technical support during the bioassay are
gratefully acknowledged.
-------
232
Short Papers anc! Notes
LITERATURE CITED
BUTLER. P. A. 1963. Pesticide-Wildlife Studies-A
review of Fish and Wildlife Service investigations
dunnc 1961 and 1962. U.S. Fish and Wildlife Ore.
167:f 1-25.
DEWITT, J. B., D. G CRABTREE. R. B. FINLEY,
AND J. L. GEORGE. 1961. Effects on wildlife. In:
Effects of pesticides on fish and wildlife in 1960.
U.S. Fish and Wildl. Serv. Ore. 143:4-15.
ELDER, W. H. 1964 Chemical inhibitors of ovulation
in the pigeon. J. Wildl. Mgt. 28(3):556-575.
EROSCHENKO, V. P.. and W. O. WILSON. 1975. Cellu-
lar changes in the gonads. livers and adrenal glands
of Japanese quail as affected by the insecticide Ke-
pone. Toxicol. Appl. Pharmacol. 31: 491-504.
GOOD, E. E., G. W. WARE. AND D. F. MILLER. 1965.
Effects of insecticides on reproduction in the labora-
tory- mouse: 1. Kepone. J. Econ. Eniomol.
58(4):754-757.
GOODMAN, L. R., D. J. HANSEN, J A. COUCH, AND J.
FORESTER. 1976 Effects of heptachlor and toxa-
phene on laboratory-reared embryos and fry of the
sheepshead minnow. Proc. 30th Annu. Conf. South-
east. Assoc. Game and Fish Comm., in press.
HANSEN, D, J., A. J. WILSON, D. R. NIMMO, S. C.
SCHIMMEL. L. H. BAHNER, AND R. HUGGETT. 1976.
Kepone: Hazard to aquatic organisms. Science
193:528.
, S. C. SCHIMMEL, AND J. FORESTER 1975.
Effects of Arodor® 1016 on embryos, fry, juveniles,
and adults of sheepshead minnows (Cyprinodon var-
iegatus). Trans. Am. Fish. Soc 104(3):584-588.
-, S. C. SCHIMMEL, AND J FORESTER. 1974.
WELL. 1959. Relative toxicn> of ten chlorinated
hydrocarbon insecticides to four species of fish.
Trans. Am. Fish. Soc. 88(l):23-32.
McFARLAND, L Z.. AND P. P. LACY. 1969 Physio-
logic and endocnnologic effects of the insecticide
Kepone in the Japanese quad. Toxicol. Appl. Phar-
macol. 15:441-450.
NAS-NAE Committee on Water Quality Criteria.
1973. Water Quality Criteria. 1972. Ecof Res Ser.
xx +594 pp. U.S. Environmental Protection
Agency, EPA-R3-73-033-March 1973. U.S. Go\.
Print. Office, Wash.. D.C. 20402.
PARRISH. P. R., S. C. SCHIMMEL. D. J. HANSEN. i. M.
PATRICK, JR., AND J. FORESTER. 1976. Chlordane:
Effects on several estuanne organisms. J. Toxicol.
Environ. Health 1:485-494.
SCHIMMEL. S. C., AND A. J. WILSON, JR. 1977. Acute
toxicity of Kepone® to four estuarine animals Chesa-
peake Sci. 18(2)-.224-227.
, D. J. HANSEN, AND J. FORESTER. 1974. Ef-
fects of Aroclor® 1254 on laboratory-reared em-
bryos and frj of sheepshead minnows (Cyprinodon
variegaius). Trans. Am. Fish. Soc. 103(3):582-5S6.
-, P. R. PARRISH, D. J. HANSEN, J. M. PATRICK.
Aroclor® 1254 in eggs of sheepshead minnows: Ef-
fec'. '•>" fertilization success and survival of embryos
:na :r.. Proc. 2"th Annu. Conf Southeast. Assoc.
I;- jn;j^.s. C., Q. H. PICKERING, AND C. M. TARZ-
JR., AND J. FORESTER. 1975. Endrm: Effects on
several estuarine organisms. Proc. 28th Annu. Conf.
Southeast. Assoc. Game and Fish Comm., 1974.
p. 187-194.
SMITH, J. C., AND F. S. ARANT 1967. Residues of
Kepone® in milk from cows receiving treated feed.
J. Econ. Eniomol. 60(4):925-927.
DAVID J HANSEN. LARRY R. GOODMAN
AND ALFRED j. WILSON, JR.
U.S. Environmental Protection Agencv
Environmental Research Laboratorv
Gulf Breeze, Florida 32561
-------
EFFECTS OF KEPONE^ ON ESTUARINE ORGANISMS1
D. J. Hansen, D. R. Nimmo, S. C. Schimmel,
G. E. Walsh, and A. J. Wilson, Jr.
U. S. Environmental Protection Agency
Environmental Research Laboratory
Gulf Breeze, Florida 32561
ABSTRACT
Laboratory toxicity tests were conducted to deter-
mine the effects and accumulations of Kepone in estuarine
algae, mollusks, crustaceans, and fishes. Nominal Kepone
concentrations calculated to decrease algal growth by 50%
in static bioassays lasting seven days were: 350 vg/a,
Chlorococcum sp.; 580 pg/£, Dunaliella tertiolecta', 600
yg/£, Nitsschia sp.; and 600 pg/£, Thalassiosira
pseudonana. Measured Kepone concentrations calculated
to cause 50% mortality in flowing-seawater toxicity
tests lasting 96 hours were: 10 pg/£ for the mysid
shrimp (Mysicbpsis bahia); 120 ug/£ for the grass shrimp
(Palaemonetes pugio}', >210 yg/£ for the blue crab
(Callinectes sa.pid.us}; 70 yg/£ for the sheepshead minnow
(Cyprinoobn variegatus)', and 6.6 yg/£ for the spot
(Leiostomus xanthurus). Bioconcentration factors (con-
centration in whole animals divided by concentration
measured in water) in these tests were greatest for
fishes (950 to 1,900) and less for grass shrimp (420 to
930).
Survival, growth, and reproduction of mysids 2rd
sheepshead minnows were decreased in chronic bioassays
lasting 14 to 64 days. Growth of mysids and sheepshead
minnows was reduced by exposure to 0.07 ug/ji and 0.08
ug/£ respectively. Bioconcentration factors for sheeps-
head minnows in the chronic bioassay averaged 5,200
K)
^Registered trademark, Allied Chemical Corp., 40 Rector St., New York,
10006. Kepone was purchased from Chem Service, West Chester, PA, as
99% pure. Our analyses indicated 88% purity.
Contribution No. 311, Environmental Research Laboratory, Gulf Breeze.
20
-------
(range, 3,100-7,000) for adults exposed for 28 days and
7,200 (3,600-20,000) for juveniles exposed for 36 days.
The chronic toxicity and bioconcentration potential of
Kepone are more important factors than its acute
toxicity in laboratory evaluations of environmental
hazard. Therefore, these factors should be considered
when attempting to assess present impacts and to limit
future impacts of this insecticide on the aquatic
environment.
INTRODUCTION
Kepone (decachlorooctahydro-1,3,4-metheno-2H-cylobuta [cd] pentalene
2-one) is an insecticide that was manufactured and formulated in the United
States to control ants, cockroaches, and insect pests of potatoes and
bananas. Kepone is toxic to birds and mammals, including man (Jaeger 1976),
and acutely toxic to some estuarine organisms (Butler 1963). Recent contam-
ination of water, sediment, and biota in freshwater and estuarine portions of
the James River, Virginia, has stimulated concern about this chemical's
hazard to aquatic biota (Hansen et al. 1976). This concern was based on (1)
the continued occurrence of Kepone in many finfishes and shellfishes in
amounts that forced closure of fishing because of potential human health
hazard, and (2) laboratory studies which showed that Kepone is highly bio-
accurnulati ve and toxic to estuarine organisms, particularly in chronic
exposures. This paper describes the results of these laboratory toxicity
tests with estuarine algae, oysters, crustaceans, and fishes and chronic
tests with a crustacean and a fish.
EXPERIMENTAL PROCEDURES
Acute Toxicity
Algae: The unicellular algae Chloroaocoum sp., Dunaliella tertioleeta,
nitzszkia. sp., and Tnalassiosira pseudonzr.a were exposed to Kepone for seven
days to determine its effect on growth (Walsh et al. 1977). Algae were
cultured in 25 or 50 ml of growth media and artificial seawater of 30 °/oo
salinity and a temperature of 20 C (Hollister et al. 1975). Kepone, in 0.1
ml acetone, was added to culture media, and 0.1 ml of acetone was added to
control cultures. Photoperiod consisted of 12 hours dark and 12 hours of
5000 lux illumination. Effect on growth was determined by electrophoto-
metrically measuring optical density. Also, algae grown for 6 days in media
and then exposed to 100 yg/.n Kepone for 24 hours were analyzed for Kepone
content.
Oysters: The acute toxicity of Kepone to embryos of the eastern oyster
(Crzssostrsa virginica) was determined by measuring its effect on development
21
-------
of fully-shelled, straight-hinged veligers in a 48-hour static exposure .
Methods used were those of Woelke (1972) and U. S. EPA (1975). Test contain-
ers were l-£ glass jars that contained 900 mi of 20 C, 20 °/oo salinity sea-
water and 25,000 ± 1,000 oyster embryos. All test concentrations were
triplicated. The number of normal and abnormal embryos were counted micro-
scopically in a Sedgewick-Rafter cell at the end of 48 hours of exposure to
Kepone.
Crustaceans and Fishes: The acute toxicity of Kepone to grass shrimp
(Palaemonetes puaio], blue crabs (Callinsctss sapidus], sheepshea'd minnows
(Cyyrinodon variegatus), and spot (Leiostomus xanthurus] was determined in
96-hour flow-through toxicity tests (Schimmel and Wilson 1977). Acclimation
and testing procedures were compatible with those of Standard Methods (APHA
1971). Test animals were caught locally and 20 were placed in each 18£
aquarium. Water flow to each aquarium was 68 £/hour. Stock solutions of
Kepone in acetone were metered into experimental aquaria at the rate of 60
me/hour. Control aquaria received 60 m£ of acetone/hour. At the end of the
experiment, surviving animals were chemically analyzed for Kepone content.
The acute toxicity of Kepone to mysids. (Mysidopsis bahia} was determined
by using intermittent flows of water from a diluter (Mount and Brungs 1967)
or continuous flow of w'ater from a siphon and Kepone from an infusion pump
(Bahner et al. 1975). Thirty-two 48-hour-old juvenile mysids were placed in
chambers (4 mysids per chamber) in each test aquarium. Chambers consisted of
glass petri dishes to which a 15 cm tall cylinder of 210p mesh nylon screen
was glued. Water in the chambers was renewed by a self-starting siphon which
nearly emptied and then filled each aquarium at about 25 min intervals.
Chronic Toxicity
Mysidopsis bahia: The chronic toxicity of Kepone to this mysid was
determined 'in 19-day exposures that began with 48-hour-old juveniles. (Nimmo
et al., in press). The time permitted production of several broods for
assessment of reproductive success and survival of progeny. Exposure condi-
tions, apparatus, and number of mysids per concentration were identical to
those of the acute toxicity tests. Three tests were conducted: One to
---- -"' ' •" - _- ' -. -. ; tv/c =t "-y.-.Qr i^r.centrations
to cetern-.Tne effects on growth. Data from the two growth experiments were
pooled for statistical analysis.
Cyorinodon variegatus: The chronic toxicity of Kepone to sheepshead
minnows was determined in a 64-day flow-through bioassay—exposure of adults
for 28 days followed by a 36-day exposure of their progeny (Hansen et al.
1977). We delivered Kepone, 0.0088 y£ of the solvent triethylene glycol,
and 1.5£ of filtered 30 C seawater (average salinity, 15 °/oo; range, 8-26
°/oo) to each 70£ aquarium during each of 440 daily cycles of the dosing
apparatus of Schimmel et al. (1974). Seawater and solvent were delivered to
the control aquarium. Thirty-two adult females and 32 adult males were
This research was performed under an EPA contract by Tom Heitmuller,
Bionomics-EG&G, 'nc. Marine Research Laboratory, Pensacola, Florida 32507.
22
-------
exposed to each concentration of Kepone for 28 days. Egg production was
enhanced using injections of 50 I.U. of humar chorionic gonadotrophic hormone
on exposure day 25 and 27 (Schimmel et al. 1974). Eggs were fertilized on
day 28 and placed in chambers (glass petri dishes with 9-cm tall cylinders of
450y nylon mesh). Twenty embryos were used in each chamber. Embryos from
control fish were placed in four chambers in the control aquaria and in four
chambers in each of the six aquaria receiving Kepone. Embryos from fish in
each of the six aquaria receiving Kepone were placed in four chambers in that
aquarium and in four chambers in the control aquarium. Water in the chambers
was exchanged by the action of a self-starting siphon in each aquarium that
caused water levels to fluctuate 5 cm about 40 times per day. In the 36-day
exposure to determine Kepone's effect on survival and growth of progeny,
embryos hatched and fry grew until they were juvenile fish. Kepone content
of adult fish, their eggs, and juvenile fish was determined.
STATISTICAL ANALYSES
Probit analyses of growth and mortality data were used to determine
EC50's and LC50's. Growth data for /./. bahia were subjected to analysis of
variance (a = 0.05) and for C. variegatus, analysis of covariance and Newman-
Kuels tests (a = 0.01) was used.
CHEMICAL ANALYSES
Water from acute and chronic tests with crustaceans and fishes, and
organisms surviving these tests, were analyzed by gas chromatography.
Methods of extraction concentration, cleanup, and quantification were des-
cribed by Schimmel and Wilson (1977).
RESULTS AND DISCUSSION
Acute Toxicity
Algae: Growth of marine unicellular algae was reduced by exposure to
Kepone in static tests (Table 1). CrdoTococcus was the most sensitive of
the four algae tested with a 7-day EC5Q cf 350 'jg/t. The tnree less sensi-
tive species responded similarly to Kepone with overlapping confidence limits
for EC50's. Algae exposed to 100 yg Kepone/c of media accumulated the
chemical with Chlorococs-iMv containing 0.80 yg/g; D. tertiolecta, 0.23 yg/g;
Uizzscnia, 0.41 yg/g; and T. pseudonanz, 0.52 yg/g. Butler (1963) reported
that when estuarine phytoplankton were exposed to 1,000 yg/£ carbon fixation
was reduced by 95;i.
Oysters: The 48-hr EC50 for oyster larvae in static tests was less than
those of algae (TableJ). The EC50, calculated using nominal water concen-
trations, was 66 yg/?/. Embryos from 56 uq/i were fully shelled and straight-
hinged but appeared smaller than those from controls. The percentage of nor-
rr.al embryos in 65 ugA was 32 percent and in 87 MgA it was 0:;. The concen-
tration of Kepone calculated to reduce shell deposition of juvenile eastern
oysters by 50;^ in a 96-hour flowing water bioassay was 38 ygA in water of
14 C and 11 ygA in water of 31 C (Butler 1963)!
23
-------
•TABLE 1. ACUTE TOXICITY OF KEPONE TO ESTUARINE ORGANISMS. ALGAL AND MOLLUSK
TOXICITY TESTS WERE STATIC AND ESTIMATED NOMINAL CONCENTRATIONS
REDUCING GROWTH OF ALGAE AND EMBRYONIC DEVELOPMENT OF OYSTERS BY
50% (EC50). TOXICITY TESTS WITH CRUSTACEANS AND FISHES WERE FLOW-
THROUGHS THAT ESTIMATED THE MEASURED CONCENTRATION IN WATER LETHAL
TO 50% (LC50). NINETY-FIVE % CONFIDENCE LIMITS ARE IN PARENTHESES.
Organisms
Temperature,
C
Salinity,
%o
Exposure
Duration,
Days
EC50/LC50
ugA
Algae
Chloroccecum sp. 20
Dunaliella tertioleeta 20
Nitzsch-ia sp. 20
Thalassiosira pseudonana 20
Mollusk
Crassostrea virginiaa 20
Crustaceans
Callinectss sapidus 19
Mysidopsis bahia 26
Palaemonetes pugio 20
Fishes
Cyprinodon variegatus 18
L±i :-3tomus xanthurus 25
30
30
30
30
21
20
13
16
15
18
7
7
7
7
4
4
4
4
4
350
580
600
600
(270-400)
(510-640)
(530-660)
(500-700)
66 (60-74)
>210
10
120
(8.1-12)
(100-170)
70 (56-99)
6.6 (5.3-8.S)
Crustaceans and Fishes: Kepone, at the concentrations tested, was
acutely toxic to mysids (Nimmo et al. 1977), grass shrimp, sheepshead min-
nows, and spot, but not to blue crabs (Schim.r.el and Wilson 1977) (Table 1).
Spot and mysids were the more sensitive species with 96-hour LC50 values of
6.6 and 10 pg/£. Crabs exposed to as much as 210 yg Kepone/x, suffered no
significant mortality. Symptoms of acute Kepone poisoning in fishes included
lethargy, loss of equilibrium, and darkened coloration on the posterior
portion of the body, occasionally only in one quadrant. Crustaceans became
lethargic before death but exhibited no color change. Butler (1963) reported
48-hour LC50 or EC50 values (based on nominal concentrations) for other
estuarine organisms were: brown shrimp (Penaeus aztecus), 85 yg/£; and white
mullet (Mugil curema], 55 ug/£.
Kepone was bioconcentrated from water by all four species we exposed for
96 hours. Bioconcentration factors (concentration in tissue divided by
24
-------
measured Kepone in water) for fishes were similar (950 to 1,900). Bioconcen-
tration factors for grass shrimp ranged from 420 to 930 and for blue crabs,
6 to 10.
CHRONIC TOXICITY
Mysidopsis bahia: Exposure of this mysid to Kepone for 19 days in the
first experiment decreased its survival and reduced the number of. young pro-
duced per female (Table 2) (Nimmo et al. 1977). At the highest concentration
(8.7 pg/£) all mysids were dead within the first two days. At lesser concen-
trations (1.6 and 4.4 pg/x) mortality continued throughout the test. Eighty-
four % of the mysids survived exposure to 0.39 pg Kepone/£ water and 91%
survived in control aquaria. In addition, natural reproduction was affected.
Average number of young mysids produced per female was 15 in control, 9 in
0.39 pg/£, and 0 in 1.6 ug/£. Mysids that survived throughout the Kepone
exposure appeared smaller than those in control aquaria, therefore, two
additional experiments were conducted to measure Kepone's effect on growth.
TABLE 2. EFFECT OF KEPONE ON THE SURVIVAL OF MYSIDOPSIS BAHIA AND ON AVERAGE
NUMBER OF YOUNG PER FEMALE IN A 19-DAY FLOW-THROUGH TOXICITY TEST.
Average Measured Percentage Number of Young
Kepone Concentration Survival per Female
Control
0.39
1.6
4.4
8.7
91
84
50
3
0
15.3
8.9*
0
—
*Statistically significant at a = 0.05 using 2 sample t-test.
In these experiments, the average length (tip of carapace to end of
uropod) of mysids exposed to Kepone was decreased (Nimmo et al. 1977).
Females exposed to 0.072, 0.11, 0.23, or 0.41 pg/£ were significantly shorter
than were control mysids; average length was 8.2 mm for exposed versus 8.6 mm
for control female mysids. Unexposed and exposed males, however, were;of
similar average lengths, 7.7 to 8.0 mm.
C-jprinodon variegatus: Kepone was toxic to adult sheepshead minnows
exposed for 28 days (Table 3). Symptoms of poisoning included: scoliosis,
darkening of the body posterior to the dorsal fin, hemorrhaginq near the
brain, edema, fin-rot, uncoordinated swimming, and cessation of feeding.
'Symptoms were first observed on day 1 in 24 yg/£, 2 in 7.8 ug/£, 3 in 1.9
pg/Z, and day 11 in 0.8 pg/£. Mortalities began 5 to 8 days- after onset of
symptoms.
25
-------
TABLE 3. EFFECT OF KEPONE ON AND ACCUMULATION OF KEPONE BY ADULT SHEEPSHEAD
MINNOWS EXPOSED FOR 28 DAYS.
Average Measured
Exposure Concentration, yg/£
ND*
0.05
0.16
0.80
1.9
7.8
24.
Percentage
Mortality
5
5
0
22
80
100
100
Whole Body
Concentration, pg/g
ND
0.30
0.78
3.0
12.
*ND = Kepone not detected in control water (<0.02 yg/£) nor in control fish
(<0.02 yg/g).
Kepone affected the progeny of 28 day exposed adults. In Kepone-free
water, mortality of embryos from adults exposed to 0.05-0.8 vg/a was similar
to that of embryos from unexposed adults (range, 6-12 percent). However, in
Kepone-free seawater, 25% of the embryos from fish exposed to 1.9 yg of
Kepone/£ died; abnormal development of 13 of these 20 embryos preceded
mortality.
Kepone in water affected progeny of exposed parents to a greater extent
than progeny of unexposed parents (Table 4). Some embryos exposed to 2.0
ug/z developed abnormally and fry had more pronounced symptoms ard *>?"
began to die 10 days earlier wrier, parental fish had been exposec co i.^ -ng/\
than was observed in progeny from unexposed parents.
or
Kepone also affected growth of sheepshead minnows in the 36-day exposure
;rcteny ;rvrjre 1). The average standard lenrth of iuveri'1 es excosed to
all Kepone concentrations was less than that of unexposed control juveniles.
Lengths decreased in direct proportion to increasing Kepone concentrations in
water and were generally not influenced by parental exposure. A similar
decrease was also noted in weights, but because juveniles exposed to 0.72,
2.0, or 6.6 yg/£ were edematous, they weighed more than unexposed juveniles
of similar lengths.
Kepone was bioconcentrated by sheepshead minnow adults and their progeny
exposed to the insecticide in water. Kepone was bioconcentrated in adult
fish in direct proportion to concentration in exposure water (Table 3). Con-
centration factors averaged 5,200 (range, 3,100-7,000). Kepone concentra-
tions in females and their eggs were similar and were 1.3 times greater chan
amounts in males. Concentrations of Kepone in juvenile fish, at the end of
the 36-day progeny exposure, increased with increased concentration of Kepone
in water (Table 4). Prior exposure of parental fish apparently did not
26
-------
affect final Kepone concentration in progeny. Concentration factors for
juvenile fish averaged 7,200 (range, 3,600-20,000) and increased with decrease
in concentration of exposure.
TABLE 4. MORTALITY IN PROGENY OF ADULT SHEEPSHEAD MINNOWS THAT WERE EXPOSED
TO KEPONE AND IN PROGENY OF UNEXPOSED, CONTROL FISH. NOMINAL
EXPOSURE FOR THE 28-DAY EXPOSURE OF ADULT FISH AND THE 36-DAY
EXPOSURE OF PROGENY WERE THE SAME. PROGENY EXPOSURE BEGAN WITH
EMBRYOS AND ENDED WITH JUVENILE FISH FROM THE EMBRYOS. RESIDUES
ARE CONCENTRATIONS OF KEPONE (ug/g) IN WHOLE JUVENILES, WET WEIGHT.
Measured Exposure
Concentration
1
Parental Fish History
Progeny of Unexposed Parents Progeny of Exposed Parents
»g/.
Control (ND)
0.08
0.18
0.72
2.0
6.6
-33.
Mortal ity
10
22
12
28
40
40
100
Residue
yg/g
ND1
1.1
1.4
2.6
7.8
22.
--
Mortality
10
9
18
18
62
—
--
Residue
ND1
1.6
1.0
1.9
8.4
--
--
ND = not detectable, <0.02 ug/>>, <0.02 yg/g.
In our tests, Kepone was acutely toxic to, and accumulated by, estuarine
algae, mollusks, crustaceans, and fishes. Chronic toxicity tests with ;•/.
"o-if-.ia. and C, va.rieg3.-cus revealed that Kepone affected survival, growth, and
reproduction. Effects on growth were observed at 0.001 of the 96-hour LC50.
.-"(-• .rnulation of Kepone was also greatest in chronic tests. Therefore,
•-•,•, iic tests should be used to assess Kepone's environmental hazard and to
•'<• o decisions necessary to minimize its future impact on the aquatic envi-
•• •• int.
27
-------
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-------
REFERENCES
American Public Health Association et al. 1976. Standard methods for the
examination of water and wastewater. 14th ed. Am. Public Health Assoc.,
Washington, D. C. 1193 p.
Bahner, L. H., C. D. Craft, and D. R. Nimmo. 1975. A saltwater flow-through
bioassay method with controlled temperature and salinity. Prog. Fish-
Cult. 37(3): 126-129.
Butler, P. A. 1963. Commercial fisheries investigations. Pp. 11-25 in J.
L. George (ed.), Pesticide-wildlife studies: a review of Fish and
Wildlife Service investigations during 1961 and 1962. Fish and Wild],
Serv. Circ. 167. U. S. Dept. Int., Washington, D. C. 109 p.
Hansen, D. J., L. R. Goodman, and A. J. Wilson, Jr. 1977. Kepone^-':
Chronic effects on embryo, fry, juvenile, and adult sheepshead minnows,
(Cyprinodon variegatus), Chesapeake Sci. (In press),
Hansen, D. J., A. J. Wilson, D. R. Nimmo, S. C. Schimmel, L. H. Bahner, and
R. Huggett. 1976. Kepone: hazard to aquatic organisms. Science 193
(4253): 528.
Hollister, T. A., G. E. Walsh, and J. Forester. 1975. Mirex and marine
unicellular algae: accumulation, population growth and oxygen evolution.
Bull. Environ. Contam. Toxicol. 14(6): 753-759.
Jaeger, R. J. 1976. Kepone chronology. Science 193(4248): 94.
Mount, D. I., and W. A. Brungs. 1967. A simplified dosing apparatus for
fish toxicology studies. Water Res. 1(1): 21-29.
Nimmo, D. R., L. H. Bahner, R. A. Rigby, J. M. Sheppard, and A. J. Wilson,
Jr. 1977. Mysidopsis bahia: An estuarine species suitable for life-
- "•• - '- " - •"-- J - J-~ •.—,•: ^o ~i,!O o-i-UsI Q-F-Pc^J-^ of q nn"P uf.lt. IP
Proceedings .symposium uii nquaoic lUAicoio^ ano r.^^arj Evaluation.
(Held in Memphis, Tenn. Oct. 25-26, 1976.) American Society of Testing
Materials. (In press).
-- ^., „. ~., „. „. .,„.,.,...., -..- ~. ...... ... .. _. ._
1254 on laboratory-reared embryos and fry of sheepshead minnows
(Cyprinodon vcriegatus}. Trans. Am. Fish. Soc. 103(3): 582-586.
Schimmel, S. C., and A. J. Wilson, Jr. 1977. Acute toxicity of Kepone^ to
four estuarine animals. Chesapeake Sci. (In press).
U. S. Environmental Protection Agency, Committee on Methods for Toxicity
Tests with Aquatic Organisms. 1975. Methods for acute toxicity tests
with fish, macroinvertebrates, and amphibians. Ecol. Res. Ser. EPA-
660/3-75-009. Natl. Environ. Res. Cent., Off. of Res. & Devel., U. S.
Environmental Protection Agency, Corvallis, Ore. v + 61 p.
29
-------
Walsh, G. E., K. Ainsworth, and A. J. Wilson. 1977. Toxicity and uptake of
Kepone in marine unicellular algae. Chesapeake Sci. (In press).
Woelke, C. E. 1972. Development of a receiving water quality bioassay
criterion based on the 48-hour Pacific oyster (Crasscstrea giaas] embryo
Washington Dept. Fish. Tech. Rept. 9: 92 p.
30
-------
Reprinted •from
5 August 1977, Volume 197, pp. 585-587
Kepone-Induced Scoliosis and Its
Histological Consequences in Fish
John A. Coucn, James T. Yv'in^teau and Larry li. Goodman
Copyright £' 1977 by the American Association fnr the Advancement of Science
-------
f
1
1
I
Kepone-induced Scoliosis and Its
Histological Consequences in Fish
Abstract. Scoliosis in fish i.\ caused by several diverse cgen!s r/u: ^n>-~;'"!-.- ,-;r' •>•:
the central nervous system, neuromuscular junctions, or ionic meiabonsm. The or-
ganochiorine pesticide Kepone induces scoliosis in the sheepshead minnow. Some
effects associated with Kepone-induced scoliosis in these fish are disruption ofmyo-
tomal patterns, inter- and intramuscular hemorrhage, fractured centra of vertebrae,
and death. The histological syndrome of Kepone poisoning in fish and the clinical
syndrome in humans suggest that the nervous system is a primary target for Kepone
and that scoliosis is a secondary effect of Kepone poisoning in fish.
1
We have found that exposure of
sheepshead minnows (Cyprinodon vari-
egatus) to a relatively low concentra-
tion of the organochlorine Kepone (deca-
chlorooctahydro-1,3,4-metheno-2//-cy-
clobuta(c.c/]pentalen-2-one) produces a
syndrome in the fish in which scoliosis,
resulting in severe spinal column injury,
is one cardinal sign. Scoliosis, lateral
curvature of the spine, has been reported
to occur in several species of fish as a
result of dietary deficiencies (/). organo-
phosphate and carbamate poisoning (2),
heavy metal exposure (3), and parasitic
infections (4). This report is concerned
with the severe histological effects of
scoliosis in sheepshead minnows ex-
posed to Kepone in the laboratory.
Hansen and co-workers (5) first ob-
served scoliosis and blacktail (loss of
neurologic control of melanocytes in the
caudal region) as a syndrome of Kepone
exposure in sheepshead minnows. They
experimentally demonstrated that induc-
tion of scoliosis with Kepone was a dose-
dependent, time-related phenomenon.
-------
Scoliosis occurred in sheepshead min-
nows exposed 1 day to 24 jag of Kepone
per liter. 2 days to 7.8 /ng of Kepone per
liter. 3 days to 1.9 /ug of Kepone per liter.
and 11 days to 0.8 /u.g of Kepone per liter.
All tests were laboratory-controlled
flowing seawater exposures.
We repeated exposures of sheepshead
minnows to Kepone in the laboratory to
obtain specimens exhibiting scoliosis for
histological study. Twenty juvenile and
ten adult sheepshead minnows were ex-
posed for 17 days to flowing seawater
containing 4 ju,g of Kepone per liter at 21°
to 30°C and 23.5 to 29 parts per thou-
sand salinity. Triethylene glycol was the
solvent earner for Kepone. A second
equivalent group of control fish received
only the solvent carrier chemical in flow-
ing seawater.
All exposed fish exhibited signs of Ke-
pone poisoning by day 10 of exposure.
These fish demonstrated scoliosis, black-
tail, loss of equilibrium, sporadic hyper-
kinesis, and tetanic convulsions. Living
specimens with various degrees of sco-
liosis were selected from day 10 to day
17 of exposure for standard histoiogicai
fixation and processing. Fish were fixed
in Davidson's fixative, embedded in
I •' .<''>'4 i?- ''> "^y^o,, •''"'•-• \.'i <.\"r
I ' -:-\ fe -v ---^ .^:\VX
Fig. I. (a) Longitudinal histological section from the horizontal plane of normal control fish:
section is at level of spinal column I x? 5). (b) Longitudinal horizontal plane section offish with
advanced scoliosis: the concave region in trunk (arrow) is location of flexure (x3.5). Fig. 2.
Contorted muscle bundles (arrows) in myotome in concave region shown in Fig. Ib; the con-
torted musculature reflects the tetanic paroxysms that lead to scoliotic flexure of the trunk.
Compare Figs. 1 and 4 ( x 160). Fig 3. Xeroradiographs of normal sheepshead minnow (a)
and sheepshead minnow with incipient scoliosis (b): the region of early spinal column flexure
(arrows) shows obvious loss of vertebral periodicity. Fig. 4 Horizontal plane section from
normal fish showing normal alignment of vertebral centra and normal myotomes in the region of
trunk affected by Kepone-mduced scoliosis in exposed fish ( x |f>0). Fig. 5. Fractured centra
of vertebra in severely scoiiotic fish exposed to 4 Mg of Kepone per liter for 17 days: distortion
of myotomes and foci of osteoblastic repair tissue at points of breaks in centrum walls is shown
(arrows) (x64). Fig. 6. Horizontal section through the spinal cord of fish used in Fig. 5 with
fractured vertebra: intrusion of disonented bone (arrows) from neural arch of vertebra against
lateral funiculi of spu-.al cord is shown (x64).
paraffin, and serially sectioned at 7 /xrn—
some through a longitudinal, horizontal
plane (Fig. 1. a and b) and others through
a sagittal-parasagittal plane. Normal
control fish.-were processed similarly.
Selected fish with scoliosis and control
fish were xeroradiographed for study of
spinal column form.
Histolog'cal study revealed that fish
with incipient scoliosis had precaudal
trunk flexures resulting from long-term
rigorous contractions of skeletal muscle
in myotomes on one of either side of
their bodies (Figs, la and 2). Xeroradio-
graphs show that the major spinal col-
umn flexure occurs in the vicinity of. and
involves, vertebrae 17 through 21 (ce-
phalic to caudal count). We have
counted 26 to 27 nonfused vertebrae in
several normal specimens of the sheeps-
head minnow (Fig. 3). Normal muscle
bundle patterns are broken and myotome
boundaries (septa) are obscured by the
abnormally contorted muscle bundles
(Fig. 2).
In more advanced cases of scoliosis.
hemorrhagic foci often occurred in af-
fected myotomes, suggesting a rupture
of minor vessels or capillaries possibly
caused by severe contortion of muscle.
The most striking effect observed, al-
ways in severely scoliotic fish, was the
breaking of the centra of vertebrae at the
epicenter of flexure in the spinal column
(Figs. 4 and 5). This was accompanied by
apparent osteoblastic repair tissue (Fig.
5, arrows) suggesting that the fractures
occurred --Berime prior to fixation of
the rish ror Histology. Fish with fractured
vertebrae also had the greatest dis-
placement and disorientation of myo-
tomal musculature (Fig. 5). Obstruction
(pmchina) of the dorsal aorta or caudal
bone into the neural canal were further
concomitant results of severe scoliosis
(Fig. 6). Fish thus affected were para-
lyzed and probably would have died.
No histological lesions in the central
nervous system that could have initiated
tetany or paralysis were observed. We
propose, however, that scoliosis is sec-
ondary to tetany and paralysis of trunk
musculature initiated by an undeter-
mined molecular neurological or neuro-
muscular dysfunction (or both), prob-
ably directly caused by Kepone. The ap-
pearance of blacktail (loss of neurologic
control of caudal melanocyte patterns)
preceding, during, and after scoliosis
strengthens the possibility that the ini-
tiating lesion is neurologic (6). Another
possibility is that systemic calcium me-
tabolism may have been affected by Ke-
pone. Evidence from living and fixed fish
-------
indicate that trunk muscles are unable to
regain normal tonus and form (Fig. 2).
Tetanic convulsions or chronic muscular
rigor (or both) are associated with sco-
liosis and probably produce the fractured
vertebrae of severely affected fish. The
possible effects that Kepone may have
on fish calcium metabolism, on the cor-
puscles of Stannius (because of their cal-
cium-mediating role in some fishes), and
on muscle contraction have not yet been
evaluated.
The mechanism or mechanisms
whereby different organochlorine com-
pounds affect organisms are poorly un-
derstood. Human victims of Kepone poi-
soning have suffered tremors, nervous-
ness (hyperkinesis), loss of memory, and
slurred speech, among other effects (7).
The human response syndrome suggests
neurological lesions, some of which
probably occur at higher nervous cen-
ters, as a result of Kepone poisoning.
Tremors and other neurological-depen-
dent responses in laboratory animals in-
creased in severity with increasing Ke-
pone concentration and duration of ex-
posure (7). Hansen, el al. (5) observed
the same correlation between concentra-
tion of Kepone. duration of exposure.
and severity of scoliosis and related
signs in fish. Our observations suggest
that the severity of scoliotic effects in the
tTgheepshead minnow is related to the du-
ration of continuous exposure to a single
low Kepone concentration (4 /j.g/liter).
MBch higher concentrations of Kepone
^ to 400 mg/kg per day) are required to
elicit neuropathological, reproductive,
and tissue effects in birds or mammals
(7).
JOHN A. COUCH
JAMES T. WINSTEAD
LARRY R. GOODMAN
U.S. Environmental Protection Agency,
Environmental Research Laboratory.
Gulf Breeze. Florida 32561
References and Notes
I. D Horak, Colo. Dep. Nat Kesour. Fish. Inf.
Leafl. No. 29 (1975). J. E. Halver, Fish Nutri-
tion (Academic Press, New York, 1972).
2. F. P. Meyer. Prog. Fish Cult. 28. 33 (1966); D. I.
Mount and C. E. Stephan, Trans. Am. Fish.
Soc 96. 185(1967), J. A. McCann and R. L.Jas-
per, ibid 101. 317 (1972); P. Weis and i. S.
Weis.Environ Res. 12. 196(1976); F. L. Carter.
thesis, Louisiana State University (1971).
3. G. W. Holcombe, D. A. Benoil. E. N. Leonard.
J. M. McKim;y. Fish. Res. Board Can. 33, 1731
(1976).
4. G. L. Hoffman. U.S. Fish Wildl. Sen'. Fish.
Leafl No. 508 (1962).
5. D. J. Hansen, L. R. Goodman. A. J. Wilson, Jr.,
Chesapeake Sci.. 18. 226 (1977).
6. K. Wolf and M E. Markiw, J. Protozoa!. 23,
425(1976).
7. J. Raloff. Chemistry 49. 20 (1976); L. Z. McFar-
land and P B. Lacy, Toxicol. Appl. Pharmacol.
15, 441 (1969); N. Chemoff and E. H. Rogers,
ibid. 38, 189(1976)
8. We thank H. S. Barrett for the radiograph of the
fish.
8 March 1977; revised 13 April 1977
-------
Chesapeake Science Vcl 18, No 3, p. 299-308 September 1977
Kepone® Bioconcentration,
Accumulation, Loss, and Transfer
through Estuarine Food Chains.1
LOWELL H. BAHNER. ALFRED J. WILSON. JR..
JAMES M. SHEPPARD. JAMES M. PATRICK. JR..
LARRY R.GOODMAN, AND GERALD E. WALSH
U.S. Environmental Protection Agency
Environmental Research Laboratory
Gulf Breeze, Florida 32561
ABSTRACT: Accumulation, transfer, and loss of Kepone in estuarine organisms were studied in
laboratory bioassavs. Kepone was bioconcentrated b\ oysters (Crassosirea virginica), mysids (Mvsi-
dopsis bahia), grass shrimp (Palaemonetespugio), sheepshead minnows (Cyprinodon variegatus),
and spot (Leiostomusxanlhurus). from concentrations as low as 0.023 fj.g/\ seawater. Bioconcentra-
tion factors ranged from 10 to 340 in static exposures and 900 to 13,500 in flow-through bioassavs.
and were dependent on species and exposure duration.
Depuration of Kepone from oysters in Kepone-free water was rapid (35** loss in 24 hours);
however, depuration of Kepone was slow in crustaceans and fish, with tissue concentrations
decreasing 30-50^ in 24-28 days.
Oysters, fed Chlorococcum containing approximate!) 34 /tig Kepone/g wet weight, attained 0.21
/*g Kepone/g (wet tissue) in 14 davs. but when fed Kepone-free plankton, depurated Kepone to
below detectable concentrations (<.02 /ig/g) within 10 days.
*:-;•• --'.Sj'p-.-d -"-.••v.':- u»*»' fH ':-.-.- —',-;!'s; «'-•>> '-~.-J ••'•••.??<* • -- V:-"«-,»;«.'.-!t!i brine shrimp.
Kepone resiuue* (1.05 /J-K/S wet tissue) in these lish jpproacheu liie concentration of their food
(1.23 MS/" "et ti^ue): at the lower concentration tested. Kepone concentrations below detection
limits (<.2 jjtg/g) in prey accumulated in the predator to detectable concentrations (0.02 /*g/g>
within 30 days. Bioaccumulation factors (concentration of Kepone in predator/concentration in
prej) at 30 dajs were equal (0.85 spot/mysid; 0.53 mysid/brine shrimp) in the high and low
concentrations tested. The initial bioconcentration of Kepone from water by plankton was the
dominant source of Kepone to each member of this food chain, but significant (>859c) quantities of
Kepone transferred from prej to predatory fish.
Introduction
Contamination of the James River water,
sediments, and biota with Kepone has
prompted research to help define the routes
of transfer ot the insecticide from water
through selected estuarine trophic levels.
Biota of the James River estuary and Chesa-
peake Bay contain Kepone (Hansen e't al.
1476). apparently due to transport of the
chemical downstream from the freshwater
portion of the river. Since no convenient
•& Registered trademark. Allied Chemical Corpora-
tion. 4(1 Rector Street. New York. New York 10006.
1 Contribution No 294. Environmental Research
Laborator\. Gulf Breeze. FL.
method existed to assess the rate of Kepone
movement in the biota of the James River
and Chesapeake Bay, laboratory biocon-
centration from water and bioaccumulation
from food experiments were designed to de-
termine the rates and magnitudes of Kepone
accumulated from water and food by se-
lected estuarine organisms. It is important
to determine the accumulation of Kepone
from water and food by various estuarine
species, so that the information can be used
in the decision-making processes that may
affect the water quality for the biota in the
Chesapeake Bay region, or limit transfer of
Kepone to seafoods consumed by man. The
alga, oyster, mysid. shrimp, and fish used in
299
-------
300
L. H. Bahner, et al.
our experiments are representative of many
ecologically important species. The top con-
sumers in our food chains tested are en-
demic to both the Chesapeake Bay area and
northern Gulf of Mexico and are commer-
cially important human food items. Leiosto-
mus xanihurus, the top carnivorous species
of one laboratory food-chain, provides an
estimated 21.5 million pounds annual recre-
ational catch for fishermen of the Middle
Atlantic states (U.S. Department of Com-
merce 1975).
