vvEPA
United States
Environmental Protection
Agency
Environmental Research
Laboratory
Gulf Breeze FL 32561
EPA-600/9-78-014
June 1978
Research and Development
Research
Review
1977
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FOREWORD
The year 1977 was a milestone in the history of the Sabine Island, site of EPA's
Environmental Research Laboratory, Gulf Breeze, Florida (ERL.GB). Our new aquatic
lexicological laboratory dedicated on October 7 became a symbol of the moderniza-
tion of the island where research was originally conducted in a former quarantine sta-
tion built at the turn of the century.
In the dedicatory address, EPA Deputy Administrator Barbara Blum predicted that
ERL,GB will make a "significant contribution to many critical issues currently facing
the marine environment." "Our goals," Ms. Blum said, "must be not only to help cor-
rect the dangerous environmental mistakes that have been made in the past, but, more
importantly, to honestly assess and effectively address the difficult environmental reali-
ties that confront our generation."
U.S. Representative Robert L.F. Sikes echoed Ms. Blum's optimism that our labora-
tory will contribute to the national effort to "create a better American for tomorrow."
And Dr. Stephen J. Gage, Assistant EPA Administrator for Research and Development,
forecast a significant role for ERL.GB in EPA's transition from the problems of the
60 's to the problems of the 80 's.
A symposium held in conjunction with our laboratory dedication ceremony drew
representatives from national conservation groups, scientists, and professors who dis-
cussed the essential role of research and regulatory agencies in protecting marine eco-
systems. Participants offered their views of the special function of federal agency scien-
tists, the social responsibility of the scientists, and the need for research in support of
environmental regulation.
A highlight of our Laboratory Dedication Ceremony was the presence of a scientific
delegation from the Soviet Union, who took part in an American-Soviet bilateral ex-
change of environmental scientific technology. ERL.GB in 1977 participated in two
projects sponsored under the American-Soviet Environmental Protection Agreement
and also provided expertise for U.S. technical assistance efforts in Egypt and Poland.
With the enactment of Congressional amendments to the Federal Water Pollution
Control Act and the Safe Drinking Water Act, ERL.GB research broadened in scope in
1977. Scientists who screened chemicals to determine their toxicity to marine species
were joined by colleagues who examined how a test chemical traveled through the en-
vironment and how it may affect physical, chemical, and biological processes. One goal
was to acquire a more thorough understanding of the biochemical mechanisms that are
set in motion in organisms and in ecosystems under environmental stress.
In addition, new programs were initiated to examine the ecological impact of off-
shore oil drilling and the environmental acceptability of wastes from various manufac-
turing processes.
Investigations in 1977 focused on many subtle, but far-reaching effects of pollutants
that threaten the ability of ecosystems to support vital life processes. A deeper knowl-
edge of the intricacies of complex environmental interactions will afford new insights
into threats posed by toxicants to man and life support systems. This report reviews
ERL.GB's supportive research in 1977 for EPA's commitment to enhance the quality'
of life and to protect human health from the increasingly apparent problem of hazard-
ous substances in the environment.
* c^CtjLt^
Thomas W. Duke
Director
Environmental Research Laboratory
Gulf Breeze, Florida
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CONTENTS
EPA-600/9-78-014
June 1978
United States Environmental Protection Agency
Environmental Research Laboratory
Gulf Breeze, FL 32561
EXPERIMENTAL ENVIRONMENTS BRANCH 1
PROCESSES AND EFFECTS BRANCH 18
BEARS BLUFF FIELD STATION 35
ENVIRONMENTAL EFFECTS OF OFFSHORE DRILLING 45
KEPONE 55
ENVIRONMENTAL PATHOBIOLOGY 57
PUBLICATIONS AND PRESENTATIONS 62
NOTE: This report is for informational purposes only. All data and conclusions must be considered
provisional. Mention of trade names or commercial products does not constitute endorsement
or recommendation for use.
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Figure 1. ERL.GB scientists determine effects of pollutants on marine animals in a new toxicological laboratory
dedicated in 1977
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EXPERIMENTAL
ENVIRONMENTS BRANCH
JACK I. LOW!':, Chief
A new $1 million toxicological laboratory completed in
1977 provides KPA's Environmental Research Laboratory
in Gulf Hrcc/c, Florida, (KRL.GB) with expanded and
moderm/ed facilities for observing marine speeies exposed
to toxie chemicals in raw, or filtered, flowing seawater
(Fig. 1).
Tests conducted by the Experimental Knvironments
Branch yield data required by F.PA for registration and
reregistration of pesticides, development of water quality
standards, and the issuance of permits to Jump wastes in
the ocean.
Historically, ERL.GB research has been oriented to-
ward an assessment of the effects of pesticides and other
poisonous substances on the marine environment. In
1977, the Kxperimcntal Knvironments Branch established
toxicity concentrations for individual pollutants on the
basis of tests with single species of animals. Investigations
extended from single species to multiple species of ani-
mals established as a community, and from criteria for
lethal effects to more subtle, longer lasting effects of
chemical contaminants and their fate and transport in the
marine environment.
Fewer tests were conducted by the branch in 1977 on
"hard" or organochlorine pesticides, and more tests were
conducted on organophosphates and "third" generation
pesticides that inhibit normal growth. The branch staff
also initiated studies of complex industrial and municipal
wastes.
The new aquatic laboratory will enable branch scien-
tists to observe how marine life will respond to pollutants
under natural or controlled conditions. Test animals (in-
dividually and in communities) are exposed to toxic com-
pounds in raw or filtered flowing seawater. An intricate
pumping system can deliver up to 450 gallons of seawater
per minute from adjacent Santa Rosa Sound.
A wing of the laboratory houses F,RL,GB's analytical
chemistry section where pollutants and their concentra-
tions are identified. The unit is equipped with 10 gas and
3 liquid chromatographs (some controlled by computers)
for analyses of toxic compounds in marine water, sedi-
ment, and biota.
During 1977, the analytical chemistry section analy/.ed
3288 samples for pesticide and related organics. Analyti-
cal procedures were validated for trifluralin and two de-
alkylated products; Sevin and 1-naphthol; mono- and
dehydro-Kepone ; l.epthophos; pentachloronitrobenzene;
Dimilin ; and FPN. In 1978, methods will be validated
for determination of dimethoate, trichlorofon, Guthiqn1*,
phorate, DKF , trithion, methylparathion, AltosidR ""A
tnchlorophenol in marine water and biota.
and
Acute Toxicity Tests (Dynamic)
STKVF.N C. SCI11MMEL, Research Aquatic Biologist;
JAMFS M. PATRICK, Jr., Biological Lab Technician
Data from acute toxicity tests have played a crucial
role in KPA's development of Water Quality Criteria, eval-
uations of new, "substitute" chemicals and in Effluent
Limitation Hearings.
In the past, research results have appeared on the cau-
tion label of pesticides tested with methods developed at
KRL,GB. In 1977, acute toxic effects of sodium penta-
chlorophenate (PCP), Kepone, Leptophos, FPN, trifluralin
and Sevin were investigated in flowing seawater tests with
estuarine molluscs, crustaceans, and fish. Studies of the
herbicide trifluralin and the insecticide Sevin will be com-
pleted in 1978; a manuscript reporting results of
Leptophos/F.PN research is in preparation.
Pentachlorophenol (PCP) and sodium pentachlorophe-
nate (NA-PCP), found in defoliants, herbicides, insecti-
cides, wood preservatives, and other products, were tested
with several estuarine animals collected near ERL.GB.
The 96-hour (96-h) LCSOs (concentration estimated to be
lethal to 50% of test organisms) for each organism tested
were: grass shrimp (Palaemonetes pugio), 515 micrograms
per liter (pg/jj,); brown shrimp (Penaeus aztccus), > 195
pg/j),; longnose killifish (Fundulus similis), > 306 Ug/£;
pinfish (Lagodon rhomboides), 53.2 yg/ {_; and striped
mullet, (Mugil cephalus), 112 pg/£.
Oyster shell deposition data were analyzed by linear
regression with probit transformation to determine an
EC50 (concentration of PCP effective in reducing shell
deposition of exposed oysters to 50% of that of control
oysters) and 95% confidence intervals.
Sodium pentachlorophenate was acutely toxic to
oysters (EC50 = 76.5 pg/O exposed for 96-h (Table 1).
Due to the low seawater temperature, control oysters de-
posited an average of only 1 mm shell/oyster. Therefore,
oysters were exposed for an additional 96-h, but the
F.C50 value remained unchanged.
Exposure of brown shrimp to Na-PCP concentrations
as high as 195 pg/£ and the grass shrimp to concentra-
tions up to 515 Pg/£ did not lead to significant mortal-
ity.
Sodium pentachlorophenate, at the concentrations
tested, was acutely toxic to striped mullet and pinfish
but not to longnose killifish (Table 1). Pinfish were the
R
Registered trademark
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Table 1. Acute toxicity of sodium pentachlorophenate (Na-PCP) to several estuarine organisms
Species
Crassostrea virginica
Penaeus aztecus
Palaemonetes pugio
Fundulus similis
Lagodon rhomboides
Mugil cephalus
Sizea
("x", mm)
45
66
18
42
80
58
96-h
Nominal
104.0
(54-158)c
> 320.0
> 560.0
> 560.0
107.6
(93.7-122.0)
221.6
(92.3489.6)
g/£b
Measured
76.5
(37-116)
>195.0
>515.0
>306.0
53.2
(42.4-65.4)
112.1
(44.0-210.4)
Temp.
(T, °C)
8.4
25.0
24.8
24.4
25.0
24.7
Salinity
("x", °/oo)
20.3
26.5
24.3
22.9
20.8
25.5
aSize is height (umbo-distal valve edge) for oysters; rostrum-telson length for shrimp; standard length for fishes.
Effect measured is shell deposition for oysters and mortality for shrimp and fishes.
^Fhe 95% confidence intervals are in parentheses.
most sensitive species tested: the 96-h LC50 was 53.2
Bioconcentration Tests
STEVEN C. SCHIMMEL, Research Aquatic Biologist;
JAMES M. PATRICK, Jr., Biological Laboratory Technician
The purpose of these tests is to determine the rate of
uptake (mainly from water) of toxic organics and the rate
at which these substances are depurated from estuarine
mollusc and fish tissues. Resulting data can be used to in-
dicate the bioconcentration factor (BCF) of toxic chemi-
cals. BCF, the chemical concentration found in tissues of
organisms divided by the exposure concentration in sea-
water, is useful in predicting the potential of a compound
to persist and accumulate in marine food webs.
In 1977, two long-term studies were conducted to
determine toxicity, uptake, and depuration of Kepone in
blue crabs (Callinectes sapidus) after the James River in
Virginia was contaminated by Kepone and closed to com-
mercial fishing.
In the first study, Kepone was administered to crabs in
seawater (0.03 or 0.3 yg/X,) or food (eastern oyster,
Crassostrea virginica, containing 0.25 yg/g Kepone). Up-
take of Kepone in 28 days was primarily through the con-
taminated oysters. When the crabs were held in Kepone-
free seawater and fed Kepone-free oysters for 28 days, no
loss of the insecticide was evident. Adverse effects on
molting and survival were observed in crabs fed oysters
that contained 0.25 y g/g Kepone.
The second study investigated: (1) the depuration of
Kepone over a 90-day period in blue crabs fed oysters
(containing 0.15 Mg/g Kepone) from the James River,
Virginia; and (2) the effects of Kepone on molting and
survival of blue crabs fed James River oysters or labora-
tory-contaminated oysters that contained 0.15 or 1.9
yg/g Kepone. Crabs fed Kepone-contaminated oysters fol-
lowed by a diet of Kepone-free oysters for 90 days had
detectable concentrations of the insecticide in tissues.
Blue crabs that ate oysters containing Kepone in concen-
trations similar to those found in oysters from the James
River died in greater numbers or molted less frequently
than crabs fed Kepone-free oyster meats.
In bioconcentration studies with PCP, eastern oysters
exposed to measured PCP concentrations of 25.0 and 2.5
yg/5, f°r 28 days accumulated the chemical in their tis-
sues an average of 41 and 78 times, respectively (Fig. 2).
Pentachlorophenol reached an apparent equilibrium in
oysters within the first four days in both exposure con-
centrations and remained relatively constant throughout
the uptake portion of the study. The mean concentration
of PCP in oysters from the 25 yg/£ aquarium was 1060
pg/kg and 180 pg/kg for the 2.5 yg/£ aquarium. After
the PCP delivery was discontinued, however, the oysters
purged themselves of the pesticide within four days.
In summary, Na-PCP reduced shell deposition of east-
ern oysters at concentrations >34 ]jg/£ during a 192-h
exposure. Concentrations of the pesticide as high as 195
and 515 yg/Jl, however, were neither toxic to brown
shrimp nor grass shrimp, respectively, in 96-h exposures,
nor did the animals bioconcentrate the chemical. Pinfish
were the most sensitive of all species tested (96-h LC50 =
53 yg/£). Oysters bioconcentrated PCP 41 to 78 times
the amount measured in water and, when held in PCP-
free seawater, depurated the chemical to nondetectable
concentrations in four days. Compared with other organo-
chlorine insecticides (such as toxaphene, chlordane, and
BHC) tested on estuarine animals, Na-PCP is relatively less
toxic and is bioconcentrated to a lesser extent.
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10,000
1,000
in
UJ
25.0»ig/l
o—o 0__.
100
7
I/
XI
14
-UPTAKE-
21
28
35
TIME (doyi)
Figure 2. Uptake of pentachlorophenol (PCP) by eastern oysters (Crassostrea virginica)
exposed 28 days, then allowed to depurate in PCP-free seawater *
Chronic Toxicity Tests
DAVID J. HANSEN, Research Aquatic Biologist;
LARRY R. GOODMAN, Research Biologist;
WALTER BURGESS, Jr., Assistant
Long-term toxicity tests with saltwater species primar-
ily have used juveniles that are found inshore and are
easily collected. Use of saltwater fish and invertebrates in
life-cycle tests has been limited because of difficulties re-
lated to their culture in the laboratory.
In 1977, ERL.GB researchers successfully maintained
sheepshead minnows (Cyprinodon variegatus) in life-cycle
tests to determine maximum acceptable toxicant concen-
trations (MATC) of pollutants and application factors that
can be used to provisionally estimate a chronically safe
toxicant concentration applicable to species when only
acutely lethal concentrations are known. (The application
factor [AF] is the ratio between the MATC in a life-cycle
test and the acute or incipient LC50).
Sheepshead minnows were used in 1977 in embryo/
juvenile and partial and entire life-cycle tests (Fig. 3) to
determine effects of long-term exposures during sensitive
life stages to Leptophos, trifluralin, trifluralin decomposi-
tion products, and Kepone.
Leptophos - Sheepshead minnows were exposed to Lepto-
phos for 28 days in beginning with embryos and lasting
through hatching and growth of fry to the juvenile stage.
Survival of embryo, fry, and juvenile sheepshead minnows
exposed to Leptophos for 28 days did not differ signifi-
cantly from that of controls (Table 2). However, fish in
9.8 Vg/fc were markedly less active from day 7 to 19,
and were significantly shorter than control fish; survival
seemed to be reduced. The highest concentration at which
no effects were apparent (2.9 yg/£) was greater than 0.1
of a concentration acutely lethal to 90% of the juvenile
fish (20 yg/£). This finding suggests that the chronic
toxicity of Leptophos may not be excessive in relation to
other insecticides.
Trifluralin - Sheepshead minnows were exposed to triflu-
ralin (2,6-diriitro-N, N-dipropyl-a,a,a-trifluro-p-toluidine)
for 28 days from the embryonic stage through hatching
and growth of fry to the juvenile stage. Acute toxicity
tests also were conducted on trifluralin, trifluralin III, and
other decomposition products.
Trifluralin was found to be more toxic to sheepshead
minnows in acute, or in embryo-to-juvenile tests, than the
decomposition products tested. The 96-h LC50 of Triflu-
ralin to juvenile fish in static tests was 143 yg/£ (95%
confidence limit = 117-175 P g/il). Survival and growth of
•Reprinted by permission of Plenum Publishing Corp., NY. From: Pentachlorophenol, Environ. Sci. Res. Vol. 12,
K. Rango Rao, Ed. 1978. p. 154.
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BROOD STOCK
ACCUMATE ADULTS
AMBIENT SALINITY
30* C
NATURAL SPAWNWG
APPROX. 12 WEEKS
TERMINATE
EXPERIMENT
LIFE CYCLE TOXICITY TEST
SHEEPSHEAD MINNOW
INJECT FEMALE
WITH HC6
PARENTAL
GENERATION
STRIP GAMETES
FERTILIZE EGGS
EXPOSURE BEGINS]
DAYS 4-6
MEWLY-HATCHED FRY
DAY 28
CONTROL FISH
APPROX. K>mm S.L.
Figure 3. Flow chart of a life-cycle toxicity test with the sheepshead minnow
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Table 2. Toxicity of Leptophos to sheepshead minnows (Cyprinodon variegatus) exposed continuously in a 28-day test.
The test began with 80 embryos in each concentration and lasted through hatching and growth of fry to
juvenile fish
Nominal
Control/
Carrier
0.93
1.9
3.8
7.5
15.
Concentration
Measured
ND
0.33
2.3
2.4
2.9
9.8
Percentage
Hatching
69
67
69
60
57
70
Combined Embryo/
Fry Survival, %
68
66
68
59
56
62
Average
Standard
Length, mm
9.4
9.1
9.0
9.7
9.6
8.0
ND = non-detectable < 0.02 yg/
fish in 20 or 31 y g/ & of trifluralin in the embryo-to-
juvenile test was significantly reduced, but exposure to
1.2, 2.7, or 5.5 y g/ & affected neither growth nor survival.
Signs of poisoning (lethargy, upset equilibrium, edema,
and melanism of the posterior one-third of the body) in-
creased in severity and frequency of occurrence as con-
centrations increased from 2.7 to 31 Vg/£. In the initial
test and a second experiment, light microscopy revealed
pathological effects in fish that showed external signs of
poisoning.
Concentrations of trifluralin plus trifluralin II, (2,6-
dinitro-n-propyl-a,a,a-trifluoro-p-toluidine) in juvenile
sheepshead minnows on day 28 ranged from 500 to 2,200
times the concentration of trifluralin measured in the ex-
posure water.
Trifluralin III was toxic to juvenile sheepshead min-
nows in acute static tests at nominal concentrations of
about 1 mg/ I but exposure to measured concentrations
of from 1.8 to 29 Ug/£ in the 28-day tests produced no
visible signs of poisoning, decrease in growth, or decrease
in survival that were not observed in control fish. Concen-
trations of trifluralin III accumulated by fish were similar
to those measured in the exposure water.
Kepone - The effect of Kepone on survival, growth, and
reproduction was studied in sheepshead minnows through-
out an entire life cycle in these concentrations: 0.0 (con-
trol), 0.01, 0.02, 0.06, 0.12, 0.37, or 0.77 yg/£ (Fig. 3).
Signs of Kepone poisoning were scoliosis, lordosis, black-
ened tails, edema, decreased growth, a reduction in eggs
spawned, and death. Kepone was found in the fish tissues
in all lifestages. (Pathological effects of Kepone in this
test and a second exposure are described on p. 55.
Chronic Toxicity of Methoxchlor, Malathion, and
Carbofuran to Sheepshead Minnows (Cyprinodon
variegatus)
P. R. PARRIS, Principal Investigator. EPA Contract
68-03-0264. Bionomics, EG&G, Inc.
DAVID HANSEN, Project Officer
Sheepshead minnows (Cyprinodon variegatus) were ex-
posed to each of three pesticides-methoxychlor, malathi-
on, and carbofuran-in flowing seawater to determine the
acute and chronic (partial life-cycle) effects. The calcu-
lated 96-h LCSO's and 95% confidence limits, based on
measured concentrations, were: methoxychlor, 49 micro-
grams per liter (ug/J,), 37-65 Ug/£; malathion, 51 y g/£,
41-63 yg/£;and carbofuran, 386 ug/£, 311480 yg/£.
Mortality of adult sheepshead minnows exposed to
mean measured concentrations of methoxychlor^23
yg/£ was significantly (P 0.05) greater than mortality of
control fish during the 140-day study. Further, hatching
success of fry from eggs spawned by fish exposed to 23
yg/£ was significantly less than hatching success of con-
trol fry. The maximum acceptable concentration (MATC)
was estimated to be >12<23 yg/£ and the application
factor limits were 0.24-0.47.
Mortality of adult sheepshead minnows exposed to
mean measured concentrations of malathion >_18 yg/£
was significantly greater than mortality of control fish
during the 140-day study. Mortality of fry hatched from
eggs spawned by fish exposed to 9 and 18 yg/& was sig-
nificantly greater than mortality of control fry. The
MATC was estimated to be >4<9 yg/2, and the applica-
tion factor limits were 0.08-0.18.
Mortality of adult sheepshead minnows exposed to
mean measured concentrations of carbofuran >_49 yg/il
was significantly greater than mortality of control fish
during the 131-day study. Hatching success of fry from
eggs spawned by fish exposed to 49 yg/ J, was signifi-
cantly less than hatching success of control fry. Also,
mortality of fry hatched from eggs spawned by fish ex-
posed to 49 yg/& was significantly less than hatching suc-
cess of control fry. Also, mortality of fry hatched from
eggs spawned by fish exposed to 23 and 49 yg/& was
significantly greater than control fry mortality. The
MATC was estimated to >15<23 yg/ j,and the applica-
tion factor limits were 0.04-0.06.
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Results of the study were published in the EPA Eco-
logical Research Series, EPA-600/3-77-059, May 1977.
(See Table 3,below).
Chronic Toxicity of Chlordane, Trifluralin, and
Pentachlorophenol to Sheepshead Minnows
(Cyprinodon variegatus)
P. R. PARRISH, Principal Investigator. EPA Contract
68-03-0264. Bionomics, EG&G, Inc.