This study provides information about:
(1) the rates and magnitudes of Kepone
accumulation from water by estuarine biota;
(2) rates of Kepone depuration by animals
in Kepone-free water; and (3) rates of Ke-
pone transfer through laboratory food
chains.
Methods
BlOCONCEKTRATION OF KEPONE FROM
WATER BY ESTUARINE ORGANISMS
Oysters
Eastern oysters (Crassostrea virginica)
were exposed to Kepone (88% pure) in a
56-day, flow-through bioassay to determine
the rates of uptake and depuration of this
insecticide. Seawater (mean temperature
14.2 C. range 9-20 C; mean salinity 15 %o,
range 4-22 %o) was pumped from Santa
Rosa Sound, Florida, into a-constant-head
trough in the laboratory. Approximately
440 I/hour was delivered by siphons to each
of three 166 1 aquaria. Oysters (100/aquar-
ium) were not fed but could obtain plankton
from the unfiltered seawater in which they
were held. Stock solutions of Kepone in
triethylene glycol (TEG) were metered into
the two experimental aquaria at the rate of
10 ml/day. Measured concentrations of Ke-
pone in the two experimental aquaria were
0.39 and 0.03 fj.g/1 seawater. A control
aquarium received 10 ml TEG/day.
Oysters (56 mm to 92 mm. umbo to distal
valve edge height; x = 71.8 mm) were col-
lected, acclimated to laboratory conditions
for ten days, exposed to Kepone for 28
days, and then held for 28 days in Kepone-
free seawater. Five oysters were sampled
from each aquarium at 4 hours. 8 hours. 1
day, 8 days, and twice weekly thereafter to
day 28. During the 28-day depuration por-
tion of the test, oysters were sampled at
similar intervals. Analysis methods for Ke-
pone in water and tissues (whole-body, wet
weicht) were those of Schimmel and Wilson
(1977).
Crustaceans
Mysids (Mysidopsis bahia), collected
from laboratory cultures (Nimmo et al.
1977), were exposed to average measured
concentrations of 0.026 or 0.41 ^g Kepone/
1 seawater for 21 days (mean temperature
27.2 C. range 26-29 C; mean salinity 18 %o,
range 12-26 %«) and in a second experi-
ment, grass shrimp (Palaemonetes pugio),
seined and acclimated to experimental con-
ditions for 10 days, were exposed to average
measured concentrations of 0.023 or 0.40
(j.g Kepone/1 seawater in a 28-day, flow-
through bioassay (mean temperature 27 C,
range 26-29 C; mean salinity 25 %o. range
21-28 %o). Experimental methods used
were those of Bahner et al. (1975). The
grass shrimp were held for an additional 28-
day period in clean seawater to assess de-
puration of the insecticide. Filtered seawa-
ter at a "rate of approximately 60 I/hour
flowed through each aquarium containing
mysids or grass shrimp. Mysids and shrimp
were fed 48-hour-old Anemia nauplii daily.
Kepone content of mysids and shrimp was
determined weekly during exposure and de-
puration. In a third experiment, grass
shrimp were collected by seine from the
Lafayette River estuary near Norfolk, Vir-
ginia, and were held in flowing seawater
(mean temperature 25.5 C; mean salinity 15
%o) in the laboratory to determine the extent
of depuration of Kepone from field-exposed
shrimp. These shrimp were analyzed for Ke-
pone concentrations on days 7, 11. 17, and
21 after being transferred to flowing Ke-
pone-free water in our laboratory.
Fishes
Sheepshead minnow (Cyprinodon varie-
gatus) adults, acclimated to laboratory test
conditions, were exposed to an average
measured concentration of 0.05 fj.g Ke-
pone/1 of water (mean temperature 30 C;
-------
Kepone Accumulation and Food Cham Transfer
301
mean salinity 15 %o. range 8-26 %o) for 28
days, using the methods of Hansen et al.
(1977) and were held in Kepone-free \\ater
for an additional week. Ten fish, generally
five females and five males, were sampled
on days 0,1,3,7.14.21. and 28 of expo-
sure to Kepone and on day 7 of depuration.
Spot (Leiostomus xanthurus) were
seined, acclimated for 10 days, exposed to
average measured concentrations of 0.029
or 0.4 /j.g Kepone/1 of filtered flowing sea-
\sater (mean temperature 23 C. range 21-
24 C: mean salinity 18 %o. range 9-24 "/<«,)
for 30 days and allowed to depurate the
chemical for 24 days. Composite samples of
three fish were sampled each week for resi-
due analysis. Spot, exposed to 0.4 /xg Ke-
pone/1 seawater and allowed to depurate for
24 days, were dissected into liver, brain,
gills, muscle, and offal (rest of body tissues)
and analyzed for Kepone.
Fillets (including scaleless skin) and the
remaining portions of sheepshead minnows
and spot (wet weight) were analyzed for
Kepone content. The data were summed-to
calculate concentrations in whole fish.
BlOACCUMULATION OF KEPONE IN FOOD
CHAINS CONSISTING OF ESTUARINE
ORGANISMS
Algae-Ovster Food Chain
The green alga, Chlorococcum sp., con-
taminated with Kepone was used as food for
oysters to determine if contaminated phyto-
plankton could be a significant source of the
pesticide to oysters. Chlorococcum sp. was
grown for 6 days in one liter of culture
medium in 2800-ml Fernbach flasks accord-
ing to the method of Hollister et al. (1975).
Random cultures were dosed with 0.1 mg
Kepone in acetone, while others served as
controls after treatment with acetone alone.
After 24 hours of exposure, algal cultures
(mean wet weight = 0.15 g) were harvested
by centrifugation and washed three times by
resuspension in clean growth medium and
centrifugation. The cells were resuspended
in 5 1 of seawater and fed to oysters by using
the methods of Bahner and Nimmo (1976).
Samples of the algae were analyzed daily for
Kepone.
Rate of Kepone accumulation was deter-
mined by allowing oysters to feed on control
or Kepone contaminated green algae in
flowing seawater. Oysters for this study
were collected and acclimated for 10 days to
laboratory conditions in flowing seawater.
Twenty-four oysters \\ere placed in each of
two aquaria (one control and one experi-
mental) that received 60 1 filtered seawater/
hour (mean temperature 22 C. range 21-23
C; mean salinity 19 %o, range 15-24 %o) and
were fed approximately 50 ml of the appro-
priate (control or contaminated) algal sus-
pension at 15-minute intervals for 14 days.
A 10-day depuration period followed the
feeding study during which the oysters re-
ceived raw, unfiltered, flowing seawater and
no additional Chlorococcum. Oysters (n = 3
per sample) were analyzed for Kepone con-
tent on daysO. 7. 10/14. 17. and 24 of the
experiment.
Plankton-Mvsid-Fis/i Food Chain
Transfer of Kepone from water to plank-
ton to mysids to fish was investigated by
feeding living brine shrimp nauplii that were
contaminated with 2.33 ,ug Kepone/g tissue
to mysids. that were then fed to spot, the
top predator of this laboratory food chain.
Juvenile spot were seined and acclimated
according to the methods previously de-
scribed for sheepshead minnows and spot.
Mysids (Mysidopsis bahia). the intermedi-
ate food organism, were collected from lab-
oratory cultures. Commercially available
brine shrimp eggs were hatched during 48
hours in clean seawater or in seawater to
which 0.005 or 0.1 mg Kepone/1 was added
(Bahner and Nimmo 1976). The brine
shrimp were harvested daily and served as
the "planktonic'' food for the mysids. Ap-
proximately 40 mysids were distributed
among each of six compartments of two 30-1
aquaria. The compartments were separated
by coarse nylon screen that allowed for flow
of water and Anemia throughout each
aquarium while confining the mysids to sep-
arate compartments. Mysids that had fed on
Artcmia for 72 hours were harvested from
one compartment of each holding aquar-
ium, rinsed with seawater. and fed to the
spot. Each compartment was refilled with
mysids to provide for subsequent feeding
-------
302
L. H Bahner, et al
periods. By this method, each of 12 juvenile
spot, (average length 40 mm), in each of
three 30-1 glass aquaria, were fed 3 to 5
control or contaminated mysids daily.
Aquaria containing spot received 60 1 of sea-
\vater/hour to prevent anoxia and to mini-
mize bioconcentration of Kepone depurated
from the mysids. Water averaged 19 C
(range 16-21) and 18 %o salinity (range 13-
23 %o). Brine shrimp. 30 to 45 mysids. 2 to
3 spot, and water from each aquarium were
analyzed weekly for Kepone.
Results and Discussion
BIOCONCENTRATION FROM SEAWATER
Kepone was bioconcentrated from water
by oysters, mysids. grass shrimp, sheeps-
head minnows, and spot in all concentra-
tions tested (Figs. 1. 2. 3. 4, and 5; Table
1). and all species showed nearly equili-
brated tissue concentrations of Kepone
within 8 to 17 days after exposure to Ke-
pone began in water. Bioconcentration fac-
tors for Kepone in these species ranged
from 2.300 to 13.500 in long-term (>96
hrs) flow-through bioassays (Table 2). Ke-
pone bioconcentrated in oysters to approxi-
mately 10.000 times the concentration in
the exposure water within 19 days. Mysids
bkvcncjr,;riac-i Kcpor.c up to 13.000 time".
the amount measured in the exposure wa-
039u»/l
OOJjJj/l
iND
10 20
-ACCUMULATION —
TW£ {40)1)
Fig. 1. Bioconcentralion of Kepone from water con-
taining average measured concentrations ol 0.03 or
0.39 /ug/l by oysters (Crassostrea virgmica) exposed for
28 days, and its depuration b> oysters placed in Ke-
pone-free water for 28 days (mean temperature
14.2°C. mean salinity 15 "/i»). ND = not detectable.
<0.02 pig/g wet weight.
ter. Each mysid (mean live-weight = 2.5 mg
for 66 aduits), exposed to 0.026 ^tg Ke-
pone/1 for 14 days contained approximately
5.9 ng Kepone; therefore, this amount of
the chemical could enter food chains of estu-
arine predators that consumed each mysid.
Stomachs of flounders from Chesapeake
Bay (standard length 25 to 174 mm) con-
* KT'
\
04liig/l
12 16
TIME (days)
24
2B
Fig. 2. Bioconcentration of Kepone from water con-
taining average measured concentrations of 0.026 or
0 41 jig/1 by mysids (Mysidopsis bahw) exposed for 21
days (mean temperature 27.2°C; mean salinity 18 %o).
_ '°r
• WHOLE BOOT
* FIELD EXPOSED
Fig. 3. Bioconcentration and depuration of Kepone
in crass shrimp (Palaemonetes pugio) during 56-day
study. Circles indicate concentrations of Kepone accu-
mulated from water containing average measured con-
centrations of 0.023 or 0.4 /ig/1 by grass shrimp ex-
posed in the laboratory for 28 days, and its depuration
by shrimp placed in Kepone-free water for 28 d.iys
(mean temperature 27°C. mean salinity 2? V). Trian-
gles indicate concentrations in shrimp collected from
Lafayette River. Norfolk. Virginia and held in clean
flowing seawater at the ERL, Gulf Breeze, for 21 days
(mean temperature 25.5°C; mean salinity 15 %o).
Dashed line represents extrapolation to initial concen-
tration at beginning of depuration.
-------
Kepone Accumulation and Food Chain Transfer
303
ID''
J3
E
to
bj
—i
• WHOLE BODY
• FILLET
10 20
-ACCUMULATION —
40
-DCPUOATION
Fig. 5. Bioconcentration of Kepone from water con-
taining average measured concentrations of 0.029,
0.40, 1.5*. 3.4*, 44'. 12.0*, and 16.0* Mg/l by spot
(Leioswmu<; xanthurus) exposed for 4 or 30 days, and
its depuration by fish placed in Kepone-free water for
24 days (mean temperature 23°C; mean salinity 18 %o).
*Data from Schimme! and Wilson (1977).
fj.g Kepone after 28 days of exposure to
0.05 (tig Kepone/1 seawater. Kepone con-
centrations were slightly higher in female
sheepshead minnows (0.35 jug/a) than male
fish (0.25 /ig/g).
Spot, a commercially valuable food fish,
bioconcentrated Kepone from 0.029 /xg/1
seawater; each fish (mean weight 1.4 g) con-
tained approximately 0.13 /u.g Kepone. The
bioconcentration factors for Kepone in fish
were similar to those of other chlorinated
hydrocarbon insecticides (Schimmel et al.
1975; Schimmel et al. 1976). Kepone accu-
mulated in edible fillets to near the whole-
body concentrations in fish (Figs. 4, 5,& 6);
therefore, one of the largest reserves (22 %)
of Kepone in absolute weight is in the edible
portion of contaminated fish. Although the
greatest body burdens of Kepone on a wet-
weight basis are in the brain, liver, and gill
tissues, the relatively large size of muscle
and offal tissues contributes large Kepone
reserves to higher trophic levels (Fig. 6).
Depuration of Kepone was not consistent
among the species tested. Clearance of the
chemical from oysters was relatively rapid.
with no Kepone detected within 7 to 20 days
after exposure ceased (Fig. 1). Depuration
of Kepone from laboratory exposed grass
shrimp (Fig. 3) and fish (Fig. 4 & 5) was
slower; Kepone concentrations were re-
duced 30-50% in 24 to 28 davs. Grass
-------
..1
TAHl H
exposed
1 . Concentrations of Kepone (^
to 0.026 or 0.4 1 /ug/l for 2 1 d.iys.
:/£ wet tissue) measured in oysters (C. virginica) exposed to 0 03 or 0.39 /u.g/1 for 28 days, mysids (M. ba/iui)
fji.iss shrimp (/' piigii)) exposed to 0 023 or 0 40 yitg/l for 28 days, sheepshead minnows (C. viiru'Ktiliif) exposed
to 0 05 /jg/l for 28 days, and spot ( /, mntliiuitt ) exposed to 0 02V or 0.4 /j.g/1 for 30 days in flowing water experiments. Animals were allowed to depuiate
Kepone
Duia-
tion of
l.xpo-
sure
(Days)
1/6
1/3
1
2
3
4
7
8
9
1 1
12
14
15
19
21
25
28
30
Duration
of De-
puration
1/6
1/3
1
2
4
7
11
14
21
24
28
lot up to
Oysters
0 0.1
MP/I
Whole
Body
0012
0 031
0.036
_
—
0.11
—
0.18
—
—
0.21
—
0.20
0 29
0.23
0.19
0.21*
-
0.30
0 30
0.14
-
0.074
v.inj> seawater.
Mysids Shrimp
041 0 02.1
Mg/l Mg/l
Whole Whole
Body Body
- —
<0.02
_ _
2.0
3.3 0.038
0 072
6.3
4.7 0.088
_ _
4.8* 0.12
0.087*
-
0.1
0.1
0.084
0.055
_
0.035
Shrimp
040
Mg/1
Whole
Body
_
0.27
_
0.47
1.79
2.18
_
3.15
4.57*
-
2.62
2.5
1.78
1.5
—
0..84
Minnows Minnows Spot Spot Spot Spot
Dura- 0.05 005 0029 0.029 0.40 040
tion of /jig/i /ig/l Mg/' Mg/' Mg/' Mg/l
sure Whole Whole Whole
(Days) Muscle Body Muscle Body Muscle Body
1/6
1/3
1 <0.01 <().()!
2
3 0.019 0.031
4
7 0.086 0.14 <0.02 0.037 0.50 0.73
8
9
11
12
14 0.19 0.22
15 - 0.055 0.072 0.86 0.76
19
21 0.21 0.24
25
28 0.29* 0.37*
30 - - 0.048* 0.093* 0.99* 0 94*
Duration
of De-
puration
1/6
1/3
1
2
4
7 0.21 0.22 0.074 0.093 0.87 1.04
11
14 - - - 0.57 0.72
21
24 - 0.031 0.0469 0.38 0.52
28
CO
o
I
CD
1'inal day of exposure.
-------
Kepone Accumulation and Food Cham Transfer
305
TABLE 2. Bioconcentration factors for selected species exposed to measured concentrations of Kepone in water
Species
CMorococcum sp.
Chlorococcum sp.
Crassostrea \ irgmica
Crassostrea virginica
Anemia sa/ina
Anemia sahna
Mysidopsis bahia
Mvsidopsis bahia
Paliiemoneies pugio
Palaemoneles pugio
Cypnnodon variegatus
Leiostomus xanlhurus
Leiostornus xanlhurus
Leiostomus xanthurus
Leiostomus xanlhurus
Leiostomus xanlhurus
Leiostomus \unthurus
Leioslomus xanthurus
Exposure
Concentration
(Mg/D
100. (static)
40. (injection)
0.03
0.39
5. (static)
100. (static)
0.026
0.41
0.023
0.4
0.05
0.029
0.4
1.5*
3.4*
4.4*
12.0*
16.0*
Duration of
Exposure
(days)
1
2
19
21
2
2
21
21
28
28
28
30
30
4
4
4
4
4
Mean
Bioconcentration
Factor
340
6.000
9,354
9,278
10
23
5.962
13,473
5,127
11.425
7,115
3.217
2,340
1,120
941
1.591
900
1.050
* Data from Schimmel and Wilson (1977).
shrimp from the Lafayette River depurated
Kepone at rates similar to those of labora-
tory exposed shrimp with approximately
20% of the Kepone lost during 21 days in
seawater containing no Kepone. Spot that
were exposed to Kepone for 30 days and
allowed to depurate for 24 days, contained
highest Kepone concentrations in brain, fol-
lowed bv liver, gills, and muscle (Fig. 6).
Many chlorinated chemicals are highly con-
centrated in liver and other fattv tissues
(Parrish et al. 1974; Parrish et af. 1975).
but the unusual distribution of Kepone
might be explained by its water solubility
relative to other insecticides.
BlOACCUMULATlON OF KEPONE IN FOOD
CHAINS CONSISTING OF ESTUARINE
ORGANISMS
Algae-Oyster Food Chain
Chlorococcum. grown in media enriched
to 0.1 mg Kepone/1, bioconcentrated the
chemical to a mean of 34 /ig/g in whole
cells: the bioconcentration factor in these
static exposures was 340X (Table 2). In
preliminary tests, continuous infusion of 2.1
pig Kepone/hour into static cultures of Chlo-
rococcum produced cells with bioconcentra-
tion factors near 6000 x within 48 hours;
adsorption of Kepone by the cells and ves-
sels limited the average measured concen-
_.^ - •_
Fig. 6. Distribution of Kepone in selected tissues of
spot (Leioslomusxanlhurus). Spot were e\po>ea io U 4
/xg Kepone/1 tor 30 days and allowed to depurate and
equilibrate vr an additional 24 > prior to ^jmrim;
(mean temperature 23°C; mean salinity 18 %o). Per-
centage oi whole-body Kepone burden in five tissues is
on left. Measured residues (wet weight) are on right
tration in the media to approximately 0.04
mg/1. Continuous exposure in this test is
probably more representative of those oc-
curring in an estuary, and places the impor-
tance of bioconcentration of Kepone by
phytoplankton in proper perspective.
Oysters bioaccumulated Kepone to 0.21
pig/g when fed Chlorococcum sp. containing
an average of 34 ptg Kepone/g for 14 days
(Fig. 7; Table 3). Kepone in feces and pseu-
dofeces from these oysters averaged 1.78
/ug Kepone/g (dry weight). Kepone was not
detectable in the unfiltered water samples;
shell growth was evident; and all oysters
gained weight during the test. Weight gain
-------
306
L H Bahner, et al.
TIME (doyt)
Fig. 7. Bioaccumulation of Kepone by oysters
(Crassostrea virginica) that consumed algae (Chloro-
coicum sp.) with residues of 34 ^g Kepone/g (wet
weight) Oyster1; fed on contaminated algae for 14 days
(mean water temperature 22°C; mean salinity 19 %o),
and were then fed uncontammated plankton for an
additional 10 days to allow depuration of the chemical.
ND = not detectable, <0.02 /ig/g wet weight
in exposed oysters was not different than
control oysters (analysis of covariance. a =
0.05). Kepone concentrations in oysters fed
the contaminated algae appeared to almost
reach equilibrium in 14 days; but quantity of
K.T;-T.;- trnn^fj-rco :rom rhoc aigac to oys-
ters was limited, probably due to rapid de-
ouratirn of tb; cr^rv.io'.1.! fr^-"' ''.•; •'• -t;-r-.
•xepone was not actectable (<0.(J2 /u.g/g) 10
days after the oysters received no contami-
nated food. Most Kepone was depurated
from oysters within 96 hours; therefore, if
•"•yster^. in the n:::i:rai > ^ '• •••'•.:•; ^ •".',., ••
measurable Kepone residues, a recent or
continuous source of Kepone from water
and/or food has been available.
The maximum overall accumulation and
transfer of Kepone or food-chain factor
from \\ater to algae and finally to oysters
was 2.1 (Table 4). The food-chain factor
was obtained by dividing the concentration
of the contaminant in the final consumer by
the concentration of the contaminant in the
water of the priman producer. The concen-
tration measured in each consumer can be
compared uith a lower trophic level to de-
termine the bioaccumulation factor of the
contaminant for that predator-prey pair.
Bioaccumulation factor is similar to biocon-
centration factor, but the contaminant is in
food and is consumed by the predator. The
TABLE 3. Concentrations ot kepone (^g-g whole
body, wet tissue) measured in oysters (C virgmica) fee
algae (Chlorococcum sp.) containing 34 /xe/g for 14
days and in spot (L. xanrhiirus) fed mysids (.V/. bahia
containing 0.02 jig/g (estimated) or 1.03 /ug-g for 30
days in flowing water experiments. Oysters were fed
uncontaminated plankton for an additional 10 days tc
allow depuration of the chemical.
Duration
of
Exposure
(Days)
0
5
7
10
14
20
30
Duration
of
Depur-
ation
3
10
Oysters
fed 34 p.g/g
Hg/'g
Whole Body
<0.02
—
0.10
0 18
0.21*
_
-
0.075
<0.02
Spot fed 0.02 M&'E
(Esumated)
^g/g
Whole Body
<0.02
<0.02
—
<0.02
—
<0.02
0.015.0.024*
_
-
Spot fed
1.03 ^ig/g
Mg/g
W hole Body
<0.02
0.095
_
0.15
—
0 59
1 0.1.1*
—
-
* Final day of exposure.
TABLE 4. Kepone transfer in algae-oyster tood chain.
Algae (Chlorococcum sp.) grown in Kepone enriched
media for 24 hri was fed 'oo\sters(C. virj;i>!;rfl)for 14
days in flou-throush feeding experiment
Control
food chain
\ij rvepone Dingle
dose) in algal media
(mg/l)
(21 Kepone residues in
algae after 24 hrs oi
factor from water
lorn]
(4) Kepone residues in
oysters after 14
days of feeding
(5) Bioaccumulation
factor from algae to
oysters j(4)/(2)]
(6) Food chain factor
Control
Control
(ND)"
Control
(ND)*
Evposed
food chain
0.1
.007
2.1
* ND = non-detectable (<0.02 mg/kg).
bioaccumulation factor for oysters consum-
ing algae under these test conditions was
only IK007 (Table 4). These data indicate
that transfer of Kepone from algae by oys-
ters was inefficient, or that the uptake was
masked by the oyster's ability to depurate
the chemical quickly.
-------
Kepone Accumulation and Food Crta.n Transfer
307
Plankton-Mysid-Fish Food Chain
Spot accumulated Kepone by consuming
live mysids that had grazed on Kepone-
laden brine shrimp (Fig. 8; Tables 3, 5).
Brine shrimp exposed to 0.1 mg Kepone/1
seawater contained whole-body residues of
2.33 ju.g/g after 48 hours. Mysids that fed
for 72 hours on these brine shrimp con-
tained 1.23 /j.g Kepone/g. Kepone concen-
trations in spot that consumed the mysids
ACCUMULATE* TMHOUGM fOC» LEVEL fOOO CHA*
Fig. 8. Block diagram of four-level tood chain.
Nominal concentrations of Kepone in water (left) were
control (bottom), 0.005 mg/1 (center), and .1 mg/1
(top). Anemia salina naupln. hatched in these concen-
trations ot Kepone. were fed to mysids (Mysidopsis
biitud). and m\sids were fed to spot (Leiostomus xan-
'.'':,':• > • - Average mea^v.-j concentrations
of Kepone. . »JL.I .rop/nc specie^ is _.'•tv.itT ;crr": -*oiarc was 1S*~C
and mean salinity 18 %«.
TABLE 5. Kepone transfer in plankton-mysid-fish food chain. Brine shrimp (A. salina) were hatched during 48
hrs in Kepone enriched seawater and were fed to mysids (M. bahia) for 72 hrs. Mvsids were then fed to spot (L.
(1) Kepone (single dose) in hnne shrimp me-
dia (mg/l)
(2) Kepone residues in brine shrimp after 48
hrs of exposure (mg/kg)
(3) Bioconcentration factor from water [(2)/
(01
(4) Kepone residues in mvsids after 72 hrs of
feeding (mg/kg)
(5) Bioaccumulalion factor from brine shrimp
to m\sid- |(4)/(2)J
(6) Kepone residues in spot after 30 days of
teedins! (mg/kg)
(7) Bioaccumulation factor from mysids to
spot [<6)/(4)]
(8) Food chain factor |(6)/(1)]
food chain
Control
Control
(ND)*
-
Control
' (ND)'
—
Control
(ND)*
—
-
food chain
0.005
0.044
0.043
0.058
x = 0.050
10.
x = 0.023
(estimated)
0.5
(estimated)
0.015
0.024
x = 0.0195
>0.85
(estimated)
>3.9
food chain
0.!
1.3
2.4
3.3
x = 2.33
23.3
0.89
1.0
1.8
x = 1.23
0.53
1.0
1.1
\ = 1 .05
>0.85
>10.5
* ND = non-detectable (<0.02 mg/kg).
-------
308
L H. Bahner, et al
: ^•£'.-t&'f-~-f~£
Mvsids. which consumed Anemia with
residues of 0.05 or 2.33 /u.g Kepone/g (wet
weight), attained 0.023 (estimated) or 1.23
/jig Kepone/g whole-body residues within 72
hours. The estimated 0.023 ju.g Kepone/g
whole-body mvsids. obtained by assuming
the bioaccumulation factor of 0.85 (Table
5) for Kepone transfer from mysids to fish
as observed in the food chain beginning with
0.1 mg/1. also occurred in the food chain
that began with 0.005 mg Kepone/1. The
residue in mysids was then estimated to be
0.023 yu.g Kepone/g. which was consistent
with the estimated bioaccumulation factor
of 0.5 for Kepone transfer from Anemia to
mysids for 72 hours. The food-chain factors
were different for this food chain (3.9 com-
pared to 10.5). since the bioconcentration
factor for Kepone by brine shrimp from the
lower concentration in water (0.005 mg/1)
was less than that from the higher concen-
tration (0.1 mg/1). The initial bioconcentra-
tion of Kepone from water by planktonic
food organisms was the dominant source of
Kepone to each member of this food chain.
since bioaccumulation factors were less than
unity.
In the field, lower concentrations of Ke-
^One if! ^e^'-V '* ~ '•"••'1'^ t->->^nl* -p • -'•- -1- '^_
tion or Kepone residues, tound in the fish of
this food chain because the plankton could
be expected to be chronical!) exposed to the
contaminant. Thus, the food-chain factor
would be expected to increase in the natural
environment since bioconcentration factors
.\>r a chionnatcu iisarocaroon pesticide
(DDT) in feral plankton ha\e been shown
:.; exceed 4nnn\ /CoN ;^~:). H;y.\cwr.
bioconcentration of Kepone could oversha-
dow the amount of the chemical received
from food by the animals in this food chain.
Approximately 3.000 times as much Ke-
pone in food as in water was needed to
produce similar concentrations in spot in 28
to 30 days. Therefore, bioconcentration of
Kepone was dominant in this food chain.
but significant quantities (>85%) of Ke-
pone transferred from prey to predatory
fish. Rapid uptake from water and food.
slow depuration, and appreciable solubility
in water indicate that Kepone will transfer
through food \\ebs and pose threats to con-
sumers.
LITERATURE CITED
BAHNER. L. H.. AND D. R. NIMMO. 1976. A precision
She-feeder for flow-through larval culture or food
chain bioassays. Prog. Fish-Cult. 38:51-52.
. C. D 'CRAFT. AND D. R. NIMMO 1975 A
saltwater flow-through -bioassay method with con-
trolled temperature and salinity. Proq. Fish-Cult.
37:126-129.
Cox. J. L. 1971. DDT residues in seawater and panic-
ulate matter in the California current system. U.S.
Natl. Mar. Fish Serv. Fish. Bull. 69:443-450.
HANSEN. D. J.. A. J. WILSON. D R. NIMMO. S. C.
SCHIMMEL. L. H. BAHNER. AND R. HUGGETT. 1976.
Kepone: Hazard to aquatic organisms. Science. 193:
528.
. L. R. GOODMAN. AND A J. \\ILSON.JR 1977
Kepone: Chronic effects on embryo, fry. juvenile.
and adult sheepshead minnows (Cyprinodon vane-
gaius). Chesapeake Sci. 18(2).227-232.
HOLLISTER. T. A.. G. E. WALSH. AND J. FORESTER.
1975. Mirex and marine unicellular algae: accumula-
tion, population growth and oxygen evolution. Bull.
Environ. Cnmam. To.iicoi. 14-753-759.
MARKEL. D. F.. AND G. C. GRANT 1970. The summer
food habits of young-of-the-year striped bass in three
Virginia rivers. Chesapeake Sci. ll(l):50-54.
NIMMO. D. R.. L H. BAHNER. R A. RIGBV. J. M.
SHEPPARD. AND A. J. WILSON 1977. "Mysidopsis
balna": An estuanne species suitable for life-cycle
bioassays in determining sublethal effects of a pollu-
tant. Presented at the Symposium on Aquatic Toxi-
cology and Hazard Evaluation. American Society of
Testmc Materials. Memphis. Tennessee. October
25-26'1976.
"•• , ° R . J. A CuLLh. J. FuKLMtk. J M.
PATRICK. JR.. AND G H COOK. 1974 Dieldnn
Effects on Se\eral Estuanne Organisms. Proc.
27th Annu Conf. Southeast Assoc. Game Fish
Comm. 1973. p 427-434.
PARRISH. P. R.. G. H COOK. AND J M. PATRICK. JR.
1975. Hexachlorobenzene: Effects on Several Estua-
nne Animals. Proc. 2Sth Annu Conf Southeast
.-V-.K. O..mc F:sn v_\irnm.. i-J~4 p. IT1—1,S~
SCHIMMEL. S. C , AND A. J WILSON. JR. 1977. Acute
toxicity of Kepone to t
-------
Effects of Kepone on Estuarine Microorganisms
A.W. Bourquin, P.H. Pritchard, and k.R. Mahaffey
U.S. Environmental Protection Agency
Environmental Research Laboratory
Gulf Breeze, FL 32561
Developments in Industrial Microbiology
(Society for Industrial Microbiology)
(In press, Volume 19)
ERL-GB Contribution No.
-------
INTRODUCTION
Aquatic microorganisms undoubtedly play a critical role in the metabolic
transformation of organic and inorganic compounds which enter lakes,
estuaries, and oceans. Some of these compounds, particularly the chlorinated
hydrocarbon group, degrade very slowly in nature and consequently their fate
and accumulation are of great importance in determining environmental quality.
Since many chlorinated hydrocarbons accumulate in terrestial and aquatic
ecosystems, they could eventually alter basic microbial degradation processes
if the compounds were toxic to the microorganisms. For example, interferences
in the competition among bacteria in a natural population, due to the
elimination of an important member of that population, could reduce trans-
formation efficiency and even effect the extent of transformation. The
possibility that pesticide residues may have deleterious effects on
microorganisms and their activities has received considerable attention
(Alexander 1968, Edwards 1972, Tu and Miles 1976, Ware and Roan 1970). The
emphasis of these studies, however, has been on the effects of pesticide
residues on soil rather than aquatic microorganisms. Although the effects of
pesticides on phytoplankton and photosynthesis has been docu-
mented (Ware & Roan 1970), information is lacking on the effects of organo-
chlorines on aquatic microorganisms and the processes they mediate. Several
important organochlorine compounds have been tested for their toxicity to
aquatic microorganisms. Bourquin et al. (1975) and Bourquin and Cassidy
(1975) have shown polychlorinated biphenyls to be toxic to a variety of
bacteria isolated from estuarine waters. Using the agar-disc diffusion
method, the investigators found that 0.1 mg of Aroclor 12^42 per disc elicited
definite zones of inhibition. They also showed that, by documenting
physiological characteristics relative to toxicant-sensitive and
-------
toxicant-insensitive groups, certain characteristics correlated with one group
more than another. Richards (1977) has shown that pentachlorophenol in
flowing sea water systems caused the selection of PCP-tolerant microorga-
nisms. Brown et al. (1975b) have shown that mirex was nontoxic to aquatic
bacteria but Kepone, a contaminant of mirex, was toxic. Kepone may be a
chemical breakdown product of mirex (Carlson et al. 1976).
Kepone^ (decachlorooctahydro-1,3,4-roetheno-2fi-cyclobuta (cd) pentalene-
2-one), an insecticide, has been shown to be an extensive contaminant of the
James River System in Virginia (U.S. EPA, 1975). Its introduction into the
James River was due to industrial effluents. Recent studies have shown that
Kepone is toxic to algae (Walsh et al. 1977) and several estuarine animals
(Schimmel and Wilson 1977). This paper reports on the toxicity of Kepone to
estuarine bacteria.
MTERIALS AND METHODS
Bacterial Cultures
bacterial isolates obtained from Range Point salt marsh (Pensacola Beach,
Florida), Escambia Bay surface water, and Sabine Island disposal pond were
used in disc-agar sensitivity assays. These pure cultures of bacteria and
fungi were originally isolated from batch culture enrichments on various
carbon sources and maintained as part of our laboratory stock culture
collection. Samples of water and sediment from these waters and the Gulf of
Mexico were used in all mixed-culture toxicity testing. Isolates from these
environments were used to determine the effects of Kepone on growth and the
oxidation of specific substrates.
Registered trademark, Allied Chemical Corp., hew 1'ork. Mention of trade
names in this publication does not constitute endorsement by the U.S.
Environmental Protection Agency.
-------
Low concentrations of the insecticide Kepone, approaching those found in
contaminated James River sediment, were shown to be inhibitory to the growth
and oxygen uptake of microorganism randomly selected from estuarine
environments. No significant correlations were noted between growth
inhibition by Kepone and cell morphology, aliphatic hydrocarbon utilization,
pesticide tolerance, selected enzyme activities, nitrate reduction, and urea
hydrolysis. Oxygen uptake by pure cultures grown on glucose or hydrocarbons
at cell densities equivalent to 103 - 101* cells/ml was decreased by 60-100£ at
Kepone concentrations of 0.02 -2.0 mg/1. Total viable counts from estuarine
water or sediments grown aerobically on agar media containing 0.02 mg/1 Kepone
were reduced by 6 - 78 °/oo. Ihe inhibitory effect was partially eliminated
when sediment populations were grown anaerobically.
-------
Reagents and Chemicals
Kepone (98$ pure) was obtained from Chem Services, Inc., West Chester,
PA. N,N-dinethylforamide (DMF) was purchased from Aldrich Chemical Co.,
Milwaukee, Viisc. Microbiological media and substrates were obtained from
Difco Laboratories, Detroit, MI.
Zobell's 2216 marine medium Z-15 (Aaronson 1970) was prepared at a final
salinity of 15 °/oo using fiila Marine Mix (Rila Products, Teaneck, N.J.).
Solid medium was prepared with 2% Bacto-agar. Minimal salts basal medium
(MSB) was prepared according to Stanier et al. (1966). Glucose (0.2%) was
added to MSB as a growth substrate and $.01% yeast extract (Difco) was added
to compensate for any auxotrophic cultures.
Kepone-harine agar medium was prepared by adding desired concentrations
of Kepone (in acetone carrier) to Zobell's medium after autoclaving. Acetone
control plates (no Kepone) were used in all experiments.
Pure Culture Inhibition Studies; Disc Sensitivity Assays
Pure.cultures, grown in Zobell's (Z-15) broth for two 18 hr growth cycles
were spread on Z-15 agar plates. Three filter paper discs (Schleicher &
Schuell, 12.7 mm, No. 7^0-E, Keene, NH), designed for antibac-
terial substrate testing, were saturated with acetone or DMF solutions
containing 0.01, 0.1, or 1.0 rag Kepone per milliliter, dried, and then placed
on the inoculated agar plate. A fourth disc contained only DMF or acetone.
The actual volume of Kepone solution per disc was calculated to be 0.11 y£ by
determining the average increase in weight of a DMF or acetone saturated disc.
Mixed Culture Inhibition Studies
Water and sediment samples collectd from James River, VA., and various
estuarine and marine areas near Pensacola Beach were serially diluted in water
-------
blanks of the appropriate salinity and plated on 2.-15 agar plates with and
without Kepone. The plates were incubated aerobically at 28°C and colonies
were counted at 4- and 8-day intervals. Sediment samples were similarly
plated and incubated anaerobically in BBL Disposal Anaerobic Systems (EBL,
Baltimore, hd.) at 28° for 8 days. Dominant colony types from the aerobic
Kepone containing plates were picked with sterile toothpicks and spotted onto
Z-15 agar. The resulting colonies were replicated onto various differential
and selective media for physiological characterization.
Oxygen Uptake Studies
Selected pure cultures aerobic Kepone plates were grown in 100 ml of
either 2-15 broth or MSB broth with glucose or succinate (0.2%) for 18 hr
prior to harvesting by centrifugation (10Xg, 10 min.). The cells were washed
once with 0.05 M ^HPOij buffer (pH 7.5) containing 1.5$ NaCl and finally
resuspended in 2 ml of saline buffer. Suspensions were kept in an ice bath
until used. Oxygen uptake was determined by a Gilson Oxygraph (Model K-ICT-C,
Gilson Electronics, hiddleton, Vvisc.) equipped with a Clark electrode. The
reaction vessel contained 1.7 - 1.8 ml phosphate buffer, 10 - 50 y£ washed
cell suspension (100-300 yg protein), and 10 - 50 mg oxidizable substrate.
After a baseline level of oxygen uptake was established, 10 y£ of Kepone
solution (0.19 - 19.0 yg Repone/£ DKF) was added to the reaction vessel and
the oxygen uptake observed for any resulting effects. A positive control, 10
y& of a pentachlorophenol solution (20 mM), was then put in the same reaction
vessel.
hethods
Physiological characterization of isolates on differential and selective
media was carried out according to the methods of Colwell and Weibe (1970).
The ability of isolates to grow on hydrocarbons (hexadecane, undecane,
-------
octadecane, benzene, naphthalene, biphenyl, xylene, toluene) was determined by
placing hydrocarbon-saturated filter paper discs in the lids (for volitale
hydrocarbons) or on the agar (for nonvolitale hydrocarbons) of MSB agar plates
streaked with the appropriate isolate. The appearance of growth on the agar
whicn was greater than the control plates was taken to be hydrocarbon utili-
zation by the isolate since no other carbon source was supplied. Pesticide
tolerance tests (using toxaphene, Arochlor 1242, methoxychlor, heptachlor,
DDT, raalathion, pentachlorophencl) were performed using the disc-aear
diffusion method and recording effects on growth of isolates streaked on MSB
agar containing glucose or succinate and yeast extract.
Protein determinations were performed according to the methods of Lowry
et al. (1951).
RESULTS
Pure Culture Toxicitv Studies
The toxicity of Kepone to a variety of pure cultures from our laboratory
collection was determined by the disc agar diffusion sensitivity method.
Table 1 shows the degree of toxicity as expressed by the size of the zone of
inhibition. Only the sensitive cultures are shown. Several of the isolates
(MO.'S 1, 3, ^i 11, 1^, 15, 29) were particularly sensitive (as judged by the
size of the zone of inhibition) to Kepone. Chemical analysis has shown Kepone
to be 99!» pure with no trace (less than 0.5 ng/ml) of other chlorinated
compounds. Of the 26 isolates tested, 33% were inhibited at the 3.65 yg/disc
concentration and 50? were inhibited at the 1M.6 yg/disc concentration.
Fifteen percent of the isolates were inhibited at the 1.U6 yg/disc
concentration. Higher concentration of Kepone (20 yg/disc) inhibited 100? of
the isolates.
-------
Of the six fungal cultures tested, one was poisoned at the 14.6 yg/disc
concentration. Two yeasts, Candida maltosa and Candida lipolvtica. were both
sensitive but at higher concentrations of Kepone than used with the bacteria
tested.
Kepone-sensitive cultures (i.e., showing zones of inhibition with disc
assay) showed no significant correlation (a = 0.05) with morphology, gram
stain, hydrocarbon utilization, pesticide tolerance, amylolytic, lipolytic,
and proteolytic activity, nitrate reduction, sugar utilization and urea
hydrolysis. These results are summarized in Table 2.