DAVID HANSEN, Project Officer
Sheepshead minnows (Cyprinodon variegatus) were ex-
posed to each of three chemicals—chlordane, trifluralin, or
pentachlorophenol-in flowing, natural seawater to deter-
mine acute and chronic (full life-cycle) effects. The cal-
culated 96-h LCSO's and 95% confidence limits, based on
measured concentrations, were: chlordane, 12.5 micro-
grams per liter (yg/£), 3.4-45.9 yg/£; trifluralin, 190
yg/£, 128-282 yg/£; and pentachlorophenol, 442 yg/£,
308-635 yg/£.
In a chronic test, sheepshead minnows were exposed to
mean measured concentrations of chlordane (0.5-18.0
yg/£) for 189 days. Exposure to concentrations^_2.8
yg/£ caused significant (P<0.5) mortality of parental
fish. Exposure to chlordane concentrations ^_0.8 yg/&
significantly reduced hatch of embryos spawned by paren-
tal fish and exposure to concentrations > 1.7 yg/£ caused
significant mortality of second generation fish. The esti-
mated maximum acceptable toxicant concentration
(MATC) of chlordane for sheepshead minnows was
>0.5<0.8 yg/£; the application factor (AF) limits were
0.04-0.06.
Sheepshead minnows were exposed to mean measured
concentrations of trifluralin (1.3-34.1 yg/£) for 166 days.
Exposure to concentrations M7.7 Pg/£ caused significant
mortality of parental fish. Exposure to trifluralin concen-
trations ^9.6 yg/£ significantly reduced growth of paren-
tal fish and exposure to concentrations M.8 yg/£ signif-
icantly reduced fecundity of parental fish. Exposure to
concentrations ^_9.6 yg/£ significantly reduced hatch of
embryos spawned by parental fish, and survival of and
growth of second generation fish. The estimated MATC
of trifluralin for sheepshead minnows was >1.3<4.8 Mg/p-
the AF limits were 0.007-0.025.
Sheepshead minnows were exposed to mean measured
concentrations of pentachlorophenol (18-389 yg/&) for
151 days. Exposure to concentrations ^88 yg/J, caused
significant mortality of parental fish. Exposure to penta-
chlorophenol concentrations M95 yg/£ significantly re-
duced hatch of embryos spawned by parental fish and
survival of second generation fish. The estimated MATC
of pentachlorophenol for sheepshead minnows was
>47<88 yg/£; the AF limits were 0.11-0.20.
Results of these experiments will be reported in the
EPA Ecological Research Series, EPA-600/3-78-010, in
January 1978. (See Table 3.)
Table 3. Concentrations (yg/£) of Six Pesticides Toxic to Sheepshead Minnows in Acute and Chronic Tests,
and the Relationship of Acute Toxicity to Chronic Toxicity
Pesticide
Methoxychlor
Malathion
Carbofuran
Chlordane
Trifluralin
Pentachlorophenol
96-h LC50
(95% confidence
limits) MATC limits
49 >12<23
(37-65)
51 >4<9
(41-63)
386 >15<23
(31M80)
12.5 >0.5<0.8
(3.4-45.9)
190 >1.3<4.8
(128-282)
442 >47<88
(308-635)
Application
factor limitsa
0.24-0.47
0.08-0.18
0.04-0.06
0.04-0.06
0.007-0.025
0.11-0.20
aDerived by dividing the Maximum Acceptable Toxicant Concentration limits by the 96-h LC50.
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Experiments at, or supported by, ERL,GB have de-
monstrated that sheepshead minnows are suitable salt-
water fish for life-cycle toxicity tests. Because no other
estuarine fish has been used in toxicant exposures contin-
uing from reproduction through the growth of progeny,
the usefulness of sheepshead minnows for determining ap-
plication factors for other estuarine fish has yet to be
evaluated. Results of experiments with diazinon, endrin,
heptachlor, malathion, and trifluralin demonstrated strik-
ing similarities between application factors for sheepshead
minnows and freshwater fish exposed to these chemicals.
These data suggest that application factors for freshwater
and saltwater fish are similar.
Physiology
D.R. NIMMO, Research Ecologist;
E. MATTHEWS, Biological Laboratory Technician;
T.L. HAMAKER, Biological Technician
Concentrations of pollutants lethal to marine life are
more easily determined than concentrations that have
long-term effects on an aquatic animal's life span, growth,
or movements. A prime objective of ERL,GB research is
to develop new methods for evaluating subtle, sublethal
effects of contaminants on the marine environment.
In 1977, ERL.GB investigators successfully used a
tiny, shrimp-like crustacean (Mysidopsis bahia) through-
out its life cycle (14 to 17 days) in toxicological and
physiological studies during sensitive stages of develop-
ment. Commonly called opossum shrimp (the female car-
ries young in a brood pouch), mysids are an important
element of estuarine plankton and an integral in estuarine
and marine food webs (Fig. 4).
The species was first reported from West Bay,
Galveston, Texas, and also has been observed in South
Florida. The animals feed on 48-h-old brine shrimp
(Artemia salina); their culture and maintenance in the
test aquaria demands minimal time and effort. However,
as required in the unautomated culture of most labora-
tory animals, continuous monitoring is necessary.
In 1977, 96-h toxicity tests and life-cycle tests with
mysids demonstrated a sensitivity to cadmium and eight
pesticides that was equal to or greater than the degree of
sensitivity displayed by other estuarine biota (Table 4).
Indicators monitored for effects in life-cycle tests include:
susceptibility on the basis of sex, time required for for-
mation of brood pouch, time required for release of
brood, number of young released per female, survival of
young to the F-. and ¥^ generations, and growth rate.
Table 4. Results of toxicity tests with Mysidopsis bahia
Compound
Cadmium
D
Diazinon
DimilinR
EPN
KeponeR
Leptophos
Methyl Parathion
SevinR
Toxaphcne
96-h LC501
15.5
4.83 (Adults)
2.06
3.44 (Adults)
10.1
3.16
0.78 (Juv.)
7.7
3.19 (Adults)
6.32 (Juv.)
MATC1
4.8 - 6.4
1.15 -4.4
«0.42
0.44 - 3.44
0.026 -0.39
0.64 - 1.77
_3
2.8 - 7.7
0.067 - 0.14
AF
0.31 -0.41
0.24 - 0.91
«0.192
0.13 - 1.0
0.003 - 0.04
0.20 -0.56
-
0.36 - 1.0
0.02 - 0.04
Micrograms per liter (Ug/Jl)
Effects on reproduction were observed at an estimated 0.025 ]jg/£.
In progress
-------
Figure 4. Mysid shrimp, 7 to 8 mm long, are cultured at ERL.GB in 38-2, glass aquaria containing filtered (20u)
flowing water (salinity = 10 to 27 parts per thousand, ppt). The species can be cultured continuously without fluc-
tuations in population density on a diet of 48-h-old Artemia salina larvae
-------
Table 5. Effect of Dimilin on reproductive success of Mysidopsis bahia at 22-26°C
and
Nominal Test
Concentrations
Measured Test
Concentrations
22-28 parts per thousand salinity
Seawater TEG*
Control Control
ND** ND**
Ug/Jl
0.075 0.25 0.50 0.75
ND** ND** 0.55 0.91
Total females
Juveniles produced
Juveniles per
female, X
16
343
21.4
26
547
21.0
22
298
13.5'
19
193
10.2"
19
136
7.2'
15
36
2.4*
"Triethylene glycol control
*Not detected; detection limit = 0.4 tag/8,.
'Significantly different from controls at = 0.05 (Dunnett's test).
Dimilin, a new insecticide that inhibits chitin synthe-
sis in insects, was tested for the first time at FRl.,(iB in
1977 and found to be acutely and chronically toxic to
mysids. During life-cycle studies, exposure to Dimilin
affected the number of young produced by female my-
sids: only 13.5 young/female were produced in an esti-
mated concentration of 0.075 ug/£, whereas 21.4 and
21.0 were produced in controls (Table 5). As concentra-
tions of Dimilin were increased, direct suppression of re-
production followed.
Tests were also conducted with toxaphene to deter-
mine life-long survival rates of young produced by expos-
ed females. Parental mysids (24-h-old at outset) were ex-
posed to toxaphene continuously until progeny (F^s)
were produced. Survival of young produced by parents
exposed to 0.14 and 0.39 (jg/J, toxaphene was equal to
that recorded for control mysids; however, reproduction
by young of exposed parents was either completely in-
hibited or drastically reduced. On the basis of reproduc-
tion as a criterion, investigators estimated that 0.067
yg/£ is a no-effect concentration of toxaphene (Table 6).
Table 6. Effects of toxaphene on survival and reproduction of mysid shrimp after a 14-day exposure
followed by a 6-day depuration
Average Measured
Toxaphene Concentration
Survival
Per Female
Seawater Control
Triethylene Cilycol Control
0.14*
0.39
1.3
4.2
81
69
88
67
19
0
6.8
7.8
1.2**
0
0
0
'Temperature range, 20° to 26°C; salinity, 20 to 26 ppt
**Additional testing showed no significant decrease in number of young per female in an estimated
0.067 yg/fc toxaphene.
-------
Table 7. Concentrations (Mg/&) of toxaphene acutely and chronically toxic to Mysidopsis bahia adults and juveniles
Days
Juveniles
4 6.32
14** 0.70
20 0.66
LC50 (yg/£)*
95% Fiducial
Limits
--
0.38 - 1.47
0.32 - 1.58
Adults 95% Fiducial
Limits
3-19 2.41 -4.38
0.85 o.61 - 1.21
°-59 0.37-0.99
Temperature range, 20 to 26°C; salinity, 20-26 ppt.
* Estimated by probit analysis
"Exposure halted
In the toxaphene tests, this pesticide was found to be
slightly more toxic to adult mysids than to juveniles ex-
posed for 96 h. However, continued exposure to toxa-
phene for 14 days, followed by 6 days without exposure,
did not cause any apparent difference in susceptibility
(Table 7).
In addition, mysids were used in a 96-h static test, fol-
lowed by a 10-day study of reproduction success, to de-
termine toxicity of a chemically contaminated sewage.
The mysid shrimp was cultured at ERL.GB for transport
by air to a mobile bioassay unit conducting field surveys
of industrial waste in EPA Region IV (Southeast). Vari-
ous laboratories, government and private, have reared
mysids from stock obtained from ERL.GB.
Bioassay (Crustaceans)
DANA BETH TYLER-SCHROEDER, Research Biologist
Marine toxicologists have demonstrated that crusta-
ceans are often more sensitive to organic pollutants than
many other marine and estuarine organisms. However,
few toxicity studies have been conducted with larval
stages of shrimp because laboratory attempts to induce
reproduction have been generally unsuccessful.
In 1977, experiments at ERL.GB demonstrated the
usefulness of the grass shrimp (Palaemonetes pugio
Holthuis) to monitor sublethal effects of a pollutant.
These tests showed that grass shrimp are easily cultured
in the laboratory, sensitive to toxicants, and can be
maintained in flowing seawater systems for toxicity tests
throughout a life cycle. Spawning was induced by tem-
perature/light controls.
Initial tests with grass shrimp at ERL.GB determined
the toxicity of endrin and its effects on shrimp develop-
ment. Test animals were exposed to varying concentra-
tions of endrin: 0.0 (control), 0.03, 0.05, 0.11, 0.18,
0.38, and 0.79 yg/J, in seawater.
Juveniles reached sexual maturity during the first two
weeks of the exposure. Larvae spawned by control (unex-
posed) and exposed parents were continuously exposed
until the juvenile stage (7 to 20 mm, rostrum-telson
length).
Ability of the grass shrimp (Palaemonetes pugio) to
complete life-cycle functions was seriously impaired by
exposure to concentrations of endrin significantly lower
than the 96-h LC50 (Table 7). The 96-h LC50 for expos-
ed juvenile grass shrimp was 0.35 pg/£. In the life-cycle
toxicity test, gonadal development and spawning were in-
hibited at 0.03 ug/jj. Effects on reproduction were ob-
served in the life-cycle toxicity test at all exposure con-
centrations tested. Effects on survival, larval development
and growth were observed at life-cycle test concentrations
Survival of the parental generation was greatly affected'
after 2 weeks exposure to endrin concentrations of 0.38
yg/£ and above. At test termination, survival of parental
shrimp was less than that in controls at concentrations of
0.11 yg/£ and above.
The onset and duration of spawning were significantly
delayed and lengthened for female grass shrimp at all ex-
posure concentrations (Fig. 5). Egg production and hatch-
ing success apparently were not affected at concentrations
tested.
Larval mortality, length of time to metamorphosis, and
growth of juvenile shrimp were impaired by endrin con-
centrations of 0.11 yg/jl and higher. Final length (rostrum-
telson) of juvenile shrimp exposed to concentrations of
0.11 yg/£and above was 12 to 65% shorter than control
shrimp. Final weight of juvenile shrimp exposed to con-
centrations of 0.05 yg/Jl and above was 26 to 94% less
than control shrimp. However, growth of parental shrimp
was unaffected.
10
-------
33-
50-
45
to
5 40'
UJ
u_
35
030
UJ
o
O 20
23 33 43 53 63 73
DAY OF EXPOSURE
83
93
103
113
Figure 5. Effects on reproductive success of grass shrimp exposed to several concentrations of endrin in a life-cycle
toxicity test. Exposures began with 100 shrimp per test concentration; the natural sex ratio for P. pugio is 50% female,
50% male.
In addition to the life-cycle tests with grass shrimp,
ERL.GB staff in 1977 determined the acute 96-h LC50 of
endrin-exposed first-day larval, juvenile, and adult grass
shrimp and the rate of shrimp uptake and depuration of
endrin. The specific application factor for grass shrimp
was found to be less than 0.08, approximately one order
of magnitude lower than for saltwater and freshwater fish
(Table 8). These data were used by EPA to establish
water quality criteria for endrin.
Table 8. Comparison of application factors generated for endrin
Concentration, Vg/&
Species
Cyprinodon variegatus
Jordanella floridae
Palaemonetes pugio
Habitat
Saltwater
Freshwater
Saltwater
96-h LC50
0.34
0.86
0.35
MATC
>0.12<0.31
>0.22<0.3
> ? <0.08
Application
Factor
0.35
0.25
0.08
11
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Community Bioassays
MARLIN TAGATZ, Research Aquatic Biologist;
JOEL IVEY, Biological Technician
Community bioassays offer another facet to scientific
analyses of the environmental impact of man-produced
pollutants. These tests enable investigators to determine
simultaneously the relative sensitivity to toxicants of
many different types of organisms (more than 100 species
of 9 phyla have been collected). Animals are exposed to
lest compounds in their early development stages, rather
than in their later, and often more resistent, juvenile or
adult stages.
Community bioassays conducted in 1977 at ERL.GB
sought to determine effects of oil drilling fluids on the
development of estuarine communities. Planktonic larvae
in flowing seawater from Santa Rosa Sound were allowed
to colonize in a group of aquaria. After about 10 weeks,
the number and species of animals (marcoinvertebrates)
exposed to various concentrations of the test fluids were
compared statistically to those in unexposed aquaria.
Four experiments with drilling muds were completed
during the reporting period. Pentachlorophenol and sodi-
um pentachlorophenate, used to control bacteria in drill-
ing muds, altered development of exposed estuarine com-
munities. Total numbers of individuals and species were
significantly fewer in aquaria exposed to 76 fJg/£ (parts
per billion) pentachlorophenol or 161 Mg/£ sodium penta-
chlorophenate than in unexposed aquaria. As little as
15.8 Mg/£ sodium pentachlorophenate reduced the total
number of animals but not the total number of species.
Molluscs, arthropods, and annelids were particularly affect-
ed by these compounds.
Barite (BaSO^, weighting agent and primary compo-
nent of drilling muds) and a lignosulfonate type of whole
mud (obtained from a drilling operation) also affected the
composition of estuarine communities. Significantly fewer
animals and species developed in aquaria sand covered by
5 mm of barite or 2 mm of the mud than in unexposed
aquaria. Settling organisms were also affected when these
toxicants were mixed with the sand. A ratio of 1 part
barite to 3 parts sand, or a ratio of 1 part drilling mud to
10 parts sand, significantly reduced the total number of
animals. Annelids and molluscs were the most sensitive to
barite; annelids and coelenterates were the most sensitive
to whole drilling muds.
Results of ERL.GB community bioassays will be com-
pared with concurrent field studies on a platform in the
Gulf of Mexico. Future tests of drilling muds, pesticides,
and other toxic organics should provide additional param-
eters of toxicity and more data for broader ecological
application.
Food Chain Studies
LOWELL BAHNER, Research Aquatic Biologist
The uptake of pesticides by estuarine animals through
bioconcentration from water or bioaccumulation from
food is a useful indicator of pesticide movement in the
natural environment. Field surveys indicate that food is a
prime source of the pesticide contamination of certain
estuarine species.
Food Chain Studies at ERL.GB seek to evaluate the
magnitude of pesticide transfer to estuarine animals via
water, food, or bottom sediment. Results of these studies
are used in (1) interpreting field test data; (2) predicting
sublethal effects of pesticides on the natural environment;
(3) developing ecosystem models to predict long-range
movements of pesticides in ecosystems.
Tests in 1977 exposed 10 species of estuarine organ-
isms to toxaphene, trifluralin, Kepone, diazinon, and
Halowax 1014 in water, food, or sediments. Results in-
dicate that: (1) toxaphene, trifluralin, and Halowax 1014
accumulate in tissues of grass shrimp and spot, and (2)
trifluralin will transfer to spot when fed copepods con-
taminated with the chemical. Grass shrimp exposed for
28 days to 0.1 or 10.0 yg/£ diazinon contained no de-
tectable diazinon residues, but all shrimp exposed to 10.0
yg/Adied by day 15 of exposure. Sheepshead minnows or
pinfish that fed on grass shrimp or mysids exposed to
diazinon had no measurable diazinon residues, but cholin-
esterase activity in the fish was suppressed by 15%, com-
pared to control fish fed uncontaminated living shrimp or
mysids.
Six species-oysters, grass shrimp, polychaete worms,
fiddler crabs, blue crabs, and spot-were exposed to James
River sediments containing 0.2 to 0.4 Hg Kepone/g. The
fiddler crabs and polychaete.worms ingested the fine sedi-
ments as indicated by residues of 0.3 yg/g in whole-body
samples. The other four species contained residues com-
parable to concentrations of Kepone in animals that re-
ceived Kepone (0.02 yg/g) from water only. Results indi-
cated that field-exposed animals (with residues greater
than water-exposed animals) obtain significant portions of
Kepone from food.
Additional laboratory food chains will be developed by
testing and culturing alternate food chain organisms.
These organisms will be used to determine if quantity and
quality of various foods affect pesticide availability to
predatory fish.
Algal and Protozoan Studies
NELSON R. COOLEY, Microbiologist
Protozoa, algae, and bacteria form the broad base of
aquatic food chains. Ciliated protozoa are the most nu-
merous organisms of the estuarine benthos and may be
more important than bacteria as nutrient regenerators
particularly of nitrogen and phosphorus.
Further, some ciliates are able to concentrate certain
persistent pesticides and thereby aid in translocating them.
Thus, effects of these toxicants possibly could be exerted'
at higher trophic levels either through the disruption of
nutrient cycles or through their translocation and biologi-
cal concentration in the food chain.
12
-------
Algal and protozoan studies at ERL,GB in 1977 ex-
amined the effects of toxicants on population growth of
marine unicellular algae and ciliate protozoans and the
toxicant bioaccumulation/bioconcentration by test
organisms.
The ERL.GB investigation evaluated changes in bio-
mass of algae due to toxicants on the basis of changes in
optical density (OD). (OD values are relative; i.e., they
are not absolute numbers of cells or of volume of cellular
material per unit volume of culture.)
In addition, changes in cell counts/ml (determined
optically or electronically) were used to measure popula-
tion changes. In certain instances, cell numbers can de-
crease while cell volumes simultaneously increase, thus
causing no significant change in biomass.
Population growth of two species of algae (10 random-
ly chosen cultures of each species) was measured daily for
10 days. The OD of each culture was read on a photom-
eter, then cell counts and cell volumes were determined
with a Coulter ZB1 Counter and P-64 Size Distribution
Analyzer. Data were subjected to linear regression analy-
sis and ANOVA (analysis of variation).
For Dunaliella tertiolecta, mean cell volume per ml
was linearly correlated with mean OD (r = 0.941), the
F-value for regression being 761.065 (a<0.001), and with
mean cell volume (r = 0.959), the F-value for regression
being 1117.885 («< 0.001). For Chlorococcum sp., simi-
lar results were obtained. One other species of different
body form will be tested before this study is concluded.
Studies of (1) interaction of 2,4-D and metals with
and (2) effect of Sevin on population growth of marine
unicellular algae were performed in cooperation with
Dr. Gerald Walsh; results are reported on p. 14.
The ciliate protozoan (Tetrahymena pyriformis) was
exposed in flask cultures at 26°C to chrysene, a
petroleum-related polynuclear aromatic hydrocarbon. Test
concentrations that ranged in 10-fold steps from 0.0024
to 2.4 parts per million (ppm) exerted no significant
effect (a = 0.05) on either 24-h growth rate or on 96-h
population size.
However, in a 24-h test, this ciliate bioaccumulated
and bioconcentrated chrysene 97 times the initial concen-
tration in the medium (from 1 ppm in the medium to 97
ppm in the cells). In 24-h and in a test lasting 120-h, it
bioaccumulated and bioconcentrated chrysene 144 times
the initial concentration in the medium (from 5 ppm in
the medium to 722 ppm in the cells). Similar bioaccumu-
lation/bioconcentration in nature would permit the chem-
ical's entry into aquatic food chains. Test results indicate
that chrysene might serve as a useful model to determine
how other polynuclear aromatic hydrocarbons are bio-
accumulated and bioconcentrated.
In earlier continuous-flow tests at ERL.GB, a simple
homemade chemostat was used to study the effect of
chrysene on cultures of T. pyriformis at room tempera-
ture. Although bioconcentration of chrysene by the ciliate
could be demonstrated, growth data were equivocal be-
cause of the many uncontrolled environmental variables
inherent in this chemostat. A new commercially made
chemostat was put into operation in 1977 to control
medium flow-rate, rates of agitation and aeration, and
temperature, as well as record pH and dissolved oxygen.
A simple device was also added for aseptic delivery of
measured amounts of sterile Antifoam B to prevent foam-
ing of the medium. This system will be used to develop a
continuous-flow bioassay for toxicants and possibly to
study the fate of the toxicants and to supply contami-
nated organisms for food chain studies.
Toxicity of the carbamate insecticide, Carbaryl
(Sevin ) to Tetrahymena Pyriformis W (grown axenically
in Tetrahymena medium) also was studied in 1977. Test
concentrations of Carbaryl ranged from 0.001 to 30 ppm.
Greatest observed decrease in 24-h growth rates occurred
at 30 ppm, but were only 7.0% to 10.5% less than con-
trol growth rates. Reductions of population sizes at 96 h
when exposed to 30 ppm did not exceed 12.0%. Liquid
chromatographic analyses of cells grown for seven days in
the presence of Carbaryl revealed no significant biocon-
centration of Carbaryl, but considerable 1-naphthol was
recovered. The latter compound is a breakdown product
of Carbaryl.
Effects of Industrial Wastes on Selected Estuarine
Flora and Fauna
G.E. WALSH, Research Biologist
A mobile laboratory was established by ERL.GB in
1977 to conduct "at-the-pipe" field surveys of industrial
waste disposal sites. The mobile bioassay unit provides
data supplemental to ERL.GB laboratory analyses and re-
quired by the Surveillance and Analysis Division of EPA's
Region IV.
A trailer containing 125 square feet of floor space
serves as the mobile laboratory (Fig. 6). It is equipped
with two proportional diluters for bioassays with mysid
shrimp and juvenile sheepshead minnows and with instru-
ments for monitoring pH, Eh, temperature, salinity, and
dissolved oxygen.
Bioassays of wastes from seven industrial plants (pro-
ducing textiles, paper, and chemicals) were completed dur-
ing the reporting period. Field analyses and algal bioassays
at ERL.GB of samples showed that both algal and animal
tests are essential for a complete analysis of the biological
effects of wastes. For example, in ERL.GB tests, certain
effluents did not affect survival of fish or mysid shrimp,
but caused algae to grow rapidly and attain high popula-
tion densities.
In conjunction with the investigation of industrial
wastes, a new method for a rapid screening algal bioassay
was developed at ERL.GB and was successfully tested
with waste samples from 16 plants associated with the
textile industry. The method allows Skeletonema costatum
grown in optically matched tubes to be measured by
spectrophotometry up to 96 h. In general, algal tests were
more sensitive than comparable tests in which animal
13
-------
Figure 6. Dr. Alan Auwarter examines aquaria in
ERL.GB's mobile bioassay laboratory used in field
surveys of industrial effluents.
Sevin was not highly toxic to Chlorella sp., Chlorococ-
cum sp., Nitzschia sp., or Skeletonema costatum. The
average ECSOs, in ppm, were: Chlorella. 0.9; Chlorococ-
cum, 23; Nitzschia, 1.0; and Skeletonema, 1.5. Uptake of
Sevin or its metabolite, l.napthol, was not found when
Skeletunema was exposed to 0.05 ppm for 24 h. Future
work will be limited to studies on chemicals that require
a rapid assessment.
The Dynamics of an Estuary as a Natural Eco-
system
F.J. VERNBERG, Principal Investigator, EPA R804407,
Univ. of South Carolina, Columbia, S.C.; G.E. WALSH,
Project Officer
In this study, investigators attempted to (1) establish
data on an undisturbed estuary for comparative studies
on effects of various stresses of pollutants on other estu-
arine environments; and (2) develop ecosystem models for
predicting probable effects of environmental perturbation.
The principal objective of a microecosystem study was to
develop and test replicate experimental units to be used
to assess long- and short-term effects of pollutants on the
Spartina alterniflora salt-marsh community.
An interdisciplinary team of marine scientists develop-
ed a conceptual model of energy flow for a marsh-
estuarine ecosystem, using three subsystems (water col-
umn, intertidal marsh zone, and benthic subtidal zone).
A linear dynamics system with 22 states was chosen as
the mathematical model.
In addition, investigators developed a linear systems
model of the intertidal oyster community. Results were
reported in the EPA Ecological Research Series, EPA-600/
3-77-016, January 1977.
methods were- used. Some wastes were found to contain
high concentrations of plant nutrients.
Bioassays of industrial plants with the mobile labora-
tory will be continued in 1978, and laboratory studies on
effects of complex wastes will be initiated on phytoplank-
ton communities.
Effects of Pesticides on Population Growth of
Marine Unicellular Algae
GERALD WALSH, Research Biologist
Effects of Sevin, 2,4 - D + nickel, 2,4 - D + aluminum,
Kepone, and Leptophos on marine unicellular algae were
studied in 1977.
Water Quality and Mangrove Ecosystem Dynamics
S.C. SNEDAKER, Principal Investigator, EPA R804355,
University of Miami, Miami; G.E. WALSH, Project Officer
A report on results of work will be published in 1978.
The project will provide data on biomass of several com-
partments of estuarine forests, their turnover and produc-
tivity rates, amount of selected pesticides in the compart-
ments, and the rate of pesticide exchange and loss.
Effects of Selected Wastewater Chlorination
Products and Captain on Marine Algae
H.C. SIKKA, Principal Investigator, EPA Grant R803943)
Syracuse Research Corporation, Syracuse, NY; G.E.
WALSH, Project Officer
I •
-------
Tlic release ol potentially toxic chlorinated organics in
the aquatic environment is an environmental concern.
Further understanding of the effects and fate of ehlorina-
tion products in the biota is required to evaluate the im-
pact of waste-water chlorination. This research grant ex-
amined effects of stable organic compounds produced
during chlorination of sewage effluents and the pesticide
captan on phytoplankton, which contribute oxygen to
the aquatic environment.
Test results showed that:
3-Chlorobenzoic acid (1 or 10 ppm) had either no
or a slight effect on growth of Dunaliella or Porphyrid-
ium. It inhibited growth of Skeletonema at 10 ppm, but
had no marked effect at 1 ppm.
5-Chlorouracil at 1 or 10 ppm did not affect
Skeletonema, but stimulated growth of Dunaliella initially.
4-Chlororesorcinol had no effect on Dunaliella at
1 ppm, but 10 ppm of the chemical caused a small de-
crease in growth. The chemical produced an initial stimu-
lation in growth of Porphyridium, followed by an inhibi-
tion. Growth of Skeletonema was inhibited by 4-chloro-
resorcinol at concentrations ranging from 1 to 10 ppm.
3-Chlorophcnol stimulated growth of Dunaliella.
Skeletonema growth was inhibited at concentrations high-
er than 2.5 ppm, but showed some stimulation at 1 ppm.
At 1 ppm it stimulated growth of Porphyridium, but was
slightly inhibitory at 5 ppm.
. A combination of 3-chlorophenol and 4-chloro-
resorcinol interacted synergistieally to reduce Skeletonema
growth.
. Captan suppressed growth of Dunaliella and For-
phyridium at a concentration of 5 ppm. Slight stimulation
in growth of the two organisms was noticed in the pres-
ence of 0.1 and 1 ppm of the fungicide. Captan was in-
hibitory to Skeletonema at concentrations ranging from
0.25 to 5 ppm. Treatment of Skeletonema with 0.5 ppm
of Captain for 30 min caused a substantial reduction in
photosynthetic ^CO2 fixation.
These findings are reported in EPA-600/3-77-029,
Ecological Research Series, March 1977.
Table 9. Acute Toxicity of Louisville Sludge
Acute Static Bioassay
PATRICK W. BORTHWICK, Research Biologist
Acute static bioassays (<_96 h) are used to rapidly
assess the immediate environmental hazards of toxic com-
pounds to single marine species. Static tests effectively
measure toxicity of little-known substances and contami-
nants available in limited qualities.
In 1977, these bioassays were performed at ERL,GB
to evaluate hazards of complex wastes, such as industrial
effluents, sewage treatment plant outfalls, and materials
dumped in the ocean.
In response to an emergency request from officials in
Louisville, Kentucky, ERL,GB tested sludge from the
city's main sewage treatment plant to determine if its
disposal in the ocean would threaten marine life. The
Louisville treatment plant was contaminated in April 1977
by a substantial quantity of hexachlorocyclopentadiene.
The treatment plant was forced to cease operation until
the toxic sludge could be removed.
ERL.GB static bioassays showed that the sludge was
toxic to marine life (Table 9) and that the poisonous sub-
stance would bioaccumulate in the aquatic food web.
These findings were corroborated by marine copepod
toxicity tests conducted by Bionomics Marine Research
Laboratory in Pensacola and by tests with marine diatoms
conducted by Dr. G.E. Walsh at ERL,GB. Test results
lead to a decision for on-land disposal of the sludge.
In another series of tests, ERL,GB conducted bioassays
on undiluted effluents from four ocean outfalls in re-
sponse to a request by EPA's Office of Water Programs.
Tests showed that none of the effluents were toxic to
sheepshead minnow fry.
Future studies will attempt to develop static toxicolog-
ical methods to determine hazards of industrial wastes to
fish and macroinvertebrates.
TEST ORGANISM
PERCENTAGE LOUISVILLE SLUDGE BY SERIAL DILUTION
96-h LC50 (95% CONFIDENCE INTERVAL)
Marine copepods
Mysid shrimp
Grass shrimp
Sheepshead minnows
Marine diatoms
0.0025
0.047
0.073
0.32
*0.068
(0.0013-0.0046)
(0.024-0.100)
(0.054-0.105)
(by interpolation)
(0.052--0.088)
*96-h EC50
15
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Figure 8. Dr. H.P. Pritchard examines a continuous-flow system developed at ERL.GB to monitor how a pollutant
moves and is transformed in an estuarine environment.
If.
-------
PROCESSES AND EFFECTS BRANCH
FRANK G. WILKES, Chief
The development and validation of a Ha/.ard Assess-
ment Model to predict risks caused by toxicants entering
the marine environment are primary goals of F.PA's Office
of Research and Development. Two major components
comprise the model: (1) the toxicity component that de-
scribes effects of specific pollutants at given concentra-
tions on organisms and ecosystems; (2) the exposure com-
ponent that describes and predicts concentrations and
duration of toxic exposures.
Research conducted by the Processes and Effects
Branch in 1977 was directed toward the development of
data for an Exposure Assessment Model capable of pre-
dicting toxicant concentrations released in marine eco-
systems. The model will predict modifications of toxicant
concentrations brought about by biological, chemical,
and physical interactions with a chemical and its by-
products. This model eventually will he linked or com-
bined with other models that predict toxicity of pollut-
ants after differing duration and exposure concentrations.
Data used in the development of the Exposure Assess-
ment Model are derived from tests using "microcosms" or
miniature ecosystems supported by life processes. Micro-
cosms represent segments of the environment that can be
monitored to evaluate integral interactions among marine-
organisms and alterations of their physical/chemical sur-
roundings.
Another objective of the Processes and Effects Branch
is to develop new methods for evaluating changes in eco-
system compartments subiected to stress caused by ha/.-
ardous organic or inorganic pollutants. In 1977, branch
scientists worked on a broad spectrum of tests varying in
complexity and purpose (Fig. 9). Selection of a test or
combination of tests is determined by the pollutant, the
aquatic environment under study, and the intended use
of the data generated by the test. Microcosms also pro-
vide data related to Water Quality Criteria and analyses of
the environmental risk of toxic substances.
XENOWOTIC
NONBIOLOGICAL
\
ItOLOGtCAL
Phot
Metab
Physio
Patho
1
Tramfo
olysif Hydrc
Fate
1
rmation
•lysis Ctwh
Mtcn
1
olism Accumulation
logical
ogical
f^—^B—^V^MkA
MMntjeTuTii
titan Volatility Adm
1
Mo/ogictl
1
1
Behavioral
1
1
Effc
ICtt
MriiMta.
1
rptton Sotubility
1
Fata
1
1
MAnflDOltWl AOCUfW
Predator-
LjbMicr
1
ilation DKw
1
Community
1
icoam
ftanthic
Manhland
Ettuarina
Gulf
Effwns
Carbon
rsity dioxide Population
1
Toxicity
1
1
Biochemical Activity Avoidance Preference prey Diversity Succession Acute Chronic
Figure 9. Tests in diagram are used to determine the effects of xenobiotics on ecosystem processes and components.
17
-------
Microcosms
The impact of pollutants on estuarine environments
can be assessed in reproducible laboratory systems ("mi-
crocosms") that isolate the physical or biological compo-
nents under study.
Tests under development in the Processes and Effects
Branch range from systems that investigate the fate of
pollutants in an estuarine environment to a system that
focuses on how pollutants affect specific behavioral re-
sponses. During 1977, ERL.GB scientists studied the fate
of a pollutant to determine its transport route, availabil-
ity, and its transformation. For example, the insecticide
methyl parathion was found to degrade into a number of
chemicals demonstrating different toxicities and charac-
teristics (Fig. 10).
Investigators developed the following systems, with
methyl parathion as the pollutant:
. Environmental Fate Screening System. The effects
of a pollutant are closely related to its availability and
chemical form. This system allowed investigators to deter-
mine the environmental processes and compartments that
influence the movement and transformation of pollutants.
. Eco-core System. This technique isolated indigenous
microorganisms from intact environmental sediment-water
cores and evaluated their potential to degrade pollutants.
. Continuous-flow Systems. These large- and small-
scale systems incorporate flowing water and allowed in-
vestigation of dynamic environmental processes.
. Aquatic Gradient Avoidance Response System
(AGARS). This system used behavior as an indicator of
chemical exposure and allowed quantification of the
avoidance responses of estuarine organisms to different
pollutant concentrations.
. Benthic Bioassay System. Changes in sediment sur-
face features produced by lugworms (Arenicola cristata)
exposed to toxicants were used to monitor effects of pol-
lutants on a benthic infaunal organism.
Environmental Fate Screening System
R.L. GARNAS, Investigator
The Environmental Fate Screening System was design-
ed to determine ecosystem substrate exchange coefficients
HHS
CONJUGATES
HUMIC ACIDS
C02
OH
CH3Ov» /=
^o>-°<\ /r ^
Figure 10. Projected pathway for the breakdown of methyl parathion (I) in aquatic systems. Compounds are:
methyl paraoxon (II), jv-nitrophenol (III), amino methyl parathion (IV), and £_-aminophenol (V).
18
-------
and rates of pollutant alteration. Environmental factors
that influence pollutant fate are: sediment type (sorption
processes: clay, organic), water quality (salinity, Eh, pH),
physical forces (temperature, sunlight, volatilization), and
biological factors (microbiological processes: metabolism,
degradation).
In the analysis for the pollutant, the system is fraction-
ated into water, suspendible particulate, and heavier, non-
suspendible sediment or sand. These fractions are extract-
ed separately with an organic solvent and analyzed by
thin layer chromatography/autoradiography, with confir-
mational analysis by gas or liquid chromatography. The
extracted sand and particulate fractions are combusted at
900°C to liberate COj to determine unextractable
(bound) residues. All exit routes and system components
are sampled; therefore, a materials balance can be calcu-
lated.
In 1977, ERL.GB investigators used sterile and non-
sterile systems to study the movement of the insecticide
methyl parathion in a simulated salt-marsh environment
(Table 10). The water compartment (W) in the non-sterile
systems demonstrated rapid degradation to amino methyl
parathion and unextractable products. After 14 days, the
parent compound was undetectable. The radioactivity in
Table 10. Methyl parathion: percentage3 radioactivity distribution
Day
Non-sterile Systems
0
4
7
11
14
18
21
25
Sterile Systems
0
7
14
21
W
67.3
30.7
28.2
27.4
20.5
18.8
19.4
18.3
68.8
46.9
49.0
50.0
Se
0.9
0.7
0.5
0.4
0.3
0.2
0.3
0.3
0.9
1.5
0.9
1.3
Sc
0.1
2.2
1.1
1.8
2.3
2.3
2.4
1.6
0.1
0.2
0.2
0.3
Pe
22.6
6.5
5.4
3.9
2.9
2.7
2.2
2.5
29.3
45.1
33.7
30.8
PC
5.6
48.5
55.4
55.2
63.7
64.2
64.5
63.9
0.7
2.9
4.8
5.1
R
0
0.3
0.2
0.3
0.1
0.3
0.3
0.3
0
0.2
2.2
3.4
Total
96.5
88.9
90.6
89.0
89.8
88.5
89.1
86.9
99.8
96.8
90.8
90.9
a!00% =8.8 g = 2,649,100 dpm
W - water
Se - nonsuspendible sediment extract
Sc - nonsuspendible sediment combustion
Pe - suspendible particulate extract
PC - suspendible particulate combustion
R - volatilization
19
-------
these systems moved quickly to the detrital paniculate
fraction (Pc) where it became irreversibly bound; volatili-
zation (R) and interaction with nonsuspendible sediments
(Se) were of minor importance in the fate of this chem-
ical. Sterile systems displayed little transformation of
methyl parathion; the chemical was either in the water
column or extractable from the suspendible particulate
(Pe). These data suggest that the irreversible binding of
methyl parathion to the suspendible particulate fraction
of a salt-marsh ecosystem is partly a result of microbial
action. This phenomenon has been reported previously
for agricultural soil and may be of general occurrence for
many organophosphorus insecticides.
Due to the static nature and small size of this system,
further validation of these results is required. The system,
however, has proven to be a valuable screening tool be-
cause of its simplicity.
Eco-core System
A.W. BOURQUIN, Research Microbiologist
The main agents for returning organic carbon com-
pounds to the carbon cycle are bacteria and fungi. These
organisms affect mineralization of natural compounds and
might be expected to degrade those of synthetic origin.
Therefore, microbial considerations are essential in the re-
sponse and recovery of natural and artificial ecosystems
from the stress of chemical perturbations.
The Eco-core, an artificial microbial microcosm, closely
mimics natural conditions for studying microbial inter-
actions. The system evaluates the degradative potential of
indigenous microorganisms and the effects of chemical
perturbation on microbial ecology. The system utilizes
sterile glass cylinders to extract intact sediment and water
cores from the environment. Degradation products are
continuously monitored in the water column by thin-layer
chromatography and autoradiography.
Methyl parathion (MPS) fate and effects were examined
in a salt-marsh Eco-core. Table 11 shows the disappear-
ance of MPS from the water column for three types of
cores: (1) sea water (water), (2) water and sediment (sedi-
ment), and (3) sterilized water and sediment (sterile). The
sterile cores showed only a slight decrease of MPS (82%
extractable after 30 days). All other cores showed rapid
disappearance of MPS from the water column. Radioactiv-
ity in all sediment cores decreased little after 20 days in-
cubation; however, analysis of the radioactivity indicated
the presence of a major degradation product, amino-
methyl parathion (AMPS). The decrease in MPS concen-
tration in water cores was due primarily to adsorption to
the glass in both sterile and non-sterile systems. Degrada-
tion of MPS was considerably less in water cores than in
cores containing sediment.
Polar products (Table 11) represented the organic
solvent-unextractable radioactivity in the water column
and occurred to the greatest extent in the cores contain-
ing sediments. Production of CO2, another indication
of aromatic ring cleavage in the parent molecule (MPS),
occurred to a greater extent in the sediment cores than'
in the cores with water only (Table 12). No significant
CO2 evolved from the sterile systems.
Total recovery of the radiolabel amounted to greater
than 90% in all cases. Combustion of the extracted sedi-
ments to CO2 showed 7% of the radioactivity to be un-
extractable from the sterile sediment systems. Results in-
dicate sediments may be necessary for rapid metabolism
of methyl parathion.
Table 11. Metabolism of methyl parathion in a salt-marsh Eco-core
Day
Percentage of MPS remaininga
Sterile Sediment
Water
Percentage of polar products
Sterile Sediment Water
0 100
2 92
4 85
8 84
14 84
31 82
100
80
57
15
4
0.01
100
85
66
27
15
6
0
4
6
11
16
21
0
8
13
23
40
54
0
3
7
9
10
9
aMPS added = 1 mg/& containing 22.2 MCi.
20
-------
Table 12 Evolution of CO2 from C-methyl parathion
Day
Total dpm added
Radioactivity (PPM x ICr)
Sterilea
0.02
Water
Sediment
0 0
2 0
4 8
8 3
14 5
24 4
31 2
34 2
0
0
113
133
380
716
990
554
0
2
1520
910
830
1956
1550
789
Water and sediment treated with 2% formalin
As a technique for assessing biodegradation of xeno-
biotics, the Eco-core system can: 1) determine interactive
processes contributing to the metabolism of the test com-
pound, 2) monitor changes in microbial populations in-
duced by the toxicant, and 3) easily facilitate system rep-
lication. The core, however, is a static system with a dis-
advantage of possible artifactual changes that can be in-
duced by accumulating metabolites.
Continuous-flow Systems
H.P. PRITCHARD, Environmental Scientist
Continuous-flow systems (Fig. 8, p. 16 ) provide infor-
mation on the rate and extent to which a pollutant: a)
moves into the various compartments of an aquatic eco-
system and b) is transformed by biological and non-
biological forces.
Small-scale System - A small-scale continuous-flow sys-
tem (500-ml reactor vessel volume) was used to study the
fate of methyl parathion under conditions typical of an
aquatic estuarine environment. This system (Fig. 11) was
designed with multiple stages to study transformation
processes as a function of dilution. It used fresh seawater
and artificial seawater, supplemented with radiolabeled
methyl parathion (50-500 ppb). Transformation products
were quantified by thin layer chromatography, autoradi-
ography, and gas chromatography. Radio-labeled MPS was
added to the system either as a spike into the primary re-
actor vessel (wash-out kinetics followed) or continuously
from a reservoir (quasi-steady kinetics followed).
Table 13 shows the stability of MPS in sterile and un-
sterile continuous-flow systems without sediment. Very
little chemical or biological degradation of the substrate
occurred under varied conditions (differing flow rates,
nutrient concentrations, seawater, and organic supple-
ments). However, MPS was much less stable when sedi-
ment was added to the system. Aeration was adjusted to
give an aerobic water column and an anaerobic sediment-
water interface. Table 14 shows that MPS stability, as in-
dicated by CO2 and polar product production in the un-
sterile system, decreased with time. This presumedly re-
flects the enrichment of an MPS degrading population of
bacteria. MPS was also transformed in the sterile control
but to a lesser extent. Amino methyl parathion (AMPS)
was the principle transformation product in both systems,
but p_-nitrophenol was also detected. The presence of sedi-
ment, therefore, promoted the transformation of MPS in
estuarine seawater environments. Calculations from these
initial experiments indicated a half-life of MPS of 400 to
500 hours for the conditions tested. If methyl parathion
input to the continuous-flow system is eliminated (but
seawater flow is maintained), residual MPS in the sedi-
ment (shown in the other systems to be tightly bound) is
slowly released with time.