Nixed Culture_Toxicity Studies
To determine the toxicity of Kepone to mixed populations of bacteria from
a variety of marine habitats, total viable counts were performed with Zobell's
seawater agar (2-15) containing dissolved Kepone. Results of these assays
(Table 3) are expressed as the percentage reduction in colony forming units
(CFU) normalized against control plates that contained no Kepone. Ihe results
demonstrate that Kepone, in concentrations as low as 20 Vg/&, is inhibitory to
the development of colonies on an agar plate. Different degrees of inhibition
(concentrations of Kepone which would give the same reduction in CFU's) were
noted with samples taken from the same area at different times. In many
cases, concentrations below 20 ug/£ were inhibitory. Within each population,
some microorganisms were quite resistant to Kepone (i.e., grew in its
presence).
Twenty cultures, which grew in the presence of Kepone, were selected
(based on predominance) for further study. The cell types and enzymatic ac-
tivities of these purified isolates showed no significant correlation (a =
0.05) with Kepone sensitivity.
-------
Because Kepone is known to be present in sediments and detritus in con-
centrations higher than that found in the water column (U.S. Environ.
Protection Agency, 1975), we attempted to determine the toxicity of Kepone to
bacteria in sediments. The results of viable plate counts on Zobell's marine
agar containing Kepone using samples taken from Range Point marsh sediment is
shown in Table 4. Under aerobic conditions, CPU's decreased as a function of
increasing Kepone concentrations as expected from earlier results. Under
anaerobic conditions, however, the inhibitory effect of Kepone was reduced.
For example, at 0.2 mg Kepone/1, a slight reduction (0 - 12%) in CPU's
occurred under anaerobic conditions but a larger reduction (3^ - 80%) occurred
under anaerobic conditions. Colonies originally grown aerobically, also
showed decreased Kepone sensitivity under anaerobic conditions. Sediment
samples taken at different times generally showed different degrees of
inhibition at the various concentrations.
The effect of Kepone on the anaerobic microbial populations in
contaminated James River sediment was different. These sediments contained 0.1
ppm Kepone (A.J. Ivilson, ERL-GB) when collected (8/29/77). Results of viable
plate counts under aerobic and anaerobic conditions are presented in Table 4.
As opposed to Range Point sediment, the James River sediments contained
bacteria which did not show decreased sensitivity to Kepone under anaerobic
conditions.
Oxygen Uptake Studies
To further assess the sensitivity of bacteria to Kepone, we conducted
oxygen uptake studies. As seen in Table 5, oxygen uptake was a sensitive
indicator of Kepone toxicity. The isolates showed varying degrees of sen-
sitivity: most cell suspensions (10^ - 10? cells/ml) were inhibited at the 20
mg/1 concentration, whereas relatively little inhibition occurred at the 2
-------
mg/1 concentration. Inhibition at 20 mg/1, in many cases, was substantially
greater than at the 200 mg/1 (see isolates 32, 43, and 56). It should also be
noted that some isolates actually increased oxygen uptake in the presence of
Kepone (isolate 49).
DISCUSSION
Our results indicate that Kepone is toxic to microorganisms. Kepone
concentrations as low as 0.02 mg/1 reduced the number of colony forming units
on Zobell's marine agar by 23-92 °/oo. This is ten to a hundred times more
toxic than the PCE's (bourquin 1975), chlordane (Trudgill et al. 1971) and
heptachlor (Shamiyeh and Johnson 1973). Kepone levels in James River sediment
have been shov/n to be 0.05 - 0.5 mg/kg, particularly in areas of high organic
content (U.S. EPA, 1975). Since Kepone is resistant to chemical and
biological degradation (Garnas et al. 1977) and since Kepone appears to be
washing cut of the James River system at a very slow rate, residual Kepone
could have an inhibitory effect on bacteria in the river.
However, this compound was not universally toxic to all cultures at the
concentrations tested. Since only certain members of the bacterial
populations were inhibited by Kepone at concentration levels equal to those in
the James Hiver, it is important to know if the tolerant species will replace
the sensitive species and thus maintain the metabolic integrity of the
ecosystem. In general, this replacement process occurs in both soil (Tu and
Miles 1976) and aquatic (ttare and Roan 1970) environments. Unpublished
results from our laboratory, however, have shown that Kepone, at low
concentrations, decreases the rate at which the pesticide methyl parathion is
degraded, thus implicating a possible interference with the metabolic
integrity of an aquatic system.
-------
Of the three methods used to determine toxicity in this study (i.e.
sensitivity disc, plate counts and oxygen uptake), each appear to yield
similar toxicity results. Plate counts are the easiest method to assess toxic
effects on a mixed population of bacteria, and the minimum inhibitory
concentration (MIC = 0.02 mg/1) gives a good reflection of the effects
possibly occurring under natural conditions. Little can be said about the MIC
from agar-disc diffusion experiments except that low concentrations of Kepone
are inhibitory.
Much higher concentrations of Kepone would appear to be necessary to
inhibit oxygen uptake. However, these studies employed high densities of
cells (1()6 - 10? cells/ml). These levels are, in fact, proportional to an
enivornmental level. Escambia Bay waters have a cell density of 103 - 10^
cells/ml. Vie have determined, in plate assays, that the growth of this
population is inhibited at 20 vig/A Kepone. By concentrating both factors
1000X, observe that 10^ - 10? cells will require 200 mg Kepone/1 to affect
inhibition, which are the same levels employed in oxygen uptake studies.
The use of physiological characteristics (sugar fermentation, enzyme
activities, urea hydrolysis, hydrocarbon oxidation, etc.) as a possible tool
to determine the toxic effects on specific physiological processes has not
been used extensively (Brown et al 1975a). Our study demonstrates no
significant correlation between these characteristics and Kepone toxicity.
Amylolytic activity was prevalent in some Kepone-sensitive organisms as was
the case for in PCB-sensitive bacteria (Bourquin and Cassidy 1975). These
effects apparently cannot promote the enrichment of amylase-positive organisms
in a mixed population, because neither static nor flow-through systems exposed
to Kepone in our laboratory showed this type of enrichment.
-------
The nontoxic effect of Kepone on bacteria grown anaerobically points to a
possible involvement with electron transport and respiration. Widdus et al.
(1971) and Trudgill et al. (1971) have shown that the inhibition of KADK
oxidase activity in Bacillus subtilis by chlordane occurred indirectly through
the apparent disruption of membrane-mediated electron transport. Similar
mechanisms could account for the affect of Kepone in anaerobic versus aerobic
conditions. Trudgill et al. (1971) did not study the effect of chlordane on
anaerobic growth, but he showed that a Streptococcus species (which would have
minimal cytochrome-mediated electron transport activity) was unaffected by
chlordane. Kepone, therefore, could interfere with membrane-mediated
transport.
A possible explanation for the increased oxygen consumption in the
presence of Kepone, is an oxygen uncoupling effect which would generate
hydrogen peroxide. Hydrogen peroxide would then be split by catalase,
releasing C>2 gas. The mechanism accounting for the release of oxygen by cells
is still under investigation.
-------
Table 1. Sensitivity of pure cultures to Kepone using the agar-disc diffusion method3
Concentration Kepone (yg) per disc
GBERL
Isolate Acetone
# Control 1.46 3.65 7.3 14.6
1 0 0 4-4-4- 4- 4- 4- 4- 4-4-
3 + + + 4- + + + -+ + H
4 0 + + + + + + + + + + H
5 ± ± ± ± +
8 ± ± ± ± +
11 + ++ + + + + + + + + 4
14 0 0 + ++ + + 4
15 0 0 ++ + + +_.+ + 4
29 0 + ++ + + + + +
36 + + + + +
42 + + +-++ + + 4
47 0 0 + + +
54 + ' -^ 4- + 4-4- 4- 4-
aO = no zone; + = 0-1 mm; 4- = 1-2 mm; 4- 4- = 2-3 mm; 4- 4- 4- = 3-4 mm;
4- 4- 4- 4- = > 4 mm.
-------
Table 2. Physiological activities of cultures used in Kepone toxicity study_._
Culture
Number
I
3
4
5
11
Kepone ^4
^nsitive
29
42
47
54
8
36
15
10
18
20
23
40
(epone 45
nsensitive
46
56
59
2
7
27
44
Organism
Type
rod
rod
cocc-bac
cocc
cocc-bac
rod
rod
coccus
cocc
rod
yeast
yeast
fungus
rod
pleo
rod
rod
cocc-bac
rod
rod
cocc-bac
rod
yeast
yeast
fungus
fungus
Gram Alkanea Aromatic2 Physiological Function0
Stain Utilizer Utilizer Lipo Amylo Proteo
pos 4- + - + +
pos - - - 4 +
neg - -4-4-4-
neg - -4-4-4-
pos 4- 4-44--
pos - -44--
pos - - - - 4-
pos - - 4- - -
neg - -
pos - 4- - 4- 4-
4- + 4- -+
4- -4-4-4-
-4-4- N.D.C N.D. N.D.
neg - +
neg 4 - + - -
neg '4- - N.D.C N.D. N.D.
pos 4- 4- N.D. N.D. N.D.
pos 4- - 4- - 4-
neg - - 4- - 4-
neg - 4-
neg - - 4- 4- -
neg - -
1 4- 4-
4-4-4
- - 4- N.D. N.D. N.D.
- N.D. N.D. N.D.
Pesticide
Tolerance
+
-
-
-
+
•"•
-
-
-
-
-
-
-
-
-
+
+
4-
+
-
-
-
+
+
aHydrocarbons and pesticides listed in text
bLipo = lipolytic activity; Amylo = amylolytic activity; Proteo = proteolytic activi
CN.D. = not determined
-------
Table 3. Effects of Kepone on total viable counts of bacteria from estuarine wate
Sample
source Date3
Range
Point
Es cambia
Bay
Laboratory
Pond
Gulf of
Mexico
A
B
C
A
B
C
A
B
C
A
B
D
Salinity
(°/oo)
15
15
20
11
15
18
20
15
20
33
33
33
Temp.
(C)
21
24
23
22
24
22
20
26
27
18
22
24
3-15
181
86
1
267
13
12
479
104
544
270
13
15
CPU's x 102
0.02b
108(41)
65(25)
12(28)
81(70)
7(50)
11(8)
448(7)
49(53)
501(8)
62(78)
10(21)
6(59)
ner ml water
0.20b
c 73(60)
35(60)
13(23)
233(13)
10(26)
9(23)
409(14)
437(20)
32(89)
10(25)
1(92)
2.00b
35(81)
33(61)
13(25)
130(52)
5(66)
9(23)
396(18)
19(82)
309(43)
46(83)
8(40)
1(95)
Sampling Dates. A = 4/10/77; B = 4/17/77; C = 4/29/77; D = 5/7/77.
mg Kepone/c. used in Zobell's marine agar.
cNumbers in parentheses are percentage reduction in CPU's normalized against
growth on control plates of Zobell's marine agar containing no Kepone.
-------
Table 4. Typical Effects of Kepone on the production of colony forming units
from Range Point narsh and James River sediments3
Sediment
Source
Range
Point
James
River
Incubation
conditions
anaerobic
aerobic
anaerobic
aerobic
Control
5-15
34
210
43
15200
CFU'
'0.02
41(0)C
200(5)
28(35)
16500(0)
2
s x 10 /gm sediment
Kepone (mg/£)
0.2
41(0)
140(34)
24(44)
14200(7)
2.0
37(0)
30(86)
9(79)
9400(36)
ajames River sediment contained 0.1 ppm Kepone
"CPU's are the mean of the results of triplicate platings. Experimental error in
these assays averaged + 8 CFU's
cNurabers in parentheses are percent reduction in CFU's relative to 2-15 control
-------
Table 5. Effect of Kepone on oxygen uptake by resting cell suspensions of
isolates from estuarine environments
Isolate
no.
49
51
61
56
32
35
43
Source0
EB
EB
EB
EB
RP
RP
RP
Kepone
Enrichment
Concentration
0.00
0.02
0.20
2.00
0.00
0.02
2.00
Percentage
2
+25
0
0
66
0
11
-
Reduction
Kepone (mg/
20
100
100
100
75
83
100
64
in 09 L'Dtake3
'£)°
200
100
100
100
66
31
100
33
an>I 02/ml/min/yg protein or glucose
"Kepone concentration required to show inhibition of concentrated cell suspension
(10° - 10 cells/ml). One thousandth of this concentration would inhibit 02
uptake by bacterial populations in Escambia Bay (10 - 10 cells/ml)
CEB = Escambia Bay, RP = Range Point
-------
LITERATURE CITED
Aaronson, S. 1970. Experimental Microbial Ecology. Academic Press. New
York.
Alexander, M. 1968. Degradation of pesticides by soil bacteria. In Ecology
of soil bacteria. (Gray and Parkinson, eds.). Univ. of Toronto,
Toronto.
Eourquin, A.W. and S. Cassidy. 1975. Effect of polychlorinated biphenyl
formations on the growth of estuarine bacteria. Acpl. Hicrobiol. 29:
125-127.
Eourquin, A.W., L.A. Kiefer, N'.K. Eerner, S. Crow, and D.G. Ahearn. 1975.
Inhibition of estuarine microorganisms by polychlorinated biphenyls.
Dev. Ind. Microbiol. 16:256-261.
Brown, L.R., E.G. Alley, and D.W. Cook, 1975a. The effect of mirex and carbo-
furan on estuarine microorganisms. Ecological Research Series, U.S.
Environmental Protection Agency, Cincinnati, Ohio.
Brown, L.R., G.W. Childers, and D.D. Vaishnav. 1975b. Effect of Mirex and
related compounds on estuarine microorganisms. Dev. Ind. \
Microbiol. 16:267-266.
Carlson, D.A., K.D. Konyha, W.B. Wheeler, G.P. Marshall, and K.G. Zaylskie,
1976. Mirex in the environment: its degradation to Kepone and related
compounds. Science. 26:939-9^1.
Colwell, R.R. and W.J. Wiebe. 1970. "Core" characteristics for use in
classifying aerobic, heterotrophic bacteria by numerical taxonomy.
Bull. Georgia Acad. Sci. 28:165-185.
Edwards, C.A., 1972. Insecticides. In Organic Chemicals In the Soil
Environment (Goring and Kamake, eds.) Vol. 2, p. 513. Marcel Dekker,
New lork.
-------
Garnas, R.L., A.W. Eourquin and P.H. Pritchard. 1977. The fate and degra-
dation of 1^C-Kepone in estuarine microcosms. In preparation.
Lowry, O.K., N.J. Rosebrough, A.L. Farr, and R.J. Randall. 1951. Protein
measurement with the Folin phenol reagent. J.. Biol. Chem. J_9_3_: 265-275.
Richard, W.W. 1977. Personal Communication.
Schimmel, S.C. and A.J. Wilson. 1977. Acute toxicity of Kepone to four
estuarine animals. Chesapeake Sci. 16:224-226.
Stanier, R.Y., N.J. Palleroni, and M. Doudoroff. 1966. The aerobic pseudo-
monads - a taxonoinic study. J.. Gen. Microbiol. ^3: "159-271.
Trudgill, P.W., R. Widdus and J.S. Rees. 1971. Effects of organochlorine
insecticides on bacterial growth,' respiration and viability. J. Gen.
Microbiol. 69:1.
Tu, C.M. and J.R.N. Miles, 1976. Interaction between insecticides and soil
microbes. Residue Reviews. 64:17-66.
U.S. Environmental Protection Agency. 1975. fact sheet on Kepone levels
found in environmental samples from the Hopewell, Va. area. Health
Effects Research Laboratory. EPA. Research Triangle Park, N.C. Un-
published, 15 p.
Walsh, G.E., K. Ainsworth, and A.J. Wilson. 1977. Toxicity and uptake of
Kepone in Marine Unicellular Algae. Chesapeake Sci. 16:222-224.
bare, G.'w. and C.C. Roan, 1970. Interactions of pesticides with aquatic
microorganisms. Residue Reviews. 33:15.
hiddus, R., P.W. Trudgill, and D.C. Turnell. 1971. Effects of technical
chlordane on growth and energy metabolism of Streptococius faecalis
and Mvcobacterium phlei: a comparison with Bacillus subtilis. J.
Gen, hicrobiol. 69:23-31.
-------
Test of Model for Predicting Kepone Accumulation
in Selected Estuarine Species
1 2
Lowell H. Bahner and Jerry L. Oglesby
U.S. Environmental Protection Agency
Environmental Research Laboratory
Gulf Breeze, Florida 32561
2
University of West Florida
Department of Mathematics and Statistics
Pensacola, Florida 32504
In: Proceedings of the ASTM Second Symposium on
Aquatic Toxicology. October 31-November 1,
Cleveland, Ohio. 1977. (in press)
Gulf Breeze Contribution Number 356
-------
Abstract
•\
Extensive testing has shown that Kepone is rapidly accumulated by
estuarine animals when administered in water or food. Flow-through
laboratory experiments with oysters, shrimp, crabs, and fish indicate
•
that food-chain transfer of Kepone is important in predicting Kepone
residues in estuarine organisms. The rates of Kepone movement
through estuarine organisms were previously unknown; rates of uptake
and depuration by these organisms were determined with a regression
model that describes mathematically the uptake and depuration of
Kepone by these organisms. The model describes biological data as a
single equation, thus allowing variations due to many physical,
chemical, biological, and random error factors to be analyzed
simultaneously.
The direct application of this model to cautious extrapolation
will aid administrative decisions that affect water quality. The
rates calculated by this single-species model also can be used in
developing models that can predict the long-term fate of Kepone or
other pollutants in an estuarine environment.
Key Words: Kepone, estuarine, food-chain, uptake (bioconcentration),
model (mathematical).
-------
Introduction
^
Kepone has been shown to accumulate and depurate from estuarine
animals at rates that vary with the species and the source of
contamination—water or food (Banner et al. 1977). These
laboratory-derived results complement field surveys by determining
whether water, food, or both contribute significant quantities of
Kepone to estuarine animals. However, variability in the laboratory
and field survey data from the James River obscures trends and limits"
full utilization of these pesticide residue data.
Several reports -are available that describe methods of analyzing
laboratory derived fish residue data using kinetic models where
exposure concentrations in water are known and closely regulated
(Blau and Meely 1975, Branson et al. 1975). With minimal uptake
data, these kinetic models can accurately predict equilibrium
residues in fish, however, use of these models for invertebrate
residue data has yet to be demonstrated.
In many instances, such as for field survey residue data, exposure
concentrations, length of exposure, temperature, pH, and salinity are
not known accurately and often several representative invertebrate
and vertebrate species are sampled or tested. Field-exposed animals
-------
can depurate in a clean environment and time-series analyses of these
\
residues are valuable in determining how long biota will retain a
chemical. There is a need to analyze these field and laboratory
uptake and depuration data, so that differences in exposure
•
concentration, differences due to species or size-,lass, or route of
uptake can be statistically examined. Therefore, a generalized
mathematical equation was developed that: 1) describes the available
data and delineates rates; 2) describes uptake from water data; 3)
describes uptake from food data; 4) describes uptake singly; 5)
describes depuration singly; 6) describes uptake and depuration
simultaneously; 7) is expandable to multi-dimensions (describes
uptake and depuration simultaneously by a species exposed to several
concentrations, or describes uptake and depuration simultaneously by
one or several species exposed to one or several pollutants); and 8)
is one equation. This uptake/depuration equation can be incorporated
_J.nto a stochastic, dynamic estuarine ecosystem model to assess
movement of Kepone in James River biota.
-------
Methods
%
Model-Building Process
Initial uptake of persistent chemicals by estuarine animals is
»
normally rapid, but net accumulation diminishes until a constant
concentration is reached (Fig. la). On the other hand, depuration of
chemicals does not follow a consistent pattern, and rate of loss is
dependent upon the nature of the chemical and the test species.
There is variability in most experimental data due to fluctuation in
physical, chemical, and biological factors such as temperature,
chemical concentration, animal size, molt cycle, and seasonal
spawning cycles. Chemical concentrations in water and food (the
variables most critical in determining uptake/depuration) are easily
controlled in the laboratory, however, molting and spawning cycle
variations are difficult to control, and often are affected by a
pollutant's toxic properties. We developed a statistical model to
analyze mathematically the uptake of pollutant chemicals by estuarine
animals in laboratory or field exposures. This method of analysis
aids comparisons of laboratory with field data and permits use in
larger ecosystem models to predict movement of pollutants in
estuarine biota.
-------
A nonlinear statistical model was designed (Daniel and Wood, 1971)
to describe the uptake and depuration of chemicals because: 1)
transformations of the residue data failed to provide an acceptable
model that was linear in the parameters, and 2) one equation was
desired to describe simultaneously both uptake and depuration since
these are not mutually exclusive events. Effects, such as sublethal
toxicity, should be distinguishable by statistically significant
changes in parameter values of this model.
The linear equation, Y=a+bX, would describe uptake of pollutants
if uptake were a linear relationship. Y would be the pollutant
residue at time = X, and a and b would be the intercept and slope
parameters to be calculated by the regression analysis. In a similar
manner, the pollutant residue, Y, and time, DAY, are used by a
non-linear regression procedure to calculate parameters of our
non-linear uptake/depuration equation.
__ The general form of our model found to best fit residue data is:
(1) Y = [ A + 1QC-C x DAY)j-1 _[F + G x e(-D x (DAY - E))]-1f where
A, C, D, F, and G are parameters estimated with the natural logarithm
of pesticide residue, Y, found at time, DAY. The effects of
parameters A and C are graphically illustrated (Fig. 1a). Parameter
A has the value of the uptake curve. The asymptotic residue
-------
concentration (in the animal at the end of exposure), RESIDUE, is
determined by:
In(HESIDUE) = I/A or RESIDUE = e(1/A).
Parameter.D (Fig. 1b) determines the slope of depuration, where D^_0
indicates no depuration and CKLX.7 indicates increasing rapidity of
depuration. E is not estimated from the data but is fixed at DAY =
'beginning of depuration'. In extreme cases, parameters F and G are
required to modify the shape of the curve so the model will describe
the data with greater accuracy. We found that parameters F and G
usually can be replaced by A and 10, respectively, yielding the
model:
(2) Y = [A + 10<-C * DAY)]-1 . [A + 10 x e(-D x (DAY - E))]'1.
Results of this equation are shown graphically to indicate
applicability to depuration data of different rates (Fig. Ic, slow
depuration; Fig. Id, rapid depuration).
-------
Figure 1. (a) Plot of generalized uptake equation. Parameter A
(A=0.125) in this illustration denotes upper asymptote; parameter A1
illustrates effect of increase in value of A to 0.2. Parameter C
determines initial uptake slope; normally, C varies from 0.1 (slow)
to 2.0 (rapid).
(b) Plot of generalized depuration equation: parameter D
determines loss slope and varies from 0.1 (top), 0.5 (mid), to 0.65
(lower); parameter E denotes onset of depuration.
(c) Plot of generalized equation, indicating slow depuration.
(d) Plot of generalized equation, indicating rapid depuration.
-------
lOOOOr
2
<
UJ
oc
•f " A— '•
Asymptote
-/— *—
0 14 28
UPTAKE—H
42
TIME (days)
56
IOOOO
0
14 28 42 56
UPTAKE—H DEPURATION -
TIME (days)
IOOOO
1000
- 100
a
10
14 28 42 56
|—DEPURATION
TIME (days)
lOOOOr
I 1000
UJ
Q
05
UJ
o:
14
28 42 56
-I — DEPURATION ^
TIME (days)
-------
Both models (1) and (2), are nonlinear in the parameters and
require an iterative method to estimate those parameters. The method
of numerical analysis developed by Marquardt (1953) was chosen
primarily because of its wicie acceptance and general use. A typical
program dataset (exclusive of job control), using the S.A.S. liLIiJ1
procedure, follows:
DATA KEPOHE; INPUT SPECIES $ COI.'C TISSUE $ RESIDUE DAY;
IF RESIDUE EQ 0 THEN Y = 0;
IF RESIDUE UE 0 THEN Y = LOG (1000*RESIDUE);
CARDS;
0.00 0
0.50 7
0.86 15
0.99 30
0.87 37
0.57 44
0.38 54
0.000 0
0.038 7
0.072 9
O.OS8 14
0.120 21
0.037 28
0.100 30
0.100 35
0.084 42
0.035 49
0.023 56
PROC SORT; BY SPECIES CONC TISSUE;
PROC PHIirf; BY SPECIES COUC TISSUE;
PROC iJLIU BEST = 05 ITER = 200 METHOD = MARQUARDT; BY SPECIES CONC
TISSUE;
FARMS A = 0.1 TO 0.3 by 0.1
C = 0.1 TO 0.7 by 0.2
D = -.1 TO 0.3 by 0.2;
0 = (A+(10**(-C*DAY))); E = 28;
R = (A+10**EXP(-D*(DAY-E)));
MODEL Y = Q**(-1) - K**(-1);
DER.A = -Q**(-2)) + R**(-1);
DLH.C = (Q**(-2))*(10**(-C*DAY))*DAY*LGG(10);
SPOT
SPOT
SPOT
SPOT
SPOT
SPOT
SPOT
SHRIMP
SHRIMP
SHRIMP
SHRIMP
SHRIMP
SHRIMP
SHRIMP
SHRIMP
SHRIMP
SHRIMP
SHRIMP
.4 FILLET
. 4 FILLET
.4 FILLET
.4 FILLET
. 4 FILLET
.4 FILLET
.4 FILLET
.023 WHOLE
.023 WHOLE
.023 WHOLE
.023 WHOLE
.023 WHOLE
.023 WHOLE
.023 WHOLE
.023 WHOLE
.023 WHOLE
.023 WHOLE
.023 WHOLE
-------
LO.D = -10*(DAY-E)*EXP(-D*(DAY-E))*R**(-2);
WEIGHT = DAY;
^Statistical Analysis System, SAS Institute Inc., Raleigh, li.C.
Mention of coriirnercial products does not necessarily constitute
endorsement by the U.S. Environmental Protection Agency.
-------
Results and Discussion
%
Our uptake/depuration statistical models (Equations 1 and 2) were
validated using raw data from uptake and depuration of
di-2-ethylhexyl phthalate by fathead minnows (Pi::iphales pronielas)
(Dranson, Blau, and Mayer 1977). Nine sample periods were
represented with four replicate single-fish samples for the seven
concentrations tested. The parameter estimates for each
concentration tested were generated from the five-parameter (Equation
1) and three-parameter (Equation 2) model equations; standard linear
lack-of-fit tests were applied using the calculated parameters and no
lack-of-fit was evident (Table 1). Dioconcentration factors (ECF),
the concentration in animal at chemical equilibrium * concentration
in exposure water, were calculated using our statistical models. The
BCFs from our models and the Dow Chemical DIOFAC model (Elau et al.
1975) are listed for comparison.
Dioconcentration factors and bioaccumulation factors (3AF,
concentration in animal at chemical equilibrium ^ concentration in
exposure food) are shown for six species of estuarine animals exposed
to Keporie in water, food, or both (Table 1). Uptake-only and
depuration-only data were analyzed using the appropriate portions of
-------
Table 1. Parameter estimates for five-parameter model3 or three-parameter model that describe uptake and
depuration of di-2-ethylhexyl phthalate by fathead minnows and uptake and depuration of Kepone for
six species of estuarine animals exposed to Kepone in water, food, or both.
Exposure Route of
Species Concentration Uptake
(ug/£ or yg/kg)
fathead3
minnow
"
"
"
"
It
"b
oyster^
oyster3
mysidsc
n
grass shrimpb
n
1.9 fresh water
2.5
4.6
8.2
14.0
30.0
62.0
.03 sea water
.39
.026 sea water
.41
.023 sea water
.40
" Lafayette R. unknown
blue crab"
"
"
n
n
sheepshead
minnows
spot'3
M
.03 sea water
.3
250.0 food
250.03 food + water
250.30 "
.05 sea water
.029 sea water
.40
, A
0.141
0.133
0.124
0.125
0.120
0.114
0.110
0.184
0.122
0.197
0.118
0.212
0.124
0.155
no
"
0.211
0.217
0.202
0.177
0.216
0.144
C
1.392
1.455
1.416
1.763
1.741
1.699
- 1.680
1.099
5.136'
.293
.465
.183
.220
1.
detectable
n
.136
2.624
.123
.354
.168
.304
Parameters
D E
.702
.202
.261
.238
.688 t
.703 .
.074
.621
.303
-
—
.0980
.0994
.0827
up cake
"
-.0571
-.125
-.0324
-.0561
.0923
.0850
56
56
56
56
56
56
56
28
28
-
—
28
28
—
28
28
28
28
30
30
Model
F G BCFd
or
BAF
.751 76. 820 f 630
.504 6.153 ' 733
.564 11.911 680
.674 11.079 371
.773 165.856 304
.752 73.976 220
141
7641
.194 1.083 9305
6160
11686
4862
7949
— — —
.457
.401
.564
5684
3534
2593
Q
Biof ac
BCF
774
915
838
467
364
252
204
aY = 1/((A-HO**(-C*DAY)) - (F + G*EXP(-D*(DAY-E)))); five parameter model.
bY = 1/C(A+10**(<-C*DAY)) - t(A+10*EXP(-D* (DA.Y-E)))) J three parameter mod.cl.
CY = 1 (A+10**(-C*DAY)); uptake only.
, (I/A)
dBCF=Moconcentration Factor = e
(cone, in water); BAF = Bioaccumulation Factor = e ''I (cone, in food)
M-ii
1Q77
-------
the three-parameter model (Table 1). The three-parameter model
(Equation 2) was also used to describe the laboratory depuration of
Kepone from grass shrimp collected in the Lafayette River, near
Norfolk, Virginia. Application of this model to depuration-only data
gave excellent results, indicating that data from field-exposed
animals can be used to derive all parameter estimates except C, the
uptake rate parameter; C was arbitrarily set to 1.0 for these data (C
should be chosen so that 10^~c*DAY)is 10.00001).
Parameter estimates for uptake and depuration of Kepone in six
species of estuarine animals were calculated with our five-parameter
(Equation 1) and three-parameter (Equation 2) statistical models
(Table 1). The three-parameter model (Equation 2) sufficiently
described (by graphical analysis) all uptake from water data except
that for oysters exposed to 0.39 yg Kepone/i. In this instance, the
five-parameter (Equation 1) model was necessary for generation of
adequate parameter value estimates. The three-parameter model was
also sufficient to describe the uptake and retention of Kepone in
blue crabs that were fed 0.25 yg Kepone/g, regardless of whether 0.03
or 0.3 yg Kepone/£ seawater were also administered to the crabs.
Graphs of the computed models and data for several of the species
tested are shown (Figs. 2-6).
-------
Fig. 2. Model representation of bioconcentration of Kepone from water containing average measured
concentrations of 0.03 or 0.39 ug/fc by oysters (Crassostrea virginica) exposed for 28 days, and its
depuration by oysters placed in Kepone-free water for 28 days. Parameter estimates for oysters exposed
to 0.39 pg/JZ. were: A=0.122, C=5.136, D=.303, F=.194, and G=1.083. For oysters exposed to 0.03 Mg/%
A=0.184, C=1.099, and D=.621. Lower limit of analytical detection was 10 yg/g.
-------
CP
c
C/)
cr
UJ
I-
co
>
o
UJ
z:
o
Q.
UJ
lOOOOr
1000
100
0
14
-UPTAKE
28
42 56
DEPURATION
TIME (days)
-------
Fig. 3. Model representation of bioconcentration of Kepone from water containing average measured
concentrations of 0.026 or 0.41 yg/£by mysids (Mysidopsis bahia) exposed for 21 days. Parameter estimates
for mysids exposed to 0.026 pg/£ were: A = 0.197 and C = .293. For mysids exposed to 0.41 yg/JL, A = 0.118
and C = .465. Lower limit of analytical detection was 20 ng/g.
-------
en
c
C/)
O
2
LJ
O
Q.
UJ
10000
1000
100
10
0
14
UPTAKE-
21
TIME (days)
28
-------
Fig. 4. Model representation of bioconcentration of Kepone from water containing average measured
concentrations of 0.023 or 0.40 ug/£ by grass shrimp (Palaemonetes pugio) exposed for 28 days, and its
depuration by shrimp placed in Kepone-free water for 28 days. Intermediate curve represents depuration
of Kepone from grass shrimp collected in the Lafayette River, Norfolk, Virginia, and held in Kepone-free
seawater at the ERL, Gulf Breeze, for 21 days. Parameter estimates for shrimp exposed to 0.023 ^Jg/2. were:
A=0.212, C=.183, and D=.098. For shrimp exposed to 0.40 yg/Jl, A=0.124, C=.22, and D=.099. For Lafayette
River shrimp, A=0.155 and D=0.083. Lower limit of analytical detection was 20 ng/g.
-------
O»
v.
o>
c
I
CO
en
o:
10000
o
0.
UJ
1000
100
10
0
0
14
UPTAKE-
o
v
\
A— -
Lafayette River
\
0.023 pg/l
28 42
H DEPURATION
TIME (days)
56
-------
Fig. 5. Model representation of bioaccumulation of Kepone from food and water by blue crabs
(Callinectes sapidus) exposed for 28 days followed by 28 days of consuming food and water containing
no Kepone. Parameter estimates for crabs exposed to 250 ng/g were: A=0.211, C=.136, and D=-.057. For
crabs exposed to 250 ng/g plus 0.3 ug/£> A=0.202, C=.123, and D=-.032. Lower limit of analytical detection
was 10 nc/a.
-------
lOOOOr
o>
en
c
(/)
CD
<
o:
o
UJ
ID
CO
UJ
2
o
Q.
UJ
1000-
100-
0
14
UPTAKE
42
56
TIME (days)
-------
Fig. 6. llodel representation of bioconcentration of Kepone from water containing average measured
concentrations of 0.029 or 0.4 }ig/l by spot (Leiostomus xanthurus) exposed for 30 days, and its depuration
by fish placed in Kepone-free water for 24 days. Parameter estimates for spot exposed to 0.029 yg/£ were:
A=0.216, C=.168, and D=0.092. For spot exposed to 0.4 yg/£, A=0.144, C=.304, and D=.085. Lower limit
of analytical detection was 20 ng/g.
-------
"5 loooo
o>
c
1-
CL 1000
y
o
X
^ IOO
^* \ \J \J
z
LL)
1 10
LJ
_
(
•— • J
/
/
/
/
/ C
/ /^°
.//
f /
I /
* / i
'/ »
»
3 14 28
i ir>TA istr -*»
0.4 ^g/l
k _ ^P /
^"^•^.^^^
^ n
0.029 ^g/l 0<^^^
42 56
r\r"ni ir»A-ri/^M w
TIME (days)
-------
Extrapolation from Model
%
Assuming the parameter values generated sufficiently describe the
uata, extrapolations to the time required for grass shrimp or spot to
depurate 90* of their Kepone burden can be made. 3y applying the
•
parameter values (Table 1) to the three-parameter model, 39 or 35
days are required for grass shrimp exposed to 0.023 or 0.4 vg/£,
respectively, to depurate 90* of their Kepone burden and 43 days for
grass shrimp collected from the Lafayette River. Spot would require
41 or 42 days to depurate 90£ of their Kepone residues if previously
exposed to 0.029 or 0.4 pg Kepone/&, respectively.
«r •""
Extrapolation was not necessary for the oyster data, since
non-detectable concentrations were reached. Insufficient data exist
to determine the time needed for sheepshead minnows to depurate to
nondetectable concentrations. Data and the model demonstrate that
Kepone was not depurated from blue crabs.
-------
Conclusions
^
The stochastic uptake/depuration model sufficiently described
uptake and depuration of Kepone by estuarine oysters, shrimp, crabs,
•
and fiiih, regardless of whether the pesticide was in water or in
food. In addition, the model described depuration-only data, so that
field-exposed animals could be utilized to project depuration rates
and to allow estimation of the time required for residues to reduce
to non-detectable concentrations. The ultimate residue concentration
in exposed animals is predicted to depurate to zero concentration, if
** •*"
parameter D of the three-parameter model is greater than zero and
given that time is sufficient. The exponential depuration portion of
the model gives an accurate fit to data regardless of depuration
rate, and given sufficient data, extrapolations can be made
cautiously. All parameters in the model have confidence intervals;
therefore statistical inferences (statistical significance) can be
judged with joint confidence region comparisons. For example, the
model can be used to determine if pesticide residues from laboratory
exposures are statistically different than those from field
exposures.
-------
If expanded, the present single-species, single-concentration
\
model should produce multidimensional models that describe
cheraical-species-dose-time interactions. Expansion of the number of
parameters in the model will be limited by the number of replicated
»
data points that are available. Because thorough, replicated
sampling schemes testing multiple concentrations are not, possible for
all pollutants, attempts should be made to categorize parameter
estimates for chemicals, species, and exposure concentrations.
Successful categorization of the model parameters would allow use of
generalized parameter estimates in dynamic ecosystem models.
-------
^ References
[1] Banner, L.H., Wilson, A.J., Jr., Sheppard, J.M., Patrick, J.H.,
Jc., Goodman, L.R., and Walsh, G.E., Chesapeake Sci.,
Vol. 18, 1977, pp. 299-308.
[2] Blau, G.E. and Neely, W.B., Adv. Ecological Research,
Vol. 9, Academic Press, 1975, pp. 133-163.
[3] Branson, D.R., Blau, G.E., Alexander, H.C., and Neely, V.'.B.,
Trans. Arn. Fish. Soc., 1975, pp. 785-792.
[4] Daniel, C., and Wood, F.S., Fitting Equations to Data,
John Wiley and Sons, New York, 1971, pp. 5-115.
[5J Marquardt, D.W., J_. Soc. Indust. ApjxL. Math.,
Vol. 11, 1963, pp. ^31-441.
[6] Branson, D.R., Blau, G.E., and Mayer, F.L., "Bioconcentration
kinetics of di-2-ethylhexyl phthalate in fat head minnows,"
— Environmental Contamination by Industrial Organic Chemicals
Symposium. Assoc. of Official Anal. Chemist 1977 national
Meeting. Washington, D.C. Oct., 1977.
Blau, G.E., Neely, W.B., and Branson, D.R., AIChE Journal,
Vol. 21, 1975, pp. 854-861.
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The Fate of ^C-Kepone in Estuarine Microcosms
*R. L. Garnas, A. W. Bourquin and P. H. Pritchard
U.S. Environmental Protection Agency
Environmental Research Laboratory
Gulf Breeze, Florida 32561
Presented at 175th National Meeting of the
American Chemical Society, Anaheim, California
March 16, 1976 - Pesticide Chemistry, paper 59-
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Kepone was developed by the Allied Chemical Corporation in the early
1950's and was manufactured in Hopewell, Virginia intermittently until 1974.
In 1973 Allied Chemical subcontracted the .production of Kepone to Life Science
Products Company, which operated from a building located adjacent to Allied
Chemical's plant on the James River, which empties into Chesapeake Bay.
Their plant on the James River operated 24 hours a day, seven days a
week, and manufactured 3,000 to 6,000 pounds of Kepone per day. Over 90$ of
»
the 1.7 million pounds of Kepone produced by Life Science during its 16 months
of active operation was exported to Latin America, Europe, and Africa for
»
control of insect pests. The effluents from the plant were hooked directly to
Hopewell's sewage treatment plant. Kepone in the effluents disrupted the .
normal biological treatment process and put the plant out of operation. Later
investigations revealed that sediment from adjacent Bailey's Creek and waste
water from the sewage treatment and landfill areas contained Kepone at levels
/
between 0.1 and 10 ppm. Sludge samples taken from the holding pond and from
the landfill near the Hopewell sewage treatment plant contained 200-600 ppm of
Kepone.
In August 1975 the Life Science plant was closed. EPA collected
monitoring data showing widespread dissemination of Kepone in the James River
system. Kepone in the water was detected at levels between 0.1 and 4 ppb;
residues in fish and shellfish were between 0.1 and 20 ppm. The Food and Drug
Administration set the allowable limit of Kepone in fish and shellfish
consumed as food at 0.1 ppm. The James River was closed to fishing in
December 1975.
In October 1976, Allied Chemical Corporation was fined $13.2 million for
its role in polluting the James River with Kepone. Life Science Products
Company itself was fined $3-8 million. It is now estimated by the scientists
-------
constituting the Kepone Task Force that approximately 200,000 pounds of Kepone
have been disseminated into the James River system.
SLIDE ONE: Conceptual Fate of Kepone
Following the contamination of the James River system with Kepone, the
Environmental Research Laboratory at Gulf Breeze responded with necessary data
about the toxicity of Kepone to estuarine organisms and its potential for
bioaccumulation. and biomagnification. Serious questions arose concerning the
fate of Kepone in the river. The fate of a pollutant is closely related to
its toxicity; forces such as volatilization, sorption, metabolism, and abiotic
•
transformation (photolysis, hydrolysis, chelation) affect the availability and
toxicology of pollutants to aquatic species. A knowledge of the sites of •
Kepone concentration and rates of exchange associated with these sites is
necessary for long term regulatory actions. Sorption and transformation data
are needed to determine whether the ecosystem can remove the pollutant by
degradation or eventual washout, or whether physical assistance from dredging
or damming is necessary.
The conceptual fate of Kepone shown in this slide was developed and
modeled by the Gulf Breeze Laboratory through a research project with
Manhattan College. The projection of time required to reduce the levels of
Kepone by various natural processes such as adsorption-desorption and trans-
formation are included as an important phase of the project. Unfortunately,
insufficient data are available for Kepone.
A variety of laboratory microcosms have been developed to supply fate
information for this modeling effort. The following presentation is a brief
summary of the research effort we conducted with Kepone in these systems.
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SLIDE TWO: Kepone
Kepone is an extremely stable member of the cyclodiene insecticides, with
a molecular structure similar to Mirex. However, due to the presence of an
oxygen atom in the molecule, Kepone is more water-soluble through hydrogen
bonding than Mirex. Within a pH range of 4-6, the solubility of Kepone ranges
from 1.5 to 2.0 ppm. An increase of pH to 9-10 increases the solubility to
5-70 ppm. Although Kepone has a molecular configuration similar to Mirex, it
*
should not be assumed to behave like Mirex in the environment.