Large-scale Systems - Large-scale, continuous-flow sys-
tems (40-1 reactor vessel volume) are designed to study
21
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I SINGLE FEED, DUAL STAGE CONTINUOUS CULTURE SYSTEM |
CRUSHED OYSTER
SHELL FLTER
' ?•?->?•
VESSEL-2
0000 ml«)
HgO OaOmli)
DETRITUS (5.0 em)
-T8em-
Figure 11. Schematic design for a continuous-flow microcosm. A and B represent two options for systems developed at
ERL,GB to assess fate and transport of pesticides in estuarine environments.
the fate of a pollutant affected by marine macrobiota.
Thirty liters of seawater were added to a vessel containing
14-£ methyl parathion and a 9-cm layer of marsh sedi-
ment. The systems were continuously fed fresh filtered
seawaier and all water exited through polystyrene resins.
Toxicant movement into the sediment was the major
transport process.
Sediment-burrowing lugworms (Arenicola cristata)
caused a more rapid dispersal of radioactivity throughout
the sediment column of reactor vessels. These worms en-
hanced the movement of radioactivity from the water col-
umn to the sediment, but accumulated no radioactivity.
Radioactivity budgets of the systems suggested accumula-
tion of unextractable residues in the sediment, a process
verified by the other systems discussed. Analysis of ex-
tractable radioactivity indicated both £_-nitrophenol and
amino-methyl parathion as degradation products. The
amount of degradation was affected by microbial activity
and not by lugworms.
The selective nature of continuous-flow systems accom-
modates the study of many complex biological processes
separated into their component parts. Rates of transfor-
mation and compartmental exchange obtained from con-
tinuous-flow studies are considered more appropriate for
environmental studies than similar rates derived from
static tests.
Aquatic Gradient Avoidance Response System
R.L. GRIPE, Research Biologist. EPA Grant R804458,
University of West Florida, Pensacola; C.N. D'ASARO
Principal Investigator; F.G. WILKES, Project Officer
Behavioral tests facilitate the study of subtle, sublethal
effects of certain pollutants. Long-term ecological conse-
quences of avoiding lethal or sublethal toxicants in an
area near a pollutant source may affect the marine en-
vironment, either overtly (a kill) or through less obvious
mortalities at the lower trophic level. Inability of aquatic
organisms to avoid contaminants also may cause harmful
substances to bioaccumulate in the aquatic food chain
Most apparatus designed to detect avoidance of pollut-
ants by aquatic organisms require visual observations of
22
-------
Table 13. Stability of methyl parathion in continuous-flow systems without sediment.
Retention
Volume1
1.3
4.2
8.3
11.3
23.6
Concentration of Methyl Parathion (mg/l)
Sterile
Reservoir
1.10
1.23
0.87
1.08
1.15
Control
Vessel
0.97(81%)b
1.06(85%)
0.97(90%)
0.92(85%)
1.01(88%)
Unsterile
Reservoir
0.97
1.07
1.10
1.03
0.94
System
Vessel
0.92(94%)
0.89(83%)
1.04(94%)
0.90(87%)
0.83(88%)
aTurnover of one retention volume requires 11 h.
Numbers in parentheses are percentage MPS recovered.
test organisms that are subjected to steep pollutant grad-
ients. In 1977, an Aquatic Gradient Avoidance Response
System (AGARS) was developed at ERL.GB to eliminate
these limitations.
The system allows animals to choose between one un-
contaminated zone and three increasingly toxic zones in a
gradient trough that is monitored for extended time per-
iods by infrared light sources, sensors, and a microproces-
sor (Fig. 12). Baseline data are obtained by comparing
the time test animals spent in trough areas (avoidance or
attraction) in the absence of a pollutant.
In initial tests with the system, a group of 4 pinfish
(Lagodon rhomboides) were monitored in the trough for
(1) 3 days in clean water; (2) 1 day in chlorination-
Table 14. Transformation of C-methyl parathion in continuous-flow systems with sediment.
Hours
48
72
96
120
144
168
192
216
Unsterile
14co2b
374
2720
3200
6952
6903
17693
23079
26314
System
Polar Products0
7.2
15.3
13.1
22.1
27.3
30.7
32.2
31.8
14co2
0
63
0
96
0
0
0
0
Sterile System1
Polar Products
5.7
8.6
10.0
11.4
16.0
16.0
15.1
15.8
treated with 2% formalin in reservoir and sediment.
CC>2 (expressed as dpm) in alkaline traps per day.
cPercentage of total radioactivity in water column not extractable with methylene chloride.
23
-------
HIGH
MEDIUM
LOW
CONTROL
OVERFLOW
PHOTOTRANSWTOR
Figure 12. The Aquatic (Gradient Avoidance Response System (A(iARS), above, was developed to monitor behavior
of marine animals exposed to toxicants.
[•'igure 13. Results of 9-day AGARS test to monitor response of pinfish to toxicants. Mean hourly total of time spent
iti four /ones are recorded. Chlorination-produced oxidants (CPO) were used on days 4 and 7; no toxicant was used
during remaining test days.
24
-------
produced oxidant (CPO) concentrations that progressively
increased downstream (control, 0.02, 0.07, and 0.21 mg/£
for /.ones 1-4, respectively); (3) 2 days in clean water-,
(4) 1 day in CPO concentrations decreasing downstream
(O.O9, 0.04, 0.02, and 0.01 mg/£ in /.onc.s 14)-, and (5)
2 days in clean water. Trough temperature was between
28.5° and 29.0°C and salinity from 22 to 26 parts per
thousand (ppt) for the 9-day test.
On day 1 fish spent the longest time in /one 1 where
current was lowest; fish were more uniformly distributed
throughout the four /ones on days 2 and 3 (Fig. 13). No
measurable CPO was present in the trough during this 3-
day acclimation period.
On the day after CPO was added to the water (day 4),
pinfish spent significantly (Q<_().()5) less time in /ones 3
(0.07 mg/£ ) and 4 (0.21 mg/£ ) where CPO concentra-
tions were highest. No avoidance of /.one 2 (0.02 mg/£)
was evident. More residence time was recorded in /.ones
3 and 4. The avoidance threshold was indicated to be be-
tween 0.07 and 0.02 mg/£. Return to CPO-free water on
days 5 and 6 resulted in a continued high presence in
/.ones 1 and 2.
On day 7, the second toxicant exposure day, zones 1
(0.09 mg/2.) and 2 (0.04 mg/£) were significantly (a- 0.05)
(a''0.05) avoided; no avoidance was shown in zone 3
(0.02 mg/£), indicating a fish avoidance threshold of ap-
proximately 0.02 to 0.04 mg/£ CPO. For days 8 and 9
(CPO-free), fish spent more time in /.one 4 than in any
other area.
Initial tests with AGARS showed that pinfish will
avoid chlorination-produced oxidants at concentrations of
0.02 to 0.04 mg/SL. Future studies with AGARS will
monitor the number to entries into /ones by test species
and time spent in each zone. Other possible uses for the
device are tests for thermal or salinity preferences.
The system is currently being used at F,RL,GB to moni-
tor burrowing habits of pink shrimp exposed to toxicants.
F.arlier acute bioassays indicated that this species, which
normally burrows during daylight and emerges at night,
reduces time spent in burrows after exposure to certain
poisonous compounds. This altered behavior could cause
shrimp to become more vulnerable to predation. Prelimi-
nary tests have demonstrated the usefulness and versatility
of AGARS in determining sublethal effects of toxicants
on marine species.
Benthic Bioassay System
N.I. RUBINSTFIN, Research Biologist FPA Grant
R804458, University of West Florida; C.N. D'ASARO,
Principal Investigator; F.G. WII.KFS, Project Officer
Bioassays currently used by regulators' agencies and in-
dustry to assess the potential impact of pollutants on
marine biota utili/e a variety of sensitive pelagic and epi-
benthic species, but rarely incorporate infaunal organisms.
This practice is due in part to the relative lack of sensi-
tivity displayed by many infaunal species and the diffi-
culties encountered in observing biological effects while
organisms are buried in bottom sediment.
However, many infaunal species are deposit feeders
that affect the marine environment by their substrate re-
working activity. These organisms influence benthic com-
munity trophic structure and sediment stability and pro-
vide pathways for cycling nutrients, organic material, oxy-
gen, and pollutants between the sediment and water col-
umn. Consequently, an accurate appraisal of the environ-
mental effect of contaminants must include information
regarding their impact on representative benthic species.
The Benthic Bioassay System was developed to exam-
ine the effect of low pesticide concentrations on the ac-
tivity of an infaunal polychaete, Aremcola cnstata
Stimpson (commonly referred to as the lugworm). Lug-
worms are tube-dwelling deposit feeders found in littoral
habitats throughout the world. Their activities are some-
what analogous to those of earth worms and are respons-
ible for bioturbations of the substrate to depths as great
as 40 cm. Populations of the Furopean lugworm
(Arenicola marina) have been observed to turn over nearly
500 tons of sediment (dry weight) per acre per year. Con-
sequently, lugworms play an important role in sediment-
water column dynamics and are of interest in evaluations
of the impact of toxic substances on the benthic com-
munity.
Lugworms rework sediments as a function of their
feeding mode, producing distinct topographical features
in the form of funnel-shaped depressions on the substrate
surface. Feeding is an integral part of an activity cycle
believed to be controlled by internal pacemakers and
therefore to be independent of normal environmental
variables. A decrease in the production of feeding funnels
indicates an interruption in the lugworm activity cycle.
The test developed at KRL.GB in 1977 monitors sub-
strate reworking activity of lugworms by time-lapse pho-
tography. Rates of feeding funnel formation (surface area
|cm21 turned under per hour) were determined with lug-
worms exposed to a toxicant (.methyl parathion) and non-
exposed (control) lugworms.
Fxperiments were conducted in aerated 125-it glass
tanks (0.25 m surface area) containing 25 cm of clean
beach sand (particle size 200 to 800 ym) and 75 £ of fil-
tered seawater (20 pm filter). All tests were conducted
under static conditions; salinity ranged from 22 to 25 ppt
and temperature fluctuated between 23° and 25°C.
Six lugworms of similar si/e were introduced into each
of two tanks and allowed 48 h to acclimate to test con-
ditions. Seventy grams of ground seagrass added to each
tank served as food and also provided contrast for photo-
graphs against the white sand in areas disturbed by feed-
ing animals. The test compound (methyl parathion) was
then introduced into one tank while the non-dosed tank
served as the control. A 35-mm automatic camera was
positioned above each tank and photographs of the sub-
strate surface were taken at 12-h intervals for a 144-h
25
-------
period. The photographs were then analyzed to deter-
mine the surface area (cm ) disturbed by feeding animals.
The total surface area turned under (disturbed) was plot
ted against time to give a substrate modification rate for
exposed and control animals. Kau-s 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.
After initial tests with methyl parathion, the procedure
was used in 1977 to determine how three additional pesti-
cides (sodium pentachlorophenol, Kepone, and Sevin)
affect lugworm substrate reworking activity. Kepone was
found to be most toxic to lugworms: mortality occurred
after 144-h exposure to 29.5 Ug/fc seawater; a significant
effect on substrate reworking was observed at concentra-
tions as low as 2.8 pg/£ seawater. Methyl parathion af-
fected lugworm activity at concentrations between 75 and
150 yg/2, seawater (F''ig. 14). Sodium pentachlorophenol
inhibited lugworm activity at 45 to 80 yg/X, seawater.
Sevin was found to be least toxic to lugworms. A reduc-
tion in substrate reworking activity was observed at 1,000
U(?/£, seawater, although mortality did not occur until
10,000 yg/Ji seawater.
Figure 14. Lugworm feeding-funnels illustrated in above
photographs were produced in (1) 24 h; (2) 72 h; (3)
144 h. X indicates action of lugworms exposed to 150
parts per billion methyl parathion; C indicates control
lugworms.
Test results indicate that this technique can demon-
strate a behavioral effect on infaunal organisms at sub-
Ictlial concentrations of pollutants.
Estuarine Laboratory Systems
C.N. D'ASARO, Investigator. EPA Grant R804458, Uni-
versity of West Florida; F.G. WILKKS, Project Officer
A microcosm, designed to simulate some trophic rela-
tionships in an estuarine grassflat community, was assem-
bled to provide a vehicle in which fate and effects of
xenobiotics could be tested. Selection of species for the
microcosm (assembled in 39-i, aquaria) was based on
these criteria: wide geographic distribution, abundance
simple culture methods and sensitivity to xenobiotics
Bcnthic diatoms, the estuarine grass Kuppia inaritima and
detrital Ruppia formed the basis for the trophic structure
An isopod, Ericsonelja attenuata, grazed on the grass and
epiphytic microorganisms. An infaunal polychaete,
Arenicola cristata, which is a detrivore, occupied the sub-
strate. After acclimation, aquaria stocked with animals
and operated at constant temperature conditions did not
show evidence of environmental degradation in terms of
the parameters measured (dissolved oxygen, pH, NH,-N
algal cells/ml) for two weeks.
After behavior of the organisms in the system was
determined, mortality or loss of specimens prior to termi-
nation of the experiments was prevented. In the final
series of experiments, other invertebrates were success-
fully introduced into various tropic niches in the micro-
cosm. A polychaete, Polydora ligni, was used to filter par-
ticulatc material from the water. A herbivorous-
detritivorous prosobranch. Bittium varium, scoured the
grass blades and the aquarium walls. An apodid holothur-
ian, Leptosynapta inhaerens, competed for detritus. Prior
to termination of the experiment, juvenile portunids
Callinectcs sapidus. were introduced as carnivores. The
microcosm is now available for future tests with xeno-
biotics.
Predator/Prey System
J.A. FARR, Research Biologist. EPA Grant R804458,
University of West Florida; C.N. D'ASARO, Principal'ln-
vestigator; F.G. WILKES, Project Officer
Estuarine ecosystems contain vital species interactions
many of which may be vulnerable to stress by pollutants
A test was developed to measure the effect of a pollutant
on an important estuarine predator/prey relationship.
Grass shrimp, Palaemonetes pugio, were exposed to sub-
lethal concentrations of methyl or ethyl parathion to
determine if their ability to escape predation by the gulf
killifish, Fundulus grandis, was impaired.
26
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Table 15. Summary of the behavior of Fundulus grandis when preying on Palaemonetcs pugio exposed to methyl and
ethyl parathion as compared to undoscd shrimp. Values differ from the control at the 0.05 (a) or 0.01 ( )
level of significance, as determined by the VVilcoxon sign rank statistic*
72-h exposure to 24-h exposure to 72-h exposure to 24-h exposure to
0.1 ug/J, ethyl 0.5 ug/iU-thyl 0.1 yg/J, methyl 0.5 ijg/toiicthyl
parathion (N = 20) parathion (N = 19) parathion (N = 18) parathion (N = 19)
Control Exposed Control Exposed Control Exposed Control Exposed
Mean number of minutes
taken to capture second
and third shrimp 6.2 4.0b 6.5
Mean number of shrimp
consumed in fifteen
minutes 3.7 4.3a 3.4
Mean number of shrimp
consumed in three
hours 8.1 8.1 8.1
4.2C
4.4L
5.9
3.3
7.1
3.9L
4.1'
3.4
7.4
4.51
3.9
7.1
Fstuanne ecosystems contain many vital species inter-
actions, many of which may be vulnerable to stress by
pollutants. A test was developed to measure the effect of
a pollutant on an important estuarme predator/prey rela-
tionship. Grass shrimp, l}alaemonetes pugio, were exposed
to sublethal concentrations of methyl or ethyl parathion
to determine if their ability to escape predation In the
gulf killifish, Fundulus grandis, was impaired.
Two qualitative changes were noted in the behavior of
the shrimp. Typically, in the presence of a predator, un-
dosed shrimp remained motionless in a corner of the
aquarium, but exposure to parathion altered this behavior.
Rather than remaining immobile for long periods, the ex-
posed shrimp continually moved around the tank and
were thus more easily observed by the fish.
A second apparent effect was a decrease in the actual
physical endurance of the shrimp. While actually fleeing a
pursuing fish, exposed shrimp appeared to fatigue more
quickly than unexposed shrimp. Their tail-snap escape re-
flex was less vigorous and continued for shorter time per-
iods.
Shrimp exposed to low doses of parathion were caught
more quickly than undoscd shrimp (Table 15). More fish
were consumed by the fish in the initial 15 minutes of
the test, but no difference occurred in the total exposed
and unexposed shrimp consumed throughout the experi-
ment. These data indicate that exposure to sublethal
doses of parathion renders shrimp more easily captured
by killifish, but does not alter total shrimp consumption.
However, grass shrimp, if more easily captured than other
prey, would be expected to fulfill a greater proportion of
a predator's diet.
Thus, predictable results are: (Da reduction in detri-
tal breakdown and in food chain efficiency at the lower
trophic levels and (2) an increase in density of other prey
species. Such changes in a multi-prey community could
result in altered predator preferences and subsequently
change community structure.
SUBSTITUTE CHEMICAL PROGRAM
Fate and Effects of Atrazine in Salt-marsh Eco-
systems
DONALD F. DAVIS. Principal Investigator, KPA Grunt
R803835, Auburn University; F.G. WILKES, Project
Officer
In the third year ilc>77) of this research, the investi-
gator examined the accumulation, degradation, and effects
ot atra/ine in salt-marsh ecosystems. Model ecosystems
were developed to study effects of atra/.me on the grass,
Spartma alternitlora, tiddler crab, periwinkle snail, mussel,
and detritus. Effects of atra/ine stress on a salt marsh will
be compared with effects observed in the model ecosys-
tems.
"•Reprinted with permission of the American Fisheries Society. Trans. Am. Fish. Soc. Vol. 106, No. 3. May 1977, p. 289.
27
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During 1977, the rate of conversion of dried S. alterni-
flora was determined in a microcosm. After a 30-day test,
the average percentages of plants converted to detritus
were: 78, 68, and 67 for grass doused with 0, 10 , and
10 M concentrations of atrazine, respectively. Results
suggest that atrazine may inhibit detrital formation.
In a second experiment. S. alterniflora was grown for
2 days in a nutrient solution containing 14C-ring-labeled
atra/ine, followed by 3 days in an atrazine-free solution.
S_ alli-rniflora was dried for 2 h at 105°C and placed in a
microecosystcm for 30 days. The partially decomposed
material (detritus) was collected, dried, and extracted
with 80% methanol. Percentage totals of C in the ex-
tractions have yet to be determined.
In a third investigation, 3 groups of 15 crabs for 30
days fed on detritus derived from uncontaminated _S.
alterniflora. After drying, the detritus was wetted with
0, 10-6, and 10-4 solutions of atrazine. Neither atrazine
solution had any significant effect on crab mortality, be-
havior, or general appearance. In another 30-day period,
45 crabs fed on detritus from plants grown in l4C-ring-
labeled atrazine. Radioactivity of the feces increased
steadily throughout the 30 days. Then 15 crabs were
finely ground in a mortar by a pestle, and the ground
material was extracted with 80% methanol. This solution
was separated into chloroform and aqueous fractions and
an insoluble residue that was hydrolyzed for 2 h with
0.5 N HCL. The hydrolyzed material was extracted with
diethyl ether and then water. Percentages of radioactivity
in crabs were: 4% in chloroform; 29% in first water ex-
tract; 2% in diethyl ether; 39% in second water extract;
and 25% in residue. The remaining 30 crabs will be anal-
yzed in the next reporting period.
In addition, research was initiated in 1977 on the ef-
fect of atrazine on the edaphic diatoms of the salt marsh.
Mixed cultures of the diatom, Amphora sp., and a unicel-
lular blue-green alga were isolated from soil samples from
a Georgia salt marsh and exposed to 0, lO'8, 10"7, 10"6,
5 x 10-6, and 10'SM for 2 h. Effects of these short-term
treatments on subsequent photosynthesis (light fixation
minus dark fixation) were measured: 91% inhibition of
photosynthesis was caused by lO'Sjvl atrazine and 50% in-
hibition by approximately 5 x lO-^M. Some tests showed
a slight stimulation of photosynthesis at the two lowest
atrazine concentrations. No effects were evident on dark
fixation.
Thus far, the investigation has established that even
fairly high levels of atrazine have little, if any, direct ef-
fect on £. alterniflora or crabs, or on the conversion of
S. alterniflora to detritus.
J.W. DAY, Jr., S.P. MEYERS, and R.P. GAMBRELL,
Principal Investigators. EPA Grant R804976, Louisiana
State University; F.G. WILKES, Project Officer
The prime objective of this research is to find a means
for solving land-use conflicts caused by chemical dis-
charges from agriculture into wetlands. Investigators seek
to determine: 1) fate of substitute chemical compounds
(initially Guthion^) in brackish wetlands, correlating re-
sults with past productivity, nutrient flows, application
rates, and runoff patterns; 2) fate and effects of substitute
pesticides applied to test plots; 3) persistence of Guthion
under varying conditions. One year of a projected three-
year study has been completed.
Investigators will examine the persistence of Guthion
in swamp soil, using controlled soil physiocochemical
parameters (pH, oxidation-reduction conditions, salinity).
In preliminary field studies, selected microbial popula-
tions were identified: aerobic heterotrophs, chitinoclastic
bacteria, cellulolytic forms, proteolytic bacteria, amylo-
lytic forms, NO3 reducers, and yeast fungi. Studies were
initiated on the effect of Guthion on 1) the growth re-
sponse curve for selected heterotrophs, 2) chitin degrada-
tion, and 3) chitin solubilization.