SLIDE THREE: Static Fate System
•
This system consisted of a 125 ml Erlenmeyer flask fitted with a stopper.
A capillary glass inlet allowed introduction of air or nitrogen; the gas exit
was fitted to a disposable Pasteur pipette filled with XAD-4 resin (Rohm and
Haas, Philadelphia, Pa.) to trap volatile compounds. The small size of the
system allowed maximum replication and examination of different environmental
substrates and processes. Similar systems were sampled sequentially with time
to indicate rates of transport and transformation. ^C-Kepone was used to
minimize involved analysis and to facilitate simulation of environmental
levels. The environmental parameters tested in this study were sediment type,
salinity, aerobicity, pH, temperature, sunlight concentrations,
volatilization, and biological forces.
Standard experimental conditions included 10 gm (wet weight) of sediment;
100 ml of Santa Rosa Sound water (18-24 ppth); constant temperature of 25°C;
12/12 hr. diurnal lighting (G.E. Vita Gro); water saturated air (Silent
Giant); 19.9 yg 1l
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SLIDE FOUR: l4C-Kepone Distribution in Static Fate System
In the standard analytical procedure, the system was fractionated into
water suspendable particulate, and unsuspendable sediment (i.e., sand) by
repeated rinsing of the system with equivalent salinity water; James River
sediments were all suspendable (very little sand). Following centrifugation
(3000 RPM), an aliquot of the water was examined for radioactivity by
scintillation counting. The sediment fractions were extracted repeatedly with
*
acetonitrile (Pe), with aliquots taken for scintillation. Following the
addition of 2% sodium sulfate water to the acetonitrile extracts (^1:1
•
water/solvent), the aqueous fractions were extracted with 1:1 petroleum
ether/diethyl ether and analyzed by thin layer chromatography (3:1 diethyl .
ether/n-hexane; Quanta Gram LQGDF, Quanta Industries, Fairfield, New Jersey)
and autoradiography (Birchover Radiochromatogram Spark Chamber, Hitchin,
England). Periodically, the extracts were cleaned on florisil columns (2 gm,
hexane washed; 20 ml rinse of 5% diethyl ether/hexa-^e; final elution with 50
ml of 1/t methanol/benzene) and analyzed for Kepone, octachloro-Kepone, and
nonachloro-Kepone (standards provided -by A. J. Wilson, EPA, Gulf Breeze,
Florida) by electron capture gas chromatography (Hewlett-Packard GC Model
5830A; Ni63detector; Model 1&850A GC terminal; 2 mm id x 2 m glass, 2% OV-101
on Gas Chrom Q, 100/120). Extracted sediments (Pc) were dried and combusted
at 900° (Harvey Instrument OX-200 Combustion System, Hillsdale, New Jersey) to
liberate and trap residual radioactivity as ^CC^.
The experimental results revealed that within one to two days, approxi-
mately 75 to 80$ of the total radioactivity added to the system was found
associated with the sediment. Most of this radioactivity (Pe) was adsorbed"to
the suspendable particulate material. Kepone, which could not be extracted
from this material by organic solvent (Pc), constituted approximately 2 to 6%
-------
of the total radioactivity. Very little Kepone was associated with the
heavier sand material in the sediment. Virtually no Kepone volatilized out of
the flasks (R) (no radioactivity in resin traps). The remainder of the
radioactivity, that is 15 to 20% of the Kepone initially added, remained in'
the water column (W). In most experiments, total recovery of the
radioactivity from the flask was between 90 and 95J. No evidence for the
production of degradation products at any time was seen. Long-term incu-
•
bations did not change this basic distribution of Kepone in the flask. When
similar systems were sterilized with 2% formaldehyde or forced to become
•
anaerobic by bubbling with nitrogen gas, a similar distribution of Kepone was
apparent. Similarly, changing salinities, exposure to sunlight, varying
concentrations of Kepone, and differing temperatures, all showed very little
effect on this basic Kepone distribution pattern. Under all of these
conditions, no evidence (total recovery of radioactivity as Kepone) was
obtained for degradation of the pollutant.
In further tests with these screening systems, a sediment washing step
was included to determine the desorption capability of Kepone from particulate
material. This procedure consisted of washing Kepone-containing sediment
•
three times with clean seawater. The washed sediments were then placed in a
flask, covered with a column of fresh seawater, and incubated for three days.
At the end of this incubation, the amount of radioactive Kepone in the water
column was determined. This procedure was followed on test systems for the
final three week sampling period. Typically, 9 to 11? of the radioactivity in
the sediment desorbed into the water column in the first week wash. Another M
to 8? of the radioactivity in sediment leached into the water column during.
each of the second and third week periods. Overall, 20 to 40? of the total
radioactivity in the sediment was desorbed.
-------
From these experiments, we conclude the following:
A. Kepone has a propensity for adsorption to and desorption from
sediment. Most of the Kepone in the sediment was associated with the
lighter suspendable particular matter and not with heavier sand
particulate.
B. Kepone does not volatilize out of an aquatic ecosystem and there was
no transformation or degradation of the Kepone.
*
C. The distribution of the Kepone between the water and the sediment
(i.e., the Kp value), was not altered by changing environmental
•
conditions such as aerobicity, temperature, Kepone concentrations,
sunlight, and salinity.
SLIDE FIVE: Adsorption Isotherms for Kepone in Sediments
Adsorption isotherms are shown on this graph for various environmental
and reference substrates. The adsorption data for all systems fitted linear
isotherms over a broad range of water phase concentrations. The Kp's of
Kepone show an increase with increasing organic carbon content from quartz
sand (O.C. r 0.01$) to Range Point salt marsh detritus (O.C. = 25%). Quality
of organic carbon, as well as quantity, influenced the Kp of Kepone. Ground
seagrass (Thalassia), which was aged for several weeks in raw flowing
seawater, displayed an organic carbon content of 60?. The Kp for Kepone with
this substrate was lower than that for sediment from a local salt marsh with
lower organic carbon content (O.C. = 25%). The presence of animal detrital
matter in the saltmarsh sediment could account for this anomaly.
Field samples from the James River System (open and closed symbols)
showed the same correlation with organic content of the sediments. With
sediments of lower organic content, insufficient Kepone was present in the
water to determine Kp values.
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SLIDE SIX: Schematic of Continuous Flow System
Continuous-flow microcosm studies allow the dynamic nture of an ecosystem
to be investigated in the laboratory. They provide information on the rate
and extent to which a pollutant will a) move into the various compartments-of
an aquatic ecosystem and b) be transformed by biological and nonbiological
forces.
The system consists of a reactor vessel containing the substrates and .
•
biota of interest. Raw seawater can be fed continuously to the system. The
aeration apparatus samples exiting air for volatile organics and ^CCt2~
•
To follow the fate of pollutants resulting from the biological activity
of aquatic macro biota, two continuous flow systems were structured with 9 em
of Range Point sediment and 24 liters of Santa Rosa Sound water. The systems
were allowed to acclimate statically with aeration for 48 hrs. Twelve
lugworms (Arenicoj.a cristata) were added to one of the tanks and flow was
/
started through both systems (D = 0.04 hr'"1); after four days, the water flow
was stopped and each system was spiked with 1 mg (1 x 10^ dpm) of ^C-Kepone
in 0.5 ml acetone. After two days the water flow was again started. Aliquots
of water (3 ml) were sampled directly for radioactivity until background
levels were obtained. At that point larger sample volumes of water were
required for detection of radioactivity. Thereafter, the radioactivity was
concentrated on two beds of XAD-4 resin (75 ml wet volume in 250 ml separatory
funnel). Following removal from a system, the resin was extracted with
methanol in a Soxhlet extractor overnight and analyzed directly for
radioactivity.
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SLIDE SEVEN: Desorption Kinetics for Continuous Flow Systems
The resin was efficient in the removal of 1i4C-Kepone from raw seawater
(99/t); the Soxhlet extraction procedure removed all radioactivity from the
resin throughout these studies (95% recovery).
The graph shows the washout of radioactivity, plotted as a function of
the radioactivity remaining in the sediment at any time. By Day 5 all of the
lugworms were dead, with fungal mats forming over the tissues. One lugworm
•
was removed from the system and found to contain high levels of radioactivity
in its tissues (3% of total spike to system); all of the radioactivity was
•
Kepone. At these levels of accumulation, the lugworms added to the system
contained a large fraction of the original fortification (3$ x 12 = 36%). The
other dead lugworms were left in the system, where they eventually decayed and
completely decomposed. Desorption followed simple first-order kinetics and.
gave a desorption rate of .002 hr~1 (that is, .002 of the original Kepone in
s
the sediment desorbed per hour). This desorption rate was a reflection of the
diffusion of Kepone off the sediment material and was, therefore, independent
of flow rate. Numerous water samples were checked for the presence of
particulate matter containing adsorbed radioactive Kepone. In all instance^,
of the radioactivity in the water, only 5 to 7% was associated with this
particulate material and, therefore, washout was not a function sediment
scouring. Thus, the radioactivity in the water was a true reflection of the
dissolved Kepone present.
Since desorption of Kepone is probably a simple diffusion process, its
ultimate distribution within the James River system will depend on the
hydrodynamics of the river itself. However, ingestion of Kepone by a bentMc
organism could decrease the rate of desorption due to an additional
sequestering compartment. These experiments indicate that the presence of •
-------
polychaete worms in the sediments of the flow-through systems increased the
amount of Kepone adsorbed into the sediments relative to worm-free control
systems. This was presumably due to a bioaccumulation. In this particular
experiment, Kepone was toxic to the polychaete worms and their eventual death
and decomposition also resulted in a slower release of Kepone from the system.
The slower rate of desorption is presumedly not due to increased amounts of
organic material from the worm (not greater than 0.01^ of the natural organic
*
matter present), but is more likely a different form of sequestered Kepone
that was desorbed slower. These data indicate that desorption of Kepone from
•
sediment was a function of the quality of organic matter present and not the
quantity. For additional sorption studies ^C Kepone-containing water (0.1.
ppm) was continuously passed over a sediment bed at a dilution rate = 0.01
hr~1.
SLIDE EIGHT: Conclusions for Kepone Transport.
The adsorption capacity was large and would probably not reach saturation
levels within reasonable time limits. The amount of Kepone adsorbed to the
sediments in these experiments was a function of the rate at which the water
passed over the sediment. Kepone adsorbed to the top 2-3 cm of sediment in,
these studies and little diffusion down the sediment column was observed. The
removal of Kepone from inflowing water (60% at 0.01 hr~1) was constant with
time over 600 hours of flow. If the flow to the reactor vessel was stopped,
preventing additional Kepone from entering the system, the residual Kepone .in
the water continued to be adsorbed rapidly into the sediment.
SLIDE NINE: Schematic of Intact Core System
The final system used to study the fate of Kepone in the James River •
estuary was a series of microcosms referred to as Eco-cores. These systems
consist of glass tubes (30 mm in diameter and 38 cm long) which can be
-------
inserted into the water and into the underlying sediment layer to extract an
intact core. These cores are then stoppered into the glass column, brought
back to the laboratory, and tested for their biodegradation capacity. In the
case of the James River, direct coring was not possible and thus sediment and
water samples collected from the James River were mechanically placed in the
glass columns to simulate a cored sample. In addition to the James River
sediment studies, simultaneous cores were taken from a local salt-marsh near
•
Pensacola Beach and studied in a similar manner. The advantage of these
Eco-cores, besides their potential for looking at processes in intact core
•
samples, is that they allow a large number of environmental parameters to be
tested on a particular degradation process taking place within the cores.
These degradation processes were monitored as follows: Once the core has been
taken and brought back to the laboratory, the glass column was outfitted with
an aeration device and the water in the core spiked with radioactive Kepone at
/
a concentration of 500 ppb. The aeration of the water column disperses and
mixes the Kepone and ensures contact with the sediment. These cores were then
incubated at 25°C. Degradation was measured two ways: (a) Exiting gas from
the cores was bubbled through alkaline solutions to trap radioactive carbon
dioxide released from the radioactive Kepone; (b) Samples were taken from the
water column and analyzed (by the same basic procedures used in the static
system) to obtain the amount of Kepone remaining at any time and the presence
and amount of any degradation products formed. After sufficient periods of
incubation (i.e., diminished activity), the distribution of radioactivity in
the cores was analyzed. Vilater and sediment were removed from the core and
each component extracted and analyzed for the presence of radioactivity and
the presence of any degradation products from the Kepone.
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SLIDE TEN: Kepone Degradation .Studies
SLIDE ELEVEN: Data from Coring System
A variety of conditions were varied in all of the systems described to
observe the effect on the degradation of Kepone. Extremes in salinity,
temperature and light did not alter Kepone. Analysis by GLC showed no
detectable dechlorinated products of Kepone in any of the systems examined.
Since some chlorinated hydrocarbons are known to undergo dechlorination
»
reactions under anaerobic conditions, cores were set up under a variety of
environmental conditions. For example, cores were made anaerobic by bubbling
•
with nitrogen gas; cores were made anaerobic and supplemented with glucose and
other organic materials; and cores were maintained aerobically and
supplemented with organic substrates which might stimulate the microbial
degradation of Kepone. In all cases no evidence for Kepone degradation was
obtained. In the analysis of several cores maintained under anaerobic
conditions, thin layer chromatography revealed extraneous radioactive products
which chromatographed separately from Kepone.
SLIDE TWELVE: Kepone Analysis
With anaerobic conditions in Eco-core experiments, thin layer auto-
radiography analysis revealed two radioactive spots, one of which was Kepone.
Radioactive profiles obtained with high pressure liquid chromatography also
showed similar radioactive distribution. When rechromatographed on TLC or
HPLC the unknown radioactive spot migrated with the same Rf. In contrast,
extracts of exposed lugworms displayed two radioactive areas; however, when
rechromatographed, the unknown spot migrated at the Rf of Kepone. An isolate
from estuarine sediments (corynebacteriurn-like) previously grown on minimal •
media and camphor as the sole carbon source, also displayed the unknown
product. Mild acid treatment of the unknown would cause it to chromatograph-
-------
like Kepone. The unknown spot failed to elicit a response on GLC-ECD at the
Rt of Kepone and did not chromatograph favorably under most conditions. At
this time the material obtained from the anaerobic sediment/water systems and
the pure culture study are being examined in greater detail by GCMS and
selective detectors.
CONCLUSIONS
At this time, we believe that Kepone does not degrade in our experi-
•
mental systems. The reversible quality of the unknown back to Kepone causes
us to suspect that a strong association is occurring from the presence of the
•
oxygen atom in the molecule with other chemicals in the extracts and not
degradation. Kepone did not volatilize from any of the systems described.
Kepone displayed a dynamic movement potential in sediment/water systems.
The rate of this movement was related to the organic content of the sediment
and to the quality of the organic fraction.
/
These systems were used to indicate the major environmental components
affecting the fate of Kepone. Extrapolation of these data to the James River
system is hampered by severe scaling problems. However, these studies do
validate current field monitoring practices and offer direction for future
field work to supplement the existing data base for mathematical modeling.
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A BENTHIC BIOASSAY USING TIfC-LAPSE PHOTOGRAPHY
TO MEASURE THE EFFECT OF TOXICANTS ON THE FEEDING BEHAVIOR
OF LUGWORMS (POLYCHAETA:ARENICOLIDAE)
Norman I. Rubinstein
Faculty of Biology
The University of West Florida
Pensacola, Florida
IN: Symposium on Pollution and Physiology of Marine Organisms
(Eds) W.S. and J.F. Vernberg. Academic Press. 1977. (in press)
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ABSTRACT
A benthic bioassay was developed utilizing time-lapse photography to •
measure the feeding activity of a lugworm, Arem'cola cristata. Automated 35 mm
cameras were used to record formation of feeding funnels at 12-hour intervals.
Substrate surface area reworked by lugworms held under identical conditions in
separate aquaria was plotted against time to" determine substrate reworking rates
for each group. Rates were subjected to linear regression analysis and compared
to demonstrate that no significant difference between the slopes of the calculated
lines existed. Therefore, a difference in slope when one group is. exposed to a
toxicant could provide a measure of effect on lugworm activity. To demonstrate
the applicability of this approach, lugworms were exposed to the pesticide,
Kepone, and their rate of substrate reworking was compared with non-dosed lugworms.
Kepone was acutely toxic to lugworms at a concentration of 29.5 yg/£. A
significant difference in substrate reworking rates was observed following exposure
to concentrations as low as 2.8 yg/£ Kepone in seawater. It is suggested that a
behavioral response to toxicity testing provides a sensitive and realistic
approach for evaluation of ecological impact of pollutants on the marine environ-
ment.
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INTRODUCTION
Federal legislation (i.e. the Marine Protection, Research and Sanctuaries
Act of 1972) requires that permits for the discharge of materials into coastal
waters be evaluated on the basis of their ecological impact on the marine
environment. Under this legislative mandate the U.S. Environmental Protection
Agency (EPA) has been delegated the responsibility to establish guidelines for
conducting bioassays used to define the types and amounts of materials that
may be released into the marine environment. The bioassay, therefore, serves
as a regulatory tool used by federal agencies and private industry to assess
the ecological impact of pollutants on the marine environment.
Bioassay procedures recommended by EPA (1976) for conducting-toxicity
evaluations utilize a variety of sensitive epibenthic and pelagic species but
do not include representative infaunal organisms. This is due, in part, to the
relative lack of sensitivity displayed by many infatmal species and the difficulty
in observing biological effects while organisms are buried in sediment. However,
many macrofaunal invertebrates are deposit feeders that have a great effect on
the benthic community as a result of their substrate reworking activity. These
organisms have been shown to in/luence benthic community trophic structure and
sediment stability (Rhoads and Young 1971). In addition, sediment processing
organisms provide a pathway for cycling organic material, nutrients and pollutants
between the sediment and the water column (Rhoads 1973, Meyers 1977). Therefore,
meaningful evaluations of the impact of a pollutant on the marine environment
must include information regarding effects on representative members of the
infaunal community. The objective of this study was to develop a sensitive and
practical method that could be used to assess biological effects of pollutants
on estuarine and marine infaunal organisms.
-------
Various benthic organisms including holothurians, crustaceans, pelecypods
and polychaetes, produce distinct, characteristic topographical features on the
substrate surface as a result of their normal activity (i.e. feeding, burrowing
and excretion). These features in some cases may serve as in situ indicators of
the organisms' activity (Rowe et_ aj_. 1974). With the aid of time-lapse photography,
surface features can be monitored and analyzed statistically to determine the
relative effects of xenobiotics on organisms selected for study.
The benthic bioassay described here utilizes time-lapse photography to
measure the formation of feeding funnels produced by the lugworm, Arenicola
cristata Stimpson. Comparisons of rates of feeding funnel formation between
exposed and control lugworms serve as the test criterion.
Ecological Significance
Lugworms are sedentate polychaetes distributed throughout the world in
most littoral habitats. Their activities, which are^omewhat analogous to
those of the earthworm, are responsible for bioturbations of the sediment to
depths as great as 50 cm. Populations of the european species, Arenicola marina,
have been observed to turn over nearly 500 tons of sand (dry weight) per acre
per year (Blegvad 1914).
Lugworms form u-shaped burrows in a variety of substrates ranging from
silt and mud to coarse gravel and mud. Arenicola cristata normally builds its
burrows in muddy sand (partical size 200-700 ym) at depths of 20-25 cm and in
densities as great as 20 per square meter (D'Asaro 1976). The burrow consists
of a tail shaft, a horizontal gallery and a head shaft. Periodically, the
lugworm moves forward in the gallery and ingests sand along with associated
organic material (living and dead). The resulting displacement of the overlying
sediment produces a subsiding column of sand marked by a funnel shaped depression
on the substrate surface. When the organic content in the region of the head
-------
shaft is depleted, A_. cristata forms new feeding funnels in adjacent areas.
Figure 1 illustrates the progressive formation of feeding funnels produced by
one lugworm at 12 hour intervals.
Feeding and the consequent formation of feeding funnels is an integral
part of an activity sequence which also incorporates excretion and peristaltic
pumping of water through the burrow for respiration and ventilation. These
combined activities comprise the "Normal Cyclical Pattern" which is believed to
be controlled by internal pacemakers and is therefore independent of normal
environmental variables (Wells 1966). A decrease in the formation of feeding
funnels would indicate an interruption in this activity cycle.
As the lugworm feeds and pumps water through its burrow, it mixes organic
material and oxygenated water into the substrate (especially in the vicinity of
the head shaft). This process is undoubtedly beneficial to other infaunal
organisms living in association with the lugworm, for it provides them additional
sources of food and oxygen. However, during periods" of environmental perturba-
tion, contaminating agents are also transported into the substrate. Garnas et al.
(1977) demonstrated that lugworm activity affected movement of the pesticide
methyl parathion from the water column into the sediment. The fate of such
compounds as they interact with infaunal organisms raises questions of great
complexity. The role of the lugworm in this regard is not yet fully understood.
However, the lugworm has been observed to suspend substrate modifying activity
when environmental stress reaches a threshold level (Rubinstein 1976). This
type of behavioral response to environmental contaminants could serve as a
sensitive indicator for various polluting agents.
Feeding Activity of Unstressed Lugworms
To insure that comparisons between exposed and non-exposed lugworms could
be made, I conducted preliminary tests to determine if lugworms of similar size
-------
rework the substrate at equivalent rates when subjected to the same conditions.
Due to a limitation in available time-lapse photography equipment, only two
groups of lugworms could be compared at a given time.
Bioassay Procedure
Two 125 £ aquaria were used as test habitats. Both aquaria contained
0.25 m2 of sand (particle size 200-700 urn) to a depth of 25 cm and 72 i of 20 ytn
filtered seawater. All tests were conducted at salinities between 20 and 23 °/oo
and temperatures between 22 and 25°C. Water was aerated by airstones except
when automatically turned off prior to taking photographs.
Lugworms were obtained from stocks cultured at the University of West
Florida Marine Laboratory, Sabine Island, Pensacola, Florida by the method of
D'Asaro. (1976). Six worms 8.5 to 9.5 cm long (measured fully contracted) were
placed in each aquarium and allowed 48 hours to establish burrows and acclimate
to test conditions. Following acclimation, 50 grams^of ground seagrass (pre-
dominantly JJiaJj!_as_i_a_ tes_tud_i_num) was added to both aquaria. The seagrass formed
a dark mat on the sediment surface and served as the detrital component of the
benthic system. It was used as food by lugworms and also provided photographic
contrast against the white underlying sand when turned under by feeding animals.
A 35-mm single lens reflex camera was positioned above each aquarium
(Figure 2). The cameras were equipped with an automatic advance mechanism,
24-hour timers and an automatic lighting system consisting of four strobes and
two floodlights. Photographs of the substrate surface were taken at 12-hour
intervals for 72 hours and then analyzed to determine the surface area disturbed
by feeding lugworms. Surface area was calculated by using a system of point
counting in a coherent grid modified from Hyatt (1973). The outline of feeding
funnels was traced onto a 0.25 cm grid overlay, all points on the 5 mm intersections
-------
2
were counted and then converted to actual surface area (cm ). Total surface
area turned under by feeding lugworms was plotted against time to determine the
substrate reworking rate of lugworms in both aquaria. Rates were subjected to
linear regression analysis and the slopes of the calculated lines were compared;
differences due to treatment were considered significant at » = 0.01.
Six replicate tests were conducted with a different group of lugworms for
each test. Results are shown in Figure 3. Variability in the magnitude of
surface area disturbed between tests can be attributed to slight variations in
size, age and condition of lugworms. For this reason comparisons of substrate
reworking rates were made within a test and not between separate tests. Rates
of feeding funnel formation were not significantly different (t[97.5; 8]) between
aquaria within each of the six tests. Therefore, the use of two aquaria, one
experimental and one control is valid because the rates of substrate reworking
between lugworms of similar size and condition are comparable.
• /
Appl i cation _pf_ Feedijig^Acti vi ty To Toxicvty Testing - " -
Following preliminary tests using no toxicant the sensitivity of lugworm
feeding activity was tested with the insecticide Kepone (dodecachlorooctahydro-1,
3, 4-metheno-2H-cyclobuta [cd] pentalene). Kepone was selected because of the
extreme hazard it poses to aquatic life in the James River Estuary and Chesapeake
Bay (Hansen e_t aj_. 1977), areas in which the lugworm is endemic. Although the
acute toxicity of Kepone to several estuarine fishes and invertebrates has been
investigated (Hansen e_^ a]_. 1977, Schimmel and Wilson 1977, Nimmo e_t aj_. 1977)
the sub-lethal effects of this compound on infaunal organisms have not been
evaluated.
-------
METHODS
A series of six tests using diminishing concentrations of Kepone was
conducted using the bioassay procedure previously described. A stock solution
of Kepone (88% pure) in nanograde acetone was dispersed in the water of one
aquarium; the second aquarium received an equivalent amount of the acetone
carrier and served as the control. Aquaria were dosed following lugworm
acclimation and approximately one hour after the ground seagrass was added.
From this point on photographs of the substrate surface were taken at 12-hour
intervals for 144 hours. Measured concentrations of Kepone tested were 29.5,
7.4, 6.6, 4.5 and 2.8 vg/£. A non-detectable level (<0.02 yg/&) was also tested.
One liter water samples taken from aquaria one hour after introduction of the
test compound were analyzed by gas chromatography as in the method of Schimmel
and Wilson (1977).
RESULTS AND DISCUSSION *
Kepone was acutely toxic to lugworms at the highest concentration tested.
All lugworms exposed to 29.5 yg/£ died while no mortalities occurred at the
lower concentrations during the 144 hour period. Significant inhibition of
lugworm feeding activity in both magnitude and rate was observed at all detectable
levels of Kepone tested (F >^ 91, d.f. 2, 10; » = 0.01). Comparisons of substrate
reworking rates between exposed and control lugworms for selected tests are
shown in Figure 4.
Lugworms were sensitive to Kepone at sub-lethal levels as low as 2.8 yg/£ .
During the first 48 hours of exposure to concentrations ranging from 7.4 to 2.8
yg/£ exposed and control lugworms displayed similar rates of feeding funnel
formation. However, between 60 and 144 hours a significant reduction in the
amount of surface area disturbed by exposed lugworms was observed. This latent
effect suggests that lugworms may gradually accumulate Kepone until a threshold
-------
level is reached which then interrupts the "Normal Cyclical Pattern."
The relative toxicity of Kepone to several estuarine species has been
determined (Schimmel and Wilson 1977, Nimmo e_t al_. 1977). The species examined,
and their 96-hour LC50 values were: grass shrimp (Palaemonetes pugio), 121_ ug/r,
blue crab (Callinectes sapidus), 210 ug/&; mysid (Mysidopsis bahia), 10.1 yg/£;
sheepshead minnow (Cyprinodon variegatus), 69.5 yg/£; and the spot (Leiostomas
xanthurus), 6.6 yg/£. Although the 96-hour LC50 value for the lugworm was not
determined, complete mortality was observed within 48 hours at 29.5 ug/£. Based
on these findings it is apparent that the lugworm is sensitive to Kepone when
compared with other estuarine species.
General Conclusions
I have presented a bioassay system for quantifying the effects of a pollutant
on a marine infaunal polychaete. The toxicity tests conducted with Kepone serve
to illustrate applicability of the bioassay technique". At sublethal concentrations
(7.4 to 2.8 yg/£) the Normal Cyclical Pattern of the lugworm was interrupted,
resulting in a decrease in substrate reworking activity. Although the consequences
of reduced lugworm activity are speculative at this time, it is possible that a
suspension of substrate reworking by the lugworm and other infaunal organisms
with similar deposit feeding habits could reduce the exchange of pollutants
between the sediment and water column thereby prolonging the residence time of
a pollutant in the water. Long term effects of reduced lugworm feeding activity
could eventually result in the depaupuration of lugworm populations. Such an
event would affect the overall transport of nutrients and pollutants through the
benthic system as well as alter food chains of which the lugworm forms a part.
Whatever the environmental significance of this deviation from normal
activity caused by pollutant stress, it is clear that this technique can
demonstrate a behavioral effect on an important infaunal organism. Such a
-------
test which demonstrates biologi-cal effects of low levels of pollutants on an
important ecological process such as sediment reworking will be of value in
determining the potential impact of a contaminant on the marine environment.
ACKNOWLEDGMENTS
This study was supported by an EPA grant (R804458) to the University of
West Florida. I would like to thank Dr. C. N. D'Asaro (University of West
Florida) for giving me the opportunity to carry out his original suggestions,
Dr. N. R. Cooley and Mr. D. J. Hansen (EPA) for editorial comments and Mr.
Lowell Banner (EPA) for help with the statistical analysis. Facilities to
complete the research were made available by Dr. T. Duke of the Gulf Breeze
Environmental Research Laboratory.
-------
Figure 1. Feeding funnels produced by one lugworm at 12-hour intervals,
-------
-------
Figure 2. Photo-Bioassay System (A-24-hour timer, B-35 mm camera with automatic
advance, D, E aquaria with 25 cm of sand and 75 i of seawater).
X
-------
-------
Figure 3. Comparison of the rates of sediment turned under by groups of "lugworms
of similar size. A different group of lugworms was used for the six
replicate tests. S
-------
* » '%
-------
Figure 4. Comparison of the rates of sediment turned under by lugworms. C:
control; E: experimental group exposed to Kepone. Each group
consisted of six lugworms. • /
-------
SURFACE AREA DISTURBED (cm^)
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-------
LITERATURE CITED
Blegvad, H. 1914. Food and conditions of nourishment among the communities of
invertebrate animals found on or in the sea bottom in Danish waters. Rep.
Danish Biol. Sta. 22:41-78.
D'Asaro, C.N. 1976. Lugworm aquaculture Part I. A preliminary plan for a
commercial bait-worm hatchery to produce the lugworm Arenicola cristata
Stimpson. Report 16; State University System of Florida. A Sea Grant
College Progarm.
EPA. 1976. Bioassay Procedure for the ocean Disposal Permit Program. U.S.
Environmental Research Laboratory Office 'of Research and Development,
Gulf Breeze, Florida.
Garnas, R.L., C.N. D'Asaro, N.I. Rubinstein and R.A. Dime. 1977. The fate of
methyl parathion in a marine benthic microcosm. Paper #44 in Pesticide
Chemistry Division, 173rd ACS meeting, New Orleans, Louisiana, March 20-25,
1977. . ~ .-
Hansen, D.J., A.J. Wilson, D.R. Nimmo, S.C. Schimmel, L.H. Banner and R. Huggett.
1976. Kepone: Hazard to aquatic organisms. Science. 193:528.
Hyatt, M.H. 1973. Principles and techniques of electron microscopy. Vol. 3.
van Nostrand Rheinhold Co., New York, pp 239-289.
Meyers, A.C. 1977. Sediment processing in a marine subtidal sandy bottom
community: I Physical aspects; II Biological consequences. J. Mar. Res.
35(3):609-647.
Nimmo, D.R., L.H. Banner, R.A. Rigby, J.M. Sheppard and A.J. Wilson. 1977.
Mysidopsis bahia - An estuarine species suitable for life-cycle toxicity
tests to determine the effects of a pollutant. Aquatic Toxicology and
Hazard Evaluation, ASTM STP 634, F.L. Mayer and J.L. Hamelink Eds. ppl09-116.
-------
Rhoads, D.C. 1973. The influence of deposit-feeding benthos on water turbidi
and nutrient recycling. Am. J. of Sci. 273:1-22.
Rhoads, D.C. and O.K. Young. 1971. Animal sediment relations in Cape Cod Bay,
Massachusettes. II. Reworking by Molpadia oolitica (Holothuroidea).'
Marine Biology 11:225-261.
Rowe, G.T., G. Keller, H. Staresinic and N. Macllvaine. 1974. Time lapse
photography of the biological reworking of sediments in Hudson Bay submarin
canyon. J. Sed. Patrol. 2:549-552.
Rubinstein, N.I. 1976. Thermal and haline optima and lethal limits affecting
the culture of Arenicola cristata (Polychaeta: Arenicolidae). Masters
Thesis, University of West Florida.
Schimmel, S.C. and A.J. Wilson, Jr. 1977. Acute toxicity of Kepone to four
estuarine animals. Cheasp. Sci. 18(2)224-227.
Wells, G.P. 1966. The lugworm (Arenicola) a study in adaptation. Ne'th. J.
Sea Res. 3:294-313.
-------
•n
Kepone : Toxicity and Bioaccumulation in Blue Crabs
Steven C. Schimmel, James M. Patrick, Jr.,
Linda F. Faas, Jerry L. Oglesby, and Alfred J. Wilson, Jr.
U.S. Environmental Protection Agency
Environmental Research Laboratory
Gulf Breeze, Florida 32561
Estuaries (In Press)
-------
^Registered Trademark for decachlorooctahydro-1,3, 3-metheno-
2H-Cyclobuta (cd) pentalen-2 one. Allied Chemical Company,
%
JJO Rector Street, New York, New York. Mention of commercial
products does not constitute endorsement by the Environmental
Protection Agency.
^Contribution No. 3^9, Environmental Research Laboratory,
Gulf Breeze.
-------
ABSTRACT
Two long-term studies were conducted to determine toxicity,
uptake and depuration of Kepone in blue crabs (Callinoctes
sapidus). In the first, Kepone was administered to crabs in
seawater -(0.03 or 0.3 yg Kepone/£) or food (eastern oyster,
CrassQstrea virgjLnica, containing 0.25 ug/g Kopono). Uptake of
Kepone in 28 days was primarily through the contaminated
oysters. When these crabs were held in Kepone-free seawater
and fed Kepone-free oysters for 28 days, no loss of the
insecticide was evident. There were adverse effects on molting
and survival in cj?abs fed oysters that contained 0.25 ug/g
Kepone,
A second study was conducted to determine•/(1) the
depuration of Kepone over a 90-day period in blue crabs fed
oysters from the James River, Virginia (containing 0.15 yg/g
Kepone); and (2) the effects of Kepone on molting and survival
of blue crabs fed James River oysters or laboratory-
contaminated oysters that contained 0.15 or 1.9 ug/g Kepone.
Crabs fed Kepone-contaminated oysters followed by a diet of
Kepone-free oysters for 90 days had detectable concentrations
-------
of the insecticide in tissues. Also, blue crabs that ate
oysters containing Kepone in concentrations similar to those
found in oysters from the James River, died or molted less
frequently than crabs fed Kepone-free oyster meats.
-------
INTRODUCTION
Contamination of Virginia's James River estuary by Kepone in
the late 1960's and early 1970's, and its transport into
Chesapeake Bay, raised questions about the chemical's
persistence in and effects on the area's estuarine biota.
Kepone residues in James River biota, in particular blue crabs
(Callinectes sapidus Rathbun), bluefish (Pomatomus salatrix
Linnaeus)1 and eastern oysters (Crassostrea vireinica Gmelin).
were found by the U.S. Food and Drug Administration (FDA) to be
sufficiently high to limit the commercial harvesting of these
and other species -captured in the estuary.
Kepone residues in James River blue crabs coincided with a
decrease in commercial crab landings. Residues in males was
0.81 ug/g; in females, 0.19 yg/g (Bender et al., 1977). Total
blue crab catch from the James River indicated a 90£ decline
from 1972 through 1975 (U.S. Dept. Commerce, 1968-1975).
Laboratory exposures of blue crabs in seawater to Kepone
indicated relatively low acute toxicity and bioconcentration
(Schimmel and Wilson, 1977). This, in contrast to James River
-------
residue data, suggested that the major route of Kepone entry in
crabs may be through contaminated food.
>
In this paper, wo report the results of two long-term
studies to determine: (1) uptake and depuration rates of Kepone
in blue crabs exposed to the insecticide in seawater and food
•
(eastern oysters); and (2) effects of Kcpono in food on molting
and survival of blue crabs.
The authors thank Mr. Steve Foss for the illustrations, Kr.
Johnnie Knight for chemical analyses on water samples, and Mr.
Monte Tredway for help in bioassays. V*e also thank the staff
of the Virginia Institute of Marine Science, particularly Mr.
•*t "
Robert Huggett, for collecting James River oysters and Mr.
Chuck Taylor of the Environmental Protection Xgency's (EPA)
Environmental Research Laboratory, Athens, Georgia for analyses
of heavy metals in oyster tissues..
-------
METHODS AND 'MATERIALS
animals
Blue crabs for both studies were collected from Santa Rosa Sound,
Florida; oysters, used as food for blue crabs, were collected either
from Santa Rosa Sound (local oysters) or from Wreck Shoals in the
James River estuary, Virginia. Eastern oysters were appropriate
food in these bioaccumulation studies because: (1) oysters
bioconcentrate Kepone under laboratory conditions (Banner et al.,
1977); (2) oysters contaminated with Kepone have been reported from
the James River (Bender et al., 1977); and (3) oysters are a natural
food of blue crabs_ (Anonymous, 19*11; Loosanoff, 19**8; Lunz, 19^7;
Menzel and Hopkins, 1956; and Menzel and Hichy, 1958).
/
Approximately 95£ of the crabs collected were Callinectes sapidus:
the remaining, .C. simills (Williams). No effort was made to
separate the latter species because of the limited numbers of
animals available and the need to avoid excessive handling. Locally
collected oysters (Santa Rosa Sound) and blue crabs contained no
Kepone detectable by gas chroraatographic analysis. Blue crabs were
acclimated to laboratory conditions for at least 14 days prior to
testing.
-------
Study No. 1
Approximately ^150 juvenile blue crabs (19-^9 ram carapace width,
= 36.7 mm) were collected from Santa Rosa Sound, in July 1976.
During acclimation, crabs were fed meats of eastern oysters from
Santa Rosa Sound ad, libitum.
A 56-day bioconcentration (uptake directly from the exposure
water) and bioaccumulation (uptake primarily from ingested food)
study was conducted, using the diluter described by Schimmel et al.
(1977). Seawater was pumped from Santa Rosa Sound through a sand
filter and into a reservoir in the laboratory. It was then heated
to a mean temperat-ure of 22.1°C (range, 20.0 to 23.5°C); salinity
was allowed to vary with sound conditions (range 20.0 to 31.0 °/oo;
S
x = 25.5 °/oo). From the reservoir, seawater was pumped to the
diluter mixing box by LambdaR pumps. The diluter provided two
Kepone concentrations in seawter (0.03yg/£ and 0.3 u/?/£ » nominal
^Registered Trademark, Harvard Apparatus, Co., Inc. 150 Dover Road,
Millis, Massachusetts 0205M.
-------
concentrations), each concentration duplicated, as well as seawater
fres of Kepone, also duplicated. Six exposure aquaria measured ^6
cm x 71 cm x 26 cm (volume = 86&), and the diluter delivered one
liter to each aquarium each cycle. There was an average of 300
cycles each day (10 turnover volumes). Fifty-six blue crabs were
placed simply in individual compartments (b.5 cm x ^.7 cm x 10.0 en)
in each aquarium. At least 12 holes (6 mm diameter) were drilled in
each compartment to provide adeuqate circulation.
The 5b-day study was divided into a 2B-day uptake period and a
2o-day depuration period. In the uptake period, Kepone was
administered to tfre crabs; (1) in the water delivered to the
experimental aquaria; and (2) in contaminatedyeastern oysters as a
food source. Adult oysters were contaminated by exposing them to
0,1 yg/il Kepone for 42 hours; afterwards oyster meats were removed
from their shells, pooled, and analyzed quantitatively for Kepone
content. Approximately 0.5 g of the oysters, contaminated with 0.25
vg/g Kepone, were fed to the crabs twice each week in a control
aquarium, and to crabs exposed either to the 0.03 y?/£ or 0.3 yg/£ .
Approximately 0.5 g of uncontaminated, Santa Rosa Sound oysters (<
1
0.02 ug/g Kepone) were fed to crabs in the remaining control and
experimental aquaria (Table 1). Two crabs were sampled from each
-------
aquarium during the uptake portion of the test at 1 day, 2 days,
<
and, after the second day, twice each week to the end of the 28-day .
>
exposure period. Identical sampling intervals were used for the
28-day depuration portion of the study. When sampled, each crab was
dissected into muscle and remaining tissues including exoskeleton.
•
Muscle tissue was obtained from the thoracic region and contained
small amounts of chitin. Remaining tissues consisted of whole crab
not included in the muscle tissue sample. Molting and mortality of
crabs were monitored daily throughout the study.
Study No. 2
Approximately 580 juvenile blue crabs (9 to ^2 mm carapace width,-
x = 28 mm) and approximately 1000 adult eastern oysters were
collected from Santa Rosa Sound in February and March^1977. -
Uncontaminated local oysters, the control food, were removed from
their shells, cut into 0.5 g portions, and frozen.
Two hundred local, Uncontaminated oysters were placed in an
aquarium and exposed to 1.0 pg/£ Kepone for 20 hours to obtain an
average of 1.9 Vg/g Kepone in their tissues. Another 200 oysters
were exposed to 0.1 yg/Jl to obtain an average of 0.15 yg/g Kepone.
Oysters from each aquarium were then cut into 0.5 g pieces and '
frozen.
-------
Additional eastern oysters were collected in the James River
(near Wreck Shoals) in March 1977 by the staff of the Virginia
\
Institute of Marine Sciences and shipped by air freight to the Gulf
Breeze Environmental Research Laboratory. Gas chroraatographic
analysis of pooled oysters showed a Kepone concentration of 0.15
•
yg/g wet weight. These oysters wore also cut into 0.5 p piocos and
frozen.
4
Study No. 2 with blue crabs was conducted to determine: (1) the
effects of Kepone on molting and survival; and (2) the rate of
depuration of Kepone beyond the 28-day period tested in Study No. 1.
The same exposure apparatus used in Study No. 1 was used except that
no Kepone was administered in seawater. Six aquaria were used, each
containing 68 blue crabs (held in individual'compartments as in the
previous study). Crabs in aquaria Nos. 1 and 4 were used to
determine uptake and depuration of Kepone administered through their
food; those in aquaria Nos. 2, 3, 5, and 6 were observed for effects^
on molting or survival (Table 2). Crabs in aquarium No. 1 were fed
0.5 g of Kepone-free oysters twice each week. Crabs were sampled
twice each week for 28 days, three per sample, for Kepone residue
analyses; thereafter, crabs were sampled less frequently for an *
additional 90 days. Crabs were dissected into muscle and remaining
tissues, as in the first study.
-------
Chemical methods
Methods of chemical analyses of Kepone were those of Schimmel and '
Wilson (1977). The average recovery rate of Kepone from fortified
tissue was 87$; from water, 85$. Residue concentrations were
calculated on a wet-weight basis without correction for percentage
recovery. All samples were fortified with an internal standard
(dichlorobenzophenone) prior to analysis to evaluate the integrity
of the results.
Statistical methods
Blue crab molting data were analyzed by one-way analysis of
variance followed._by the Student-Newman-Keuis post hoc tests to
determine significant differences (ct= 0.05); mortality data were
/
analyzed by Student's t (a= 0.05).
10
-------
RESULTS
Stufiy No. 1
Blue crabs fed oysters contaminated with 0.25 pg/g Kepone
accumulated the insecticide readily in their muscle and remaining
tissues in 28 days. However, after 28 days in a Kepone-free
environment, no depuration of the compound was evident (Fig. 1).
When fed 0.25 pg/g Kepone in oysters for four weeks, crabs
accumulated the insecticide to an average of approximately 0.1 pg/g
in both their muscle and remaining tissues. Uptake of Kepone by
crabs fed contaminated oysters only (aquarium No.l) was nearly
identical to that"of crabs fed contaminated oysters and provided
Kepone in seawater (aquaria Nos. 3 and M), indicating that very
little of the insecticide was taken in directly from water. No . -
measurable concentrations of Kepone were detected in crabs from
aquaria supplied with 0.3 pg/£ (0.08 pg/£ measured) or 0.03 yg/£
(0.01^jjg/£ measured) Kepone (aquaria Nos. 5 and 2 respectively).
Blue crabs in all aquaria were monitored for molting and
mortality throughout the 56-day test. Although no statistical tests
could be applied to these data, we observed that crabs fed control
t
oysters molted a total of 63 times, and 22 crabs died (Table 3).
Crabs fed Kepone-contaminated oysters for 28 days, and control
11
-------
oysters for 28 additional days, had fewer molts (n=48) and more
deaths (n=^9) than crabs fed only control oysters. The data
indicated that Kepone in crab food may reduce their ability to molt
or survive. Therefore, we conducted Study No. 2 to investigate this
possibility and to determine the depuration rate of Kepone in crabs •
over a longer period than in the previous study.
Study No. 2
Blue crabs in aquarium No. 4 that were fed James River oysters
(containing 0.15 yg/g Kepone) accumulated the insecticide in muscle
and whole-body tissues (muscle and remaining tissues) to a maximum
of 0.069 pg/g in.2-8 days (Fig. 2). After a Kepone-free diet for 90
days, crabs showed some loss of the insecticide. However, 0.025
yg/g Kepone was detected in remaining tissues, and approximately -
0.01^ pg/g remained in muscle tissues. Some of this apparent loss
of Kepone must be attributed to growth of the blue crabs. No length
or weight measurements were made at the end of the 28-day exposure
period, and consequently the exact growth could not be determined.
Molting by crabs fed James River oysters (aquarium No. 3, Kepone
concentration = 0.15pg/g), oysters containing Q.15ug/g Kepone
(aquarium No. 5), or 1.9 Mg/g Kepone (aquarium No. 6) was
significantly reduced (ct = 0.05) compared with those fed control
12
-------
oysters (aquarium No. 1, Fig. 3). The average molts per crab in 56
days were: control, 1.4; 1.9 yg/g Kepone, 0.56; 0.15 yg/p Kepone,
^
0.97; and James River oysters, 0.48.
Percent mortality in crabs fed oysters containing 1.9 yg/g Kepone
and those fed James River oysters was significantly greater (ct=
»
0.05) than those fed control oysters (Fig. 4). Blue crabs which ato
oysters containing 1.9 yg/g Kepone exhibited extreme excitation,
especially during feeding. In the advanced stages of Kepone
poisoning, the crabs were generally lethargic for several days
before death. No crabs in any other aquaria displayed these signs.
Percent mortality of crabs fed oysters containing 0.15 yg/g Kepone
was not significantly different from that of crabs fed control
oysters.
13
-------
DISCUSSION
The uptake and depuration of Kepone by blue crabs in both studies
help explain why relatively high Kepone residues are found in crabs
from the James River, Virginia. No discernible depuration of Kepone
in crab nfuscle or remaining tissues was evident in 28 days but some
loss of Kepone was apparent beyond 28 days. However, detectable
Kepone was present in tissues of crabs held 90 days in a Kepone-free
environment. In the James River, Bender et al. (1977) reported that
average Kepone concentrations in estuarine vertebrates and
invertebrates ranged from 0.09 to 2.0 yg/g. Many, if not all, of
the species listed" are included in the diet of the blue crab.
Therefore, it is reasonable to conclude from our laboratory studies
and James River field data that Kepone residues will persist in blue
crab tissues as long as detectable concentrations remain in the
food.
Blue crabs fed oysters containing 1.9 yg/g Kepone exhibited signs
of poisoning similar to crabs fed fish contaminated with 1.0 yg/g of
mirex (Lowe et al., 1971). Mirex is an organochlorine insecticide
chemically similar to Kepone. Lowe et al. (1971) also reported
t>
that juvenile blue crabs exposed to 100 yg/£ mirex in flowing
seawater for 96 hours showed no signs of poisoning. Schimmel and
14
-------
Wilson (1977) exposed blue crabs to Kepone for 96 hours at
concentrations as high as 210 ug/£ and reported no significant
mortality.
Blue crabs fed oysters from the James River (containing 0.15 yg/g
Kepone) died in significantly greater numbers and molted fewer times
than those fed uncontarainated oysters; crabs fed oysters containing
0.15 yg/g Kepone (by laboratory exposure) moulted fewer times but
did not die in significantly different numbers than those fed
uncontaminted oysters. Reasons for the difference in mortality are
unclear. However, it should be noted that a 0.15 yg/g Kepone
residue appears to' be the threshold level for effect: blue crabs fed
local oysters contaminated with 1.9 yg/g Kepone suffered 80%
mortality, and those fed local oysters contaminated with 0.25 yg/g
Kepone died in greater numbers than those fed control oysters. It
is possible that the difference in mortality in the crabs fed James
River oysters (0.15 yg/g) and those fed local, contaminated oysters
(0.15 yg/g) may be due to a toxicant not detectable by our methods
of chemical analysis or that our methods of exposure and duration
were different than those of oysters from the James River. To
investigate the possibility of some metal in the oysters, samples of
local and James River oysters were sent to the EPA Environmental
15
-------
Research Laboratory, Athens, Georgia, for analysis. Results of
these analyses (multielement analysis by plasma emission and spark
source mass specrometry) indicated that oysters from the James
River contained approximately 5 x more aluminum (70 ug/g) , 10 x more
copper (2-9 ^g/g), and 9 x more zinc (415 ug/g) than local oysters.
Concentrations of other metals in oysters from both areas were
similar. We do not know the effects of these motals and Kepone on
the survival of blue crabs. S
Annual crab fishery statistics (U.S. Dept. Comm., 1968-197^) from
the James River show that from 1968 to 1972 the commercial catch
ranged from 691,000 kg to 1,211,000 kg ("x = 899,000 kg), but from
1972 through 1975 the catch decreased more than 9Q£ to 14,700 kg.
(A moratorium on harvesting of blue crabs in the James River was in
effect in 1976), The blue crab catch in the adjacent Rappahannock
River estuary from 1968 through 1975 did not show a significant
decline. From these catch data it appears that some pertubation is
causing a particularly adverse affect on blue crab abundance in the
James River estuary.
If the James River estuary had been affected by only one
pollutant, it would have been comparatively easy to determine a
cause and effect relationship with that pollutant and the decrease
16
-------
in the crab fishery. As in nearly all instances in the natural
<
environment, this is not the case in the heavily industrialized
^
James River area. However, based on our laboratory data, we believe
that Kepone may be a factor in the decline of the James River crab
fishery.
17
-------
LITERATURE CITED
ANONYMOUS. 1941. Atlantic Coast blue crab, an important enemy of
oysters. Oyster Institute of North America, Trade Rpt.
No. 44, 2p.
BAHNER, .LOWELL H., ALFRED J. WILSON, JR., JAMES M. SHEPPARD, JAKES '
M. PATRICK, JR., LARRY R. GOODMAN, AND GERALD E. WALSH. 1977.
Kepone bioconcentration, accumulation, loss, and transfer through.
estuarine food chains. Chesapeake Sci. 18(3):299-30b .
BENDER, M.E., R.J. HUGGETT AND W.J. HARGIS. 1977. Kepone* residues'
in Chesapeake Bay biota, Kepone Seminar II, September 20 and 21,
Easton, Maryland.
LOGSANOFF, V.L. 1948. Crabs as destroyers of oysters, Notes from
A.F. Chestnut. Pastor Institute of N or t1? America, Trade
Rep. No. 9B, 2p.
LOWE, J.I., P.K. PARRISH, A.J. WILSON, JR., P.O. WILSON, AMD T.W.
4
DUKE. 1971. Effects of mirex on selected estuarine organisms.
Trans. ^6th No. Amer. Wildl. Nat. _Res. Conf.
171-1S6.
LUNZ, G.R. 1947. Callinectos versus Ostroa. J.. of the Elisha
Mitchell Sci. Soc. 63:61.
MENZEL, R.W. AND S.H. HOPKINS. 1956. Crabs as predators of oysters
18
-------
in Louisiana. Proc. Natl. Shellfish Assoc. 46:177-184.
MENZEL, R.W. AND F.E. NICHY. 1958. Studies of the distribution and
>
feeding habits of some oyster predators in Alligator Harbor,
Florida. Bull. Mar. Sci. of the Gulf and Caribbean. 8:125-145.
SCHIMMEL, S.C., J.M. PATRICK, JR., AND A.J. WILSON, JR. 1977.
* .
Acute toxicity to and bioconcentration of ondosulfan by estuarino
animals. Aquatic Toxicology and Hazard Evaluation. ASTM STP
634, F.L. Mayer and J.L. Haraelink, Eds., American Society for
Testing and Materials. 1977.
AND A.J. WILSON, JR. 1977. Acute toxicity of
Kepone to four estuarine animals, Chesapeake JSci..
18:224-227.
U.S. Department of Commerce, 1968-1975. Virginia landings. Cujrr.
JFjjj3h. J3ta_t.
19
-------
Table 1. Concentrations of Kepone used in Study No. 1 for 28-day exposure
of blue crabs (Callinectes spp.) to the insecticide in seawater
> or in food (eastern oyster, Crassostrea virginica) .
AQUARIUM
»
1
2
3
4
5
6
1
KEPONE
WATER (yg/£)
ND2
0.03
0.03
0.3
0.3
ND
FOOD (yg/£)
0.25
ND
0.25
0.25
ND
ND
Uptake period was followed by a 28-day depuration period in which no
Kepone was administered in food or in seawater.
2
ND - non-detectable: < 0.02 yg/£ in water, <0.02 yg/g in oyster tissue.
-------
Tabel 2. Experimental design for 56-day exposure of blue crabs (Callinectes _sp_p_.) to the insecticide,
Kepone, in eastern oysters (Crassostrea virginica) (Study No. 2)
AQUARIUM
SOURCE OF
OYSTERS
CONCENTRATION
OF KEPONE
i,N OYSTERS
(ug/g)
OBSERVATION
1
2
3
4
5
6
Santa Rosa Sound, Florida
Santa Rosa Sound, Florida
James River, Virginia
James River, Virginia
Santa Rosa Sound, Florida
Santa Rosa Sound, Florida
ND
ND
0.15
0.15
0.15
Molting and Survival
Uptake/Depuration
Molting and Survival
Uptake/Depuration
Molting and Survival
Molting and Survival
ND = non-detectable; <0.02 yg/g in tissues.
-------
Tabel 3. Total molts and mortality of blue crabs (Callinectes spp.) in Kepone Study No. 1.
Fifty-six crabs were placed in each aquarium at the beginning of the test. f
. I : - .• - • — — j
Aquarium No. Test Conditions Molts Mortality
1 Control water ; ', 18 15
0.25 yg/g Kepone in oyster
2 0.03 yg/£ Kepone in water;
Control oysters 20 6
3 0.03 yg/g Kepone in water; 12 20
0.25 yg/g Kepone in oysters
4 0.3 yg/£ Kepone in water; 18 14
0.25 yg/g Kepone in oysters
5 0.3 yg/2. Kepone in water; 22 9
Control oysters
\
6 Control water;
Control oysters 21 7
Control water, <0.02 yg/£ Kepone; Control oysters, <0.02 yg/g Kepone.
-------
K^ps.™
'-: Sh«•'„•-/ • :•
;*£.;'*: -
0»
0>
UJ
_J
O
05
CD
a:
o
UJ
o
Q.
UJ
0.01-
V
Detection Limit
42
DEPURATION
56
TIME (days)
-------
Figure 2. The 28-day accumulation of Kepone by blue crabs (Calllnectes spp.) fed James River
' oysters that contained 0.15 yg/g Kepone. In the depuration period, crabs were fed
Kepone-free (<0.02 yg/g) oysters. Kepone residue for muscle on day 118 is estimated
to be 0.014 yg/g. ."
-------
0.100
0>
—
o»
JD
O
O
0>
CO
•E 0.020
CD
C
O
ex
-------
l\
»
Figure 1. Bioaccumulation of Keponc in muscle tissues of blue crabs (Callincctcs spp.)
fed oysters contaminated with 0.25 wg/g of the insecticide for 28 days
f followed by a 28-day period for depuration. The uptake curve and the 95%
confidence interval represented arc a composite of three homogeneous curves
representing uptake in crabs fed: (1) 0.25 yg/g Kepone in oysters and control seawater,
(2) 0.25 ug/g Kepone in oysters and 0.03 ug/t, in seawater, and (3) 0.25 ug/g in
oysters and 0.3 ug/Jl in seawater.
-------
Total Moults
-------
> \
I
Figure 3. Molting rate of blue crabs (Callinectes spp.) fed uncontaminated (<0.02 ug/g Kepone)
or Kepone contaminated eastern oysters (Crassostrea virginica) for 56 days on a flow-
through bioassay. Molting rates are for crabs that survived the entire study in each
test aquarium. The test conditions and numbers of surviving crabs are: control, 57 crabs;
-------
ioor
l.9|ig/g
(Laboratory)
0.15 pg/g
(Jamas River)
0.15 ng/g
^(Laboratory)
? O.O^g/g
(Laboratory)
60
70
Time (days)
-------
Figure 4. Mortality of blue crabs (Calllnectes spp.) fed uncontaminated (<0.02 yg/g Kepone) or
Kepone contaminated eastern oysters (Crassostrea virginica) for 56 days in a flow-through
bioassay.
-------
John D. Costlow
QUARTERLY PROGRAM REPORT
June - September 1977
Environmental Protection Agency
Grant £R8Q3833-02-0
Effects of Insect Growth Regulators and
Juvenile Hormone Mimics on
Crustacean Development
Effect of Juvenile Hormone Mimics and Insect Growth Regulators
on Larval Development of Brachyura
Four different experiments were undertaken as continuations
of earlier work on the effects of DimilinR (TH-6040) on larval
development of several marine Brachyura. The first experiment
was to determine the rate of deterioration of Dimilin in seawater
through bioassay and, subsequently, chemical analysis. The
second experiment was designed to determine the degree of
sensitivity of individual larval stages to Dimilin. ' The third
experiment was designed to examine differential sensitivity to
Dimilin during particular times of the intermolt period of the
larvae. The fourth experiment involved, preparation of larvae
exposed to Dimilin during specific stages of development for
electron microscopy studies of cuticle* ?
As indicated in the previous'quarterly report (March-May,
1977) volumes of 10 ppb Dimilin were prepared in 20 ppt seawater,
stored in natural daylight at room temperatures, and used for a
period of approximately 70 days as the culture solution for
successive hatches of larvae of Rhithropanopeus harrisii. As
indicated in Table I, there was a high mortality of larvae during
the first stage of development in all series cultured up to 69
days following preparation of the Dimilin solution. All larvae
succumbed, in some cases after successfully completing zoeal
development, by varying times, ranging from eight days following
hatching to 17 days. When the solution was 29 days old, 25%
survived to the second zoeal stage but these larvae died before
or during molting to the third zoeal stage. Nearly all larvae
appeared normal in morphological and behavioral respects until
they began to noIt to the second zoeal stage. At that time, the
majority died during molting, frequently without being able to
remove the old exoskcleton. Those that did manage to complete
the molt were abnormal with deformed swimming setae and were
in capable of normal swimming. There were other morphological
defects, including rostral and antennal spines. In all controls,
survival ranged from 39 to 99 percent with an extremely small
percentage (1-4) of abnormalities throughout all of the zoeal
staces.
-------
-2-
Earlier experiments in this laboratory have shown that 10 ppb
Dimilin is lethal to R. harrisii larvae when they are exposed at
the time of hatching and that only a few larvae will survive beyond
the first zoeal stage. As shown in Table II, 10 ppb Dimilin is
lethal to other stages as well. One hundred percent mortality
occurred during molting when the larvae were exposed to 10 ppb
Dimilin in the second stage, the third stage, and the fourth zoeal
stages, whereas 97-99% survived in the control series without
Dimilin. The megalopa, however, did not appear to be as sensitive
to this compound as the zoeal larvae. When megalopa are exposed
to Dimilin, 44% did survive to the crab stage; within the control
series, 91% survived.
First zoeal larvae of Rhithropanopeus harrisii larvae were
exposed to 10 ppb Dimilin at various times within one intermolt
period. At 25°C, molting to the second stage begins on day 4.
Individual larvae were exposed to 10 ppb Dimilin on day 1, day 2,
or day 3, on days 1 and 2, on days 2 and 3, and on days 1 through
3. Results, as shown in Table III, indicate that the larvae were
more sensitive to the compound late in the intermolt period than
at the beginning of the intermolt period. The variations in
survival of replicates are probably due to the time exposure
relative to the time of hatching. Exposure to Dimilin was initiated
in the mornings whereas the larvae could have hatched at any time
between the previous evening and the early morning when exposure
was initiated. /
A few zoeal larvae exposed to 10 ppb Dimilin, as well- as"larvae
from the control series, were fixed for electron microscopy studies
in glutaraldehyde and OsO^. Some exposed first stage zoeae were
fixed during molting to the second zoeal stage. Sections of the
dorsal spine have recently been prepared and preliminary studies
under the electron microscope indicate that the cuticle is
considerably altered in those larvae that have been'exposed to
Dimilin when compared with larvae within the control series.
Effects of Juvenile Hormone Mimics and Insect Growth Regulators
on Behavior of Bracnyuran Larvae
The manuscript entitled "Sublethal Effects of Insect Growth
Regulators upon Crab Larval Behavior" was accepted for publication
by Water, Air and Soil Pollution. This work clearly indicated
that sublethal concentrations of Dimilin and hydroprene alter
phototaxis and swimming by Rhithropanopeus harrisii larvae. Thus
work this summer has focused on considering the functional signi-
ficance of responses to light and other environmental variables.
The main study determined whether the change in phototactic
pattern upon light and dark adaptation could serve as the basis
for a diurnal vertical migration pattern. The accumulated evidence
indicated that this probably does not occur. This work was pre-
sented at the 12th European Marine Biological Symposium (September,
1977).
-------
-3-
A second area of intensive investigation was a field study
of R. harrisii larval distributions in relation to environmental
factors^At a fixed station in the Newport River Estuary hourly
vertical samples were taken of larvae, light intensity, tempera-
ture, salinity and currents. This was carried out twice with the
first session lasting four days. The second was terminated after
two and a half days due to a mechanical breakdown. The collected
samples are now being analyzed. It is anticipated that this
information will indicate which environmental factors are important
in establishing crustacean larval distributions within an estuary.
Effects of Kepone on Development of Callinectes sapidus and
Rhithropanopeus harrisii
Preparation for Experiments
Disposal of Kepone Solutions. In response to a telephone
conversation to CHEM-DYNE CORPORATION, Bruce A. Whitten, President,
sent me a letter July 15, 1977 stating that their disposal fee
in 55-gallon drum quantities would, be $50 per drum F..O.B./ Hamilton,
Ohio. He suggested that we pour our Kepone contaminated brine from
finger bowls into one-gallon plastic jugs and cap the jugs. The
filled jugs could then be packed into 55-gallon open-top steel
drums. The spaces between and above jugs could be filled with
VERMICULITE. The drums should be labeled/Waste Brine Solution"
and shipped to CHEM-DYNE CORP., Hamilton,'Ohio. Our drums will
be consolidated with others to form a full drum truckload which
will be disposed of in full compliance with all Federal, State,
and local environmental regulations by secured chemical landfill
at the same site used by the U.S. Government for radioactive
wastes.
On July 18, 1977 Mr. William L. Blake, Safety Coordinator,
Duke University, and Dr. Conrad M. Knight, Radiological Officer of
Duke University-Duke University Medical Center were informed by
phone of our plans to dispose of Kepone. On July 20 the same
information was put in a letter and sent to Mr. Blake. On July
18, 1977, I also phoned the North Carolina Department of Agri-
culture, Pest Control Division, and talked to Larry D. Perry,
Pesticide Specialist, EPA coordinator, about our experiments and
methods of disposing of brine contaminated Kepone. On July 19,
1977 Mr. Larry D. Perry wrote me that his office approved of our
suggested method of disposing of waste Kepone. Mr. Blake and
Dr. Knight also approved of CHEM-DYNE CORPORATION'S plan. Mr.
Larry D. Perry suggested as an alternative that we send our wastes
to Rollins Environmental Services, P. 0. Box 221, Bridgeoort, NJ
08014.
Supplies. Orders for supplies of essential glassware, plastics
pipettors, chemicals and a utility cart were placed and goods
received. Chem Services were slow in filling our order for Kepone
and charged $120.00 for 10 grams rather than $34.58 as in order
placed by EPA, Gulf Breeze on December 15, 1975.
-------
-4-
Open-top 55 gallon steel drums and vermiculite were furnished
by Dr. Conrad M. Knight, Radiological Safety Officer of Duke Uni-
versity-Duke University Medical Center and one-gallon plastic jugs
were contributed by DUML personnel.
Preparation of Kepone Stock Solutions. Dr. Adam Zsolnay,
chemist at Duke University Marine Laboratory (DUML), prepared stock
solutions by dissolving a known weight of Kepone in pesticide grade
acetone and different concentrations were made up from, this stock
solution. The potency of some of these solutions were checked by
Dr. Zsolnay after a month and a half.
Source of Ovigerous Crabs and Hatching of Eggs. Ovigerous
Callinectes sapidus were collected in the Newport River near Beaufor
N.C. Four crabs with dark eggs, eyes, and visible heartbeats were
selected as a source of larvae. Isolated eggs were allowed to hatch
in filtered seawater with a salinity of 30°/oo in compartmented
boxes and flasks on a variable speed shaker according to the method
described by Costlow and Bookhout, 1960, and Bookhout and Costlow,
1975.
Ovigerous Rhithropanopeus harrisii were collected near Beaufort
in August and September 1977. When they were returned to the
laboratory each crab was placed in a large glass finger bowl (19.4
cm. d) containing filtered seawater with a salinity of 20°/Oor
the salinity to be used during rearing of/larvae for experiments
with Kepone. The ovigerous crabs were maintained in a constant
temperature culture cabinet at 25°C and with a light regime of 12 h
light and 12 h darkness until hatching occurred.
Rearing of Larvae in Range-finding Experiments. The methods of
rearing larvae of R. harrisii and C. sapidus in 1 'ppt acetone contro
and five concentrations of Kepone were essentially the same as out-
lined in the July 14, 1977 proposal, p. 4-7.
Direction of Work, Including Number and Kind of Experiments Conducte*
and Latest Results ._.
Range finding experiments using Callinectes sapidus larvae as
bioassay organisms were designed to run for 20 days. Four replicate:
were run with 50 freshly hatched C. sapidus larvae in each of the
six media of four series (I-IV). The percent survival for each
series is given in Table IV. There was differential survival with
increase in concentration, but the survival in acetone control was
far from satisfactory. Apparently the eggs from each of the four
mother crabs were poor. In previous experiments on the effect of
mirex on blue crab larvae (Bookhout and Costlow, 1975), the effects
of methor.ychlor on larvae of Rhithropanopeus harrisii and C. sapidus,
ana the effects of malathion on larvae of the same species (Bookhout
and Monroe, 1977), the average survival of blue crab"larvae to the
crab stage over a period beyond 40 days was over 50% in acetone con-
trol. The blue crab larvae were reared earlier than those reported
-------
-5-
here. As was mentioned in the proposal, July 14, 1977, blue crab
larvae in the latter part of August do not survive as well in the
laboratory as those hatched in the beginning of the breeding season
the later part of April and May. Our current experiments were
conducted during the first two-thirds of August. The high tempera-
tures throughout the summer may have been partly responsible.
Regardless of the cause, it is obvious that other range-finding
experiments will have to be conducted in May 1978 and definitive
experiments done afterwards.
Range-finding experiments using Rhithropanopeus harrisii
larvae as bioassay organisms were initially designed to run from
the time of hatching to the first crab stage. Each of the first
five series of R. harrisii larvae used came from the eggs of a
single female; the sixth series came from eggs hatched from two
females.
Since running four series of C. sapidus larvae (1200) took
an exorbitant amount of time, two series of larvae (600) were run
concurrently in case of R. harrisii. Based on the results of the
previous experiment with C. sapidus larvae, it was decided to use
the following concentrations of Kepone to test the viability of
R. harrisii larvae, Series I and II: 0.05, 0.10, 0.20, 0.40 and
0.80 Ppb. The results are given in Table V. Since survival to
the crab stage in Series I ranged from -94 to 100% in the concen-
trations of Kepone used compared to 92% in acetone control, it
was concluded that concentrations of 0.-05 ppb to 0.80 ppb Kepone
were not toxic to R. harrisii developmental stages.
In Series III and IV concentrations of Kepone used were 0.1,
2.5, 5.0, 7.5 and 10.0 ppb. The percent survival for 11 days in
these concentrations was from 96 to 100% in Series III and 92 to
100% in Series IV (Table VI). Hence, these concentrations do not
affect the survival of R. harrisii zoeae..
In Series V and VI the range of concentrations of Kepone
used were: 10.0, 20.0, 40.0, 80.0 and 160.0 ppb. Survival of
zoeae for 11 days in these concentrations is given in Table VII.
It appears from these experiments that survival of zoeae is not
affected by 10.0 and 20.0 ppb Kepone, whereas a concentration of
40.0 ppb is sublethal and 80.0 ppb and 160.0 ppb is acutely toxic.
Predictions and Plans for the Next Reporting Period
In the next range-finding experiments the following concen-
trations of Kepone will be used: 20.0, 35.0, 50.0, 65.0, and 80.0
ppb. This experiment should be the last range-finding one for R.
harrisii larvae and should furnish reliable information concerning
the concentrations to use in definitive experiments. Florida
crabs will be used for the next range-finding experiments, because
local R. harrisii femlaes have reached the end of their breeding
season. Arrangements will be made with a Florida supplier to ship
-------
-6-
ovigerous crabs to us in January for range-finding experiments
and two other shipments for definitive experiments in February
and March. Thereafter we will concentrate our efforts on range-
finding and definitive experiments with blue crab larvae.
Because it will not be possible to obtain ovigerous R. harrisi.
and C. sapidus in the best condition during the next quarterly
report we will have little to report.
Problems Encountered
1. Blue crab eggs hatched from four females on July 27, 28 and
29 proved to be less viable than those from 1972-76.
2. Our constant temperature culture cabinet broke down in
October and will not be fixed until the end of October.
Current Status of Publications
Latz, M.I. and R.B. Forward. 1977. The effect of salinity upon
phototaxis and geotaxis in a larval crustacean. Biol. Bull.,
153:163-179. /
Forward, R.B., Jr. and J.D. Costlow, Jr. 1977. Sublethal 'effects
of insect growth regulators upon crab larval behavior.
Water, Air and Soil Poll. In Press.
Forward, R.B., Jr. and T.W. Cronin. 1977. Crustacean larval
phototaxis: possible functional significance.. In: 12th
European Marine Biological Symposium. In Press.
Publications in Preparation
Christiansen, M.E. and J.D. Costlow. 1973. - Effects of Dimilin
on the Development of the Mud-Crab Rhithropanopeus harrisii.
Christiansen, M.E. and J.D. Costlow. 1978. Bioassay studies on
rate of deterioration of the compound Dimilin.
-------
Age of solution
when exposure
was started
EXiys
Number of
Replicates
Control 10 ppb
Initial Number
of Larvae
Control 10 ppb
TABLE I
Survival (%)
to Megalopa
Control 10 ppb
Days after
hatching when
all larvae were
dead in 10 ppb
Mortality (%) of develop-
mental stages in 10 ppb
I II III IV
0
1
3
5
7
9
12
1G
21
29
40
51
59
69
3
3
3
2
3
2
3
3
3
3
3
3
3
3
3
3
3
2
3
2
3
3
3
3
3
3
3
3
150
150
120
70
150
100
150
150
150
150
150
150
150
150
150
150
120
70
150
100
150
150
150
150
150
150
150
150
98.0
92.7
97.5
94.3
94.0
93.0
99.3
94.7
89.3
93.3
*
83.3
88.7
76.0
0
0
0
0
0
0
0
0
0
0
0
74.7
78.0
78.0
12
16
8
13
12
10
10
9
9
17
12
-
95.3
91.3
99.2
90.0
90.7
99.0
97.3
100
100
74.4
2.7
7.3
0.8
8.6
9.3
1.0
2.7
24.6
Days
2.0
0.7 0
1.4
0 0
40 - 69
.7
.7
- Data not analyzed
^
as
yet
Controls Day 40 were terminated at Day 12 when all 10 ppb Larvae Died.
-------
Table II
Larvae Number
Exposed of
In Replicates
Control Exposed in 10 ppb
Initial Survival Initial Survival
Number (%) Number (%)
1st stage
2nd stage
3rd stage
4th stage
Megalopa
3
3
3
4
4
120
146
144
167
109
97
98
99
98
91
.5
.6
.3
.8
.7
120
150
139
170
111
0
0
0
0
44.1
-------
TABLE III
Survival (%) to the megalopa stage v;hen larvae were exposed at
various days during the first zoeal stage.
Control 10 ppb Dimilin
Exposure of Replicate Replicate
10 ppb on 123 123
Day
Day
Day
Day
Day
Day
1
2
3
1 and 2
2 and 3
1 to 3
92
92
92
92
92
92
86
86
86
86
86
86
86
86
48
68
28
62
8
2
90
18
0
4
.0
88
46
10
-------
TABLE IV
Species: Callinectes sapidus
Bowl Size: 8.9 cm diam
~ of Bowls: 5 per concentration
Larvae per bowl: 10
Total r Larvae: 50 per
concentration
Date Hatched:
Series I 7/27/77
Series II & III 7
Series IV 7/29/77
Salinity: 30°/QO
Temperature: 25°C
Light: 12-12
Diet: Artemia nauplii and
Arbacia embryos
SERIES I
Percent survival to day 20, or day when there was no survival
Acetone
Control
Day 20
20,
*o
0.05 ppb
Kepone
Day 20
20%
0.5 ppb
Kepone
Day 16
0%
1 . 0 ppb
Kepone
Day 17
0%
5 . 0 ppb
Kepone
Day 10
0%
10.0 ppb
Kepone
Day 8
no.
U -o
SERIES II
Percent survival to dav 20, or day when inhere was no survival
Acetone
Control
Day 20
4%
0.05 ppb
Kepone
Day 20
205 •
0 . 5 ppb
Kepone
Day 20
0%
1 . 0 ppb
Kepone
Day 15
0%
5 . 0 ppb
Kepone
Dav 12
0%
10.0 ppb
Kepone ' "
Day 11
0%
SERIES III
Percent survival to day 20, or day when there was no survival
Acetone
Control
Day 2 0
18 °6
0.05 ppb
Kepone
Day 20
18 5
0 . 5 ppb
Kepone
Day 15
0%
1 . 0 ppb
Kepone
Day 14
0%
5 . 0 ppb
Kepone
Day 13
0%
10.0 ppb
Kepone
Day 10
0%
SERIES IV
Percent survival to day 20, or day when there was no survival
/'icetone
Control
Day 20
12%
0.05 ppb
Kepone
Day 20
143
0.5 ppb
Kepone
Day 17
0°o
1.0 ppb
Kepone
Day 17
0°,
5 . Q ppb
Keoone
Day 11
0?0
10.0 ppb
Kepone
Day 9
0%
-------
TABLE V
Species: Rhithropanopeus harrisii Date Hatched: 8/18/77
Bowl Size: 8.9 cm diam
= of Bowls: 5 per concentration
Larvae per bowl: 10
Total = Larvae: 50 per
concentration
Salinity: 20°/oo
Temperature: 25°C
Light 12-12
Diet: Artemia nauplii
SERIES I
% to Megalopa
% to Crab
% Megalopa to
Crab
Acetone 0.05 ppb
Control Kepone
92 100
92 100
100 100
0.10 ppb
Kepone
100
100
100
x
0.20 ppb 0.40 ppb
Kepone Kepone
96 100
94 100
98 100
0.8C
Kepc
1C
1C
1C
SERIES II - '" -
% to Megalopa
S to Crab
Acetone 0.05 ppb
Control Kepone
92 96
90 56
0.10 ppb
Kepone
98
92
0.20 ppb 0.40 ppb
Kepone Kepone
92 94
92 92
0.8C
Kepc
c
I
% Megalopa to
Crab
98
60
94
100
98
-------
TABLE VI
Species: Rhithropancpeus harrisii
Bowl Size: 8.9 cm diam
£ of Bowls: 5 per concentration
Larvae per bowl: 0
Total = Larvae: 50 per
concentration
Date hatched: 9/16/77
Salinity: 20°/oo
Temperature: 25°C
Light: 12-12
Diet: Artemia nauplii
SERIES III
Acetone 0.1 ppb 2.5 ppb 5.0 ppb 7.5 ppb 10.0 pp
Control Kepone Kepone Kepone Kepone Kepone
% Survival
for 11 days
100
100
96
100
100
96
SERIES IV
Acetone 0.1 ppb 2.5 ppb 5.0 ppb 7.5 ppb" ~ '10.0 pp
Control Kepone Kepone Kepone Kepone Kepone
% Survival
for 11 days
100
100
96
98
92
96
-------
TABLE VII
Species: Rhithropanopeus harrisii
Bowl size: small, 8.9 cm diam
% of Bowls: 5 per concentration
Larvae per box/1: 10
Total = Larvae: 50 per
concentration
Date Hatched: 9/27/77
Salinity: 20°/oo
Temperature: 25°C
Light: 12-12
Diet: Artemia nauplii
SERIES V
Acetone 10.0 ppb 20.0 ppb 40.0 ppb 80.0 ppb 160,
Control Kepone Kepone Kepone Kepone Kepc
Survival
for 11 days
82
86
88
34
SERIES VI
Acetone
Control
10.0 ppb
Kepone
20. 0' ppb
Kepone
40.0 ppb
Kenone
SO.O ppb
Kepone"
160
Keo;
Survival
for 11 days
86
100
90
64'
-------
The Role of Sediments in the Storage,
Movement and Biological Uptake of Kepone
in Estuarine Environments
Annual Report
to:
The Environmental Protection Agency
From:
Robert J. Huggett, Project Manager
The Virginia Institute of Marine Science
For the period
10/20/76 to 10/20/77
Grant Identification Number
R804993010
-------
Preface
Included in this document are three sections which
describe the efforts of the Virginia Institute of Marine
Science's staff on the Role of Sediments in the Storage,
Movement and Biological Uptake of Kepone in Estuarine
Environments. The first section is entitled: "Kepone in
James River Sediment," by Maynard M. Nichols and Richard
C. Trotman. The second, "Kepone Water-Sediment Elutriates,"
by Robert J. Huggett and the third, "Uptake of Kepone From
Suspended Sediments by Oysters, Rangia and Macoma,." is
by Dexter S. Haven and Reinaldo Morales-Alamo.
Also attached is a progress report on the EPA funded
James River Hydrographical Survey Study which was conducted
in the late summer of 1977.
-------
KEPONE IN JAMES RIVER SEDIMENTS
An annual progress report to EPA
by
Maynard M. Nichols and Richard C. Trotman
October 1977
1. Purpose.
This study aims to determine where kepone has accumulated in
the bottom sediments; that is, where are the sediment sinks for
kepone? A second aim is to trace the routes and rates of trans-
port; that is, what happens to kepone-bound sediment when released
from its source? Finally, how long will i£ take to reduce levels
of kepone in the sediment by natural processes?
Results emerging from the study are of use to advise state
and federal authorities how to clean-up kepone pollution through
natural processes. They provide basic data on sedimentary pro-
cesses for benthic ecosystem models; they are of use for evalu-
ating the effects of dredging kepone-rich sediments. As a
tracer of sediment, kepone provides new information on sediment
dispersal and the circulation of fine-grained material in a
classic estuary.
2. Highlights of Activities.
Efforts during the period were highlighted by the following:
•Review of James River sediment data to predict fate of
kepone for program formulation.
- 1 -
-------
•Presentation of paper on results historical review,
First Kepone Seminar, at VIMS, October 1976.
•Preliminary field sampling of surface sediments along
length of James in three periods, September, December
1976, and March 1977; 37 to 52 stations sampled during
each period; 18 cores obtained.
•Co-ordination conferences with EPA program manager,
Dr. Tudor Davies, Gulf Breaze and Virginia State Water
Control Board, October through December, 1976.
•Employment of project personnel, Mr. Richard Trotman,
completed April 1977; sedimentologic effort in full
swing. j
•Liason with Battelle Northwest, Dr. Onishi, on field
programs and math model formulation.
•Liason with Manhatten College, Dr. D. O1Conner and
R. Thomann, concerning formulation of a math model
for sediment and kepone transport.
•Development of structure for mathematical model of
sediment-kepone transport with Dr. Kuo.
•Formulate plans for suspended sediment-kepone field
study, May 1977.
•Follow-up sampling of bottom sediments and selected
cores of dredge disposal sites, July 1977. Continued
lab analyses of these samples and previous samples.
- 2 -
-------
'Preparation for field study; filters, field equipment,
and field labs for processing suspended sediment, June
through July 1977.
•Field observations, sampling and measurement of kepone
on suspended sediment, currents, and related parameters,
August 1977.
•Laboratory analyses of suspended sediment samples, total
concentration, organic content, September through
October 1977.
•Participation in Second Kepone Seminar and'kepone
Symposium at the 4th International Conference on
Estuaries.
/
•Follow-up sampling of bed sediments in Hampton Roads
and lower Chesapeake Bay in conjunction with closing
of area to crabbing; 12 stations occupied.
•Field sampling of bed sediments curtailed in October
1977. Data reduction largely complete.
3. Approach.
Efforts during the period mainly consisted of field sampling,
laboratory analyses, and data reduction. First, historical data
on kepone and James River sediments were reviewed to identify
probable kepone sediment sinks and relative rates of deposition.
Sampling stations were sited throughout the estuary in relation
to water depth, bathymetry, oyster grounds, deposition patterns,
dredge and disposal sites, and in relation to the kepone source.
- 3 -
-------
Field procedures were worked out to sample freshly deposited sedi-
ment on the bed as well as in cores at selected sites. Laboratory
procedures were set up to process samples for particle size and
organic content. The horizontal and vertical distributions of
kepone were delineated graphically and evaluated with time over
one year in relation to basic information concerning sedimentary
processes and transport of fine-grained sediment. An attempt was
made to determine from field samples the distribution of kepone
in relation to particle size and organic content.
4. Methods and Procedures.
Bed sediments were obtained by a Petersen grab with a 0.05 m£
bite area or a 7.6 cm (3-inch) diameter cocer. The corer was
especially constructed for obtaining soft mud with minimal- dis-
turbance. Approximately 30 ml of sediment was obtained from the
top sediment surface and returned to the laboratory for analyses.
Stations were closely positioned by ranging or sextant bearings
on buoys and landmarks. Samples were frozen prior to laboratory
analyses.
In the laboratory bulk sediment samples were processed for:
(1) kepone content, (2) organic matter by loss on ignition, and
(3) particle size (percentage sand, silt and clay) by sieving
and pipette. Additionally, the sieved fraction, less than 63u
of samples collected in September and December 1976, was analysed
for both kepone content and for particle size by a Coulter Counter.
Laboratory methods follow conventional procedures described in
- 4 -
-------
ORIGINAL
SAMPLE
FREEZER
STORAGE
PARTICLE SIZE
ANALYSES,
SIEVE AND PIPETTE
PARTICLE
SIZE
< 63U,
COULTER
COUNTER
SAND
FRACTION,
STORED
SILT AND CLAY
FRACTION,
KEPONE ANAL.