Understanding the effects of pesticides in wetlands re-
quires knowledge of the influence of hydrology, chemis-
try, temperature, and community structure. As important
members of the food web, invertebrates are useful tools
for detecting environmental perturbations. Some species,
such as the crayfish (Procambarus clarki), are consumed
by man. Invertebrates occupy virtually all trophic levels
and as recycling agents can reflect environmental condi-
tions for extended time. Therefore, field studies in this
project focused on the effects of Guthion on invertebrate
populations. The nature of the changes within these com-
munities will need further study before conclusions can
be drawn.
In addition, investigators initiated an attempt to meas-
ure the metabolism of communities occupying the
swamp's floor (the floating vegetation layer, the water
column, and sediments). In the forthcoming reporting
period, controlled field applications of Guthion will be
undertaken in isolated wetland areas. Studies will be con-
ducted on the degradation of Guthion and other substi-
tute chemicals (malathion and methyl parathion) in a
salt marsh. Pesticide levels will continue to be measured
in sediment, water, and biota.
Determination of the Environmental Impact of
Several Substitute Chemicals in Agriculturally
Affected Wetlands
MICROBIOLOGY
Aquatic microorganisms play a critical role in the met-
abolic transformation of organic and inorganic compounds
entering lakes, estuaries, and oceans. Many of these com-
pounds, particularly the chlorinated hydrocarbon group,
28
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originate from man's industrial and agricultural enter-
prises. Often, they have no close counterpart in nature;
therefore, their fate and accumulation are important in
determining environmental quality.
Kepone Toxicity to Aquatic Microorganisms
A.W. BOURQUIN, Research Microbiologist;
P.H. PRITCHARD, Environmental Scientist
Chlorinated hydrocarbons and pesticides are known to
accumulate in humus soil and aquatic ecosystems, due in
part to their resistance to biological and non-biological
degradation.
Questions remain unanswered regarding the potential
of these-compounds to alter basic microbial transforma-
tion processes. Therefore, the impact of Kepone on mi-
crobial transformation processes in the James River,
Virginia, was investigated. This study was conducted in
four phases: 1) pure culture toxicity; 2) natural popula-
tion toxicity; 3) effects on oxygen uptake; 4) effects on
naphthalene and methyl parathion transformation.
In the first phase, toxicity of Kepone to laboratory
stock cultures was determined by the disc agar diffusion
sensitivity method. The stock cultures were originally iso-
lated from batch culture by selective enrichments on a
wide spectrum of substrates. Of the 30 isolates tested,
33% were inhibited at the 3.65 ug/disc concentration,
and 47% were inhibited at the 14.6 ug/disc concentration.
Kepone-sensitive cultures showed no significant differ-
ence from randomly selected stock cultures in terms of
morphology, aliphatic hydrocarbon utilization, tolerance
to pesticides (Aroclor 1242 and 1016 methoxychlor,
heptachlor, DDT, malathion, toxaphene), lipolytic and
proteolytic activity, nitrate reduction, sugar utilization,
and urea hydrolysis. However, many of the sensitive cul-
tures (7/8) were gram positive, whereas fewer of the
Kepone-tolerant cultures (2/9) were gram positive.
The same was true of amylolytic activity: 6/8 were
positive for Kepone-sensitive cultures and 1/7 were posi-
tive for Kepone-tolerant cultures. A greater percentage
(50% versus 11%) of Kepone-sensitive bacteria metabo-
lized one or more aromatic compounds (phenol, naphtha-
lene, toluene, biphenyl, and xylene). Only one of the six
fungal cultures was poisoned by Kepone at the 14.6
yg/disc concentration. Two yeasts, Candida maltosa and
lipolytica, were both sensitive, but at a higher concentra-
tion than the bacteria tested.
Toxicity of Kepone to natural mixed populations of
bacteria from a variety of marine habitats was determined
by standard total viable counts and Zobell's seawater agar
containing dissolved Kepone. Kepone concentrations (as
low as 20 Ug/fc) inhibited development of colonies on
the agar plate. Different degrees of inhibition were noted
in samples taken from the same area at different times;
frequently, concentrations below 20 yg/jj, appeared in-
hibitory. These levels of Kepone toxicity almost equaled
those found in James River sediment.
A total of 17 colony-forming units, which grew in the
presence of Kepone, were selected on the basis of pre-
dominance for further study. When compared with 20 iso-
lates randomly selected from Zobell's marine agar (no
Kepone), 75% of the Kepone-tolerant isolates displayed
amylolytic and lipolytic activity, whereas only 55% of
the non-tolerant isolates exhibited such activity; 90% of
the Kepone-tolerant isolates were gram negative, com-
pared to 55% of the isolates from Zobell's marine agar
(no Kepone).
The toxicity of Kepone to bacteria isolated from sedi-
ment was studied by using standard viable plate counts
on Zobell's marine agar containing Kepone; colony-form-
ing units incubated under anaerobic conditions showed no
significant change in the number relative to aerobic con-
trols, thus implying a resistance to Kepone under anaero-
bic conditions.
The inhibition of oxygen uptake appeared to be a
sensitive indicator of Kepone toxicity. Isolates showed
varying degrees of sensitivity: most were inhibited at the
20 mg/jl concentration, whereas relatively few were in-
hibited at the 2 mg/X, concentration. Additionally, studies
showed that Kepone in some cases actually stimulated
oxygen uptake.
Eco-core experiments have shown that both the rate
and extent of degradation of naphthlene and methyl para-
thion in the presence of Kepone were reduced relative to
non-Kepone containing controls.
Overall, these experiments indicate that Kepone is
toxic to microorganisms. To date no chemical or biolog-
ical degradation of Kepone has been demonstrated, and
Kepone appears to be only very slowly washing out of
the James River system. Residual Kepone possibly could
inhibit bacteria and the processes they mediate.
Surface Microlayer Studies
A.W. BOURQUIN, Research Microbiologist
Surface microlayer studies are concerned with the mi-
crobial ecology and the chemistry of organic material that
accumulates on water surfaces. Past investigations at
ERL.GB have shown these surface films to be active bi-
ologically and rich in organic pollutants, i.e., chlorinated
aromatic hydrocarbons and polychlorinated biphenyls
(PCBs). Experiments in 1977 focused on: (1) the move-
ment of organic compounds from the water column into
the surface film and (2) artificial laboratory surface
microlayers.
In the first phase, special vessels were constructed to
sample both a hexadecane surface film by the NucleporeR
membrane technique and the water beneath the suface
film. Movement of the compound into the surface film
was studied by spiking the water with radioactive com-
pounds. Tests with methyl parathion and DDT showed
that pollutants rapidly concentrate in surface microlayers:
in less than 48 h, methyl parathion concentrated in the
surface film 150 to 200 times the amount observed in the
water column (on a weight for weight basis); DDT con-
centrated from 250 to 300 times the amount found in
the water column for this time period. Concentrations
29
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rapidly reached equilibrium; however, the amount accum-
ulated was directly proportional to the partitioning co-
efficient of the compound between seawater and the hex-
adecane film.
In the second phase of the research, tests using hex-
adecane as a basic surface film showed that the addition
of alkyl benzene sulfonate, tannic acid, or Tween-80 all
increased the concentration of DDT from the water col-
umn, i.e., 260 to 320 times that seen in the water
column.
Effects of Pollutants on Microbial Activities in
Estuarine Surface Films
DONALD AHEARN, Principal Investigator. EPA Grant
R804477, Georgia State University; A.W. BOURQUIN,
Project Officer
A group of compounds that earlier had reacted posi-
tively in mutagenicity tests (Ames systems) were used in
studies of 9 surface slick isolates. In tests using the disc
diffusion method, pentachlorophenol (PCP) and Captan
inhibited the growth of 9 and 8 of the 9 isolates, respec-
tively. Further tests with discs saturated with 0.1 ml of
DMSO containing 100 yg of test substance showed that
PCP inhibited only 3 of 9 isolates, whereas Captan and
Captafol inhibited all 9 cultures. Future studies will estab-
lish the dose responses for selected inhibitory compounds
on Ames strains and on surface slick isolates.
Previous studies suggest that heptachlor may alter the
transport of hydrophobic materials into the cell. There-
fore, studies undertaken in 1977 assessed the synergistic
action of heptachlor and mutagenic compounds in the
Ames system. Subinhibitory levels of chlordane (20 ppm)
were found to be inhibitory in the presence of heptachlor
(20 ppm). No enhanced mutagenicity was noted for com-
binations of heptachlor with BHC, Captafol, Captan,
Carbaryl, chlordane, Kepone, mirex, and PCP. Preliminary
results from surface film samples indicate the presence of
toxic and possibly mutagenic substances.
Earlier, this investigation demonstrated preferential re-
moval of naphthalene and biphenyl in a synthetic oil,
using a heptachlor (1 mg/ml) system inoculated with
either Candida lipolytica or C_. maltosa. Work in 1977 ex-
amined the disappearance of naphthalene from a simpli-
fied system containing only hexadecane or hexadecane
and biphenyl. Biphenyl appeared to stimulate utilization
of hexadecane and naphthalene by C. lipolytica. Hepta-
chlor reduced naphthalene utilization by both yeasts.
Similar results were observed for a modified synthetic oil
containing biphenyl, naphthalene, tetradecane, hex-
adecane, and pristane. Heptachlor (1000 ppm) or Kepone
(10 ppm), when added into this system, caused a reduc-
tion in utilization of naphthalene and hexadecane.
Tests on effects of sublethal concentrations of pesti-
cides on proteolytic activity showed that selected aro-
matic and chlorinated hydrocarbons caused no reduction
in this enzyme activity.
Seventeen cultures were tested for their response to
combinations of PCP, o-chlorophenol, naphthalene, 1-
chloronaphthalene, heptachlor, and methoxychlor. Syner-
gistic responses were often produced by combinations of
1-chloronaphthalene and heptachlor or methoxychlor (8
out of 10); napthalene and PCP and naphthalene and
ortho-chlorophenol also inhibited these cultures.
Biodegradation of Chlorinated Dibenzodiozins
and Dibenzofurans
D.T. GIBSON, Principal Investigator. EPA Grant
R804525, University of Texas; A.W. BOURQUIN, Project
Officer
Chlorinated dibenzo-p-dioxins have long been recog-
nized as possible by-products in the manufacture of cer-
tain chlorinated phenols (i.e. pentachlorophenol and
2,4,5-trichlorophenol). Interest in these compounds has
increased since the discovery of highly toxic 2,3,7,8-
tetrachlorodibenzo-p-dioxin (TCDD) in samples of the
herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T).
Further, chlorinated dibenzo-p-dioxins and dibenzofurans
have been detected in commercial preparations of poly-
chlorinated biphenyls. Many of these chlorinated com-
pounds are used extensively in both industry and agricul-
ture; therefore, the high level of toxicity of the chlori-
nated dibenzo-p-dioxins is an environmental concern.
This project seeks to determine reaction microorgan-
isms that degrade dibenzofuran and dibenzodioxin, and to
identify their chlorinated derivatives.
Microorganisms were collected from mud flats and
algal mats at Port Aransas, Texas. Each sample was incu-
bated in seawater containing either dibenzo-p-dioxin or
dibenzofuran (0.5 g/£). No growth was obtained after
10 weeks incubation at 25°C on a rotary shaker (250 rev/
min). Six additional estuarine samples (collected later)
also failed to yield any organisms that could grow by
using either substrate as the sole source of carbon and
energy. The same samples, however, yielded growing
organisms that were able to use hexadecane as their sole
source of carbon. Nine of these organisms were retained
for co-oxidation studies.
Other laboratory cultures, however, were found to
metabolize dibenzo-p-dioxin and dibenzofuran:
a Beijerinckia species previously isolated through its abil-
ity to grow in biphenyl; a Pseudomonas species that de-
grades naphthalene; and Cunninghamella elegans, an organ-
ism isolated through ability to degrade crude oil from
North Carolina estuary. These microorganisms would not
grow in dibenzo-p-dioxin or dibenzofuran. However, when
an alternative growth substrate was present, significant de-
degradation of both aromatic compounds occurred.
Although no organism was found to use dibenzo-p-
dioxin and dibenzofuran as a sole source of carbon and
energy, these compounds were readily metabolized when
an alternative carbon source was available.
Dibenzo-p-dioxin was oxidized to cis-l,2-dihydroxy-
1,2-dihydrodibenzo-dioxin, which then forms a catechol
Studies have yet to determine whether the molecule can
be degraded completely to naturally occurring products
Dibenzofuran is attacked at two positions on the mol-
30
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ecule, indicating that dihydrodiols are produced at the
1,2- and 3,4-positions. Identification of the degradation
products posed significant analytical problems, although
most have been solved. Preliminary evidence indicates that
two catechols and two ring-fission products are produced
by Beijerinckia. The broad specificity of this organism (in
terms of its ability to metabolize the same molecule at
different positions) heightens interest in forthcoming
studies on the effects of chlorine substitution.
The fungus Cunninghamella elegans appears to metab-
olize aromatic compounds in a manner analogous to the
mammalian liver. Mammals are known to produce arene
oxides which, in some cases, can react with nucleic acids
to initiate mutagenic changes. In the case of benzo[a]py-
rene, a metabolite, trans-7,8-dihydroxy-7,8-dihydro-
benzo[a]pyrene, serves as a substrate for the formation of
9,10-epoxy-7,8-dihydroxy-7,8-dihydrobenzo[a] pyrene
(diol-epoxide), which is both mutagenic and carcinogenic.
Although no studies have been reported on the mamma-
lian metabolism of dibenzofuran, results obtained in this
study indicate the formation of an arene oxide. These
molecules, by rearrangement, are known to given phenols
and undergo enzymatic hydration to form trans-
dihydrodiols. Because C. elegans produces 2- and 3-hy-
droxydibenzofuran and 2,3-dihydroxy-2,3-dihydrodibenzo-
furan, the prior formation of 2,3-epoxydibenzofuran is
indicated. Also, the stability of the dihydrodiol to acid
treatment indicates that it is the trans-isomer.
Metabolites isolated in sufficient amounts will be test-
ed for mutagenic activity in the Ames system by Dr.
Eugene Goldschmidt at the University of Houston during
the next reporting period.
Insecticide Persistence in Natural Seawater as
Affected by Salinity, Temperature, and Sterility
W.W. WALKER, Principal Investigator. EPA Grant
R803842, Gulf Coast Research Laboratory, Ocean
Springs, MS; A.W. BOURQU1N, Project Officer
This investigation determined the effect of tempera-
ture, salinity, and sterility on the persistence and degrada-
tion of representative organophosphorus and chlorinated
hydrocarbon insecticides (malathion, parathion, methyl
parathion, diazinon, and methoxychlor).
Surface water samples of 0, 10, 20, and 28 parts per
thousand (ppt) salinity were amended with the above in-
secticides and incubated in the dark at 30°, 20°, and
10°C under sterile and nonsterile conditions. Insecticide
abatement was followed by electron-capture, gas-liquid
chromatography.
No significant differences between sterile and non-
sterile treatments were observed for any insecticide. The
effect of increasing temperature was highly significant
with regard to increased degradation of malathion, para-
thion, methyl parathion, and diazinon. Methoxychlor re-
flected the recalcitrance characteristic of the chlorinated
hydrocarbon insecticides throughout 84 days of incuba-
tion and was not significantly affected by salinity, tem-
perature, or sterility. Salinity effects varied among the
four organophosphates: highly significant for malathion
and diazinon, significant for methyl parathion, but not
significant for parathion.
Malathion was the shortest-lived insecticide: half-lives
at 30°C varied from approximately 11 days in fresh water
to less than two days at 10, 20, or 28 ppt salinity. The
disappearance rate of methyl parathion was second only
to malathion and ranged from 27 days (half-life) in fresh
water to 16 days at 28 ppt. In fresh water, a 45-day half-
life for diazinon suggested a substantial resistance to deg-
radation, especially at 30°C. In saline water, however,
diazinon abatement was accelerated; 24 days half-life at
28 ppt salinity. Parathion, the most persistent organo-
phosphate insecticide, reflected a half-life of at least 44
days regardless of salinity.
One bacterium, tentatively identified as Moraxella sp_.,
was isolated from sediment by enrichment and proved
capable of readily degrading malathion either as a primary
carbon source or in the presence of peptone. Two bac-
teria were tested for the ability to degrade methyl para-
thion: one bacteria, possibly a Pseudomonas sp., proved
capable of utilizing the insecticide with or without pep-
tone; the other, a Moraxella, reflected no degradation of
methyl parathion as the primary carbon source and only
limited utilization of the insecticide in the presence of
peptone. Neither bacteria screened for parathion metab-
olism was capable of insecticide degradation under condi-
tions of this evaluation.
Work was completed September 30, 1977, on this two-
year study. The final will be published early in 1978 in
the EPA Ecological Research Series.
Feasibility of Using Bacterial Strains to Test for
Environmental Carcinogens
J.E. EVANS, Principal Investigator, EPA grant R804586,
JJniyersity of Houston, Houston, TX; A.W. BOURQUINT
Project Officer
A rapidly growing amount of data is available concern-
ing the mutagenic and carcinogenic properties of new
chemicals and products manufactured for commerce in
recent years. However, literature pertaining to mixtures,
such as chemical wastes, is scarce and difficult to locarr
This grant produced a review of literature related to
the feasibility of using bacteria as screening agents to de-
tect cancer-causing agents in the environment. Mutage-
nicity data were also included in the literature search be-
cause of growing experimental evidence that most chem-
ical carcinogens are mutagens and therefore many muta-
gens may be carcinogens.
Results of the investigation indicate that bacterial
strains can be used to initiate a series of studies aimed at
screening mixed wastes for potential mutagens and carcin-
ogens. Findings will be published in the EPA Ecological
Research Series in 1978.
31
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Figure 15. Marine Ecosystem Test Units (METU) at the Bears Bluff Field Station enable scientists to observe effects of
chlorination on marine communities indigenous to adjacent South Carolina salt marshes.
12
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BEARS BLUFF FIELD STATION
WILLIAM P. DAVIS. Chief
The Bears Bluff Field Station, located 25 miles south-
west of Charleston, South Carolina, on the relatively pris-
tine North Kdisto estuary, conducts and coordinates re-
search related to the impact of bioeides and disinfections
on marinc/cstuarine organisms, experimental communities.
and food webs.
Since designated a branch of KKI.,(iB in 1974, the
field station has been engaged in research related to the
effects of biocide oxidants and by-products resulting from
their application in the marine environment. In 1977, the
staff investigated new methods for chemical analyses
adaptable to testing the natural marine/estuarine waters
and related aquatic communities for toxicity, histopathol-
ogy, and behavioral responses. These studies are designed
to yield better assessment of the transfer and transforma-
tion of compounds entering the marine food web.
Analytical Chemistry, Biochemistry, and Produc-
tivity Studies
A.M. CRA\I;. Chemist;
S.I. 1 RIC'KSON, Research Aquatic Biologist;
S. KL1NCII.NSM1TH, Biologist
Chemical analyses at the Bears Bluff Field Station in
1977 were conducted primarily on by-products produced
by direct ehlorination and interactions with natural levels
of ammonia and resulting halogenated amines.
Results of a one-year study completed in 1977 showed
that continuous chlorination of flowing seawater contain-
ing marine plankton caused a measurable reduction in the
concentration of adenosine triphosphate (ATP), some-
times used as an indicator of living biomass.
Effects of continuous chlorination on planktonic life
were measured by ATP content in two flowing seawater
systems for one year. System A consisted of 96 37- £,
aquaria; System B, 40 5.5-£ aquaria.
The mean control ATP for System A was 0.55 yg/Ji;
the measured ATP \vas reduced to 87.1% of mean control
value in aquaria treated with 0.125 mg/2,. (At this nomi-
nal level, no measurable residual is present.) Concentra-
tions of 0.125 mg/H sodium hypochlorite reduced ATP
levels to 77.6% of control, and 0.5 mg/£ sodium hypo-
chlorite caused a 66.8% reduction in measured ATP levels.
In System B, mean control ATP was 0.40 pg/i. Treat-
ment with sodium hypochlorite resulted in the following
percentage reductions of measured ATP levels: 0.47
mg/fl,, 74.5%; 0.94 mg/£, 56.7%; and 1.14 mgH, 42.5%.
Table 16. Fundulus hetcroclitus: Locations of significant F-ratios for all stages of development and corresponding
P values
Develop.
Stages A
1-2 Cell 1.0001
Gastrula 1 .01
Circulation 1 .001
10-day Kmbryo 1 .0001
0-day Larvae 1 .001
7-day Larvae 1 .0001
B
NS
NS
NS
NS
1.05
1 .0001
c
NS
NS
NS
NS
< .001
1 .0001
Variables
AB AC
NS
NS
NS
NS
1.01
1 -05
NS
NS
NS
NS
£.001
1 .0001
BC
NS
NS
NS
NS
1 .001
1 .01
ABC:
NS
.01
NS
NS
1.001
1.01
R
NS
NS
NS
NS
NS
NS
A = Temperature
B = Duration of exposure
C = TRC
AB. AC, BC, ABC = Interactions
R = Replicates
NS = Not significant
-------
CD
100
IB
,1 75
of
u
25
7.5 min.
.0 .05 .10 50 1.0 0 .05 .10 50 1.0 0 .05 .10 .50 1.0 0 05 1O 50 1 O
Total Residual Chlorination (mg/l)
100
IB
I 75
^3 a
0> KQ
2
C
U
I 25
7.5 min.
• 24°C
A29°C
• 34°C
15 min.
0 05 .10 .50 1.0 0 .05 10 50 1.0 0 .05 .10 .50 1.0 0 .05 .10 .50 1
Total Residual Chlorination (mg/l)
Figure 16. Percentage survival of 0-day-old and 7-day-old mummichog larvae subjected to different TRC concentrations
at 3 temperatures for 4 time intervals
In another study conducted in 1977, the Bears Bluff
Field Station reported that certain commercial resin ion
exchange filters used in laboratories and analytical work
in South Carolina had been contaminated by cleaning
water. The investigation demonstrated the need to prop-
erly clean resins during treatment of water for laboratory
use. These findings were reported in the EPA Ecological
Research Series, "Water Softening and Conditioning
Equipment," (KCA-600/3-77-107), September 1977.