< 63u
Figure 1. Scheme for laboratory processing of bed sediments.
- 5 -
-------
Moncure and Nichols (1968), Standard Methods (1973) and Folk
(1961). Details are given in laboratory instructions on file at
VIMS sedimentological lab. Figure 1 summarizes steps in labora-
tory processing.
5. Results and Their Significance.
Spatial Variability. A special study of variations in kepone
concentrations in bulk bed sediment over a small spatial range
uas conducted at two selected stations: (1) station 15 in lower
reaches near Wreck Shoal with 3 m water depth and (2) station
40a in middle reaches at buoy 62 with 6 m water depth. At sta-
tion 15, four samples were taken at random from the top <: 2 cm
of sediment and of the top < 15 cm of sediment, all from the
same grab. Table 1 lists the results. Spatial variations within
the 0.05 m2 area of the grab are relatively small with standard
deviations less than + 7 percent.
Table 1. Variation in kepone concentrations in the top < 2 cm
and the top <- 15 cm of sediment from a single grab;
station 15, June 15, 1977.
Depth Interval Kepone, ppm
0-2 cm 0.026
0.025 Mean
0.029 Range
0.026 Std. Dev,
0-6 cm • 0.012 Mean
0.013 Ranse
0.027
0.025 - 0.029
+ .002 (+ 7%)
0.013
0.012 - 0.013
+ .001 (+ 8%)
0.013 Std. Dev.
At station 40a one sample was taken of the top < 2 cm of sed-
iment from 10 successive grabs. The grabs were obtained at random
- 6 -
-------
waile the vessel drifted over distances of 225 m downstream and
135 m upstream from the station. Results of the sampling and
analyses (Table 2) indicate a very wide range of values within a
distance less than 230 m. Despite the low bottom relief and small
textural differences of the sediment at the site, kepone concen-
trations ranged as much as 0.41 ppm. When surface samples were
taken at random from 12 successive grabs at the same station,
number 40a, (Table 2) (an anchor station with an area of about
200 ms) the kepone concentrations ranged 0.47 ppm with a standard
deviation of 44 percent.
Table 2.
Spatial variation in kepone concentrations from the
top < 2 cm of sediment of successive grabs at station
40a, July 5, 1977 (drift station) and July 20, 1977
(anchor station). . /
Drift Station
Downstream
225 m
0.062
0.074
0.081
0.067
0.096
0.110
0.130
0.340
0.360
0.470
Upstream
135 m
0.021
0.025
0.029
0.023
0.017
0.013
0.027
0.033
0.029
0.023
Mean
Range
Std. Dev.
Mean
Range
Std. Dev.
0.179
0.062 - 0.470
+ 0.151 (+ 84%)
0.024
0.013 - 0.033
0.006 (25%)
Mean
Range
Std. Dev,
Anchor
Station
0.27
0.17
0.44
0.21
0.14
0.27
0.33
0.39
0.34
0.61
0.61
0.28
0.338
0.14 - 0.61
+ 0.153 (+ 44%)
- 7 -
-------
The narked variations are partly due to the sampling process
whereby some surface sediment is necessarily washed in the grab
or disturbed at depth. However, most local variations are
inherent in the bed sediments which are affected by variations
in scour and fill, variations in texture and organic matter.
Such variations define rather broad limits which may be placed
on the kepone distribution as a function of location. They
affect "seasonal" distributions inasmuch as the navigational
capability of relocating a station is no better than a circle
130 m in diameter.
Distribution of Kepone in Surface Sediments. The sediments from
middle reaches are the most contaminated. As shown in Figure 2,
f
average kepone concentrations in bulk bed sediments from the
channel (:> 4 m depth) are higher between mile 38 and 52 than
near the source (mile 63) or farther seaward in the estuary.
This is the zone of the turbidity maximum which lies landward
of the inner limit of salt intrusion. Suspended sediment con-
centrations in this zone are higher than elsewhere most of the
year.
When longitudinal distributions of kepone are compared for
surveys in December 1976, March 1977, and July 1977, there are
no significant trends with time. Instead the concentrations
are relatively stable within a range of about 0.10 ppm. How-
ever, the average levels of concentration from December 1976
through July 1977 in middle reaches (0.15 ppm) are generally
- 8 -
-------
0.3
ex
LJ
S
O
o_
LU O.I
TURBIDITY !
MAXIMUM ZONE
40 '20
DISTANCE LANDWARD,miles
MOUTHED
Figure 2. Longitudinal distribution of average kepone concentrations
in bed sediments from the channel of the James Estuary;
mean of December 1976, March and July 1977 values.
- 9 -
-------
O.I %o SALINITY
Jamestown / (average)
OURCE
TURBIDITY MAXIMUM
< ZONE >
Warwick
River/>.20ppm
Newport
News
0 .1 0 -.2 0
ppm
KEPOIJE
SEDIMENTS
.06-.10
OTTOM
7 Hampton
'
03-.06
JAMES ESTUARY
<.03
ppm
Figure 3. Horizontal distribution of average kt-ponc- conccntriit
J ., 1 1 „ ,.,1
-------
lower than those measured earlier by VIMS in September 1976 and
by the Corps of Engineers in January 1976 when concentrations
were 0.27 to 0.48 ppm.
The zone of high sediment contamination covers both channels
and contiguous shoals. As shown in plan view, Figure 3, aver-
age concentrations are higher in the reach between Jamestown and
Weyanoke than elsewhere. The highest average concentration is
in sediment from a shoal off Dancing Point. Elsewhere, concen-
trations are locally high off mouths of tributary creeks such as
Bailey's Creek near the kepone source, Chippokes Creek, The
Thorofare, Jamestown and the Warwick River. Substantial con-
centrations, ranging 0.66 to 1.20 ppm, are found in Burwell Bay.
However, concentrations are relatively 'low in narrowed reaches
around Hog Point. Kepone content generally diminishes seaward
from Burx^ell Bay to Hampton Roads where concentrations are less
than 0.010 ppm. Twelve sediment samples from lower Chesapeake
Bay in September 1977 all had concentrations less than 0.010 ppm.
Distribution of Kepone at Depth in Sediments. Contamination of
bed sediments in zones of natural fill (undredged) extends to
about 40 cm below the bed surface (Figure 4). Greatest contam-
ination, often exceeding 0.50 ppm, occurs at depths of 10 to
20 cm below the surface. However, in cores from shoals in the
shipping channel where sedimentation is locally fast (i.e., 30a),
concentrations increase downward to a depth of 60 to 80 cm. This
trend reflects the diminished supply of kepone-rich sediment with
- 11 -
-------
KEPONE IN SEDIMENT
CORES
DEPTH KEPONE-*
o o i ot 0-3 ppm
ppm
0 O.I 0.2 03
30
cm
40
60!
SPOIL
CHANNEL
FILL
UNDREDGED
NATURAL
FILL
Figure 4. Depth distribution of kepone in cores from selected sites.
- 12 -
-------
time since the Summer of 1975. Kepone content of old dredged
material decreases slightly with depth (i.e., cores 41a, 30b,
1.9-7.5). The depth trend results from mixing of sediment dur-
ing dredging and disposal. The contaminated sediment is most
likely mixed and "diluted" by uncontaminated sediment and thus
reduces the overall concentration.
A few samples from the Jamestown-Daneing Point reach collected
in May 1967 showed dec table amounts of kepone (.038 and .018 ppm).
Although the content is low, the samples suggest that the life
span of kepone in the sediments is at least 10 years.
State of Xepone in Sediments. The concentrations of kepone are
orders of magnitude greater in the bed sediments than dissolved in
estuary water. An indication of the states of kepone storage in
the sediments is gained by examining its relation to percent^clay
content, mean particle size and organic content.
Finer-grained sediments are generally the most contaminated.
A plot of mean grain size versus kepone concentrations throughout
the estuary (Figure 5a) shows a great deal of scatter. Likewise
a plot of percent clay content versus kepone concentrations varies
widely (Figure 5b). Part of the scatter results from the great
variation in textural types throughout the estuary whereas kepone
content partly varies in relation to its source. When kepone
content of samples from a single reach of the estuary is considered,
however, there is a trend for higher kepone content in the fine-
grained sediment with high clay content.
- 13 -
-------
020
0 16
0.12
e
1 CL
CL
h-*
•F-
1 g 0 08
CL
0.04
0
*
• *
• *
•
t
*
•
-
A
-
*
.
•
•
•
* • •
y • • • - -
•
••
''
X
t
*
-
B
*
020
0.16
0.12
0.08
0.04
. 0
-
-
"
•
. « *
• "'"• 1 ' T ' " ,...—.... . . 1
. .
• ! '•
t •
* * *
• j
1 j i —
MEAN PARTICLE SIZE
80
% CLAY
100
2 4 6
% ORGANIC MATTER
igures 5a. Kepone content versus
mean particle size.
5b. Kepone content versus
percent clay content.
5c. Kepone content versus
organic matter indicated
by loss on ignition.
-------
There is a distinct trend of increasing kepone content with
increasing organic content. As shown in Figure 5c, organic-rich
sediments have higher kepone content than sediments with low
organic content. As expected, samples landward from the kepone
source or from zones of scour, display wide scatter. The trend
indicates kepone prefers organic matter, either adsorbed on
detrital particles or ingested when the organic matter was pro-
duced. As organic matter slowly decomposes in the sediment,
there is an opportunity for kepone to escape into interstitial
or overlying water.
6. Discussion.
Sedimentary Sinks for Kepone. The James Estuary is an environ-
ment where much river-borne sediment accumulates. Zones of
active deposition may be expected to be areas of. relatively high
sediment contamination. On the other hand, zones where the bed
is scoured into older sediment or zones where river-borne sedi-
ments are by-passed, are zones of relatively low contamination.
Inasmuch as sedimentary processes are relatively slow, deposi-
tion sites are indicators of long-term processes. They are an
end product of short-term variations induced by local wave and
current transport.
Kepone contamination is generally greatest in sites of active
sedimentation: (1) the Jamestown-Daneing Point reach which is
also the site of the turbidity maximum, (2) Burwell Bay, and
(3) tributary creek mouths. Zones of sedimentation have been
- 15 -
-------
cr.
i
JAMES ESTUARY
V;ZH\\V^
SEDIMENTATI
RATE
DEEPENING SHOALING
PER 70 YRS. PER 70 YRS.
THAN IM.' '
-LESS THAN I M.
SPOIL
-GREATER THAN 2
CHANNEL
40 DISTANCE UPSTREAM.Km.
r
Figure 6. Sedimentation rates in the James Estuary based on water
depth changes over 70 years, from Nichols (1972).
-------
delineated in a former study (Nichols, 1972) (Figure 6) from
differences in water depths over 35 and 70 years. The rates of
sedimentation within the zones probably change with time but
the sites of deposition persist.
Kepone concentrations are locally high off the mouth of
Bailey's Creek, the kepone source. Hoxvever, the main distribu-
tion does not display decreasing concentrations with distance
atfay from the source. Instead, the main sink is in the middle
estuary, the zone of the turbidity maximum where suspended
sediments are trapped and deposited. Sediments in this zone
are finer-grained than elsewhere, less than 8u mean size. Clay
content in this zone is also higher than elsewhere in the
estuary.
Routes of Transport. From the sedimentation patterns, kepone
distributions and existing hydraulic knowledge of the James, it
is possible to sketch the probable route of kepone-sediment
transport. Both the source of kepone and the major source of
suspended sediment come from the same direction, landward or
upstream of the estuary. Since the influx of sediment from
Bailey's Creek is very small in proportion to the influx of
sediment from the main river, it is probable the kepone was
mainly introduced in the dissolved form and bound to suspended
sediment from the main river. Since the estuary is fresh above
Jamestown most of the year, net transport from Hopewell to
Jamestown is directed seaward. When suspended sediment reaches
- 17 -
-------
the Jamestown area, transport is slowed down because net velocity
approaches zero in the null zone at the salt intrusion head. The
null zone acts as a dynamic barrier that restricts seaward trans-
pDrt of river-borne suspended sediment carried near the bottom.
Only sedLnent carried near the surface is transported farther seaward
through the upper layer. If this sediment settles downward, it
is carried back upstream to the null zone by landward density
currents through the lower estuarine layer. However, sediment
carried over the shoals may escape the estuary through the upper
layer especially during floods like Agnes. Nonetheless, the bulk
of the sediment load is trapped landward of the null zone. As
a tracer of sediment, kepone supports this^fact. Most kepone
concentrations are located in or above the null zone and they.
persist with time, both over the short-term, 8 months of sampling,
and over the long-term as demonstrated from the distributions at
dspth in cores. The data indicate that it will take a long time,
many years, to reduce levels of kepone in the sediment by natural
processes of decay and dispersal. Part of the kepone will be
buried by "new" sediment but the most significant reduction will
come by "dilution" with uncontaminated sediment introduced during
freshets and floods. This trend has already started on the floor
of the shipping channel where sedimentation is locally fast.
- 13 -
-------
KKPON1-: WATiCR-SKDIMKNT KLUTKLATHS
Many pollutants have an affinity to sediments which is goverru d
by the surface charges on particles. This is particularly true for
some of the trace metals - such as zinc - with the clay mineral
portion of the sediments. The magnitudes of the surface charges are
affected by pH and salinity (Parks, 1967). Therefore, it was necessa
to determine if Kepone behaved in a similar manner because, in the
James River, both the estuarine and the tidal fresh water portions
with their wide ranges of pH and salinity were contaminated by the
pesticide. As well the distribution of Kepone in the bottom sediment;
of the James show a marked increase in that portion usually in the
vicinity of the freshwater - saltwater interface. At this boundary
the waters change from fresh, (salinity <0.5^) to saline, (salinities
0.5'' to 20 - 25^). Also in this region the pH of the water increases
from near 7 to 8 due to the buffering capacity of seawater. With
these abrupt changes in pH and salinity coinciding with the change
in Kepone concentration, it appeared possible that fresh water
sediments, highly contaminated with the pesticide, were being "ex-
tracted" by estuarine waters as they traversed this boundary pro-
gressing seaward or that Kepone in solution was not adsorbed by
sediments in saline waters. Therefore, experimsnts were conducted
in the laboratory to determine the extractibility of sediment-Kepone
by waters with varying ranges of salinity and pH.
The experimental design included two phases. The first phase
was to determine the accuracy and precision of the analysis of water
for dissolved Kepone and the second phase was to determine the amount
of Kepone removed from contaminated freshwater sediments by waters
with pH's ranging from 6 to 9 and salinities of <0.5^> and
-------
These ranges of pH's and salinities bracket those found in the
James River.
Phase I, Water-Kepone Analysis
The mechod utilized for the Kepone-water extraction was one
developed by The Environmental Protection Agency, Research Triangle
Park (1975) . It involves liquid extraction using benzene as the
organic solvent. The extractions are carried out in seperatory
funnels with 3 successive treatments of the same water with benzene
at a ratio of 1:10 benzene to water. The extracts are combined and
then dried by passing them through anhydrous sodium sulfate. The
combined extracts are then analyzed by electron capture gas chroma-
tography.
To check the efficiency and accuracy of the procedure, Kepone
free water, (obtained either from Kepone noncontaminated estuaries
such as the York or from laboratory deion.ize'fl-double distilled
stocks), was spiked with known amounts of Kepone, extracted -and
analyzed (Table I).
Phase II, Water Extraction of Kepone From Sediments.
The experimental design for this phase involved subjecting
Kepone contaminated sediments from the James River, obtained from
the fresh water portion, near Hopewell, to waters with varying pH's
and salinities. The salinities were either fresh, (0.06^), obtained
from the James River or saline, (19.57'), gotten from the mouth of
the York River at the Virginia Institute of Marine Science's facility.
The pH's of these waters were adjusted to the desired levels by
addition of either reagent grades of hydrochloric acid or sodium
hydroxide.
After the desired pH and salinity were achieved, a portion of
wet sediment (100 g ) was placed in a flask and the water (250 ml) w;
-------
added and the mixture was agitated with a Wrist-Action Shaker for
1 hr. Following this the sediments were separated by centrifugation
and the supernatant water was extracted for dissolved Kepone by the
method previously described in the Phase I section of this report.
In all, 36 separate extraction were analyzed and the resulting
water Kepone concentrations were compared to that in the exposed
sediments. The comparisons are reported as the percent removed by
a water of a given pH and salinity in Table II.
Discussion:
The data from Phase I clearly show that the Benzene method of
extracting Kepone from water yields approximately 85fv or better of
the amount of the pesticide from solutions spiked at 1 ppb to 10 ppb.
However, at concentrations below 1 ppb the efficiency drops greatly •
for instance, 64>? yield at 0.5 ppb. These yields can be used to
judge the accuracy obtained for Kepone analyses of water by this
method. The precision estimates can be seen from the standard" devia-
tions which show + 14^ or better for spiked solutions of 1 to 10 ppb,
The precision of the method for concentrations of 0.5 ppb are in the
same range which suggests that a portion of the "spike" may be
sorbed to the walls of the glassware or lost by some other means.
Attempts were made to try solvents other than benzene, for
extraction, (ethyl acetate - toluene, methylene chloride) but with
the similar results - dissolved Kepone at concentrations less than
1 ppb may be 100" in error.
Since only at the 10 ppb Kepone concentration were the effect
varying salinities on the analysis compared, it is risky to judge
salinity effects on the method. Evenso, there is no obvious effect
using natural waters of 0.06 and 19.5^>.
- 3 -
-------
The extraction experiments, the results of which are given
in Table II and Figure I, show that there is no apparent affect
of either salinity or pH, within the ranges used which approximate
those found in the James River, on the extractibility of Kepone
from sediments by water. It must be kept in mind, however, that
the amounts of Kepone extracted were in the tenths of ppb range.
Since the analytical methodology is less than ideal at these con-
centrations some differences could go undetected. Figure I shows
that all results are within 2 standard errors of each other which
implies no difference at the 95'.- confidence interval.
The data indicate that, if the analyses are correct, the
partitioning coefficient of Kepone from sediment to wafer is approx-
imately 6 x 10" , irrespective of natural ranges of pH and salinity.
It follows then, that the relatively high concentrations of Kepone
f
at the fresh water-salt water interface and upstream are likely due
to the turbidity maximum (mentioned in the sediment section) rather
than chemical factors such as partitioning.
- 4 -
-------
References
Environmental Protection Agency, 1975, Preliminary Report on
Kepone levels from Hopewell, Va area. Briefing at Research
Triangle Park, North Carolina
Parks, G. A., 1967. Aqueous Surface Chemistry of Oxides and Complex
Oxide Minerals: Equilibrium Concepts in Natural Water Systems,
p. 121-160. In Gould, F. (Ed), Advances in Chemistry, Series
67. American Chemical Society Publications.
-------
TABLE I
Extraction Efficiencies of Kepone from
Water by the Benzene Method
Salinity
0%, Deionized H00
ii *•
it
1 1
it
it
u
it
11
M
It
II
II
II
It
It
It
It
It
II
II
11
II
It
II
II
II
0.067. James R. HoO
it
ti
u
M
it
u
Adjusted
PH
7.0
M
u
H
u
M
II
II
tl
II
It
II
II
M
It
It
II
It
II
It
II
11
11
It
II
It
M
7.0
u
ti
M
i ,
ii
it
Spiked
Keoone Concentration
lOppb
u
5ppb
it
it
u
ti
Ippb
ii
M
M
tt
II
It
" - /
II
II
It
O.Sppm
u
II
It
II
II
II
II
II
lOppb
u
it
u
u
M
ii
7o Recovery
967.
997.
877.
907.
787o
94%
957o
977.
• 727o
937.
697.
93%
56%
102%
85%
86%
- 967.
83%
72%
51%
67%
71%
67%
55%
80%
4S%
69%
86%
99%
92%
85%
83%
77%
76%
-------
["A. J I (continued)
Adjusted
Salinity pH
19.5% Ycrk R. H90 8.0
n *• M
M ii
n ii
n n
ti ii
n n
Spiked
Kepone Concentration
lOppb
11
•'
"
"
n
% Recovery
7-4%
85%
73%
99%
74%
103%
9970
-------
TABLE I
Summary
Adjusted Spiked
Salinity pH Keoone Concentration
Deionized + Distilled 7.0 lOppb
5ppb
Ippb
O.Sppb
1.06% James River H20 7.0 lOppb
i.5% York River HO 8.0 lOppb
Average yield
And Standard dev
98 + 2%
89 + 7%
85 + 147,
64 + 11%
85 + 8%
87 + 13%
-------
Elutriate Results
S n H n 1 ty
II
II
II
I!
II
II
II
II
II
(Sediment
pH
6.0
n
7.0
it
ppm Kepone)
'/•_ Re-moved
0.04J
0,06/ji?
0.11
0.11
0.09
0.12
'0.01
0.07
0.06
0.06
0.11
STD. ERROR - 0.01
0.05 + 0.01;^ of total Kepone in
sediment recovered at a pH 6.0 H
0 . 06'/',
0.03 + 0.04"- of total Kepone ir.
sediment recovered at a oil 7.0
+ 0.06,'
STANDARD ERROR 0.01
II
II
8.0
Tt
II
9.0
0.06
0.09
0.09
0.05
0.06
0.08 + .02,^ of total Kepone in
sediment recovered at pH 8.0 +
0.06^
STANDARD ERROR 0.01
. X
0.06 + 0.01" of total-Kepone in
sediment recovered at pH 9.0 +
0.06,"
STANDARD ERROR 0.005
19.51.
5.0
6.0
0.03
0.04
0.06
0.03
0.03 + ? ,
0.04 + 0.02" of total Kepone in
sediment recovered at pH 6 +
19. 5>
STANDARD ERROR 0.009
7.0
0.02
0.07
0.04
STANDARD ERROR 0.015
0.04 + 0.03".' of total Kepone in
sediment recovered at pH 7 & 19.'
-------
it
it
8.0
it
M
II
II
It
0.09
0.06
0.05
0.06
<0.01
0.05
0.04
0.06
0.02
0.05
0.05 + 0.02;:- of total Kepone
in secTiment recovered at pH
8.0 + 19.5",
STANDARD ERROR 0.007
II
II
II
9.0
M
0.07
0.05
0.03
0.05 + 0.02," of total Kepone
in secTiment recovered at pH
9 + 19. 5>
STANDARD ERROR 0.012
-------
o
£.10
o
UJ
.02
JAMES RIVER SEDIMENTS
Extracted with water
#
Av.±2 std errors
I
• 0 salinity
-- I9.5%o
7 8
PH
-------
U1TAKK OF KKPONK i'KOM SUSi'KNUKi) SKIHMICNTS
BY OYSTERS, RANG IA AND MACQMA
Introduction
Laboratory studies on the uptake of Kepone from
sediments in suspension by bottom-dwelling organisms were
undertaken by the Virginia Institute of Marine Science at
Gloucester Point, Virginia on December 1, 1976. The first
two months were spent in acquisition and preparation of
laboratory equipment and space for the experiments.
In the period of time since then, three series of
laboratory experiments were conducted with three species of
bivalves. Eight experiments were completed with the oyster
Crassostrea virginica, five with the clam Rangia cuneata and
one with the clam Macoma balthica. Most of these experiments
involved exposure of the animals to contaminated sediments
in suspension. In ;:wo of them, however, the animals were
placed in a bed of contaminated sediments with uncontaminated
river water flowing over them.
This repo;:t presents the results of three series
of experiments followed by a discussion.
Materials and Methods
Apparatus
A diagram of the basic arrangement of the apparatus
used to conduct the:>e experiments is shown on Figure 1. The
units labelled A through D were used only during the first
series of experiments when ambient river water temperature
was below 10 C most of the time. York River water was piped
-------
-2-
into a constantly-overflowing box (A) from which it was
pumped through heat exchangers (C) into a rectangular cas-
cading trough (D). The latter served to allow bubbles
created by the escape of dissolved gases to dissipate before
reaching the animal trays. This section of the system was
not used in the last two series of experiments when river
water temperatures were above 10 C. Then, York River water
was piped directly Into a rectangular trough (E) which was
suspended from the ceiling directly above the wet table
that held the experimental trays. Water depth in the trough
was maintained at 20 cm by a drain standpipe of that height.
Water to supply the experimental trays was siphoned
out of trough E with plastic tubing. In the first series
of experiments wate;: flow rates were controlled by inserting
glass flounneters (F) in the tubing siphons ahead of the
mixing chambers (I), In the last two series of experiments
the flowmeters were omitted. Instead, flows were regulated
by the bore size of the plastic tubing used for siphons.
This eliminated constrictions in the tubing caused by adjust-
able clamps which enhanced flow interruptions due to clogging.
Siphons were cleaned daily and flow measurements made before
and after the siphons were cleaned.
Water from the siphons entered a rectangular mixing
chamber made of acrylic plastic (I), 25 cm in length, 16 cm
in width and 14 cm in height, through a smaller chamber (2 cm
long, 3.5 cm wide and 14 cm high). The smaller chamber was
connected to the larger one by a circular opening with a 2 cm
-------
-3-
diameter. Contaminated sediment suspensions also entered
the mixing chamber through the same small chamber. Stock
suspensions were kept well mixed in flasks (H) by magnetic
stirrers (J). They were metered into the mixing chamber at
a constant rate by peristaltic pumps (G).
River water and sediment suspensions were mixed
in the mixing chamber by magnetic stirrers. Observation
showed that the mixing was complete before the mixture flowed
cut of the mixing c.namber. Sedimentation in the chamber
was negligible. The diluted sediment suspensions flowed into
the experimental trays (K) through a standpipe located at the
end opposite to the one through which water and sediments
entered the chamber. The system set up was the same for
trays holding control animals except for elimination of
components G and H.
In experiments with the clam Rangia cuneata, York
River water salinit/ was reduced to between 5 and 6/00 by
addition of fresh ground water pumped from a shallow veil.
A second rectangular trough (P) was suspended below the one
receiving York River water (E). York River water was siphoned
(Q) from trough E into trough P. Fresh water was also piped
into a cascading trough similar to D to eliminate gas bubbles
generated, by the change in pressure the ground water was
subjected; to before it flowed into trough P. Water of the
resulting; lower salinity was then siphoned into the trays
holding I'angia clarr.s following the same system setup labelled
F through K in Figure x.
-------
Figure 2 shows a partial view of the apparatus
used in the series of experiments.
A system of sediment traps was used -co insure
that no contaminated sediments from our experiments escaped
into the floor drain which emptied into the York River. The
first component was the wet table on which the experimental
trays were set (L in Figure 1). A standpipe about 2.5 cm
high inserted in the drain hole of the wet table converted
the table into a sediment trap. A plastic circular tank (50
cm high and 30 cm in diameter) received water from the wet
table through a pipe reaching.close to the bottom. The
tank overflowed near its top into a series of three rectangular
boxes (114 cm long and 25 cm wide), each with a 15 cm high
standpipe overflow. The third box overflowed into the floor
drain. The sediments and other excess solids obtained in
the experiments wero collected in carboys for disposal.
Experimental Trays
Two types of trays were used to hold experimental
animals. In most experiments, a tray made of acrylic plastic
49 cm long, 26 cm wide, and 8 cm high, were used. The over-
flow end was 6 cm high and that also was the depth of the
water in the tray. This tray was not compartmentalized and
the animals laid directly on the bottom (Figure 3).
A larger acrylic plastic tray, 81 cm long, 54 cm
wide and 8 cri deep was used in the third series of experiments
to hold oysters whose biodeposits were collected. A baffle
-------
-5-
at the overflow end of the tray maintained water level at
a depth of 6.5 cm. These trays were divided into 25 compart-
ments by plastic strips 2.5 cm high. Each compartment held
cne oyster. The compartments facilitated separation and
collection of biodeposits.
Eiodeposits
Biodeposits produced by oysters receiving contaminated
s.ediments in suspension in the large trays were collected
every day with a bulb pipette. The aggregates collected at
the end of each weexly period were then analyzed for Kepone.
Every tiir.e biodeposits were collected, sediments settling
out by gravity in the same tray were also collected and the
weekly accumulation also analyzed for Kepone contents. Each
day, after biodeposits and sediments had been collected,
every compartment was cleaned of any remaining sediments.
Animals Buried In Mad
A modification to the manner usually used to expose
ctnimals to contaminated sediments, i.e., by flowing sediment
suspensions over th2m, was introduced in the third series
of experiments. Oysters and Rangia were buried partially
and fully, respectively, in beds of contaminated sediments
held in the smaller of the trays described above (Figure 4).
The sediment bed was 4 to 5 cm deep. It was made up of
unsieved sediments from the same batch used in simultaneous
experiments with flowing suspended sediments.
-------
-6-
Oysters were pressed into the sediments at about
a 30° ancle. Up to one-third of their height was below the
sediment surface level. The valve area over the gills pro-
truded above the sediment surface. Rangia were pressed into
the mud so that almost the whole animal was below the sedi-
ment surface level. Within several hours they had buried
themselves fully into the sediment so that only their siphons
showed. Water flowing over the animals and the sediment
bed had no sediments added to it and was approximately two
to three cm deep.
Source of Experimental Animals
The animals used were obtained from areas -to be
p
free of Kepone". Rangia and Macoma were collected from the
Fappahannock River ,a.nd oysters came from the Piankatank
River. All three species were acclimated to the experimental
temperatures and salinities under flowing-water conditions
at least one week prior to use. Analysis before start of
each experiment showed them to be free of contamination
with KeponeR.
Preparation of Sediment Suspension
Figure 5 presents a flow chart outline of the steps
taken in preparation of Kepone^ contaminated sediment suspensions
All contaminated sediments were collected with a sediment
grab sampler at Jordan Point, in the James River at Hopewell
and represented the top 6 cm of the bottom. They were trans-
-------
-7-
ported to the laboratory in 2 or 3 large plastic bags each
containing about 20 kg of material. The contents of each
bag was mixed and transferred to smaller bags in fractions
of approximately SCO ml in volume. The smaller bags were
stored in a freezer until needed. Only sediments collected
on the Scime date were used in any one series of experiments.
When needed, a bag of sediments was thawed, mixed
with well water and shaken mechanically in flasks for 12
hours or more. The sediments were then wet-sieved through
a 63 u arid the resulting suspension diluted up to 70.GO ml
with well water. This volume was labelled as stock suspension
cind giver, an identification number. It was maintained in
suspension by continuous agitation with a magnetic stirrer
and bar. Subsequently, to insure homogeneity in dosage,
it was divided into measured portions by alternately siphoning
a small volume into each of six containers and repeating the
cycle until each container had been filled to the desired
volume.
The samples in two of the containers, with volumes
of approximately 403 and 200 ml, were used to determine the
concentration of Kepone in the suspension and the dry weight
per unit volume of the sediments in the suspension, respectively,
The suspension in the other four containers, usually with
volumes of 1200 and 1600 ml, was the material to be introduced
into the trays holding experimental animals. The suspensions
in the four containers were diluted in a ratio of 1:4, and
pumped into the mixing chambers.
-------
-8-
Sampling of Animals_j.n Trays
Samples of the animals were analyzed for Kepone
at the start of each experiment and at approximately weekly
intervals thereafter for the four-weeks duration of the
experiment. Each sample consisted usually of three or four
animals and at times of up to eight individuals in the case
of oysters and Rangia.. In the case of Ma coma the number
ranged between seven and fifteen. The shell of each animal
was carefully scrubbed after removal from the tray.
Kepone Analysis
Analysis of all samples from concentration of
Kepone were done by personnel of the Department of Ecology
and Pollution in their laboratories. The method used was
soxhlet extraction, fluorosil cleanup and electron-capture
gas croir,,atography.
Determination of Kepone Concentration in Sediments
The concentration of Kepone in the diluted sediment
suspension flowing over the experimental animals was deter-
mined by computation of the product of four factors:
Kc = (sc) (kc) (dl} (d2}
where
KC = computed Kepone concentration in diluted
suspension, in ppb
SG * sediment dry weight per unit volume in
stock suspension, in Kg/1
k «= Kepone concentration determined analytically
-------
d, = factor by which stock suspension was diluted
prior to being pumped into mixing chambers.
d = factor by which the suspension being pumped
into mixing chambers was diluted; determined
by the flow rate at which it was being pumped
and the flow rate of York River water flowing
simultaneously into the mixing chamber.
The factor &2 was controlled in each experiment by
selection of perist.altic pump settings that would deliver
a desired flow rate, of the sediment suspension into the mixing
chamber. The flow of river water into the mixing chamber
was also adjusted t.o the desired rate. Flow of river water
was maintained relatively constant while the flow rate of
contaminated sediments was adjusted so that trays would
receive sediment suspensions at different rates.
Some trays received what was labelled as low
concentrations of sediment (and, therefore, also of Kepone)
while others received medium and high concentrations.
Throughout an experiment the ratio between low, medium and
high concentrations remained fairly constant even though
Kepone concentrations in stock suspensions were variable.
As a result, the concentrations labelled as low were always
significantly lower than those labelled medium or high. The
separation of low concentrations from medium and high ones
is the main distinction made between concentrations in this
report.
Preparation of Data for Analysis
In the coarse of one series of experiments between
30 and 40 different stock suspensions (500 ml bags) were used.
-------
-10-
Scdirr.ent concentration and Kepone concentration varied from
one stock suspension to another. Consequently, the experi-
mental animals in any one tray were not being exposed to a
constant concentration of Kepone during the time they were
held in the trays. However, throughout the duration of an
experiment, the ratio between low, medium and high concentrations
remained fairly constant. As an aid in interpretation of
results, a weighted mean hourly concentration was computed
for each of the weekly periods as the sum of the products
of the concentration in each stock suspension and the length
of time (in hours) that particular suspension was used,
divided by the total number of hours in the weekly period.
Included in these computations, were short intervals during
which, for a variety of reasons, no sediments were being
added to the water flowing over the animals. These intervals
were usually few and anywhere from 15 min to 2 hour in
duration. Also involved was a final interval of eight to
nine hours at the end of a weekly period when the animals
only received river water to allow them to eliminate material
held in their digestive tract.
Despite the mixing done before the sediment sample
i-'as divided into 500 ml fractions, differences in sediment
and Kepone concentrations from one stock to another were
sometimes large. "hus, differences of significant magnitude
were encountered sometimes between the mean hourly concentrations
for the different weekly periods in one experiment. Since
there was a high correlation between the concentration of
-------
-11-
Kepone in oyster meats and its concentration in the sediments,
and to eliminate the effect of the variations between stock
suspensions, the values for oyster meats were normalized
by re-computation based on the mean hourly concentration of
Kepone in the sediments over the approximately four-weeks
duration of experiment. The new values represent the con-
centration of Kepor.,e expected in oyster meats if the con-
centration in sediments was constant. The normalized values
were computed usinc a proportional equation.
Results
Fourteen uptake experiments were completed between
February and Augus : 1977. Eight with the oyster Crassostrea
virginica, five wi':h the wedge clam Rangia cuneata and one
with the clara Macoina baltihlca. In all but two of these, the
animals were exposed to suspended contaminated sediments in
flowing water. In the ot:her two, oysters and Rang la were
exposed to contaminated .sediments by partial or total burial
in an undisturbed bed in a tray.
Exposure periods consisted of approximately one to four
weeks. Kepone concentrations in the sediment suspension were
computed for each weekly period and for each of the progressively
longer periods that represented the total duration of exposure.
Kepone levels in the sediment suspensions were classified
as low, medium and high as labels of convenience. The mean
hourly concentration for each weekly period in experiments where
levels were classified as low ranged between 0.020 and 0.058 ppb
(Tables 1-3). In experiments where levels were classified as
-------
medium and high, mean hourly concentrations ranged between
0.040 and 0.153 ppm.
Mean hourly concentrations for the total duration of
exposure (one, two, three or four weeks) in experiments where
levels vere classified as low ranged between 0.027 and 0.058 ppb
(Tables 4 and 5). In experiments where levels were classified
as medium or high the range of mean hourly concentration was
between 0.057 and 0.153 ppb.
Results are presented separately for each of the three
bivalve species. No data are presented for the Kepone concen-
tration in animals; examined before the start of each experiment
or for control animals because in every case they were under the
level of detectabllity of the analytical procedure.
Crassostrea virginica
Figures 6-8 show the concentration of Kepone in oysters
examined at weekly intervals after exposure to contaminated
sediments in suspension in three series of experiments. The
values in parentheses give the mean hourly concentration of
Kepone in the sediments for the xoeekly period that immediately
preceded removal for analysis of that particular sample of oysters
Results of the first series of experiments showed a uniform
progression in the: concentration of Kepone in oysters with time
(Figure 6). There: x^as indication that an asymptotic level had
been reached after two weeks. There also was a clear separation
between the three lines which represented high, medium and low
concentrations in sediments. A uniform progression was also
evident in the second series of experiments although the
-------
- 13 -
absolute concentrations attained in oyster meats were lower
than in the first series and there was no indication that an
asymptotic level had been reached (Figure 7). In the third
series there was neither a uniform progression nor suggestion
of an asymptotic level.
The three sets of lines in Figures 6-8 did not appear
to share a common pattern. However, they did show that the
higher concentrations in oyster meats were associated with the
higher concentrations in the sediments and vice versa. When
the values for Kepone concentration in oyster meats in the three
series of experiments (Tables 1-3) were grouped into three classes
according to selected concentration ranges it was found that
the values for Kepone in sediments also separated into three
fairly distinguishable groups with different means. Eleven
sediment values associated with concentrations in oyster meats
between 0 and 0.10 ppm had a mean of 0.038 ppb (range: 0.020 -
0.098 ppb). Twelve values for concentration in sediment associ-
ated with concentrations in oyster meats between 0.101 and 0.199
had a mean of 0.058 ppb (range: 0.023 - 0.088 ppb). Five values
for sediments associated with concentrations in oyster meats of
0.20 ppn or greater had a mean of 0.095 ppb (range: 0.070 - 0.113
ppb).
A plot of concentration of Kepone in oyster meats as a
function of concentration in suspended sediments appears in
Figure 9. Regression analysis showed a correlation between the
two sets of data (correlation coefficient = 0.781).
Having obtained this correlation, the values for concentration
in oyster meats were normalized on the basis of a constant, hourly
-------
- 14 -
concentration of Kepone in the sediments. The mean hourly
concentration of Kepone in sediments for the whole duration
of each experiment (approximately four weeks) was chosen as
the normalization constant. The computed means appear in Table 4.
Plots of the normalized values for oyster meat concentrations
appear in Figure 10 and 11. The marked dips in meat concentra-
tions after two and three weeks of exposure during the third
series of experiments have been eliminated in the normalized
curves. The normalized curves suggest that an asymptotic level
is reached after the first week of exposure in that series.
The curves for the first and second series were slightly
altered by the conversion but the original trends shown were not
appreciably changed. The curves for the first series still
indicate an asymptotic plateau. Curves for the second series,
on the ether hand, still show a trend of increasing concentration
in oyster meats with time. The high value seen for the third week
in the borken line for the first period results from a relatively
high value in the meats in the original data while the correspond-
ing value in the suspended sediments was relatively low (medium
concentration, Table 1).
There were significant differences in the temperatures at
which the three series of experiments with oysters were conducted
(Table 6). In the first series, York River water had to be
heated to raise it to desirable levels. The minimum and maximum
daily temperatures recorded near the source of our river water
supply for each of the weekly periods included in the experiment
were: 1st week, 3.2-7.6°C; 2nd week, 6.4-10.4°C; 3rd week,
9.0-12.8°C; and 4th week, 10.0-12.0°C. Water temperatures in the
-------
- 15 -
cxpe-rimontal trays ranged between 14.0 and 21.0°C during the
four weeks Included, with the average being between 17 and 18°C
for each of the weekly periods.
The second and third series of experiments were conducted at
ambient temperatures. These ranged between 18.3 and 25. 7°C
during the four weeks of the second series with an average for
each week in the range of 20.9 to 23.5°C (Table 6). During
the third series the overall range was 25.0 to 34.0°C with the
weekly average ranging between 26.6 and 29.6°C.
During the first series of experiments, daily salinities
ranged between 17.5 and 22.1,^ for the four weeks, and the weekly
average ranged from 18.4 to 20.4,'v (Table 6). During the second
series, the corresponding salinity ranges were 16.2 - 20.31 and
17•1-19.4^. Likewise, the ranges of the corresponding averages foi
the third series were 20.2-23.61- and 20.6-23.1".-.
One of the experiments in the third series involved weekly
analysis of Kepone concentration in the meats of oysters that had
been held partially buried in an undisturbed bed of contaminated
sediments. York River water flowing over the sediment bed was
unconta.-ninated by Kepone. The concentration of Kepone in the
sediments forming the bed averaged 1.77 ppm in two samples
analyzed before the oysters were introduced (Table 7). A mixed
sample from the same tray analyzed after the oysters were removed
showed a concentration of 2.89 ppm. A sample collected from
the top one centimeter layer of the tray after the oysters were
removed had a Kepone concentration of 2.24 ppm.
After one weak in the sediment bed the Kepone concentration
in two samples of oysters averaged 0.037 ppm (Table 7). The
-------
- 16 -
concentration in oyster meats decreased gradually during the
next three weeks below the detectability level of the analytical
techniques, i-e_., 0.02 ppm.
Mean sizes of oysters used in the three experiments appear
in Table 9. They ranged between 7 and 8 cm in height during the
first and third series of experiments and between 5 and 6 cm in
the second series.
Oyster Biodeposits
Oysters concentrated Kepone in their biodeposits to levels
thousands of times higher than those found in the suspended
sediments (Table 8). The concentration factors for.feces ranged
from 11,000 to 55,000. In pseudofeces, the range was between
3,000 to 20,000. The concentration in feces was always higher
than that in pseudofeces but the magnitude of the difference varied
considerably between the paired sampled compared.
Concentration of Kepone in sediments that settled by gravity
in the tray compartments was usually slightly higher than those
in pseudofeces. However, it was also significantly lower than
that in feces.
Rangia cuneata
Five experiments were conducted with the wedge clam Rangia
cuneata during the second and third series of experiments. In
four, animals were exposed to contaminated sediments in suspension
and in one th^y were buried in a bed of contaminated sediments.
The results obtained for Rangia during the second series
of expeiriments are almost identical to those obtained for oysters
during the same series (Table 2, Figures 12 and 7). Most of the
actual values found at any one weekly interval were close and the
line trends are similar.
-------
- 17 -
The data for Rangla in the thrid series of experiments
were sotiex^hat different from those for oysters (Table 3, Figures
13 and 8). Distribution of the weekly values for Rangia meats
tended t;o remain at approximately the same level after the first
week with a slight dip in the third week samples. The oyster
data showed a greater vertical displacement of the weekly values.
The data for both animals showed a fairly distinct separation
between the lines for low and high Kepone concentrations in the
sediments.
Rangia buried in undisturbed contaminated sediments
accumulated Kepone to low levels (Table 7, Figure 13). After the
first week high of 0.05 ppm there was a gradual decrease with
time to 0.03 ppm after four weeks. Rangia receiving low concen-
tration:; of Kepone in suspension accumulated slightly more Kepone
than those buried in the sediments even though the latter had a
Kepone concentration several thousand times greater (2 ppm in the
bed sediments vs. 0.02 to 0.06 ppb in the water column).
Water temperatures in the trays holding Rangia during the
second series of experiments were slightly lower than during the
third series (Table 6). The range during the second series was
between 18 and 20°C and during the third series it was between
20 and 22°C. There was substantially no difference in water
salinities during the two series.
Mean sizes and Rangia used in these experiments appear in
Table 10. They ranged between 4 and 5 cm in height.
Macoma balthica
A single experiment was conducted with the clam Macona
balthica during the second series. Tna M-Tcoma were held in the
-------
- 18 -
same tray with oysters receiving sediments in suspension at a
high concentration of Kepone. However, they were placed in the
tray one week later than the oysters and consequently, they
remained in the tray one week after all the oysters had been
removed.
The Macoma laid directly on the bottom of the tray and,
being fairly small (average height was between 1.4 and 1.7 cm;
Table 11) were in close contact with the contaminated sediments
that settled on the tray bottom. Sediments settling to the
bottom of the experimental trays were removed every two or three
days .
The Macoma accumulated Kepone at the fastest rate of the
three species studied to date. After three weeks the concentra-
tion was 0.33 ppm (Figure 14). During the fourth week there
was a slight drop to 0.30 ppm.
Mean water temperatures in the trays holding Macoma ranged
between 21 and 24°C during the four weekly periods (Table 6).
Mean water salinities ranged between 17 aid 2d/-o.
Mean sizes of Maooma used in these experiments appear in
Table 11. Thay ranged around 1.5 cm in height.
Condition index. Measurements of the meat quality of samples of
the experimental animals showad no significant differences between
those analyzed at the start of the experiments and those analyzed
after approximately four x
-------
There was little difference in the results obtained for Crass-
ostrea and Rangia. Macoma, however, accumulated Kepone in greater
concentrations than the other two species.
Crassostrca and Rangia showed similar trends in uptake of
Kepone from suspension. This showed that the two species have
similar feeding habits. As suspension feeders, they are reacting
in a similar manner to the presence of the sediments in suspension.
Such a similarity was reinforced by the experiments in which
individuals of the two species were buried partly or fully in a
bed of contaminated sediments. Neither one of the two species
accumulated much Kepone under those circumstances. Water flo-7
over the sediment beds was relatively slow and the water-sediment
interface was not disturbed. Therefore, very little of the
sediment was re-suspended. Concentrations in Rangia were slightly
higher than those for oysters and if there is any significance
to the difference it may be an indication that by being fully
buried with its siphon close to the sediment surface, Rang La
had access to sediments not available to oysters.
The data for oysters showed a strong correlation between
the mean hourly concentration of Kepone in suspended sediments,
computed for weekly intervals, and the mean concentration in
oyster samples exposed to those sediments during the same weekly
period. As illustrated in Figures 6-8, usually the Kepone in
oyster meats decreased or Increased from one week to the next
following a decrease or increase in Kepone in the sediments during
the intervening week. The validity of such a correlation is furthe
reinforced by the similarity between the patterns of the curve
for low and high sediment concentrations in each of the three
series of exoerinents.
-------
- 20 -
A weaker correlation (0.614) was also found in the data
for Rangia. Further collection of data for Macoma will be
necessary before it can be determined if the relationship holds
for that species.
This correlation indicates that, at the temperatures included,
oysters and possibly other bivalves such as Rangia and Macoma
depurate themselves of Kepone continuously at the same time that
they ingest and accumulate it. Therefore, in order for the Kepone
level to remain at a high level, the Kepone concentration in
suspension will also have to remain at a correspondingly high
level.
Consequently, disturbance of river bottoms contaminated with
Kepone by natural processes or other processes initiated by man,
which would result in an increase in "the suspended sediment load,
appear to be capable of causing a sharp increase in the levels
of Kepone in individuals of bivalve populations within reach of th
increased load. On the other hand, it would appear that such an
increase in Kepone in the affected animals would also decrease
sharply once the disturbance is terminated.
It is difficult to evaluate with the data obtained to date
the influence of temperature on the uptake and depuration of
Kepone by oysters and Rangia. More data are required to establish
that.
Further studies are planned to investigate this relationship
between Kepone in sediments and in bivalves. The effect of con-
centrations in the sediments higher than those tested so far will
be considered. The effect of higher water flows capable of
-------
- 21 -
causing suspension of surface sediments in a bed holding buried
animals will also be studied. Experiments that include combina-
tions of contamination and depuration of bivalves will also be
conducted.
The: levels of Kepone flowing over animals in experimental
trays have been fairly low - never higher than 0.15 ppb in the
water column - in the experiments conducted so far. This has
been dictated by restrictions in the capability of our system
and personnel to maintain larger quantities of sediments in stock
suspensions and flowing over the animals around the clock for
four weeks. Changes required to achieve higher sediment concentra-
tions will be implemented in the forthcoming series of experiments.
The data indicate that a leveling in the concentration of
Kepone in oysters and Rangia occurs after the first week of
exposure. This was seen best in the curves obtained by normali-
zation of the data using as a constant the mean hourly concentra-
tion of Kepone in the sediments for the duration of each experiment
Since no animal samples were analyzed for a period shorter than
one week it is quite possible that the leveling may occur sooner
than one week. Either way, this is another indication of the
efficiency of these bivalves to depurate themselves of Kepone
since it: is evidently a balance between uptake and depuration that
is responsible for the leveling off in the curves.
Analysis of oyster biodeposits indicated that Kepone is
concentrated in feces to levels many thousand times higher than
it is present in the water column. These observations re-emphasize
the importance of the effect biodsposition can have on the
physico-chemical characteristics of sediments. At the same time
-------
- 22 -
oysters accumulate Kopone In their tissues to levels up to
3000 times that in the water column, they are also re-depositing
high concentrations of the chemical on the bottom. This re-
deposition is being done in the form of material less likely
to be resuspended because of its nature as an aggregate.
Kepone concentration in oyster pseudofeces was not much
different than that found in sediments that settled by gravity
onto the tray bottom. Therefore, there appears to be no indica-
tion that pseudofeces contribute to the deposition of Kepone-rich
sediments any more than natural sedimentation would. However,
pseudofeces form an aggregate which like feces may also resist
re-suspension to a greater extent than naturally-settling
sediments.
There is no way to establish to what extent sediments
settling by gravity in experimental trays are included in the
samples of feces and pseudofeces collected. However, the concen-
trations recorded for feces are so much greater than in the
natural sediments and the bulk of the feces was so obviously
greater ~han the fine blanket of sediments on the bottom of the
tray, that it can be safely infered that their contribution to
che values recorded for feces are minimal.
Literature Cited
Haven, D. S. 1950. Seasonal cycle of condition index of oysters
in che York and Rappahannock Rivers. Proc. Nat'1 Shellfish
Assoc. 54: 42-65.
-------
Table 1. Concentration of Kepone in sediments and in the
meats of oysters during successive exposure per-
iods in first series of Kepone uptake experiments
24 February - 27 March, 1977
Exposure
Period
No.
days
Sediments
Range
(ppb) Meats
Hourly
Mean
Mean
Concentration
Factor
Low Sediment Concentration
1
2
3
4
6.9
14.8
2L.8
29.2
0.014
0.014
0.003
0.015
- 0.039
- 0.066
- 0.045
- 0.046
0.027
0.037
0.023
0.033
0.086
0.125
0.135
0.113
3185
3289
5625
3228
Medium Sediment Concentration
1
2
3
4
6.9
14.8
2L.8
29.2
0.027
0.027
0.006
0.029
- 0.083
- 0.142
- 0.091
- 0.092
0.057
0.073
0.045
0.067
0.130
0.160
0.185
0.133
2281
2078
3854
1900
High Sediment Concentration
1
2
3
4
6.9
14.8
21.8
29.2
0.040
0.054
0.008
0.044
- 0.197
- 0.197
- 0.133
- 0.137
0.082
0.104
0.070
0.098
0.185
0.250
0.210
0.257
2256
2294
2838
2495
IShort period of time when no contaminated sediments
were being added to the water flox^ing over the animals
(i.e., sediment concentration = 0) are not included
in range. However, they were used in computing the
mean. This includes the final 8-9 hours when animals
were allowed to flush out sediments in their digestive
tract prior to removal for analysis.
-------
Table 2. Concentration of Kcpone in sediments and in animal
meats during successive exposure periods in second
series of Kepone uptake experiments. 13 May - 19
June, 1977.
Exposure No.
Period Days
Range
Sediments (ppb)
Hourly
mean
Meats
Mean
(ppm)
Concentration
Factor
Low Concentration
Oysters :
1
2
3
4
Rangia:
1
2
3
4
7.3
14.8
22.0
29.0
7.3
14.8
22.0
29.0
0.024
0.024
0.017
0.028
0.024
0.024
0.016
0.028
- 0.07B1
- 0.058
- 0.040
- 0.055
- 0.077
- 0.057
- 0.039
- 0.054
High Concentration
Oysters :
1
2
3
4
Rangia:
1
2
3
4
Ma coma:
1
2
3
4
7.2
14.7
21.9
28.9
7.2
14.7
21.9
28.9
7.5
14.7
21.7
29.0
0.054
0.058
0.040
0.068
0.057
0.061
0.043
0.071
0.058
0.040
0.068
0.095
- 0.178
- 0.139
- 0.095
- 0.132
- 0.18S
- 0.147
- 0.100
- 0.140
- 0.139
- 0.095
- 0.132
- 0.131
0.042
0.035
0.026
0.038
0.039
0.034
0.025
0.037
0.098
0.086
0.063
0.093
0.104
0.091
0.067
0.098
0.086
0.063
0.093
0.098)
0.039
0.058
0.064
0.096
0.025
0.050
0.048
0.083
0.09
0.16
0.11
0.23
0.05
0.14
0.11
0.22
0.13
0.19
0.33
0.30
931
1667
2424
2526
641
1453
1912
2237
905
1860
1732
2484
521
1545
1644
2254
1512
2992
3564
3067
•"•Short periods of time when no contaminated sediments
were being added to the water flowing over the animals
(i.e., sediment concentration = 0 are not included in
range. However, they were used in computing the mean.
This includes the final 8-9 hours when arrivals were
allowed to flush out sediments in their digestive tracts
prior to removal for analysis.
-------
Table 3. Concentration of Kepone in sediments and in the meats
of oysters and Rangla during successive exposure periods
in third series of Kepone uptake experiments. 8 July -
9 August, 1977.
Exposure No.
Period Days
Sediments (ppb) Meats
Range Hourly Mean
mean (ppm)
Concentration
Factor
Low Sediment Concentration
Oysters :
1
9
3
4
Rangia:
1
2
3
4
8.0
15.4
23.4
31.0
8.0
15.4 .
23.4
31.0
0.018
0.012
0.007
0.008
0.020
0.014
0.008
0.008
- 0.0871
- 0.058
- 0.041
- 0.085
- 0.097
- 0.066
- 0.044
- 0.082
0.047
0.020
0.020
0.035
0.058
0.026
0.024
0.041
High Sediment Concentration
Oysters:
1
2
3
4
Rangia:
1
2
3
4
8.1
15.5
23.5
31.0
8.1
15.5
23.5
31.0
0.113
0.067
0.049
0.067
0.058
0.063
0.041
0.068
2404
3350
2450
2030
1000
2423
1708
1658
0.046
0.031
0.019
0.019
- 0.223
- 0.096
- 0.078
- 0.195
0.113
0.043
0.040
0.088
0.21
0.10
0.069
0.16
1858
2325
1725
1818
0.058
0.039
0.021
0.023
- 0.284
- 0.121
- 0.086
- 0.230
0.153
0.065
0.053
0.126
0.12
0.12
0.085
0.125
784
1846
1604
992
-'Short periods of time when no contaminated sediments were being added tc
the water flowing over the animals (i.e., sediment concentration = 0)
are not included in range. However, they were used in computing the met
This includes the final 8-9 hours when animals were allowed to flush
out sediments in their digestive tract prior to removal for analysis.
-------
Table 4. Normalized values for Kepone concentration in
oysters exposed in laboratory trays to suspen-
sions of sediments contaminated with Kepone.
Presented as a function of the mean.hourly
concentration in sediments for the duration
of each experiment.
Exposure
Period
Length
of
Exposure
(days)
Mean
hourly
cone .
Kepone
for each
period
(ppb)
Mean
hourly
cone .
Kepone
for
accumulated
time periods
(ppb)
Actual
cone .
Kepone
in
oyster
meats
(ppm)
Normal ized
cone .
Kepone
in
oyster
meats^
(ppm)
First series of experiments (24 Feb - 27 March 1977)
0.027
0.032
0.029
0.0303
1
2
3
4
6.9
14.8
21.8
29.2
0.027
0.037
0.023
0.033
1
2
3
4
1
2
3
4
6.9
14.8
21.8
29.2
6.9
14.8
21.8
29.2
0.057
0.073
0.045
0.067
0.082
0.104
0.070
0.098
0.057
0.066
0.059^
0.0613
0.082
0.094
0.085^
0.0903
0.087
0.125
0.136
0.113.-
0.130
0.160
0.188
0.133
0.185
0.250
0.209
0.257
0.097
0.101
0.177
0.103
0.139
0.134
0.255
0.121
0.203
0.215
0.269
0.236
Second series of experiments (13 May - 11 June 1977)
1
2
3
4
1
2
3
4
7.3
14.8
22.0
29.0
7.2
14.7
21.9
28.9
0.042
0.035
0.026
0.038
0.098
0.086
0.063
0.093
0.042
0.038
0.034Q
0.035-3
0.098
0.092
0.0830
0.0853
0.039
0.058
0.064
0.096
0.090
0.160
0.110
0.230
0.032
0.058
O.OS6
0.088
0.078
0.158
0.148
0.210
-------
Table 4, (con'td)
Normalized values in oyster meats
Exposure Length Mean
Period of hourly
Exposure cone .
(days) Kepone
for each
Third series
1
2
3
4
1
2
3
4
of experiments
8.0
15.4
23.4
31.0
8.1
15.5
23.5
31.0
period
(ppb)
(8 July
0.047
0.020
0.020
0.035
0.113
0.043
0.040
0.088
Mean
hourly
cone.
Kepone
for
accumulated
time periods
(ppb)
- 9 Aug. 1977)
0.047
0.034
0.0290
0.0313
0.113
0.080
0.066,,
0.072-3
Actual
cone .
Kepone
in
oyster
meats-"-
(ppm)
Normalized
cone.
Kepone
in
oyste^
meats"
(ppm)
0.110
0.067
0.049
0.067
0.210.
0.100
0.069
0.160
0.072
0.104
0.076
0.059
0.133
0.167
0.124
0.131
1 Determined analytically
2 Normalized value computed proportionally
3 Mean value reference used in computing normalized values in
oysters
-------
Table 5. Mean hourly concentration of Kepone in
sediment suspensions flowing over Rangia
and Macoma during the total duration of
each period of exposure in experimental
trays.
Total
duration
of exposure
(days)
Mean hourly
concentration
for each
weekly period
(ppb)
Mean hourly
concentration
for full
period (ppb)
Second series of experiments (13 May - 11 June 1977)
Low sediment concentration
7.3
14.8
22.0
29.0
0.039
0. (n 4
0.025
0.037
0.039
0.037
0.033
0.034
High sediment concentration
7.2
14.7
21.9
28.9
0.104
0.091
O.C67
0.098
0.104
0.097
0.087
0.090
Third series of experiments (8 July - 9 August)
Low sediment concentration
8.0
15.4
23.4
31. 0
0.052
0.026
0.024
0.041
0.058
0.043
0.036
0.037
High sediment concentration
8.1 0.153
15.5 0.065
23.5 0.053
31.0 0.126
0.153
0.111
0.091
0.100
-------
'able 5, Continued
Scecies
Kacona:
Total
duration
of exposure
(days)
Mean hourly
concentration
for each
weekly period
(ppb)
Mean hourly
concentration
for full
period (ppb)
Second series of experiments (8 July - 9 August 1977)
High sediment concentration
7.5
14.7
21.7
29.0
0.086
0.063
0.093
0.093
0.036
0.075
0.081
0.085
-------
Table 6. Range and mean of water temperature and
salinity in trays holding animals during
Kepone uptake experiments.
Weekly
Period
Temperature (C)
Range
1st Series
Oysters :
2nd Series
Oysters :
icoma:
Rangia:
3rd Series
Oysters :
Rangia:
Mean
(Feb. 24 - March 27, 1977)
1st
2nd
3rd
4th
(May 13
1st
2nd
3rd
4th
1st
2nd
3rd
4th
1st
2nd
3rd
4th
(July 8
1st
2nd
3rd
4th
1st
2nd
3rd
4th
14.0 -
15.0 -
16.1 -
14.8 -
- June 19, 1977)
18.3 -
21.3 -
22.3 -
20.5 -
21.3 -
22.3 -
20.5 -
20.7 -
16.6 -
13.7 -
19.0 -
18.0 -
- August 9, 1977;
26.9 -
26.8 -
25.0 -
26.5 -
20.5 -
20.4 -
19.0 -
20.0 -
20.8
21.0
20.8
19.6
25.0
25.0
25.7
25.0
25.0
25.7
25.0
25.9
21.2
20.8
22.3
21.3
)
34.0
32.0
30.0
30.9
24. 3
24.9
22.0
23.2
17.2
17.7
18.5
17.0
20.9
22.4
23.5
21.5
22.4
23.5
21.5
23.7
18.6
19.5
20.4
19.2
29.6
29.3
26.6
28.5
22 . 9
22^5
20.3
21.4
Salinity (o/oo)
Range
19.3
19.1
19.1
17.5
22.1
20.6
20.1
19.2
0.5
5.0
1.3
3.2
7.3
6.4
7.9
6.4
20.2
20.9
21.9
22.9 -
2.8 -
3.9 -
4.2 -
2.3 -
- 20.8
- 22.1
- 22.9
23.6
8.7
8.8
6.0
6.8
Mean
20.4
20.2
19.7
18.4
17.5
16.2
17.5
18.9
- 19.2
- -17.9
- 19.5
- 20.3
18.3
17.1
18.3
19.4
16.2
17.5
18.9
19.9
- 17.9
- 19.5
- 20.3
- 20.0
17.1
18.3
19.4
19.9
5.5
5.4
5.1
5.0
20.6
21.6
22
23.
5.9
6.1
5.4
5.4
-------
Table 7. Concentration of Kepone in the meats of
oysters and Rangia held in control trays
receiving no contaminated sediments and
in test trays partially or fully buried
in unsieved sediments contaminated with"
Kepone. July 8 - August 9, 1977. Means
in parentheses.
Exposure
Period
Cumulative
No.
Davs
Kepone Cone
in Animals
Buried in
Sediments
(ppm)
A. Oysters (partially buried in test trays):
2
3
8.5
15.9
23.9
31.6
0.034
0. 040
(0.037)
0.024
0".014
O.Olo
(0.016)
0.014
4.0.009
«O.OC7)
Kepone Cone,
in Control
Animals
^0.007
<0.009
^0.005
<0.004
Rangia (fully buried in mud)
1 8.5
2 15.9
3 23.9
4 31.6
0.067
0.035
(0.051)
0.053
0.039
(0.0*6)
0.029
0.033
(0.033)
0.034
0.031
(0.032)
0.011
<0.006
<0.003
<0.007
-------
Table 7 (Continued)
B. Concentration of Kepone (in ppm) in unsieved sediments
used in test trays in which animals were fully or partially
buried.
1. Mixed samples at start of experiment: 0.71
(Same sediments used in both travs) 2.83
(1.77)
2. Fractionated and mixed samples at end of experiment:
a. Mixed sample from oyster trays 2.89
b. Sample from top 1-cm layer in
oyster tray 2.24
c. Mixed sample from Rangia tray 2.12
d. Sample from top 1-cm layer in
Rangia tray 0.64
-------
.'f •()
I I'D
(f Cf/f ! )
(nuo'fjf j
(8(10'r )
OfO
rc '0
u- '0
ei-o
RIO'U
j.M'jil <<1 J'"1 ;ilill11-1>>
)
C8'l
CH'I
cr-T
6i '0
9.' '0
OS - 0
ff -0
6t T
tccro
OVO'O
oriro
f ',n'0
I 1 1 • 0
/ '. f I • O
p >|>n 'iKi-^ ur
ii.M n j |c:.^ u'.i )
•<[»r if
-------
Table 9. Moan height (in cm) of oysters in
different samples analysed for Kepone
during uptake experiments. Number of
animals in each sample appears in
parentheses.
Exposure
Period
First series
1
2
3
4
Second series
1
2
3
4
Third Series
1
2
Low Medium
Kepone Kepone
cone. cone.
in in
sediments sediments
of experiments
(4)
(3)
(4)
(3)
(4)
(3)
(4)
(3)
(4)
of
(8)
(4)
(4)
(3)
(5)
(4)
(4)
(5)
of e
(3)
(2)
(3)
(3)
6
7
7
7
7
7
7
7
7
.7
.8
.7
.4
.2
.0
.1
.2
.8
(24 Feb -
(4) 7.2
(3) 7.1
(4) 7.6
(3) 7.5
(4) 7.3
(3) 7.1
(4) 6.1
(4) 7.3
(5) 7.3
experiments (13 May
5
6
5
6
5
5
5
5
.8
.0
.4
.9
.4
.7
.1
.6
xperiments
8
7
7
7
.1
.7
.9
.6
(8 July -
High Animals
Kepone Buried
cone . in
in mud
sediments
27 March 1977)
(4) 6.1
(3) 7.0
(4)
(3)
(4)
(3)
(4)
(4)
(4)
7
7
6
6
7
7
7
- 19 June
(8) 5
(4)
(4)
(3)
(5)
(6)
(5)
9 Aug.;
(3)
(2)
(3)
(3)
5
4
6
4
5
5
•
7
7
•7
7
.1
.0
.7
.6
.8
.8
.4
1977)
.7
.6
.3
.4
.8
.1
.5
1977)
.2 (3) 6.6
.9 (3) 7.2
.6 (3) 6.1
.6 (3) 7.4
Control
Animals
(4)
(3)
(4)
(3)
(4)
(3)
(4)
(4)
(3)
(2)
(4)
(4)
(4)
(3)
(5)
(4)
(4)
7
7
7
7
7
7
7
8
7
7
6
6
4
6
5
7
7
.8
.3
.1
.7
.6
.3
.8
.4
.5
.9
.0
.3
.9
.6
.4
.5
.7
-------
T.»!)]<• ') , Con I'd)
.posure
Period
Low
Kepone
cone.
in
sediments
Medium
Kepone
cone .
in
sediments
High
Kepone
cone .
in
sediments
Animals
Buried
in
mud
Control
Animals
(3') 7.6
(3) 7.5
(4) 7.1
(4) 6.8
(3) 7.5
(3) 7.0
(3) 7.7
(4) 6.3
(3) 6.9
(3) 7.3
(4) 7.0
(3) 7.4
(4) 7.7
(3) 7.7
(3) 8.2
-------
Table 10. Mean height (in cm) of Rangia in different
samples analyzed for Kepone during uptake
experiments. Number of animals in each
sample appears in parentheses.
Exposure Low High Aniir.als Control
period Kepone Kepone buried Animals
cone . cone . in
in in mud
sediments sediments
Second series of experiments (13
1
2
3
4
Third
1
2
3
4
(8)
(4)
(4)
(4)
(4)
(8)
(8)
(8)
4.
4.
4.
4.
4.
4.
4.
4.
9
9
9
7
7
6
7
8
series of experiments
(4)
(4)
(4)
(4)
(4)
(4)
(5)
5.
4.
4.
5.
5.
5.
4.
01
99
49
00
15
03
73
(8)
(4)
(4)
(4)
(4)
(8)
(8)
(8)
(8
(2)
(3)
(4)
(4)
(5)
(4)
(5)
(6)
4.
4.
4.
4.
4.
4.
4.
4.
May •
6
8
8
7
8
7
7
6
July -
5.31
4.90
4.
4.
5.
4.
4.
4.
88
92
02
89
83
79
- 19 June 1977)
9 Aug
(3)
(3)
(4)
(3)
(4)
(4)
(5)
(5)
. 1977)
4.85
5.04
'4.92
5.02
5.12
4.96
5.00
5.24
(8)
(4)
(4)
(4)
(4)
'(8)
(7)
(5)
(6)
(6)
(5)
(5)
4.
5 .
4.
4.
4.
4.
4.
5.
4.
4.
4.
4.
8
0
8
7
8
5
7
20
74
98
88
75
-------
Table 11. Mean height (in cm) of Macoma in different
samples analyzed for Kepone during uptake
experiments. Number of animals in each sample
appears in parentheses.
Exposure High Control
Period Kepone Animals
cone.
in
sediments
Second series of experiments (13 May - 19 June 1977)
1 (15) 1.7 (10) 1.6
2 (12) 1.6 (10) 1.6
3 (12) 1.5 (11) 1.6
4 (10) 1.4 ( 7). 1.6
-------
7)
;rouiid •^>——-//
G
Water
To Floor Drain
To Floor Drain
Fifjure 1. Sot':> of apparatus used in uptake experiments with
bi veil-A.: molluscs in three series of experiments.
-------
Ksy to identification of components in Figure 1
A. Constantly-overflowing box providing York River water
supply to system.
B. Submersible pump.
C. Heat exchanger system.
D. Cascading trough used to allow escape of gases coming out
oi: suspension as result of river water being heated up.
E. Constantly-overflowing overhead trough from which water
for experimental trays was siphoned.
F. Flow meter.
G. Peristaltic pump used to meter out sediment suspension.
H. Flask holding sediment suspension.
I. Mixing chamber receiving simultaneously York River water
and sediment suspension.
J. Magnetic stirrer.
K. Experimental tray holding oysters.
L. Wet table holding experimental trays.
M. Drain pipe maintained a water level of about one-inch
on wet table. This served as first component of a series
o.t sediment trays.
N. W.ater from wet table overflowed into a series of three
other sediment traps.
0. Siphon to mixing chamber of Rangia trays.
P. Constantly-flowing overhead trough from which water of low
salintiy for experimental trays was siphoned.
Q. Siphon used to add river water from Trough E to fresh water
in Tray P.
-------
Figure 2. Arrangement of trays, mixing chambers and peristaltic
pumps in third series of experiments in which animals re-
ceived contaminated sediments in suspension. Oysters in
large trays and Rangia in small ones.
-------
"&"' ?•> % 5S2&1 -.*'•*•*»-» ".?-", V|.V |£ * ' ,S
•»WWB»,," "S^^^Sw ™ " *'^V* ' ? «S.^^4^r **«.*
Figure 3. Control oysters (A) and Rangia (B) in small trays
at start of third series of experiments.
-------
Figure 4. Oysters (A) and Rangia (B) partially buried in bed
of sediments contaminated with Kepone at start of third
series of experiments. Subsequently Rangia buried them-
selves fully.
-------
e taken for determination
ol Kopone concentration.
Grab samples collected at
Jordan Point, James River.
Mixed and divided into sub-
samples approximately 500 ml
in volume. Bagged and stored
in freezer.
Bag of sediments thawed.
Mixed with well water and shaken
mechanically for 12 hours or
more.
Wet-sieved through 63 u sieve
Diluted up to 7000 ml with
well water (stock suspension)
Divided into measured portions
Sample taken for determination
of sediment concentration
(dry weight per unit vol).
Diluted with well water 1:4
Metered into experimental trays
and mixed wi th inflowing river
water at predetermined rates to
approximate predetermined dilutions.
Figure 5. Flow chart showing stops taken in preparation of sediments
COn t ill'.i llKi t eel With Ki .>•>.. tm i n t rrwliw- i i f •> ,..i.^ ......
-------
KEPONE CONCENTRATION (PPM) IN OYSTER MEATS
p-
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EXPOSURE PERIOD (DAYS)
Figure 8. Mean concentration of Kepone in meats of oysters exposed
to contaminated sediments in suspension (broken lines)
or partially buried in bed of contaminated sediments
(solid line). Third series of experiments, 8 July-9 August
1977. FiuurOS in oa ren t-hR.qo.q a rp mr-^n hniir-lv /^nnc^nh rx t- i nn
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KEPONE IN SEDIMENTS (PPB)
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Figure 9. Regression of concentration of Kepone in oyster meats
on mean hourly concentration of Kepone in suspended
sediments for v/oekly periods in three series of experi-
-------
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NORMALIZED VALUES CRASSOSTREA VMGINICA
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O SECOND SERIES 13 MAY-19 JUNE I977{MEAN TEMP! 20 9"23. 5 C)
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EXPOSURE PERIOD (DAYS)
i—r
28
Figure 10.
Mean concentration of Kepone in meats of oysters
exposed to contaminated sediments in suspension.
-------
KEPONE CONCENTRATIM (PPM) IN OYSTER MEATS
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EXPOSURE PERIOD (DAYS)
Figure 13. Mean concentration of Kepone in meats of Raj]_g_i_a cunoata_
exposed to contaminated sediments in suspension (broken
lines) or buried in bed of contaminated sediments (solid
line). Third series of experiments, 8July-9 Auyust 1977
Figures ir purer.t MOSCS are mean hourly concentration
-------
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14
21
28
EXPOSURE PERIOD (DAYS)
Figure 14. Mean concentration of Kepone in meats of Macorna balthica
exposed to contaminated sediments in suspension. Second
M;
r_ 1 Q
1 Q"77
-------
EPA Jair.es River Kcponc Hydrop.raphical Survey Study
Progress Report (Nov. 1, 1977)
I, Hydragraphical Survey (Aug., 1977)
Four transects were occupied for the field study
with three stations included in each transect. The rr.iddle
(primary) station or primary station, measured top, middle
and bottom depth, vhile the two side channel stations
measured top and bottom depths. (figures of the transect
positions are included within).
The following is a compilation of information
concerning each station.
-------
J.ircs Riv?r Station, Rivc-r rile 46.51, sampled from 8/26/77
at 1500 to 8/23/77 at 1SOO.
Station 46.51A - total depth 17 feet
Current meter depth off the bottom:
2 feet and 7.5 feet
Current meter time in:
8/23/77 at 1015
Current meter time out:
8/29/77 at 1935
Samples taken at mid depth, included all parameters
except kepone
Station 46.51 B - total depth 19.5 feet
Current meter depth off the bottom:
3 feet and 10.5 feet
Current meter time in:
8/23/77 at 1050
Current meter time out:
8/29/77 at 1925
Samples taken at ton, mid and bottom depths, included
all parameters
Stati rr. 46.51 C - total depth 23 feet
Current meter depth off the bottom:
2 feet, 6.5 feet and 12.5 feet
Current meter time in:
8/23/77 at 0940
Current meter time out:
8/29/77 at 1915
Samples taken at mid depth, included all parameters,
except kepone
-------
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.
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I '
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-------
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',-...
-.{
-------
Station 73.24 sampled from 0800 8/27/77 to 8/29/77 1100
Station 73.24 A - total depth 15.5 feet
Current ir.eter depth off the bottom:
6 feet
Current meter time in:
8/23/77 at 1550
Current meter time out:
8/29/77 at 1523
Samples taken at mid depth, included all parameters
except kepone
Station 73.24 B - total depth 21.5 feet
Current meter depth off the bottom:
2.0, 7.5 and 13.0 feet
Current meter time in:
8/23/77 at 1512
Current meter time out:
8/29/77 at 1545
Samples taken from top, mid, bottom, included
all parameters
Station 73.24C - total depth 12.5 feet
Current meter depth off the bottom:
5 feet
Current meter time in:
8/23/77 at 1440
Current meter time out:
8/27/77 at 1600
Samples taken at mid depth, included all parameters,
except kepone
-------
Star ion 87 .67 sampled from 0900 S/24/77 to 1200 8/26/77
Station 87.67 A - total depth 33 feet
Current meter depth off the bottom:
4,'12.5 and 27 feet
Current meter time in:
8/22/77 at 1620
Current meter time out:
8/29/77 at 1423
Samples taken at mid depth, included all parameters
except kepone
Station S7.67 B - total depth 23.5 feet
Current meter depth off the bottom:
4, 9.5, and 16 feet
Current meter time in:
8/22/77 at 1705
Current meter time out:
8/29/77 at 1410
Samples taken at top, mid, bottom, included
all parameters
S i a t:'. on S 7 . 6 7 C - total depth 13.5 feet
Current meter depth off the bottom:
5 feet
Current meter time in:
8/22/77 at 1730
Current meter time out:
S/29/77 at 1404
Samples taken at mid depth, included all parameters.
except kepone
-------
Station 111 - sampled from 8/24/77 at 0900 to 8/26/77 at 1200
Station 111 A - total depth 18 feet
Current meter depth off the bottom:
4 and 11 feet
Current meter time in:
S/22/77 at 1350
Current meter time out:
8/29/77 at 1215
Samples taken at mid depth, included all parameters
except kepone
Station 111 B - total depth 20 feet
Current meter depth off the bottom:
2/7.5 and 13 feet
Current meter time in:
S/22/77 at 1140
Current meter time out:
8/29/77 at 1210
Samples taken at top, mid, and bottom depth, ir.cluded
all parameters
Station 111 C - total depth 13 feet
\
Current meter depth off the. bottom:
5 feet
Current meter time in:
S/22/77 at 1215
Current meter time out:
8/29/77 at 1225
Samples taken at mid depth, included all parameters,
except kepone
-------
Tide £aup,es were installed in the following three
locations. They were installed one week before the field
intensive survey and pulled out one week after the
intensive survey. Currently, all tide data are being sent
to Fisher and Porter for reduction.
Tide gauge stations.^
1) Wooden Pier at Ft. Eustis
2) Pier Chickahominy Holiday Inn Campground
(off Rt. 5, near mouth of Chickahominy)
3) Westover, Va. Pier (near Hopewell)
II. Data Reduction
All hydrographical and sediment intensive data are
currently being keypunched. Parameters include dissolved
oxy;;<_n, ter/.perature, conductivity, salinity, suspended
rc'iic5 and kepone concentration. It is anticipated to
finish keypunching and editing by the end of November, 1977
Current rr.eter films have been developed and are being
pic pared to be read. It is also planned to hcive the data
reduction work done by the end of November, 1977.
-------
PRELIMINARY ANALYSIS OF KEPONE DISTRIBUTION
IN THE JAMES RIVER
Donald J. O'Connor
Kevin J. Farley
Environmental Engineering and Science Program
Manhattan College
Bronx, New York 10471
Annual Report to Environmental Protection Agency
Environmental Research Laboratory, Gulf Breeze,
Florida, 1977
-------
Introduction
The general purpose of this research project is to assess
the effect of synthetic materials, such as pesticides, on the
water quality and ecology of estuarine systems. The present
phase of the project is being specifically directed to the ana-
lysis of the Kepone distribution in the James River estuary in
the vicinity of and downstream from, Hopewell, Virginia. The
ultimate goal is to provide a quantitative framework for evalua-
tion of the time required to reduce the Kepone concentrations to
acceptable levels.
Significant concentrations of Kepone are present in various
phases of the estuarine system of the James River — in solution,
in suspension, in the sediment and in the food chain, particu-
larly in various species of fish. The interrelationships, or
more specifically, the transport, uptake and release of Kepone,
as shown in Figure II, are thus affected by both physio-chemical
mechanisms, as well as bio-ecological phenomena. The former of
these includes the hydrodynamic transport through the estuarine
system, adsorption to and desorption from the suspended and bed
solids, and the settling and resuspension of these solids. The
latter incorporates the assimilation and excretion routes
through the various components of the food chain. Although
less significant for Kepone, transfer to the atmosphere, photo-
chemical oxidation and biological degradation are potentially
-------
JAMES RIVER STUDY AREA
-TO RICHMOND
N
I
10
KILOMETERS
| NEWPORT NEWS CITY
HAMPTON
ROADS
BRIDGE-
TUNNEL
NORFOLK
FIGURE !_
JAMES RIVER STUDY
-------
TRANSPORT KINETIC ROUTES WITHIN THE WATER COLUMN
I PHOTOCHEMICAL DECOMPOSITION
•0-
EVAPORATION
PHOTO OXIDATION
HYDROLYSIS
AEROBIC
BIODEGRADATION
AIR
INTERFACE:
DIRECT INGESTION
IN VARIOUS LEVELS
OF FOOD CHAIN
WATER
NEKTON 1C
ACCUMULATION
IN FOOD CHAIN
DESORPTION ABSORPTION
I *
SUSPENDED
SEDIMENT
ANAEROBIC BIODEGRADATION-
FIGURE II
TRANSPORT - KINETIC ROUTES WITHIN THE. WATER COLUMN
-------
The Distribution of Kepone on Solids
Natural clays of various types, and organic material, pos-
sess an adsorptive capacity. The rates of adsorptive reactions
are being investigated experimentally under controlled labora-
tory conditions in order to provide realistic kinetic coeffi-
cients for the Kepone analysis. The desorptive characteristics
of both the inorganic and organic fractions of the suspended
solids are also being reviewed. This phenomena of adsorption-
desorption is one of the important transfer routes in the ulti-
mate transfer of Kepone from the system. Based on the Langmuir
Isotherms, equations have been developed to predict the spatial
and temporal distributions of Kepone in an advective-dispersive
estuarine system. However, due to the preliminary nature of
this work, the less complex, advective, steady state model was
used for analysis. Equations governing the water column a,nd
estuarine bed for such a system are as follows:
1. Water
3m
Solids Ux ^- = -K^ + «Kum2
3C
Dissolved U - = --rm -f K - K ( C ) -K
;L
Particulate
- r
2. Bed
3m K
Solids U, 3—^- = + — m. - K m0
2 3x a 1 u 2
-------
Diss olved U,
- -Ko(rc-r2)m2C2+Kdr2m2+Kb(Cl-C2)
Particulate
3P.
r _
where:
the subscripts 1 and 2 denote the water column and estuarine
bed concentrations, respectfully,
and where:
U - horizontal velocity
C - dissolved Kepone concentration
x - longitudinal distance
K - adsorption coefficient
r - solids adsorptive capacity
r - Kepone concentration on the solids
m - solids concentration
K, - desorption coefficient
K, - bed diffusion coefficient
K - aeration coefficient
SL
P - solids Kepone concentration
K - solids settling coefficient
s
a - the ratio of bed volume to water column
volume
[m /sec ]
[meters ]
[l/(yg/£-day)]
[yg/g]
[yg/g]
[g/M
[I/day]
[I/day]
[I/day]
[yg/g]
[I/day]
[ dimensi onless
K - solids scour coefficient
[I/day]
-------
As a first step, this preliminary analysis was simplified
by various assumptions - subject to verification by the ongoing
field and laboratory studies. The first of these assumptions -
3m. 8m^
solids being in equilibrium i.e. -z and -r = 0, appears to be
a safe assumption for the non-saline portion of the estuary. In
addition, the bed solids concentration, m«, was said to be much
greater than the suspended solids concentration, m • the aera-
tion term, K , was taken to be negligible; and the solids
3.
adsorptive capacity, r , was assumed to be much greater than
either of the Kepone concentrations on the solids, r and r_.
The kinetic coefficients - K , K , , K , and K , were assigned
O Q 5 U
from the limited data available. Finally, for this "first-cut"
model, the Kepone concentrations on the bed solids, r^, were
assigned from data; these concentrations were in turn utilized
in predicting the Kepone water column concentrations.
Based on these assignments of coefficients, the longitudinal
distribution of total and dissolved Kepone in the water column
is presented in Figure III along with the State Water Control
Board 1976 Kepone data. The line of total Kepone concentration
fits the data quite well and although the dissolved fraction of
Kepone is high, this concentration is merely a function of
Kepone kinetic coefficients, K and K, - values which were
obtained from a minimal amount of sketchy data. Further analysis
is presently being performed which will predict both the wat-r
column and the bed concentrations of Kepone.
The above analysis will be further complicated as the
-------
BED CONCENTRATION
ppm
WATER CONCENTRATION, ppb
o p .-
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-------
saline portion of the estuary is approached. As the lighter
clay particles which are maintained in suspension in the non-
saline area encounter the saline region of the estuary, floccu-
lation and agglomeration may occur, increasing the size and
possibly the density of the particles. These factors result in
further deposition, which is enhanced by virtue of their occur-
rence in the null zone of the estuary. There are, therefore,
a variety of significant factors which may account for the accu-
mulation of solids and Kepone in the estuarine bed at the fresh
water-saline interface. These factors, along with the inability
to assume solids equilibrium in the saline region, have lead to
a detailed investigation of solid material in the estuary.