Single Species Bioassays (Fishes)
D.P. MIDDAUGH, Research Aquatic Biologist
Research conducted in 1977 determined the sensitivity
of embryos and young larvae of the mummichog
(Fundulus heteroclitus) to different combinations of chlo-
rination levels and thermal stress. Results indicate that 2
larval stages tested (0-day old and 7-day old larvae) were
more sensitive than the 4 embryonic stages (Table 16).
The response of the 2 larval stages to 4 concentrations
of oxidants produced by Chlorination at 3 temperatures
(24°, 29°, and 34°C) for 4 time intervals (7.5, 15, 30,
and 60 min) are shown in Fig. 16. In general, larvae dem-
onstrated a trend of increased sensitivity to total residual
Chlorination with increased exposure temperature and ex-
posure time.
Toxicological responses observed in tests exposing
freshwater and estuarine fish to chlorination levels appear
34
-------
to be similar. However, little information is available on
the physiological responses of marine fish exposed to
chlorinated secondary sewage treatment wastes or chlori-
nation applied as antifouling biocide in thermal electric
generating plants. An investigation was undertaken at
Bears Bluff to measure toxicological and other physiolog-
ical responses of juvenile spot (Leiostomus xanthurus) ex-
posed to various levels of chlorination.
Results showed that measured concentrations (0.09 to
0.37 mg/il) of oxidants produced by chlorination caused
rapid mortality of spot; the median lethal time (LT50)
ranged from 88 to 400 min. Slightly lower oxidant con-
centrations (0.09 and 0.13 mg/Jl) caused no deaths during
48-h exposure in flowing seawater (Table 17).
The pathobiology unit at ERL.GB studied the effects
of chlorine on the gill tissues of exposed spot. When fish
were exposed to 1.0 mg/£,-l NaOCl, sloughing of respi-
ratory gill epithelium began (Fig. 17: 3,4,5). When ex-
posed to 1.6 to 3.2 mg/£ chlorine, gill epithelium slough-
ed and fused; some gills underwent aneurysm develop-
ment (became enlarged) (Fig. 17: 6,7,8). These findings
suggest that the mechanism for death caused by chlorine
in spot may be related to destruction of gill tissues by
direct contact of chlorine on exposed respiratory surfaces.
Oyster Studies
G. SCOTT, Biologist;
D.P. MIDDAUGH, Research Aquatic Biologist
The oyster (Crassostrea virginica G.) was exposed to
chlorination-produced oxidants (CPO) in different seasons
(fall, winter, and spring) for 45 to 75 days in an investi-
gation of seasonal survival patterns in 1977.
Survival was lowest in the higher nominal exposure
concentrations (5.60 and 3.20 mg/J, NaOCl); at lower
concentrations (1.80 and 1.00 mg/Jt), survival often ap-
proached rates observed in the controls (93-100%). Tox-
icity of CPO to oysters appeared to be related to the sea-
sonal water temperature and seasonal physiological condi-
tion of the oyster.
Exposure to CPO also resulted in an avoidance re-
sponse: fecal production was significantly reduced in ex-
posed oysters, suggesting oysters utilize alternate meta-
bolic pathways to avoid direct exposure to chlorination.
Measurements of condition index (dry meat weight/
cavity volume x 100) indicated that with increased per-
iods of exposure to CPO, tissue production is significantly
decreased. Gonadal index (dry gonad weight/dry meat
Table 17. Acute toxicity data for replicate tests with spot exposed to 5 nominal NaOCl concentrations.
Replicate
Test
Nominal NaOCl
Cone, mg 1~1
Measured CPO
Cone, mg 1~1
N x s
LT50
(min)
95% C.I.
(min)
Slope of
Curve
95% C.I.
of Curve
A
B
1.0 5 0.090.005 >2880
1.0 5 0.09 0.007 >2880
A
B
1.4 5 0.12 0.01 >2880
1.4 5 0.13 0.01 >2880
A 1.6
B 1.6
A 1.8
1! 1.8
A 3.2
B 3.2
5
5
5
5
5
5
0.13 0.02
0.13 0.01
0.19 0.02
0.20 0.02
0.37 0.03
0.36 0.02
360
400
216
250
88
95
285-454
296-540
183-254
217-288
81-95
90-100
1.66
1.93
1.44
1.38
1.20
1.12
1.38-1.99
1.54-2.41
1.26-1.64
1.25-1.52
1.11-1.27
1.08-1.16
Control mortality <5% for all tests.
Measured mean (x) CPO values and standard deviation(s) are given for each test.
35
-------
Figure 17. Spot gills became enlarged and tissues show progressive damage after exposure to CPO
36
-------
CHRONIC EXPOSURE: SPRING. 1977
7 -
X
LU
Q
z
o
t 4
Q
§3
QC
UJ
£ 2
O
Mean measured
concentration CPO
• Control
o 0.65 mg/I
A 0.24
o 0.12
i
i
10 20 30 40
EXPOSURE TIME, days
50 60
CHRONIC EXPOSURE: FALL. 1977
_i 80
<
>
>
5 60
10
LU
o
£ 40
2
LU
O
EC
LU on
a_ /u
p
•V n
- \ i
• \Q 0 Mean CPO
a.9 concentration
\* Control
® 1.58mg/Z
9-9 O 0.91
a ^ 0.28
\ ° 9 o 0.14
~\ L
« o
,*
1 1 A III
0 10 20 30 40 50 60 7
EXPOSURE TIME, days
Figures 18 and 19. Results of chronic exposures of oysters to CPO in spring 1977 (60 days) and fall 1977 (45 days).
Ambient temperatures were maintained throughout tests.
37
-------
irol i.inks (without clilonnation). Colom/atioii ot benthic
i.ix.i in the Ml IV system appaicnilv reflects the ncutrali-
/.iiini) inii i .ii I ion between oxuiams and hv drogi n sultulc
hum uigaiiK t oinplcxcs produced in li'Sl unu sediments
(l-ig. 2ot In general, commumty density in high concen-
tr.n IDIIS paralleled tli-it in control. Results apparently re-
lict led i lio ihrrshohl range lur chloi malion effects on
these i oinmumiics anil on natural water.
Ml I I u:ll In modified in l'>78 for studies of long-
tcim rllnis .ind bioaicumul.ition ol by-products ot chlo-
i in.iUon.
Mini-METlJ
Al M'ON C. KAIX.I K, KcSL-arch Aquatic Biologist;
I IS. JOHNSON, Biologist
111 parallel experiments, a mm.uun/ed version of the
icsiing s\su-in was evaluated. Analyses ol' results general!}
curruboi alcd MI-.TU results. However, the Mini-MI.TU sys-
tem was less sensitive to the effects of chlorination bc-
C.IUM lcu(i organisms colom/ed in (lie smaller indoor
um I s.
Halo-organic Compounds in Estuarine Waters
l< III-I./, I'nneipal Investigator. H'A (irant KKU3839-
01/0?., University of Marylaml; W.I'. DAX'IS, Project
Ol'lieer
Halo organic ioinpouiuls resulting from chlorinatioii
were imestigaleil under laliuratorx anil held conditions.
I l.ilolornis were geni'i'ateil in the lahoi ator\ with chlorine
doses ot I IO mg/l', , the range emplu\ed \>\ main coastal
powei plains
I his stiuh . completed in 1 ''77, showed that liromo-
luriii is ilie piniiipal compound lound in halo-organic by-
products at salinities above 1 g/kg. On a molar basis,
nioie ill.in -I"- ot llie chlorine tiom cliloimalion in these
tesis \\as (•unveiled lo lialofornis. In the laboratory .
u/one also gem-rated halotorms in estuarme water at rates
similar to those obtained from chlorine. However, at a
power plant field sile, only traces of halotorms were
lound, apparently due lo halolorm-bypassmg reactions or
analv ncal leehnu|ues. The investigator did not identify
.HIV bv passing reaction, but suggested (he formation of
stable halogen.iu-d macroiiiolecules.
duction of bromate from scawatcr thlorination and veri-
fied the kinetics and rate of bromoform production from
chlonnation. Future work will emphasi/.e food-web trans-
fer of by-products of chlorinatioti and analysis of meta-
bolic transformation of compounds in organisms. A long
inventor}- of halo-organics resulting from seavvater chlori-
nation is in preparation.
Design of Experiments, Statistical Analyses, and
Evaluation of Aquatic Research Data
R.G. IX)MF;Y, Principal Investigator. KPA (irant
K805007, University of Texas, Medical School, San
Antonio, VX, VV.P. DAVIS, Project Officer
Data sampling, design consultation, and statistical anal-
yses are provided under this grant. From the summer
1975 through fall 1977, METU weather, temperature, and
chemical data were collected and processed for computer
programming.
Data related to the reproduction, spawning, and hatch-
ing patterns of the .Atlantic silversides were processed in
collaboration with D. Middaugh. The information will be
used in held tests in 1978 to validate predictions.
Statistical analyses were also conducted for studies on
response of oysters to low-level chlorination and by-
products of chlonnation.
An Investigation of the Ecological Effects of Re-
sidual Ozone to Selected Estuarine Species
D.T. Bl'KTOS', Principal Investigator. EPA (irant
RK04683, The Academy of Natural Sciences of
Philadelphia, Benedict F.stuarine Research Laboratory,
Benedict, MD; W.P. DAVIS, Project Officer
Research in 1977 indicated that o/.onation of marine
waters produced the same progression of brominntion f<)r
organics as tor chlorine. Calculation to adjust ozonation
to levels equivalent with chlorination levels and rates were
developed and applied at the outset; tests using resultant
"chlorination-equivalents" revealed no difference in rela-
tive toxicity ot exposed invertebrates or fish. Studies of
physiological effects of o/one-induced halo-organics on
selected species will continue in 1978.
Isolation and Study of Halo-organics Resulting
from Chlorination of Seawater
J.ll. CARPI \II-K, Principal Investigator. I-.I'A (irant
KHo3N'H, Rosenstiel School of Marine and Atmospheric
Science, Miami, M.; W.I'. DAVIS, Project Officer
I his investigation defined and corrected the anomaly
m marine measurements with the aid of an amperumetnc
turalor. Investigators also idcniifu-d t|u- photol} tie pro-
Fish Webs, Populations, and Productivity in a
Southeast Coastal Marine Marsh
\.W. Cl IAMBI- RUN, Principal Investigator. F.PA (irant
R8O44688, (,rice Marine Biological Laboratory, College
of Charleston, Charleston, SC; W.P. DAVIS, Project
Officer
Selected study sites in the marshes of the Leadenwah/
North I disto River estuary were monitored in 1977 to
evaluate marine communities and food webs. These waters
-------
weight x 100) measurements demonstrated that the
gonads are the site of tissue reduction and that gonadal
glycogen reserves are depleted in oysters exposed to CPO.
Mantle tissue respiration measurements showed no sig-
nificant differences in O2 uptake between control and
oysters exposed 30 days at 5°C. However, at 28°C, the
()2 uptake rate in oysters was significantly higher than in
controls, suggesting a possible synergistic response be-
tween CPO and elevated water temperatures and a possi-
ble explanation for seasonal survival patterns (Figs. 18
and 19).
Marine Ecosystem Test Units (METU)
W.P. DAVIS, Supervisory Research Aquatic Biologist;
R.I.. YOAKUM, Biology Technician;
U.K. FOUI.K, Biology Technician;
S.J. KRICKSON, Research Aquatic Biologist
Since 1975, Bears Bluff investigators have used the
Marine Ecosystem Test Units (METU) (Fig. 15, p. 32) to
observe the complex interactions between low levels of
chlorination and community structure of benthic taxa.
METU consists of 96 outdoor tanks that receive a con-
tinual flow of estuarine water containing entrained larval
organisms. The system serves as a habitat for developing
communities including algae (6 to 12 species), amphipods
(6 species), barnacles, molluscs, ascidians, worms, and
other groups. Communities are exposed to three levels of
continuous chlorination applied as sodium hypochlorite
at these levels: 0.125, 0.250, and 0.500 ppm.
Statistical analyses in 1977 revealed no significant vari-
ation within replicate samples (8) at the control level, or
within the three chlorination levels. However, statistically
significant variance occurred between the control and
chlorination levels in the majority but not all tests. The
degree of this variance was unexpected because degrada-
tion of chlorination or other oxidants was generally be-
lieved to occur rapidly in silt-laden estuarine waters.
In addition, the amperometric titrator, an analytical
instrument used to detect (and consequently regulate)
chlorination levels, failed to register at any chlorination
level tested. This evidence suggested that degradation
products from chlorination or other strong oxidants may
be more significant than the oxidant itself.
Oysters never settled in any "sublethal" concentrations
of chlorination, but were found in small numbers in con-
COLONIZATION OF BENTHIC TAXA AS A FUNCTION OF SUBLETHAL DOSES
OF Cl IN SEAWATER AND 60 DAYS DURATION OF EXPOSURE IN 6
SUCCESSIVE PERIODS OVER I YEAR IN A CONTINUOUS FLOW-THROUGH SYSTEM
DOSES
MONTHS
DATS DURATION
Figure 20. Colonization of benthic taxa as a function of sublethal doses of chlorine is observed in seawater for 60 days
in six successive test periods.
39
-------
and habitats arc colonized by many of the organisms in
.Ml-'I'll/test units. Investigations of life history patterns,
seasonal ch.mgcs of l>cnthic community structures, and
trophic mu-rrclationships among marine species contribute
to the ilai.i bast' <>t ecosystem dynamics at Bears Bluff.
Investigations in 1978 will focus on algal studies, amphi-
poiK, ami molluscs. In 1977, the spawning substrate of
tin- most common estuarine fish, the mummichog
(Fundulus hctcroclitus), was identified; a related fish spe-
cies (F. majalis) is being studied to elucidate egg-laying
cycles hatching of larvae.
Rivulus marmoratus: An Investigation of its Po-
tential as a Cancer Research and Chemical Carcin-
ogen Screening Organism
C.C. KOENIG, Principal Investigator. EPA Grant
R805469, Grice Marine Biological Laboratory, College
of Charleston, Charleston, SC; W.P. DAVIS, Project
Officer
A system using a self-fertili/.ing, hermaphroditic marine
fish (Rivulus marmoratus) to screen chemical substances
for tcratogcnic and mutagenic effects is being used to
evaluate compounds identified as EPA testing priorities
and as chlorination by-products. The system developed
under an IPA agreement administered by the Bears Bluff
Field Station will be used in 1978 at the Grice Marine
Biological Laboratory. Future experiments will examine
use of this species to monitor teratogenic/mutagenic
response.
Figure 21. KRL.GB's Bears Bluff Field Station is located on the western side of Wadmalow Island, South Carolina,
where cstuarmc animals are available for tests to determine the impact of biocides on estuarine ecosystems.
40
-------
Figure 7. Biological Laboratory Technician Edward Matthews calibrates a Lambda pump for 96-h acute toxicity tests
in ERL.GB's aquatic toxicological laboratory. Tests expose mysids and anthropods to toxicants in salinity-controlled,
flowing seawater.
-------
Figure 22. Stage I, a U.S. Navy diving platform, serves as an offshore laboratory for ERL.GB marine research on effects
"I oil-drilling fluids used in oil explorations in the Gulf of Mexico
42
-------
ENVIRONMENTAL
EFFECTS OF OFFSHORE DRILLING
ERL.GB Associate Director, NORMAN L. RICHARDS, Coordinator
Environmental questions regarding the potential impact
of chemicals used in developing offshore oil resources are
under study in an extramural ERL,GB research program
supported by EPA's Office of Energy, Minerals, and
Industry. Oil-related research funded under grants and
interagency agreements focuses on two major problems:
(1) determination of effects of offshore drilling and drill-
ing compounds on the marine environment; (2) detection
of drilling emissions believed capable of causing cancer
and cell mutations in the marine food web.
New methodology is being developed to predict direct
and indirect hazards of drilling and components on
marine life indigenous to areas leased for oil exploration.
Questions remain unanswered concerning the faculty of
fish and crustaceans to retain ingested chemicals and the
potential for health ha/.ards of seafood harvested near oil
rigs.
The nature of energy-related problems requires a sys-
tems approach to their solution. The KRL.GB investiga-
tion utili/es a multi-disciplinary research team represent-
ing these disciplines: analytical chemistry, statistics, be-
havioral biology, epidemiology, fisheries biology, toxicol-
ogy, taxonomy, parhobiology, biochemistry, microbiology,
genetics, molecular biology, and physiology.
OFFSHORE OIL AND GAS EXTRACTION
The scarcity of data on the effects of drilling fluids
can be attributed in part to their chemical complexity.
Analyses of potential effects of chemicals used in well-
drilling must take into account variations in mud and cut-
ting compositions due to the type of substrate drilled,
drilling depths, availability of mud components, tempera-
tures, and equipment.
Intensified oil explorations in the northeastern Gulf of
Mexico has heightened interest in environmental questions
regarding the impact of offshore oil development on bio-
logical and commercial resources.
During 1977, ERL,GB established an offshore labora-
tory on a U.S. Navy diving platform in the Gulf of
Mexico near Panama City, FL, to study chemicals used
in oil drilling (Fig. 22). In an unusual joint research ven-
ture involving government and industry, drilling muds for
the ERL,GB tests were furnished by Amoco Production
Company, which was drilling for oil nearby in the Destin
Dome section of the Mafia acreage. In December 1977,
Amoco Production plugged and abandoned its hole off-
shore Florida on the Destin Dome; results of the ERL.GB
study with the Destin Dome drilling fluids will be ana-
lyzed in the forthcoming reporting period. The project
was carried out in conjunction with the American Petro-
leum Institute's Offshore Operators' Committee under an
interagency agreement with the Naval Coastal Systems
Laboratory in Panama City.
Effects of Drilling Mud Components
After preliminary screening, pentachlorophenol (PCPt
and barium sulfate were selected for intensive study to
determine their potential hazards as drilling mud constit-
uents.
The environmental effects of PCP, a pesticide widely
used for wood preservation, has been the subject of a
number of investigations in recent years. Its versatility has
lead to widespread applications of PCP formulations on ,1
global scale.
Although PCP is used as an anti-microbial agent in
drilling and packer fluids for offshore oil drilling, little is
known coneerning the toxicity of PCP to marine and
estuarine organisms. Therefore, ERL.GB and the Univer-
sity of West Florida jointly sponsored an international
symposium June 27-29, 1977, on Pensacola Beach, FL,
to examine available data and recent investigations related
to the chemistry, pharmacology, and environmental toxi-
cology of PCP. Proceedings of the PCP symposium were
published in Pentachlorophenol, K. Ranga Rao, Ed., Hnv.
Sci. Res. Vol. 12, Plenum Publishing Corp.. New York,
NY.*
Toxicity studies and behavioral assays with PCP arc-
described earlier as research objectives of KRL.GB's Ex-
perimental Environments (pp. 1,10) and Processes and
Effects Branches (p. 18). Additional investigations funded
by the EPA Office of Energy, Minerals, and Industry are
summarized below.
Toxic, Sublethal, and Latent Effects of Selected
Petroleum Hydrocarbons and Barium Sulfate on
Marine Organisms
K. RANGA RAO, Principal Investigator. EPA Grant
R804541, University of West Florida, Pensacola, FL;
Norman RICHARDS, Project Officer
Periodic shedding of the exoskeleton is one of the
most fascinating phenomenon associated with the growth
of crustaceans. Changes in the permeability of the cuticle
'Figures and tables summarizing PCP research in this report are reprinted by permission of the Plenum Publishing Corp.
-------
->
I
-7
, J
'
1
.
g
! "
BQ
/u
61
1 40
H
5 30
21
i
first
molt
1 .o/t'-;"
1
second
molt
If
.
li:
third N
molt 25
25
25
•
ppmNa-PCP
0
DJ
0!
Deaths not
related to
molting
phcnau (N
occur in relation to cyclic shedding, secretion, and hard-
ening of the exoskeleton. Immediately after ecdysis, t
new thin cuticle is relatively more permeable and less pro-
tective than the thicker, calcified exoskeleton charact
istic ol the intermc.lt period. Although earlier literature
has not evaluated the toxicity of pesticides to crustaceans
II Jitterent stages of the molt cycle, previous investiga-
tions indicated that newly molted animals exhibit
creased sensitivity to toxicants.
This mvc-simation examined the toxicity c
grass shrimp at different stages in the molt cycle_
In 96-h bioassays, the shrimp in later stages of 1
nnucdysial period exhibited a greater sensitivity to ?
p, ,. lhan that exhib.ted by shrimp in the mtermolt
early proecdysial stages of the molt cycle. The shrimp in
later proecdysial stages generally molted (""derwer
ecdysis) during the 96-h test period and d.ed shortly after
ecdvsis The 96-h LC50 value obtained for these shnmp
(0 436 ppm) is the lowest of all the LC50 values reported
,,slv tor adult crustaceans and is comparabl
for fish and larval crustaceans. The increased sen
dvity to Na-PCP during the early postecdysial period
also 'apparent in a long-term test (66 days) (Fig. 23
The observed postecdysial mortality of shnmp expo:
.„ 10 ppm Na-PCP was not dependent on the duration o
.sure of shnmp to Na-PCP during the proecdysial pci
iod Studies with HC-PCP indicate that an abrupt increase
in die uptake of PCP shortly after ecdysis may cause
creased mortalities during this period.
Liiiil) Kcgi-iuTation
After a limb has been severed from the body of a crus-
tacean, a new limb may Srow to replace it. Tests wer
conducted to determine effects of Na-PCP on limb gen-
eration in the grass shrimp throughout the molt cycle.
At predetermined intervals (two or three days), the
limb bud sizes of shrimp were measured. Each data point
was calculated with a regeneration index (R value):
_
size of limb bud
carapace length
The R values permitted comparisons of the extent and
rate of regeneration among various individuals. The regen-
eration patterns of 400 shrimp subjected to different
treatments revealed that Na-PCP affects the initiation and
progress of limb regeneration, such as inhibition of regen-
eration, delay in limb bud development, or reduction of
limb bud growth without altering the intermolt duration.
By comparing the R values of control and experimental
shrimp on days preceding and following ecdysis, investi-
gators determined the extent (%) of inhibition of regen-
eration in exposed shrimp. EC50 values were computed
by probit analysis. For example, the R values of shrimp
9 days after limb removal yielded the following LC50
values (95% confidence intervals are in parentheses):
unfed shrimp, 0.473 ppm Na-PCP (0.306-0.670); fed
shrimp, 0.565 ppm (0.452-0.706). EC50 values based on
postmolt R values were: unfed shrimp, 0.615 ppm Na-
PCP (0.451-0.852); fed shrimp, 0.637 ppm (0.485-0.850).