Hydrodynamic Transport
Since the concentration of suspended solids is an important
factor as an accumulation site for Kepone, the temporal and
spatial distribution of the solids within the estuarine system
is a necessary element in the analysis. The distribution is
determined by the hydraulic transport through the estuarine
system. A two-dimensional (longitudinal-vertical) analysis has
been developed, based on the fundamental principles of momentum,
continuity and state.
-------
In this analysis, under steady state, tidally averaged
conditions, the longitudinal momentum equation for a later-
ally homogeneous estuary is:
where p = density; p = pressure; N = vertical eddy viscosity;
and u = horizontal velocity. The coordinates for Eq. 1 are
shown in Fig. IV in which the longitudinal x-axis is positive
toward the ocean and the vertical z-axis is positive toward the
bed of the estuary channel. Boundary conditions compatible
with Eq. 1 are,
~ = 0 at z = -n• (2)
-N - Cd/Ub/Ub at z - h (3)
in which -n = surface elevation and h = average depth; C, =
dimensionless friction coefficient; and u, = velocity at the
bed. The vertical component of the momentum equation is
simply the hydrostatic pressure equation:
-------
V
RIVER
FRESH WATER
PLANE OF
NO NET MOTION
OCEAN
FIGURE IV
SCHEMATIC DIAGRAM OF TWO-DIMENSIONAL ESTUARINE CIRCULATION
-------
In order to solve Eq. 1, the hydrostatic pressure, Eq. 4,
is expressed in terms of the horizontal and vertical distribu-
tion of salinity. The equation of state which specifies the
density as a function of salinity is given by:
pf (1 + otC) (5)
in which pf = the density at zero salt content and a = 0.000757
(parts per thousand) . The components of the pressure force
are then evaluated in terms of the observed vertical and longi-
tudinal salinity gradients and freshwater flow, which are
assumed known from measurement.
The solution of the above equations indicates that local
rather than boundary conditions control the magnitude and
gradient of horizontal velocity at a particular location. Be-
cause of local control, the velocity at one location is rela-
tively independent of those at other locations. This condition
occurs as a result of decoupling the equations of motion and
salt transp ort.
Results of this analysis are presented for Pritchard's June
1950 survey and Nichols1 March 1965 survey of the James River in
Figure V and VII respectfully. In addition, the solution also
indicates the depth at which the net horizontal velocity is
zero. Defining this depth at a number of stations and interpo-
lating for others delineates the plane of no net motion for the
saline intrusion zone of the estuary, Figures VI and VIII. At
the tail of the salinity intrusion, this plane meets the bed of
-------
VELOCITY CALCULATION FOR JAMES RIVER ESTUARY (JUNE, 1950)
25
O
^ 20
>
t-
1 15
-j
<
to
oi 10
u
<
u.
"
_ •
X^
H^
^^
.x^
.x^
^x^
1 _U- *
B
I I I I I
40 35 30 25 20 15 10 5 0
DISTANCE FROM MOUTH, miles
VELOCITY, fps VELOCITY, fps VELOCITY, fps
-0.4 0 0.4 0.8 -0.4 0 0.4 0.8 -0.4 0 0.4 O.I
0
r 10
T
)-
Q_
LU
O
20
i\ '
*\
h *} •/
'A •/
»A a
VA 0
- \ i
I I I
/• "
/
f
MP 26.1
u
10
20
\
K
i*
.
/
O
- A \ p
A e
\A f
\A O
^A O
A. \9
I I I I 1*
M.P. 17.4
w
10
20
& I L*
A _,•'
^A «f
\^ f
\ A J*
\A *
\A |^>
^ ^ M.P. 11.9
V
I I I
|
46 11 13 15 15 17 19
SALINITY, °/oo SALINITY, °/00 SALINITY, °/00
NOTE. SALINITY AND VELOCITY MEASUREMENTS BY THE CHESAPEAKE BAY INSTITUTE
FIGURE V
-------
f
H
2
H
o
G
H
O
o
C-l H
> CD
w
G
K
M
c A i iMi-rvx n /
SALINITY, o/
_.
U1 O
VERTICAL
VELOCITY X 10s,
00
_.
Ul
HORIZONTAL
VELOCITY,
fps
VERTICAL EDDY
VISCOSITY/DISPERSION
COEFFICIENT, cm^/s
DEPTH, ft.
o
05
O
O
r
»
B
I
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li
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rn
-a
m
oo
C
>
31
Ul
O
-------
VELOCITY CALCULATION FOR JAMES RIVER ESTUARY (MARCH, 1965)
SALINITY MEASUREMENT
11-20 MARCH 1965
FLOW. 8.SOO CFS
30 25 20 15 10
DISTANCE FROM MOUTH, miles
r 10
i"
O-
LU
Q
20
VELOCITY, fps
0.4 0 0.4 0.8
I
I
M.P. 27.1
10
20
VELOCITY, fps
-0.4 0 0.4 0.8
A
\
I I I
M.P. 21.6
10
20
1 3
SALINITY, o/00
4 6
SALINITY,
VELOCITY, fps
-0.4 0 0.4 0.8
I
M.P. 16 1
9 11
SALINITY,
NOTE: SALINITY AND VELOCITY MEASUREMENTS BY THE VIRGINIA INSTITUTE OF MARINE SCIENCE
FIGURE VII
VELOCITY CALCULATION FOR JAMES RIVER ESTUARY (MARCH, 1965)
-------
in
t1
H
H
O
f
0
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1-3
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g
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OR JAMES RIVER
W
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33
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H H
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01
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VFm'CAL HORIZONTAL VELOCITY, VERTICAL EDDY
SALINITY, o/00 x 1 0* f PS , , fPs VISCOSITY/DISPERSION DEPTH_ ft
1 _ ' fps i i 0 COEFFICIENT, cm2/s
m o'-'^^JONjoNj-i^cocx) oooc
1 1
CO Cl
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^( -0 5
— 3 $ ?
r- .^
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-------
the estuary. Upstream of this area, the horizontal velocity in
the whole water column is in the seaward direction.
The estuary is then segmented horizontally and the hori-
zontal flow in the surface layer at each vertical cross section
is first calculated. Horizontal flow difference between two
adjacent vertical planes gives the vertical flow between the
surface and bottom layer, from which the vertical velocity is
obtained by dividing by the average width of the segment. This
procedure is obviously a solution of the hydraulic continuity.
The vertical flux of salt due to dispersion between the
surface and bottom layers is described by the dispersion coeffi-
cient, e, obtained from the vertical eddy viscosity through
an empirical relationship,
(6)
where Ri (Richardson number) is defined as:
Ri = — (7)
Dfl^2
P(9z}
Equation 6 indicates the relationship between the two coeffi-
cients, whose general validity has been shown by field data,
as presented by Officer.
The tidal diffusion and velocity shear contributions, which
can be envisioned collectively as a longitudinal dispersion across
a vertical section following the classical one-dimensional estu-
arine analysis, did not exhibit themselves in the portion of the
-------
estuary that our models were concerned.
The distribution of salinity was used to test the validity
of the hydrodynamic model - bottom panels of Figures VI and
VIII. Based on these validations of the hydrodynamic model, an
analysis of suspended solids followed by incorporating the set-
tling and scour rates with the hydrodynamic transport to deter-
mine the distribution of solids. Settling rates, for the
present, were assumed constant down the length of the estuary
and this rate was obtained from the average particle size, using
a modification of Stokes' Law. Since little work has yet been
performed on scouring rates in estuaries, these rates were
assigned merely to show that a good fit can be obtained. Results
of this solids modeling, with and without the assigned scouring
rates, are presented in Figure IX.
ASSIMILATION AND DEPURATION OF KEPONE IN THE FOOD CHAIN
The transfer of Kepone from its initial discharge at Hope-
well to its accumulation in the fishery stock may occur in a
number of ways. It may be ingested directly from that which is
dissolved or suspended in the water; it may be assimilated by the
phytoplankton-zooplankton; and it may be taken in by bottom
feeders from the material which has settled in the channel bed.
The predominant sites for settling appear to be downstream from
Hopewell, in the region of the fresh water-saline interface, and
in various dead zones in the fresh and saline regions. Experi-
ments involving assimilation and depuration of Kepone by various
species are being conducted. The rates of accumulation and
-------
SUSPENDED SOLIDS CALCULATION FOR JAMES RIVER ESTUARY (MARCH. 1965)
15
O
o
°. 10
h-
~, 5
LEGEND
O SURFACE LAYER
A BOTTOM LAYER
Q = 8.800 cfs
35
25
20
15
120
LEGEND
SURFACE LAYER
40
35 30 25 20
DISTANCE FROM MOUTH, miles
D
O
10
CC X p
O X £ 5
cc
I
I
NET SCOUR
FROM BED
NET SETTLING
INTO BED
120
100
en
E
to"
g
O
to
o
UJ
Q
Z
LU
CL
CO
to
80
60
40
20
= 6 fpd
LEGEND
SURFACE LAYER
A BOTTOM LAYER
JL
I
I
40 35 30 25 20
DISTANCE FROM MOUTH, miles
NOTE SUSPENDED SOLIDS MEASUREMENTS BY THE VIRGINIA INSTITUTE OF MARINE SCIENCE
TY
-------
excretion, equilibrium conditions and concentrations, lethal
and chronic - are being analyzed in order to incorporate these
kinetic factors in a food chain analysis.
Preliminary analysis has been made in evaluating the assi-
milation and depuration kinetics on various species of fish.
Data from experimental studies performed at EPA's Gulf Breeze
Laboratory are used to evaluate the relevant coefficients. The
equation utilized in this analysis - similar to the Langmuir
kinetic equation for the adsorption to and desorption from sus-
pended solids, is as follows:
= Ko(rc-r)m(t)C -
where
r - Kepone concentration in the biomass [yg/g]
m - biomass concentration [g/&]
t - time [days]
K - assimilation coefficient [I/day]
r - biomass assimilation capacity [yg/g]
C - dissolved Kepone concentration [yg/£]
K - depuration coefficient [I/day]
The only assumption made in this analysis was that the biomass
assimilation capacity, r , was taken to be much greater than the
Kepone concentration in the biomass, r. Results of this analysis
for oysters (Crassostrea, virginica) are presented in Figure X.
-------
0 9
.0—0-
s
9
CD
3.
2
cc
K~
CO
O
co
co
^
Qc
CJ
CO
LU
D
g
CO
LU
cr
LLJ
z
O
Q_
LLI
\
••"*- * \
. «•
0.03 /ig/l
1C-2
10
-1
34 //g/g
0
5 10
ACCUMULATION
0 10 20 30 40 50 60
-«—ACCUMULATION—£»--* DEPURATION >-
TIME, days
15 20 25
-^—DEPURATION—>-
TIME, days
FIGURE X
CALCULATION FOR THE ASSIMILATION AND DEPURATION OF KEPONE IN OYSTI
-------
From these results, it can be shown that the bio-ecological
phenomena of assimilation and depuration can be modeled utili-
zing Langmuir kinetics if data for the evaluation of the relevant
coefficients is available.
CONCLUSION
The equations presented in this report appear to be suffi-
ciently realistic as a first approximation in representing the
various phenomena under consideration. At the present time, the
analysis is being extended to treat the ecological system as a
continuum using trophic length as a metric. Given the inputs
from the sources in the vicinity of Bailey's Bay, the transport
in the non-saline and saline regions of the James estuary and
the distribution of suspended solids and Kepone, the food chain
model is being enlarged to include the uptake and excretion of
Kepone in the various trophic levels and the predation and
feeding associated with these levels. At this time, the saline
and non-saline regions of the estuary are being combined into
one continuous solution. Steady state conditions, which repre-
sent average conditions during various seasons of the year, are
being assumed for these preliminary steps of the analysis.
ACKNOWLEDGEMENTS
The research work described in this report is sponsored
by Gulf Breeze Research Laboratory, Sabine Island, Gulf Breeze
Florida, Grant Number R804563. The participation of Gerald L.
-------
Schnoor is acknowledged. Various phases of the computations
were performed by Cherng-Ju Kim and George A. Leahy, research
assistants in the Environmental Engineering and Science Program
at Manhattan College.
-------
INSTITUTE OF OCEANOGRAPHY
OLD DOMINION UNIVERSITY
NORFOLK, VIRGINIA
Technical Report No. 35
SURVIVAL, DURATION OF LARVAL STAGES, AND SIZE OF
POSTLARVAE OF GRASS SHRIMP, PALAEMONETES PUGIO,
REARED FROM KEPONE® CONTAMI1
POPULATIONS IN CHESAPEAKE BAY
REARED FROM KEPONE^ CONTAMINATED AND UNCONTAM I NATED
By
Anthony J. Provenzano, Principal Investigator
Kathleen B. Schmitz
and
Mark A. Boston
Final Report
Prepared for the
Environmental Protection Agency
Environmental Research Laboratory
Gulf Breeze, Florida 32561
Under
Contract No. CF-6991106J
S'uc>mi,t'z&3. Dy tns
Old Dominion University Research Foundation
Norfolk, Virginia 23503
April 1977
-------
ACKNOWLEDGEMENTS
Tom Leggett assisted in the field collection and laboratory
rearings. We are indebted to Mark Grussendorf, Carl Kinsman,
and especially to Karen Kinsman and Kim Blake for their valuable
assistance in the computer analysis of the data. We thank
Drs. Michael Bender, Robert Huggett, and especially Mr. M. Keith
Ward of Virginia Institute of Marine Science for providing the
analyses for Kepone.
11
-------
TABLE OF CONTENTS
Page
I. INTRODUCTION 1
11 . METHODS 2
Analytical Methods 5
Statistical Analyses 6
III. RESULTS 8
IV. DISCUSSION 15
V. CONCLUSIONS 19
VI. RECOMMENDATIONS 20
REFERENCES 21
List of Figures
Figure
1 Location of collection sites 3
2 Hatching unit 4
List of Tables
Table
Concentration of Kepone (ppm) in samples of ?.
p-ugio arranged by collection site ....
Survival of reared larvae of Pa.la.enone-es pugio
from IS females collected from six sites
Anova table for survival with arcsine transfor-
mation
Mean time to metamorphosis in days for larvae
o f ?. " "j. a -i o
10
11
(cont'd.)
ill
-------
List of Tables - Concluded
Table Page
5 Anova table for time to metamorphosis ... 12
6 Length (in mm) of postlarvae of P. pugio
reared from six sites 13
7 Anova table for postlarval length .... 14
8 Extraction and analysis dates, sample weights,
and volumes of frozen samples analyzed for
Kepone 16
IV
-------
SURVIVAL, DURATION OF LARVAL STAGES, AND SIZE OF POSTLARVAE
1?)
OF GRASS SHRIMP, PALAEMONETES PUG1C, REARED FROM KEPONE
CONTAMINATED AND UNCONTAMINATED POPULATIONS
IN CHESAPEAKE BAY
I. INTRODUCTION
0_ „.. ._^ (chlordecone) into the James River at
Hopewell, Virginia has created far reaching environmental, economic,
and potential health problems for the people of Virginia and
neighboring areas. Kepone, like other chlorinated insecticides,
is highly cumulative and persists in estuarine organisms. Oysters,
grass shrimp, and fishes have concentrated Kepone from 425 to
20,000 times the concentration in surrounding water (Ha'nsen, et.
al., 1976).
Palasmonetes pugio is one of the key components in Atlantic
coast estuaries. A major fraction of the total energy flow through
the food web of these estuarine communities passes through its
populations (Welsh, 1975). In 1975, broods of larvae of P. pugio
obtained from stocks in the Lafayette River, Norfolk, Virginia,
were reared in the laboratory of the Institute of Oceanography,
Old Dominion University, in an attempt to define the environmental
requirements for the larvae and to establish conditions necessary
for consistent success. Erratic results under apparently constant
laboratory conditions were obtained. Samples of P. pugio from the
same locality sent to the Environmental Research Laboratory, Gulf
Breeze, Florida for experimentation, were found to contain sig-
nificant amounts of Kepone, suggesting the possibility that Kepone
may affect larval survival.
The present study was undertaken to determine whether, under
standard laboratory conditions, broods of larvae of this species
obtained from Kepone contaminated and uncontaminated sites in the
Chesapeake Bay might vary in larval survival, time required for .
larval development, and size of post larvae. Inasmuch as variability
-------
among females had not been established, an attempt was made to
evaluate the contribution of this source of variation as well.
II. METHODS
Ovigerous grass shrimp were collected with a pushnet from six
Virginia localities in the Chesapeake Bay region (fig. 1): (1) the
Lafayette River in urban Norfolk, (2) bridge pilings at Gloucester
Point, at the mouth of the York River, (3) Lynnhaven Inlet, Vir-
ginia Beach, at the Chesapeake Bay mouth, (4) small inlet at
Fleeton, Virginia, near the mouth of the Potomac River, (5) the
James River Bridge, Portsmouth, Virginia, and (6) the western end
of the Route 301 bridge across the Potomac River. These localities
were selected in an attempt to include Kepone contaminated and
uncontaminated sites, but the results of analyses establishing
Kepone levels in the populations from these sites were not avail-
able until well after the rearing experiments were completed.
Females with embryos in advanced state of development were
sorted from the general catch and placed 'into individual hatching
units. Each unit was maintained in a separate one-gallon (3.8-
liter) aerated aquarium containing filtered natural seawater
(25 °/oo S) at ambient temperature (22° to 26° C). Water was
changed daily, and hatching usually occurred within 48 hours
after collection. Hatching units were constructed of 10-cm lengths
of 7-cm diameter clear plastic tubing (fig. 2). A layer of plas-
tic mesh (onion bag netting) was stretched across the bottom of
each tube and attached with aquarium sealant; a second layer
(several centimeters above the bottom) supported the female.
The double mesh prevented the female from capturing and eating
her newly hatched larvae which escaped through the bottom of the
unit.
Replicate groups of 33 larvae were obtained from each of the
three isolated females from each site; each group was placed into
a 20-cm-diameter glass culture dish containing one liter of arti-
ficial seawater (Instant Ocean, Aquarium Systems, Inc. Eastlake,
-------
WASHINGTON
1 Lafayette River
2 Gloucester Point
3 Lynnhaven Inlet
4 Fleeton (near Reedsville)
5 James River Bridge (west side)
6 Potomac River (west bank at Rt. 301)
SCALE: IN MILES
0 25
37°_
VIRGINIA
NORTH CAROLINA 76C,20'
Figure 1. Location of collection sites.
-------
10 era
S cm CULTURE DISH
COVER
SHRIMP
DOUBLE BOTTOM
ONION NET
SUPPORT LEG
Figure 2. Hatching unit.
-------
Ohio) at 25 ppt prepared with tap water. Larvae from each female
were reared separately in order to estimate variation due to par-
entage. Thus, six dishes, each with 33 larvae, represented each
site.
Larvae were reared in an incubator at 25° C in the dark. Food
consisted of freshly hatched San Francisco Bay Brand Arterr.ia nauplii
at an initial daily concentration of 10 nauplii/ml of medium. Lar-
vae were observed, counted, and changed to freshly prepared bowls
each day.
Newly metamorphosed post larvae were removed daily, measured
from tip of nostrum to tip of telson (using a dissecting microscope
and a millimeter rule, estimated error = ± 0.2 mm), and frozen for
Kepone analysis.
From each collection site the following samples were frozen
and transported to the Virginia Institute of Marine Science,
Gloucester Point, for Kepone analysis:
a. Ovigerous females
b. Eggs removed from the pleopods of additional females
c. Non-ovigerous females which had recently hatched larvae
d. Larvae newly hatched in the laboratory
e. Laboratory reared post larvae.
Analytical Methods
(Adapted from report by M. Keith Ward)
Frozen samples were delivered to the Virginia Institute of
Marine Science during the period through August 1976 for Kepone
analysis. One third of the samples were analyzed during the
period July through September 1976 and the remainder in February
1977.
Each total sample was extracted with three successive 20-ml
portions of toluene in ethylacetate (1:4) using a Polytron. The
Polytron generator head was rinsed with a few milliliters of
extracting solvent to minimize Kepone loss. The sample tube was
centrifuged after each extraction and the supernatant solution
-------
was passed through anhydrous granular sodium sulfate into a 50-ml
screwcap culture tube. The extract was concentrated to less than
1 ml using a gentle stream of nitrogen and a warm water bath.
A Florisil column consisting of 1.6 gram Florisil covered by
1.6 gram anhydrous granular sodium sulfate in a glass column
(dimensions 100 mm * 30 mm, Corning ^412160) was used in an attempt
to remove components that might interfere with Kepone. The Florisil
column was wet with Solvent I (2 percent methanol, 4 percent ben-
zene, in hexane). The sample was transferred to the column and
the sample tube rinsed five times with 0.5 ml Solvent I. Seven
milliliters of Solvent I was collected as Fraction I and discarded.
This was followed by 30 ml Solvent II (Fraction II) consisting of
1 percent methanol, 2 percent acetonitrile, 4 percent benzene in
hexane. Fraction II (which contains Kepone) was analyzed for
Kepone by electron capture gas chromatography.
The P. pugio samples were quantitated for Kepone on either of
two gas chromatographs: a Varian Model 2700 equipped with two
3H(tritium) electron capture detectors operated at 220° C, and a
column temperature of 210° C; or a Tracor Model 222 equipped with
two linearized high-temperature 53Ni detectors operated at 350° C
and a column temperature of 210° C. The samples were analyzed on
at least two of the following columns: 3 percent OV-1; 1.5 per-
cent OV-17 + 1.95 percent OV-210, or 4 percent SE-30 + 6 percent
OV-210, on 80 to 100 mesh Gas Chrom Q or Variaport 30. The column
dimensions were 6 mm * 2 mm * 183 mm. Values for Kepone con-
centrations presented in table 1 represent the mean of two readings.
Statistical Analyses
Three parameters were analyzed statistically in order to
evaluate intrasite variation due to parentage and intersite varia-
tion of the laboratory-reared larvae. These were (1) survival
of laboratory-reared larvae, (2) time to metamorphosis (larval
duration), and (3) length of postlarvae (rostrum-telson) at
metamorohosis.
-------
Table 1. Concentration of Kepone (ppm) in samples of P. pugio arranged by
collection site (courtesy of Virginia Institute of Marino Science).
Site
1
Lafayette
River
°y^rous 0.039
1 eina les
Kggs 0.36
Females having n 1Q
i . i i U . JLo
hatched eggs
Newly hatched o 11
zoo at;
Post larvae 0.011
2 3
Gloucester
Point Lynnhaven
0.030 0.031
0.044 *
<0.008 0.029
<0.007 0.021
<0.007 <0.001
45 6
James Potomac
Fleeton Rivei" River
-------
Survival data were treated with a two-way nested analysis of
variance (anova) (Sokal and Rohlf, 1969) following arcsine trans-
formation of percent survival. Data for development time were
analyzed with two-way nested analysis of variance and covariance
including repeated measures (Dixon, 1975) on a DEC system 10 com-
puter. Data for post larval length were analyzed in the same
manner as developmental time.
A Pearson correlation coefficient (Nie et al., 1970) was cal-
culated in order to determine the degree of relationship between
postlarval length and developmental time.
III. RESULTS
Survival data for the 36 groups of larvae are presented in
table 2. Analysis of variance of the survival data (table 3)
indicates that there is no evidence for significant difference
in survival of larvae among broods of different females from the
same collection site; nor is there significant difference in sur-
vival of reared larvae from the different sites (p > 0.05).
Data for time required for the larvae to reach metamorphosis
are given in table 4. Analysis of variance (table 5) indicates a
highly significant variance component (p « 0.001) for larval
duration among broods reared from different females representing
the same site. The variation in larval duration among broods
reared from the six different collection sites is not significant
(p > 0.05).
Length data for postlarvae obtained from the experimental
rearings are presented in table 6, and the analysis of variance
of these data in table 7. The intrasite (parental) variation
again is seen to be highly significant (p « 0.001), and the
intersite variation is not significant (p > 0.05).
Larvae which metamorphosed earliest were generally smaller
than those of longer larval duration. Therefore a Pearson
correlation coefficient was calculated in order to evaluate the •
possible relationship between larval duration and length of
-------
Table; 2. Survival oC reaied larvae of Palaemonetes pug-to from 18 females collected from six
sites (F = identification number of individual female).
i\e;>l i c;itcs
Bow 1 A
No. ff survivors
1'ercc-nt survival
Arcs i nc /p
ikv,;l 3
No. of survivors
I'j rcfin l survival
A re SHU; Wp"
Site i
Lafayette- River
«:1 ":2 l:3
31 31 50
93.9 93.9 (»0.9
75.7 7S.7 72.4
30 33 31
90.9 100.0
-------
Table 3. Anova table for survival with arcsine transformation.
Estimates of variance components are expressed as
percentages (n = 2 bowls; a = 6 sites; b = 3 females)
Source of Variation
df
SS
MS
Groups (among sites)
5 956.66 191.33 2.36 n.s
Subgroups (among females) 12 974.34 81.20 2.32 n.s
Error (within females)
18 631.09 35.06
F>05[5,12J = 3.11
F 05[12,18] =2.34
Variance components:
Error; between measurements o2 0- r^
on each female = S 3o'06
Among females within sites = Sir.A
oL/ri
MS , - MS . , .
subgroups within
n
81.20 - 35.06
o
= 23.07 (30. 16f0)
MS - MS
Among sites = S| = groups nb '
191.33 - 81.20
= 18.36 (24^c
S2
S| = 35.06 + 23.07 + 18.36 = 76.49
10
-------
Table 4. Mean time to metamorphosis in days for larvae of ?. pugio
Site No.
I
Lafayette
River
II
Gloucester
Point
III
Lynnhaven
' - iv"
Fleeton
V
James
River
VI
Potomac
River
Female No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
No . Larvae
Metamorphosed
61
64
61
62
58
44
57
57
49
63
63
58
60
59
60
57
53
48
Mean
(days)
13.1
13.5
14.2
12.9
18.1
18.0
17.5
15.4
17.8
16.0
15.1
21.7
15.6
15.6
15.1
17.1
14.2
16.8
Standard
Deviation
0.8
1.2
2.1
1.1
3.0
1.8
1.9
1.3
1.6
1.7
1.0
3.2
1.3
0.9
0.6
2.4
1.2
2.1
11
-------
Table 5. Anova table for time to metamorphosis.
Source
Sites
Females
(w i th i n si tes
Error
Sum of Squares Degrees of Freedom Mean Square
F
],756.84201
1.84500
3,187.98019
12
1016
351.36840 1.3825 n.s
254.15375 80.99806 ***
3.13778
n.s. Not significant (p > 0.05)
*** Highly significant (p < 0.001)
F 05[5,12] = 3.11
F_001[12,«] = 2.74
10
-------
Table 6. Length (in mm) of postlarvae of ?. pugio reared from
six sites.
Site No.
Lafayette
River
II
Gloucester
Point
III
Lynhaven
IV
Fleeton
V
James
River
VI
Potomac
River
Female No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
No. of Larvae
61
64
61
62
58
43
56
57
49
63
63
58
60
59
60
57
53
48
Mean Length
(mm)
7.2
7.4
7.3
7.5
7.8
7.6
7.8
7.7
7.8
7.1
7.0
8.2
7.4
7.1
7.3
7.4
7.0
7.3
Standard
Deviation
0.2
0.2
0.4
0.3
0.5
0.6
0.4
0.5
0.5
0.3
0.2
0.6
0.4
0.4
0.4
0.4
0.3
0.4
13
-------
Table 7. Anova table for postlarval length.
Source
S
Fe
(with
K
i tes
rna
in
rr
les
sites)
or
Sum
4
1
67
16
4
of Squares
.74599
.07723
.22859
Degrees of Freedom
5
12
1014
Mean
8.
5.
0.
Square F
34920 1.49366 n.s.
58977 34.51303 ***
16196
n.s. Not significant (p > 0.05)
*** Highly significant (p < 0.001)
F
[5,12] = 3.11
-------
postlarvae. The correlation coefficient was 0.51 at the 0.00001
significance level indicating a weak, but significant relation-
ship between time to metamorphosis and size at metamorphosis.
Sample size and extraction and analysis dates for frozen
samples analyzed for Kepone are presented in table 8, and the
Kepone values for these samples, arranged by size, are given in
table 1. These results could not be analyzed statistically because
of lack of replication due to small sample size. [Although recov-
ery and reproducibility data could not be obtained for these
samples, oyster samples fortified at 0.3 ppm and fish (bream)
samples fortified at 0.1 ppm extracted with a Polytron gave
recoveries of 84 percent and 80 to 94 percent respectively
(M.K. Ward, personal communication).] Nevertheless, table 1
illustrates that samples from sites 1 and 5 (the Lafayette River
and James Ri.ver respectively) exhibited high Kepone levels rela-
tive to the other sites. Laboratory reared postlarvae from all
sites showed very low (or undetectable) Kepone concentrations.
IV. DISCUSSION
?. pug-Lo is known to be more Kepone-tolerant than several
other estuarine organisms. Acute 96-hour toxicity bioassays
showed the LCsg (expressed in micrograms per liter) to be 6.6
for spot, 70 for sheepshead minnows, 10 for a mysid crustacean,
121 for P. pug-Co, and 210 for the blue crab, Callinectes sapidus
(Hansen, et al., 1976). At least one other species of Palxenone-es
has been shown to have populations resistant to a variety of
organochlorine, organophosphorous, and carbamate insecticides
(Naqvi and Ferguson, 1970).
Chemical analyses of samples revealed Kepone concentrations
from the six sites ranging from undetectable to moderately high
levels. Intersite ranges were as follows: <_ 0.005 to 0.63 ppm
for ovigerous females; <_ 0.003 to 0.47 ppm for eggs; <_ 0.008 to
0.57 ppm for females which had recently hatched larvae; <_ 0.007
to 0.11 ppm for newly hatched zoeae; and < 0.001 to < 0.015 ppm
15
-------
Table 8. Extraction and analysis dates, sample weights, and volumes of frozen
samples analyzed for Kepone (data courtesy of Virginia Institute of
Marine Science).
Site Sample
1 Newly hatched zooea
Ovigerous females
Females having hatched eggs
Eggs
Post 1 arvae
2 Newly hatched zooea
Ovigerous females
Females having hatched eggs
UtfLJS
Pos 1 1 arvae
3 Newly hatched zooea
Ovigerous females
Females having hatched eggs
li' ir f ro
''iyh,-^
Post 1 arvae
4 Newly hatched zooea
Ovigerous females
Females having hatched eggs
Eggs
Post larvae
Extraction
Date
02-08-77
09-15-76
08-04-76
07-2G-76
02-08-77
02-03-77
10-20-76
10-20-76
02-10-77
02-09-77
02-07-77
02-01-77
09-15-76
02-10-77
02-09-77
02-03-77
02-01-77
09-08-76
02-30-77
02-07-77
Gas Chrorn
Analysis Date
02-11-77
09-21-76
08-04-76
07-26-76
02-11-77
02-11-77
02-11-77
02-11-77
02-15-77
.02-15-77
02-15-77
02-15-77
09-21-76
02-15-77
02-14-77
02-16-77
02-16-77
09-09-76
02-16-77
02-17-77
Sample
Weight
(gm)
0.
3.
2.
1.
0.
1.
3.
2.
1.
0.
1.
2.
2.
2.
0.
0.
2.
2.
0.
0.
65
11
70
75
20
37
51
28
03
37
77
88
62
07
34
85
00
89
20
24
Sample
Vol ume
(ml)
17
29
30
29
21
19
29
29
21
24
21
19
29
25
22
18
15
29
22
20
.5
.5
.0
.5
.5
.5
.0
.0
.0
.0
.0
.0
.0
. 0
.0
.0
.0
.5
.0
.0
(co'nt 'd. )
-------
Table 8. Extraction and analysis dates, sample weights, and volumes of frozen
samples analyzed for Kepone (data courtesy of Virginia Institute of
Marine Science) - concluded.
Si to
G
S amp 1 e
Newly hatched isooea
Ovlgerous females
Females having hatched eggs
J.'.'ggS
Post 1 arvae
Newly hatched zooea
Qvigerous females
Females having hatched eggs
Eggs
Post 1 arvae
Extraction
Date
02-02-77
09-06-76
09-06-76
09-06-76
02-09-77
02-02-77
09-07-76
09-07-76
02-10-77
02-09-77
Gas Chrom
Analysis Date
02-16-77
09-09-76
09-09-76
09-09-76
02-17-77
02-16-77
09-09-76
09-09-76
02-16-77
02-17-77
Sample
Weight
( gm )
2.09
2.08
1.90
1.85
0.22
1.08
2.85
3.82
1.24
0.05
S amp 1 e
Volume
(ml )
12.5
30.0
29.5
29.5
22.0
20.0
29.5
29.5
25.5
17.0
-------
for laboratory-reared postlarvae. Not surprisingly, the highest
levels were found in samples from the heavily contaminated James
River and the second highest levels from the Lafayette River,
located near the mouth of the James. While detectable amounts
of Kepone were found in at least some samples from the other sites,
the levels were generally low, and the populations remote from the
James may be considered relatively uncontaminated. In spite of
these wide ranges in concentrations we were able to detect no
difference in larval response (lethal or sublethal) attributable
to the geographic origin of the larvae. However, due to the
limitations of the Kepone analytical technique (minimum sample
size = 1 gram), individual females from which larvae were reared
could not be analyzed for Kepone concentration. The analyzed
samples were a composite of several animals collected from the
same population, and the determined concentrations were average
values. There is, therefore, no way to trace each laboratory-
reared brood to a specific parental Kepone concentration. Thus
it is possible that the reared larvae from a given collection site
may have arisen from females in which the Kepone levels were not
representative of the general population.
As mentioned earlier, this study was occasioned by the occur-
rence of erratic results in larval rearings from an area known to
be contaminated. The erratic results were not obtained, and
therefore were not explained, by this study. The purpose of our
field sampling was simply to determine whether the areas from
which the reared animals were collected were subject to Kepone
contamination. A statistical sampling plan was not attempted,
but clearly should be in order to ascertain representative contam-
ination levels and individual variability in contamination of ani-
mals obtained from a given area. We cannot eliminate the possibility
that larvae reared for this study came from relatively "clean"
individuals, and hence exhibited no immediately observable effects.
The temperature and salinity combination used for the larval
rearings in this study has been determined near optimum for this,
species, at least for stocks from lower Chesapeake Bay (Floyd,
18
-------
personal communication), and the larvae always received sufficient
uncontaminated food. Hence the laboratory-reared larvae were
relatively unstressed. Field hatched larvae from eggs, or females
with Kepone residues near the highest reported here, might be sub-
jected to environmental stresses, such as temperature and/or sal-
inity extremes, contaminated food, etc. Thus synergistic effects,
unexamined here, may be operating under field conditions. In
addition, although the Kepone concentrations detected in this
study may not have had direct observable effects on larval devel-
opment of P. pug-Co reared in the laboratory, there may well be
biomagnification effects upon predators of this species, a topic
outside the scope of this study.
Although this experiment was not designed specifically to
study parental variation, highly significant differences in larval
development time and post larval length were found among broods
from single sites. If we had not accounted for variation due to
parentage (by rearing broods separately) we might have falsely
concluded that that variation was due to site of origin.
V. CONCLUSIONS
Larvae of Palaemonetes pugio obtained from three adults
collected at each of six sites within Chesapeake Bay, when reared
under controlled laboratory conditions, showed no significant
differences in larval survival, larval duration, or length of
postlarvae attributable to site of origin.
Egg-bearing adults of P. pugio, females which had recently
hatched larvae, eggs, newly hatched larvae, and laboratory-reared
postlarvae, when analyzed for Kepone, showed variation in con-
centration ranging from undetectable to levels of 0.6 ppm. Pop-
ulations from the James River and nearby Lafayette River showed
the highest concentrations of Kepone; distant populations showed
lower levels. Laboratory-reared postlarvae representing all six
populations had very low or nondetectable Kepone concentrations.
Survival rates-among broods from individual females ranged
from 61 to 100 percent, but did not differ significantly among
19
-------
females from any one site; nor did the mean survival among larvae
differ from site to site.
Mean time to metamorphosis ranged among broods from 12.9 to
21.7 days. Parental variation in this parameter was highly sig-
nificant, whereas site variation was not.
Mean postlarval length which ranged from 7.0 to 8.2 mm
exhibited significant intrasite variation, but intersite varia-
tion was insignificant.
There was a weak, but significant relationship between time
to metamorphosis and postlarval length at metamorphosis.
VI. RECOMMENDATIONS
1. For successful continuation of analysis of Kepone in ?.
pugio , the previously described Polytron extraction method should
be evaluated for this substrate, including recovery and repro-
ducibility studies. Larger sample size would probably enhance
extraction efficiency. Other methods, e.g., extraction utilizing
a micro-Soxhlet assembly, should be examined. Finally, additional
modified means of sample cleanup may be necessary for future
routine analysis (M.K. Ward, personal communication),
2. Acute toxicity tests and long-term toxicity tests should
be determined on larvae in order to determine at which concentra-
tions Kepone has adverse effects on young stages of P. pug-lo .
3. The present study showed significant variation in responses
of larvae from different females. Obviously such effects should
not be ignored in bioassay design. A study of effects of parentage
on variation in larval responses for P. pug-Lo should be conducted
in order to establish the minimum number of females which would
allow the most reliable representation of populational response.
4. A statistically designed sampling plan should be carried
out in order to determine representative contamination levels and
individual variability in contamination of animals living in a
given area.
20
-------
REFERENCES
Dixon, W.J., ed. 1975. Biomedical computer programs. University
of California Press, Los Angeles. 792 pp.
Hansen, D.J., Wilson, A.J., Nimno, D.R., Schimmel, S.C., and
Bahner, L.H. 1976. Kepone: Hazard to aquatic organisms.
Science, 193:528.
Naqvi, S.M. and Ferguson, D.E. 1970. Levels of insecticide
resistance in fresh-water shrimp, Palaemonetes kadiakensis.
Trans. Amer. Fish Soc., 99:696-699.
Nie, N.H., Hull, C.H., Jenkins, J.G., Steinbrenner, K., and Bent,
D.H. 1970. Statistical package for the social sciences.
McGraw-Hill Book Company, New York. 675 pp.
Sokal, R.R. and Rohlf, F.J. 1969. Biometry. W.H. Freeman and
Company, San Francisco. 776 pp.
Welsh, B'.L. 1975. The role of grass shrimp, Palaemonetes pugio ,
in a tidal marsh ecosystem. Ecology, 56(3):513-530.
21
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Americal Society of Microbiology National Meeting
May 1978, Las Vegas, Nevada
Fate and Effects of Kepone^ in Artificial
Estuarine Ecosystems. A. W. BOURQUIN,* P. H.
PRITCHARD and H. L. FREDRICKSON. U.S. Environ-
mental Protection Agency, ERL, Gulf Breeze, Fl. 32561.
Fate and effects of the pesticide Kepone were studied
in artificial ecosystems, containing water and sediment
from either Range Point salt marsh, FL or the James River,
VA. Approximately 75-80% of ^C Kepone added (6.5 ppm) to
jthe systems accumulated in the detrital fraction. Using
high pressure liquid chromatography and GC-mass spectral
analysis, we detected no transformation products with a
variety of experimental regimes including anaerobic or
aerobic conditions. Neither the addition of glucose (0.1%)
nor naphthalene (0.1%) stimulated the transformation of the
pesticide in analogous systems. No ^CO^ was produced in
any experiment. James River sediment with a history of
depone exposure was likewise ineffective.
Effects of Kepone on microbial communities in these
artificial ecosystems were monitored by determining the
rate of C02 evolution and metabolite accumulation from
Mc-methyl parathion (MPS). James River sediment systems
were more active than Range Point sediment system in the
metabolism of MPS to C02- The presence of Kepone at con-
centrations of 0.6 mg/kg of sediment in either sediment
system reduced the degradation rate of MPS by 59% and 54%,
Respectively. These studies indicate that Kepone, although
resistant to microbial attack, can be inhibitory to the
microbial community responsible for the degradation of oth-
jer pollutants in an artificial ecosystem.
-------
American Chemical Society National Meeting
March 1978, Anaheim, California
THE FATE OF 14C-KEPONE IN ESTUARINE MICROCOSMS. R.L. Garnas, A.W. Bourquin, and
P.H. Pritchard. U.S. Environmental Protection Agency, Environmental Research
Laboratory, Gulf Breeze, Florida 32561.
Following the contamination of the James River with Kepone, laboratory data concern-
ing its fate in the estuary were necessary for corrective actions and mathematical model-
ing efforts. The movement and transformation potentials of 14C-Kepone were studied in
static and continuous flow estuarine microcosms. Biotic and abiotic transformation and
volatilization of the chemical were not apparent in these studies. Following its adsorp-
tion from water in these model systems, Kepone desorbed from salt marsh sediments and
James River sediments. While this desorption was independent of environmental water tem-
peratures and salinities in sediment-water systems, the Kepone concentration in the water
column was proportional to its concentration in sediment. Some James River sediments re-
tained high levels of radiolabeled chemical following conventional solvent extraction.
Burrowing polychaetes (Arenicola cristata) were added to salt marsh sediment in larg
er continuous flow systems to define the effect of macrobenthic biota on the fate of Ke-
pone. These polychaetes accumulated high residues of Kepone and died; although the tis-
sues were allowed to decompose in the system, the accumulated Kepone was not as available
for desorption and washout from the system as compared to Kepone adsorbed to sediment.
-------
|