The inhibitory' effects of Na-PCP were more pronounced
on the initial phases of limb regeneration (involving
wound healing, cell division, and dedifferentiation) than
on later premolt limb growth (involving further differen-
tiation and cellular enlargement).
These experiments showed that crustacean limb regen-
eration can be used as a sensitive bioassay for studying
effects of environmental pollutants (Fig. 24).
-------
5
1
1
Q
0
°- 3
|2
s
_i
* *
1
1
5
Mr
I,
t
g
,ta
P
1
V
1
>4
Inhibition of limb bud growth
UnM
Fed
Molt
Days alter limb removal
7 9 9
ill
Premolt Postmolt
Figure 24. A comparison of the LC50 values (based on 96-h toxicity tests) and EC50 values (based on the extent or
inhibition of regenerative limb growth) for grass shrimp, Palaemonetes pugio. exposed to sodium pentachlorophenate.
The LC50 values for shrimp are shown at different stages of the molt cycle.
Community Studies
In another phase of the investigation of drilling mud
toxicity in 1977, effects of PCP on estuarine benthic
communities were studied at ERL.GB in laboratory aquar-
ia fed by a continuous flow of Santa Rosa Sound sea-
water. The two-stage experiment, concluded March 28,
1977, used meiofauna, the most numerous benthic meta-
zoan found in marine sediments. Meiofauna is defined as
an organism that can pass through a 0.5 mm sieve of a
width smaller than 0.1 mm. Within the meiofauna group-
ing, Nematoda, the most common group, was selected as
the test species.
In the first phase of the experiment, organisms were
exposed to 7, 76, and 622 pg/Jl concentrations of PCP.
Concentrations of 1.8, 15.8, and 161 yg/£ PCP were used
in the final phase.
Concentrations of 1.8, 7, and 15.8 pg/£ PCP did not
affect the biomass and density of nematodes. An inter-
mediate concentration of PCP (76 yg/S,) caused an in-
crease (P < 0.01) in biomass and density of nematodes
compared to control aquaria. Higher concentrations of
PCP (161 and 622 pg/&) caused a decrease (P < 0.01) in
biomass and density of nematodes compared to control
aquaria. Although species diversity indices of control
aquaria did not differ significantly from those of PCP-
exposed aquaria, marked changes in nematode species
composition and shifts in nematode feeding types were
noticed in the aquaria exposed to 161 and 622 ug/S.
PCP. Nematodes classified as epistrate feeders were most
abundant in the control aquaria and aquaria exposed to
1.8, 7, 15.8, and 76 ug/£PCP. Deposit feeders were rela-
tively abundant among the nematodes in aquaria exposed
to 161 and 622 yg/.?, PCP. The alterations in nematodes
observed in this investigation appeared to be due to the
variations in macrobenthic fauna and food (algae) supply
caused by the biocidal effects of PCP and the toxic
effects of PCP on meiofauna.
Effects on Respiration
Measurements of oxygen consumption not only indi-
cates metabolic rates of test organisms but also offers an
index of stress. Another oil-related research project in
1977 sought to determine effects of Na-PCP and alkyl-
dinitrophenols such as 2,4-dinitrophenol (DNP) on the
oxygen consumption of grass shrimp.
Tests showed that oxygen consumption varied in rela-
tion to activity at different stages of the molt cycle. Oxy-
gen consumption was measured for extended periods (18
to 24 h) to minimize errors in establishing basal (control)
rates of oxygen consumption. In contrast to previously
reported progressive increases in oxygen consumption dur-
ing premolt stages of other crustaceans, oxygen consump-
tion increased significantly prior to and during the shed-
ding of exoskeleton (ecdysis) in grass shrimp. Effects of
Na-PCP on oxygen consumption by shrimp varied with
the stage of the molt cycle, the concentration of Na-PCP,
and the extent of Na-PCP pre-exposure. At concentrations
of 1.5 and 5.0 ppm, Na-PCP did not alter the oxygen con-
-------
4 2 Molt 2 4
Tim* (hour* before and itt»r mo4l)
Figure 25. lotted of Na-PCP on the oxygen consumption in Palaemonetes pugio preceding, during, and after ecdysis.
I .ait proecdysial shrimp (Stages Dj-D.j) were used and a basal rate was determined prior to addition of Na-PCP to the
medium, h'.aeh curve is based on data from a representative shrimp from each group. Shrimp were exposed to 1.5 ppm
Na-PCP (triangles) and 5 ppm Na-PCP (open circles).
sumption ol intermolt and premoh shrimp. I.ate premolt
shrimp exposed to 5.O ppm Na-PCP exhibited an increase
in oxygen consumption in relation to ecdysis at the same
level as consumed by control shrimp. However, after
ecdysis, shrimp exposed to 5.0 ppm Na-PCP exhibited a
dramatic decline in oxygen consumption and died within
3 h (Fig. 25). Increased sensitivity during the early post-
moh period appeared to be related to an increase in the
uptake of PCP at this stage, compared lo intermolt and
premoh stages. A decline in oxygen consumption (as
noted above) could be induced in intermolt shrimp by
using higher concentration of Na-PCP. Exposure of shnmp
to 10 to 20 ppm Na-PCP, or to 5 ppm followed by 20
ppm Na-PCP, caused an initial increase in oxygen con-
sumption and a subsequent decline leading to death. Sur-
vival time of intermolt shrimp pretreated with 5 ppm Na-
PCP was longer than that of shrimp exposed directly to
10 or 20 ppm Na-PCP. Although 20 ppm DNP (2,4-
dmitrophenol) caused an initial increase in oxygen con-
sumption in intermolt shrimp, there was no subsequent
decline in oxygen consumption or deatli during a 24-h
exposure.
Kftects of Na-PCP and DNP on tissue respiration _m
vilro also were studied with the blue crab (Callinectes
sapidus). At concentrations of 1 x 10'6 M and 5 x 10'5
M, these compounds did not alter the oxygen consump-
tion of the muscle, gill, or hepatopancreas. At a concen-
tration of 5 x 10-3 M, both Na-PCP and DNP caused an
inhibition of oxygen consumption of isolated tissues.
In summary, the results of this investigation indicate-
that (he biocidal effects of PCP may not be solely due to
its ability to uncouple oxidative phosphorylation but also
due to a disruption of the overall metabolic activity.
Kffects of Hepatic Enzymes
Further tests with the blue crab in 1977 evaluated
effects on Na-PCP J£ vivo and in_ vitro on certain hepato-
pancreatic enzymes. Crabs were maintained in 300 mil-
liosmole seawater at 20°C under controlled conditions.
The hepatopancreas of intermolt crabs were removed
and washed in 0.25 M sucrose. The tissue was weighed,
homogenized, and fractionated; mitochondrial and micro-
somal pellets were resuspended in 0.25 M sucrose, divided
into aliquots, and kept frozen (-40°C) until needed. The
soluble fraction also was divided into aliquots and frozen.
The microsomal fraction was used within one week after
preparation and other fractions were kept no longer than
three weeks prior to assays.
The experiments revealed that Na-PCP had a stronger
inhibitors' effect than DNP on mitochondrial enzyme
from the blue crab hepatopancreas (Fig. 25). However,
the enzyme in the cytoplasmic (soluble) fraction was in-
hibited to a lesser extent in Na-PCP than by DNP.
Calcium-activated ATPase from the microsomal frac-
tion of the hepatopancreas was inhibited by Na-PCP and
DNP, both in vivo and in vitro. (Inhibitory effects were
more pronounced ni vitro than jn vivo.) Fumarase (Fum),
malate dehydrogenase (MDH), and succinate dehydro-
genase (SDH) were inhibited by Na-PCP and DNP 'm vivo,
whereas isocitrate dehydrogenase (IDH) was stimulated.
Lactic dehydrogenase was the least affected cytoplasmic
(soluble) enzyme in vivo; glutamate-pyruvate transaminase
was inhibited to the greatest extent. The fumarase in the
cytoplasmic fraction was inhibited to a lesser extent than
that in the mitochondrial fraction. The MDH in the cyto-
plasmic fraction was inhibited to a greater extent than
46
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125
100
s75
*-
•S50
#
.'
I
-
'
1
>
i
:
'
:i
-
i
r
1
l
^
1
|
i
|
Fumlml SDH MDHIm) IDH
Figure 26. In vivo effect of Na-PCP and DNP on Tri-
carboxylic Acid Cycle (TCA) enzymes of the blue crab
that in the mitochondrial fraction. Pyruvate kinase and
glucose-6-phosphate dehydrogenase were both inhibited
50% by Na-PCP. Na-PCP and DNP had an inhibitory
effect on the various enzymes tested in vitro at concen-
trations of 10-* M or higher (Table 18).
Although considerable work has been completed on
the relationship between PCP and ATPase, no definite
mechanism of action has been found. A number of ques-
tions remain unanswered: Is the effect generalized when
the phenol is bound nonspecific-ally to membrane pro-
teins? Or, is it more specific in its interaction with each
enzyme involved? Further investigation is required, but it
appears that the uncoupling of oxidative phosphorylation
is not the sole basis for the toxicity of PCP.
Ultrastructural Changes
In the final study conducted in 1977 under EPA Grant
R804541, ultrastructural changes in the gills of grass
shrimp exposed to 1.0 ppm Na-PCP were examined.
Intermolt (stage C) grass shrimp were exposed to 1.0
ppm Na-PCP for the duration of a molt cycle. Gills, hepa-
topancreas, midgut (portion of the digestive tract sur-
rounded by hepatopancreas), and hindgut (portion of the
digestive tract in the abdomen) from control and experi-
mental shrimp at known stages of the molt cycle were
observed at the ultrastructural level.
Although signs of pathology were evident in animals
in late premolt, extensive pathological changes were not
evident until after ecdysis. The extent of pathological
changes varied with the tissue examined and the interval
between ecdysis and the time of fixation for electron
microscopy.
In addition to mitochondrial swelling, the following
ultrastructural changes were seen in the gill epithelium of
shrimp exposed to Na-PCP (Fig. 27): formation of fluid-
filled invaginations of the intermicrovillar apical mem-
brane, increase in lysosomal activity and eventual cyto-
plasmic and nuclear degeneration. The podocytes in the
gill axis, the granular cells, secretory cells, and tegumental
gland cells also exhibited mitochondrial swelling, nuclear
pyknosis, and eventual cytoplasmic degeneration.
The cells lining the lumen of the midgut and hindgut
of shrimp exposed to Na-PCP exhibited swelling of the
apical membrane often accompanied by rupture, loss of
microvilli from apical foci, and increased lysosomal
activity.
Pathological changes noted in the hepatopancreatic
cells of the experimental shrimp were: high amplitude
swelling of mitochondria including vesiculation of cristae,
presence of myelin bodies within mitochondria and rough
endoplasmic reticulum, increase of autophagic activity,
and loss of microvilli.
The simultaneous deterioration of the midgut, hindgut,
hepatopancreas, and gill tissues of shrimp exposed to Na-
PCP indicates that focal cell death in any of these regions
was not the sole perpetrator of the eventual death of test
organisms.
Effects of Drilling Fluids and Oil on Corals
Occupying Hard-bank Communities
THOMAS BRIGHT, Principal Investigator. EPA Grant
R805441. Texas A&M Research Foundation, College
Station, TX; NORMAN RICHARDS, Principal Investigator
Coral reefs represent an important resource in the Gulf
of Mexico. This research grant will address three ques-
Table 18. Effect of Na-PCP and DNP on Calcium Activated ATPase in vivo
Control
Experimental
Sample
Cone.
Activity
% Activity
seawater
0.339 ± 0.012
100%
DNP
6 Ug/g
0.322 ± 0.007
95%
Na-PCP
6 yg/g
0.279 ±0.011
82%
47
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AX
Figure 27. Light micrograph of sagittal section of a pleurobranchiate gill of Palaemonetes pugio showing the association
of lamella (L) with the main axis (AX). Note the affarent (AS) and efferent (ES) hemolymph sinuses and the close as-
sociation between the clear gland (CG) and efferent sinus. Note also the reticulate glands (RG) and the granular secretory
cells (GSC).
tions: (1) Can oil drilling operations undertaken in the
vicinity of coral reefs adversely affect their structure? (2)
Is it feasible to shunt cuttings and drilling fluids? (3)
Should discharges from oil drilling activities be removed
by barge?
In 1977, corals were collected and exposed to drilling
muds at KKI.,(ili. An evaluation of these exposures will
be completed in the next reporting period.
FUTURE GOALS
Policy decisions on the use of drilling fluids currently
arc based on static, 96-h LC50 determinations, observa-
tions of divers, and theoretical models of pollutant disper-
sion. Research on drilling-fluid constituents was initiated
at ERL.GB to provide a better data base to predict the
relative biological and human health hazards of drilling
fluids and to develop more relevant laboratory methods
for xenobiotic evaluation.
Results to date indicate that drilling-mud constituents
may affect the structure and function of ecosystems, both
directly and indirectly. Improved techniques are needed
to determine the potential for these substances to bioac-
cumulate and to contaminate organisms indigenous to
areas under lease for oil drilling operations. In addition,
further lexicological information is required to assess al-
ternative chemicals available for offshore oil production.
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MARINE FOOD CHAINS
The second phase of ERL.GB research on energy
sources is concerned with cancer-causing properties of
shale-oil fuels and their potential threat to fish caught for
the marketplace.
This program will investigate carcinogens found in
shale-oil fuels, their metabolic fate in the marine food
chain, their persistence in the environment, and their po-
tential for accumulation in seafoods (Fig. 28).
Research in 1977 examined the potential for uptake,
transfer, and depuration of polynuclear aromatic hydro-
carbons (benzo[a] pyrene and chrysene) by representative
members of a simulated food web. Tests showed that
chrysene is accumulated by protozoa, algae, mysids, and
polychaete worms. Spot bioaccumulated detectable con-
centrations of chrysene from exposed mysids; chrysene
was detected only in the liver of mangrove snapper that
consumed fish exposed to chrysene in water.
In further studies, an algae-oyster food chain was
found to accumulate only a small amount of chrysene or
benzo [a] pyrene. Lugworms did not depurate chrysene
with the rapidity observed in oysters. Related studies
funded under this program are described earlier (Grants
R804458, p. 26 ).
SIMULATED FOOD CHAIN BIOACCUMULATION
L_
HAM
SEABIRDS |
SEA MAMtALS
I
Figure 28. Simulated aquatic food web
49
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Carcinogenic Photooxidation Product from Petro-
leum PAH's at Air-Sea Interface
J.L. LASETER, Principal Investigator. EPA Grant
R804647. University of New Orleans, LA;
NORMAN RICHARDS, Project Officer
The research objective is to determine major photo-
oxidation products of petroleum derived polynuclear aro-
matic hydrocarbons (PAH). Studies will attempt to par-
tially elucidate "weathering" phenomenon of petroleum
at an air-seawater interface. Many major oxidation prod-
ucts were identified in 1977, and representative com-
pounds were selected from field samples for detailed
study. Structure of these compounds will be confirmed
by chemical synthesis. Numerous aromatic hydrocarbons
and their biologically significant oxidation products have
been supplied to the project officer and will be screened
for mutagenic properties.
Accumulation/Elimination of a Certain Aromatic
Petroleum Hydrocarbon
ROBERT FARRAGUT, Principal Investigator. Interagency
Agreement IPE-IAG-D6-0084. National Oceanic and At-
mospheric Administration, Miami, FL;
NORMAN RICHARDS, Project Officer
Selected marine species commonly consumed by man
were tested to determine how they accumulate and elimi-
nate carcinogenic aromatic petroleum hydrocarbons.
Snapper and shrimp were observed in separate control
tanks and tanks containing various concentrations of test
compounds. After exposures, the cephalothorax, gut, and
tail of shrimp and the liver, gall bladder, gut, and flesh of
snapper were analyzed.
The mangrove snapper, also known as the grey snapper
(Lutjanus griseus), was exposed to 5 pg/fc chrysene in
1690- £ tanks. Analysis did not reveal any detectable levels
in snapper flesh, but chrysene was detected in the liver.
Shrimp data will be analyzed in future work.
Subcellular Distribution of Enzymes of the
Marine Ciliate (Parauronema acutum)
DONALD G. LINDMARK, Principal Investigator. EPA
Grant R805364010, the Rockefeller University,
New York, NY; NORMAN RICHARDS, Project Officer
Ensymatic capabilities of marine protozoa to transform
or bioaccumulate polynuclear aromatic hydrocarbons were
investigated in another 1977 study related to the marine
food web.
The following enzyme activities were detected in ho-
mogenates of the symbiote-free marine ciliate,
Parauronema acutum (mU/mg protein): malate dehydro-
genase (6000), NAD(P): acceptor oxidoreductase (dichlo-
rophenol indophenol, 75), isocitrate dehydrogenase (93),
non-specific esterase (p-nitrophenol acetate, 3), and the
following acid hydrolases: acid phosphatase (300),
-galactosidase (14), -glucosidase (6), and proteinase (urea
denatured hemoglobin, 200). Lactate, alcohol, NAD(P)
dehydrogenase, and malate dehydrogenase (decarboxylat-
ing) activities could not be demonstrated.
Differential centrifugation demonstrated the sedimen-
table nature of all enzymes except esterase. Malate dehy-
drogenase and NAD(P)-. acceptor oxidoreductase possibly
localized in the mitochondria, and isocitrate dehydroge-
nase possibly localized in peroxisomes sediment at 2500
rpm for 10 min and in hydrolase sediment at 10,000 rpm
for 30 min, suggesting localization in a separate particle
population.
All particle-associated enzymes exhibited structure-
linked latency (50-80%), but lost latency and became non-
sedimentable after Triton X-100 treatment or freezing and
thawing (three times). The only exception is an acid phos-
phatase which remains sedimentable after freezing and
thawing. These data suggest the occurrence of lysosome-
like organelles in this ciliate and also give biochemical
evidence for the occurrence of mitochondria and
peroxisomes.
Detection of Carcinogens in Seawater; Use of
Hybrid Fish and Food Chains ~
DOUGLAS HUMM, Principal Investigator. EPA Grant
R804650. University of North Carolina, Chapel Hill, NC;
NORMAN RICHARDS, Project Officer
A sensitive in vivo method for the detection of carcin-
ogens in hybridized fish is under study. The investigator
has produced several hybrid fish that appear to be appro-
priate for extensive validation of the methodology. Re-
sults will be published after the histopathology is
complete.
A tentative agenda has been established for a sympo-
sium on "Mutagenic, Carcinogenic, and Teratogenic PNA
Hydrocarbons in the Marine Environment" in the summer
of 1978 at ERL.GB. Patricipants will report current re-
search on physical and chemical fate; methods for con-
centration, separation and detection; the pharmacody-
namics of activation, detoxification; accumulation and
depuration; fate in marine trophic systems; the genetic
basis for in vivo screening methods; quick screen and pre-
screen methods; laboratory and field observations on
tumor formation. The overall objective will be to assess
the potential of marine animals as models for carcinogen-
esis, their use for monitoring the aquatic environment,
and the potential risk to human health from the con-
sumption of seafoods contaminated with man-mobilized
hydrocarbons.
50
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GRANTS AWARDED IN 1977
Investigation of Enzymatic Screening Tests for
Mutagens in Environmental Pollutants from
Synfuel Operations
JOSEF SCHMIDT-COLLERUS, Principal Investigator.
EPA Grant R805671. University of Denver, Denver, CO;
NORMAN RICHARDS, Project Officer
An in vitro test using the inhibition of 4-biphenyl
hydroxylase activity will be developed to determine pres-
ence of mutagenic compounds.
Novel Techniques for the Concentration and
Separation of Toxic Substances from Estuarine
Waters
ELIAS KLEIN, Project Manager. EPA Grant R805656.
Gulf South Research Institute, Baton Rouge, LA;
NORMAN RICHARDS, Project Officer
Methods to concentrate, separate, and detect com-
pounds with toxic and mutagenic properties in estuarine
waters will be developed. Techniques, such as affinity
chromatography, reverse osmosis, and Donnan dialysis
will be used to test isolated substances for mutagenic
properties.
Genetic Variation and Resistance to Carcinogens
in Natural Waters
R. JACK SCHULTZ, Project Manager. EPA Grant
R805195. University of Connecticut, Storrs, CT;
NORMAN RICHARDS, Project Officer
The study will evaluate feasibility of using isogenic fish
as carcinogen bioassay organisms. The validated bioassay
system would be used to test the carcinogenic properties
of compounds. The importance of genetic variability of
test organisms also will be determined.
Susceptibility of Genetically Defined Stocks of
Fish to Chemical Carcinogens"
KLAUS KALLMAN, Project Manager. EPA Grant
R805389. Osborn Laboratories of Marine Sciences,
Brooklyn, NY; NORMAN RICHARDS, Project Officer
The genetic structure of fish was investigated in 1977
in relation to their susceptibility to polycyclic aromatic
hydrocarbon carcinogenesis. Genetically defined fish with
the following properties were under study: inbred homo-
zygous from natural populations, inbred homozygous
from laboratory stock, heterozygous with coadapted gene
pools, heterozygous with poorly adapted gene pools.
51
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Figure 29. Radiograph shows blue fish collected in the James River fish kill (1974). The broken vertebral column is
similar to condition induced in laboratory tests of Kepone. (See p. .)
52
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KEPONE
ERI..GB Deputy Director, T.T. DAVIES, Coordinator
Kepone, a pesticide discovered in the James River Es-
tuary in 1975, was found to be highly toxic to marine
animals, including crabs, fish, and oysters. Concentrations
of Kepone considered unsafe for human consumption
have been measured in tests of commercial marine species
indigenous to the James River/Chesapeake Bay.
In response to health and the environmental ha/.ards
posed by the contamination of the James River, Congress
directed EPA in 1976 to study the effects of toxics in the
Chesapeake Bay ecosystem. ERL.GB has assumed an ac-
tive role in the continuing scientific assessment of damage
caused by Kepone and its accumulation in the James
River Estuary.
Research coordinated by ERL.GB during this reporting
period focused on the routes and rate of the insecticide's
transport and the fate of Kepone bound to estuarine sedi-
ments. A major objective is to determine how long
Kepone will persist in bottom sediments of the James
River/Chesapeake Bay and to project the time required to
reduce Kepone residues to safe, acceptable concentrations.
The answer to this question will assist the State of
Virginia and federal authorities in formulating plans to
preserve the health and productivity of the estuary.
An investigation funded under an EPA grant with the
Virginia Institute of Marine Science (R804993) found
high Kepone concentrations in the biota of the James
River Estuary. Robert Huggett, project manager, reported
that concentrations in edible tissues of most fresh and es-
tuarine fin- and shellfish commonly ranged from 0.1 to
more than 1 mg/kg. Kepone concentrations were shown
to increase in anadromous fish in relation to the time
spent in the river.
Studies conducted thus far show that Kepone does not
degrade either biologically or chemically in simulated es-
tuarine systems, indicating that normal degradation pro-
cesses probably will not alter existing concentrations of
Kepone in James River water and sediment.
Laboratory tests at ERL.GB have demonstrated that
Kepone concentrations >0.008 ug/f. in water cause ob-
servable, detrimental effects on crabs, shrimp, or fish; a
concentration of 0.15 mg/kg Kepone in tissues of oysters
fed to blue crabs had a deleterious effect on the crabs.
Field-collected, egg-bearing grass shrimp, recently
hatched larvae, eggs, and newly hatched larvae, and labor-
atory-reared postlarvae, showed variations in Kepone con-
centrations ranging from undetectable to levels of 0.6
ppm. Populations from the James River and the nearby
Lafayette River showed the highest concentrations of
Kepone; distant populations showed lower levels. Labora-
tory-reared postlarvae (representing all six populations)
had very low or non-detectable Kepone concentrations.
Two long-term studies were conducted to determine
the uptake and depuration of Kepone in blue crabs
(Callinectes sp.). In the first, Kepone was administered to
crabs in seawater (0.03 or 0.3 ng/5,) or in food (eastern
oyster, Crassostrea virginica, containing 0.25 ^g/g
Kepone). Results indicate that uptake of Kepone in 28
days was primarily through the contaminated oysters.
When these crabs were supplied with Kepone-free sea-
water and uncontaminated oysters for 28 days, no de-
puration of the insecticide was evident. There were indi-
cations of adverse effects in crabs fed oysters that con-
tained 0.25 pg/2, Kepone.
Results of the second study indicated that blue crabs
fed Kepone-contaminated oysters, followed by a diet of
Kepone-free oysters for 80 days, had detectable concen-
trations of the insecticide in their muscle and remaining
tissues. Blue crabs that ate oysters containing Kepone in
concentrations similar to those in food of crabs from the
James River died or molted less frequently than crabs fed
Kepone-free oyster meats. In addition, long-term expos-
ures of sheepshead minnows and mysid shrimp to Kepone
concentrations less than 0.08 pg/?, reduced survival, repro-
duction, or growth.
In another series of tests, Kepone-induced scoliosis. or
lateral curvature of the spine, in sheepshead minnows ex-
posed to a relatively low concentration of the organochlo-
rine. This syndrome is caused by diverse agents that pos-
sibly act on the central nervous system. Effects associated
with scoliosis in the sheepshead minnows included: dis-
ruption of myotomal patterns, inter- and intramuscular
hemorrhage, fractured centra of vertebrae, and death
(Figs. 30, 31, p.54 ).
Bluefish exposed to Kepone in ERL.GB laboratory
tests exhibited a broken-back syndrome identical to that
observed in several species of fish examined atter the mass
kills in the James River in 1973 and 1974 (Fig. 29).
In addition, nonlinear statistical models were developed
in 1977 to describe the uptake and depuration of pesti-
cides. The models described biological data as a single
equation, thus allowing variations due to many physical,
chemical, biological, and random error factors to be ana-
lyzed simultaneously.
Under EPA Grant R804563, Donald J. O'Connor and
Kevin J. Farley, of Manhatten College, the Bronx, New
York, will develop a model for evaluating time required
to reduce Kepone to harmless concentrations. Phenomena
related to the transfer of Kepone from its initial dis-
charge point (Hopewell, VA) to fishery stock will be rep-
resented in the model.
ERL.GB research findings on the effects of Kepone
has been available to the state of Virginia and federal
authorities who are attempting to minimize the future
impact of this insecticide on the environment and human
health.
53
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•
Figures 30 and 31. A normal backbone of sheepshead minnow (left) can be compared to broken backbone (right)
induced by Kepone in laboratory tests with the sheepshead minnow.
•!
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ENVIRONMENTAL PATHOBIOLOGY
.J.A. COUCH, Coordinator
Environmental hazards attributed to toxic substances
range from mild temporary dysfunction of organisms and
ecosystems to acute disorders and death. Some toxic sub-
stances are known or potential carcinogens, mutagens, or
teratogens. ERL,GB's Environmental Pathobiology Team
investigates sublethal effects of pesticides and pollutants
that may cause neoplasms (tumors) in aquatic species. The
movement of carcinogens and mutagens through the
marine food web is also under study.
An understanding of the effects and behavior of these
toxicants will enable scientists to better relate environ-
mental phenomena to possible risks to human health, par-
ticularly in regard to cancer-causing agents.
Pesticide-related Studies
Effects of insecticides, herbicides, and fungicides on
aquatic species are studied from subcellular to higher pop-
ulation levels to determine the nature and degree of dam-
age caused by exposure to pollutants. In 1977, the En-
vironmental Pathobiology Team examined and described
major tissue and cellular changes in sheepshead minnows
exposed to the insecticide Kepone and the herbicide
trifluralin.
Kepone exposure (1 to 5 \\g/i) resulted in broken
backs in sheepshead minnows (Figs. 30, 31). Vertebral
damage was severe enough to cause death of the exposed
fish. Massive fish kills were found in the James River,
Virginia, in 1973 and 1974. Many of the dead or dying
fish recovered in these kills had broken backs (Fig. 29),
thus indicating a possible relationship between the mass
mortalities of fish and the syndrome of broken-backs in
Kepone-exposed fish in the laboratory.
Sheepshead minnows exposed to trifluralin from zy-
gote to juvenile stages developed a vertebral disorder.
Vertebral columns of young fish exposed to 5.5 to 31
pg/K, trifluralin for 28 to 51 days were 5 to 100 times
their normal size (Fig. 32). The cause of these enlarged
vertebrae was attributed to hypertrophy of the boney tis-
sue in the vertebral wall. This condition was unprecedent-
ed in animals previously exposed to chemicals. Fish suf-
fering from the disorder of vertebrae are not able to swim
or compete successfully for food or mates.
Future research will be conducted on the mechanism
of trifluralin-induced dysplasia in fishes.
S.
r
Figure 32. A backbone from young sheepshead minnow exposed to the herbicide trifluralin shows tremendous hyper-
plasia of the vertebral column. Normal backbone is illustrated in preceeding figure.
55
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DO
Go
DGL
Figure 33. Section from the American oyster illustrates its general morphology; I, intestinal loops; G. gills, DG, digestive
Figure' 34. Raciioautograph shows 9 sections of an oyster exposed to radioactive labeled benzo[a] pyrene for 7 days.
Radioactivity is concentrated in the digestive gland (DG) and gonad (GO) areas.
Carcinogen Aquatic-animal Model System
The potential use of an estuarine invertebrate in early
detection of chemical carcinogens in the environment is
under study at KRI..GB. In 1977, oysters were exposed
to 1.0 and 5.0 ppb benzol a] pyrene (BP) and 3-methyl-
cholanthrene (3-MC) eight months to one year or longer.
Periodic- samples of oysters and water in the exposure sys-
tem were analyzed for carcinogen concentration, uptake,
and accumulation. Oyster tissues also were examined his-
tologically for pathological changes indicative of carcino-
genic effects (cellular disorders) (Figs. 33, 34).
Accumulation of BP and 3-MC by oysters appeared to
relate to concentrations of these chemicals in exposure
water. Oysters exposed for 8 months to 1.0 pg/£ BP and
3-MC accumulated no more than 0.264 yg/g carcinogen
in their tissues. Oysters exposed for only 3 months to
5.0 (jg/J, BP and 3-MC accumulated from 0.440 yg/g to
6.470 tjg/g carcinogen in their tissues. Algae and sessile
bacteria, as well as sessile invertebrates attached to the
exposure tanks, probably competed for absorption and
:uKorpi inn of carcinogen with oysters when the exposure
concentration was only 1.0 pg/8. When exposure concen-
u-ations were raised to 5.0 ug/fc, oysters accumulated car-
cinogen at a relatively higher rate. This phenomenon in-
dicates that a threshold concentration of polycyclic aro-
matic hydrocarbons may be needed in estuarine waters
I HI. ire significant bioconcentration occurs in oysters.
I.) date, histological alterations found in exposed oys-
u-is have been incipient and probably indicate inflamma-
tory responses. Future studies will follow the course of
cellular alterations in exposed oysters to determine if
minors arc induced.
Interactions of Chemical Pollutants and a Virus
M..M established host-parasite or host-pathogenic rela-
tionships represent more or less balanced interactions of
long evolutionary development. Few experimental studies
of aquatic ecosystem have examined possible interactions
among such environmental factors as pollutants, host spe-
cies, and parasites or pathogens. Available data, however,
indicate an unusual adverse effect on some host aquatic
species by certain of their natural parasites when the com-
plex is exposed to pollutants or pollutant mixtures.
This research project investigated the potential use of
an aquatic animal, host-virus system developed at ERL.GB
as a bioassay tool to measure and predict possible inter-
actions among pollutants, viruses, and their hosts. The
system used penaeid shrimp as the host, a shrimp-specific
Baculovirus. and selected pollutant chemicals, including
polychlorinated biphenyls (PCBs).
Earlier experiments showed that exposure of small
groups of shrimp (8 to 35 per group) having a low natu-
ral prevalence of Baculovirus to 1 to 3 ppb of PCB
(AroclorR 1254) for 10 to 25 days resulted in an increase
in the Baculovirus prevalence. In two of three tests, the
exposed shrimp exhibited a higher prevalence and inten-
sity of infection when compared to controls during and
after exposure periods.
In a more extensive test involving large numbers of
control and experimental shrimp, the enhancement of
viral prevalence was validated.
Both exposed and control groups had the same preva-
lence of patent viral infections at the beginning of this
test (Fig. 35). The rather abrupt increase in viral preva-
lence in exposed shrimp (from 23.3 to 75% in 35 days)
probably was related to an undefined interaction of host,
chemical stressor (Aroclor 1254), and virus.
Several possible interactions could account for a more
rapid increase of viral infections in chemically stressed
shrimp than in control shrimp: loss of resistance to new
viral infections in shrimp hosts due to toxic effects of
Aroclor 1254; enhanced latent or occult viral infections
possibly carried by all or most shrimp from enzootic viral
populations; increased virulence of virus when exposed in
56
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Figure 35. Graph demonstrates viral prevalence and mor-
tality in shrimp samples exposed to Aroclor 1254*
vivo to Aroclor 1254; and greater susceptibility of intox-
IcaTed, weakened individuals to cannibalistic habits of
lesser-intoxicated exposed shrimp.
Future studies will use the Baculovirus-shrimp system
to investigate bioassays with additional chemicals. Tenta-
tive criteria for evidence of interaction are: increased
viral prevalence in exposed animals; increased intensity of
infection; increased mortality; and enhanced cytopathic
effects.
Characterization of Shrimp Baculovirus
M D SUMMERS, Principal Investigator. EPA Grant
R803395, The University of Texas, TX; J.A. COUCH,
Project Officer
Baculovirus cytopathology and ultrastructure have been
studied extensively and until recently known baculoviruses
were observed only in insects. However, Dr. J. A. Couch
of ERL.GB discovered a baculovirus in the pink shrimp,
Penaeus duorarum, in studies conducted in 1974 and
1975, thus extending the host range of this class of
viruses into the class Crustacea. Since 1975, reports have
cited possible baculovirus infections in shellfish in other
areas. Observations of baculoviruses in non-insect arthro-
pods are particularly significant in light of recent use of
insect baculoviruses as microbial pesticides for agricultural
pest control.
This investigation was undertaken to partially charac-
terize the pink shrimp baculovirus, which was found to be
morphologically similar to insect baculoviruses. Research
compared biochemical, structural, and biological proper-
ties of the shrimp virus to known properties of insect
viruses.
Results confirmed earlier evidence that the shrimp and
insect nuclear polyhedrosis baculoviruses (NPVs) are
structurally related. The project pointed out the need to
study host specificity with regard to baculoviruses in
more definitive detail. Although current use of existing
viral pesticides does not appear to warrant concern, the
discovery of marine baculoviruses suggests that a virus re-
stricted in terms of specificity to insects is not absolute
as previously thought. Results of this study were publish-
ed in the EPA Ecological Research Series, Report No.
EPA-600/3-77-130, November 1977.
Effects of Kepone on Animals in James Estuary,
Virginia
C. SINDERMANN, Principal Investigator. EPA-IAG-D6-
0124. National Oceanic and Atmospheric Administration
(NOAA); J.A. COUCH, Project Officer
Chromosomal and histopathological abnormalities in-
duced in shellfish by Kepone are under investigation in a
three-phase project:
(1) Chromosomal studies in larval offspring of oysters
(C. virginica) to determine the degree and type of chro-
mosomal abnormalities in larvae of oysters exposed to
Kepone. Larval offspring of unexposed (control) oysters
will be compared with larvae whose parents had been ex-
posed for various time periods to varied levels of Kepone.
(2) Histopathological and gametogenic studies on four
species of molluscs in Kepone-contaminated and in non-
contaminated waters. Organisms (collected seasonally) on
a transect from Bailey's Creek, VA, to the mouth of the
James River will be examined histopathologically. At least
30 animals per site of the following species will be col-
lected: R_. cuneata, C. virginica, M. mercenaria, and M.
balthica.
(3) Adult oysters exposed to Kepone under controlled
conditions will be shipped to the Milford, Connecticut,
laboratory for "conditioning" and spawning. Chromosom-
al studies will be performed on larval offspring.
Benzo[a]pyrene Metabolism in the American
Oyster (Crassostrea virginica)
R.S. ANDERSON, Chief Investigator. EPA Grant
R80443S. Sloan-Kettering Institute for Cancer Research,
D.S. Walker Laboratory, Rye, NY; J.A. COUCH. Project
Officer
Susceptibility of aquatic species to carcinogens or mu-
tagens may depend on the ability of these species to me-
*Reprinted by permission from the New York Academy of Sciences, Ann. N. Y_. Acad. Sci., Sept. 1977, Vol. 298, p. 502.
-------
tabolize xenobiotics to proximal or active intermediate
compounds. This investigation was undertaken to assess
the capacity of marine mollusks to metabolize the ubiq-
uitous pollutant benzo(a] pyrene (BP), a carcinogenic
polycyclic aromatic hydrocarbon.
The susceptibility of invertebrates to chemical carcin-
ogens is largely unknown. One objective of the study was
to determine if the potent mammalian carcinogen BP
could be considered a possible bivalve carcinogen. Aside
from detrimental effects on the oyster, presence of car-
cinogenic HP metabolites in fish commonly consumed by
man may have public health implications.
A sensitive radioisotopic system was developed to
quantify alkali-soluble and water-soluble BP metabolites
produced by oyster mono-oxygenase. An NADPH- and
O2-dcpendent aryl hydrocarbon hydroxylase (AHH) was
shown to be located in the digestive glands of oyster bi-
valves associated with the microsomal subcellular fraction.
The specific activity of oyster AHH, considerably lower
than that observed in laboratory mice, was consistently
demonstrable. Water-soluble derivatives were produced
primarily by BP metabolites.
Some indications were found that oyster AHH is in-
duced by chronic exposure to environmental carcinogens
BP and J-methylcholanthrene. Further, evidence suggested
that exposure to polychlonnated biphenyls (PCBs) caused
AHH induction.
BP metabolites produced by oyster AHH were identi-
fied by high-pressure liquid chromatography. The genera-
tion of various dihydrodiol, quinone, and hydroxy BP
derivatives was shown (production was augmented in
PCB-exposed oysters). No evidence was found regarding
the production of suspected ultimate carcinogenic BP
metabolite (7,8 diol-9, 10-epoxide); the 7,8-diol and the
mutagenic 4-5 oxide derivates were present in the oyster.
However, all metabolites are not known.
Results of the research conducted from July 1, 1976,
to June 30, 1977, are summari/.qd in KPA's Ecological
Research Series, Report No. EPA-600/3-78-009, January
1978.
Effects of Petroleum Compounds on Estuarine
Fishes ~
B.J. MARTIN, Principal Investigator. EPA Grant
R804527, University of Southern Mississippi;
J.A. COUCH, Project Officer
This investigator will attempt to characterize chem-
ically induced tumors in teleost fishes. Both marine and
freshwater species will be exposed to low levels of a
known carcinogen for 300 days. Tissue from the liver,
kidney, intestine, and gills of fish developing tumors or
any pathogenesis will be studied histologically. Tissues
from all the fish with no grossly apparent pathogens at
the end of the test will also be studied in the same man-
ner in order to detect any neoplasias or preneoplastic
conditions.
This research is expected to: (1) provide evidence con-
cerning the quantities of benz[a] pyrene necessary to in-
duce neoplasia; (2) supply additional data concerning
tumors in teleosts; (3) establish the feasibility of using
teleost fish as early indicators of carcinogenic hazards in
the aquatic environment; and (4) demonstrate usefulness
of this methodology to screen compounds for carcino-
genic properties.
Fish were exposed for several months to different con-
centrations of BP and 3-MC. Differential toxicity of these
compounds to fish have been found and histological
studies will be continued.
Studies on Environmental Chemical Carcinogens
Present in Economically Important Molluscs and
Crustaceans from Oregon Bays, Estuaries, and In-
shore Areas
C. MIX, Principal Investigator. EPA Grant R804427,
Oregon State University, Corvallis, OR;
J.A. COUCH, Project Officer
These studies focus on: (1) possible incidence of se-
lected environmental carcinogens in economically impor-
tant molluscs and crustaceans from Oregon bays, estu-
aries, and inshore areas; (2) potential public health haz-
ards from shellfish containing carcinogenic by-products of
petroleum; (3) incidence of neoplastic diseases among
these shellfish and possible correlation of these diseases
and carcinogenic disorders; (4) metabolic pathways where
benz [a] pyrene could be detoxified or modified; and
(5) acute and chronic effects of selected chemical carcin-
ogens on molluscan and crustacean gametes and ecological
consequences of these effects.
Forty-four sites were sampled in 1977; molluscs were
found to be contaminated by BP at 40 sites. Future re-
search will examine possible correlation between cellular
proliferative disorders and BP concentrations in molluscs.
Metabolism of Carcinogens by Marine Animals
W.P. SCHOOR, Research Aquatic Biologist
Chemical carcinogens entering the marine environment
can be accumulated by seafood consumed by man, there-
by providing another route of exposure. In addition,
physico-chemical and biological alterations of these com-
pounds may enhance or diminish their carcinogenic activ-
ity. The classic carcinogen, 1,2-benzopyrene, for example,
is oxidized to an epoxide by microsomal oxygenase sys-
tems before it becomes the actual carcinogen.
Studies conducted at ERL.GB in 1977 attempted to
determine whether or not estuarine and marine organisms
can activate chemical carcinogens from pro-carcinogens to
true carcinogens like their mammalian counterparts.
58
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During this reporting period, the microsomal oxygenase
systems of the mullet (Mugil cephalus) were induced by
intraperitoneal injection of 30 mg/kg 3-methylcholan-
threne in corn oil. After 48 h, the animals were sacrificed,
and the oxygenase system was isolated by centrifugation.
Incubations were carried out by using 10-40 yg of 1,2-
benzopyrene or chrysene per mg of microsomal protein.
The mixtures were extracted with ethyl acetate; samples
were analyzed by high pressure liquid chromatography.
This work demonstrated the ability of the mullet to me-
tabolize 1,2-benzopyrene or chrysene in a manner analo-
gous to the rat.
Future work will include different species of fish,
crustaceans, and molluscs. Efforts will be made to eluci-
date the mechanism of induction of the microsomal oxy-
genases and the conjugation of metabolites. The induction
studies will be complemented by studies of proliferative
effects on the smooth endoplasmic reticulum of the liver.
Mechanisms of Organophosphates in Shrimp
W.P. SCHOOR, Research Aquatic Biologist
Toxicity of organophosphates in vertebrates has been
linked to acetyl cholinesterase (AChE) inhibition of nerve
transmission. Because the neuromuscular system of crus-
tacea differs from that of vertebrates, shrimp response to
AChE inhibitors may differ from the response of verte-
brates.
In vivo tests with brown shrimp (Penaeus aztecus)
showed a 96-h LC50 of 1.9 pg/£ for methyl parathion
(MPT) and 13.6 pg/£ for methyl paraoxon (MPO). The
only significant depression of AChE activity in the ventral
nerve cords of the exposed shrimp was found in mori-
bund shrimp at an exposure of 1.3 yg/£ MPT. hi vitro in-
hibition of the isolated nerve cords was about 10% after
1 h exposure to 160 mg/i MPT, and about 60% after 1 h
exposure to 22 pg/Jl MPO.
MPO was less toxic to shrimp-than MPT. AChE inhibi-
tion was found only in moribund animals exposed to
MPO, suggesting that toxicity in shrimp is not linked di-
rectly with AChE inhibition or that AChE activity is of
little significance in shrimp.
The reason for the low toxicity of MPO in vivo could
be due to its rapid degradation before reaching the active
site. The specific active component for this inhibition
may not be present in the nerve cord, and consequently
MPO may be highly toxic only in vitro where nerve prep-
arations are directly exposed.
The relative toxicities of MPT and MPO in vitro were
the same as those observed in other systems: the MPO is
100 to 1000 times more toxic. However, neither the
LC50s nor the in vivo AChE inhibition of MPT and MPO
seem to agree. The reasons for high toxicity of MPT in
shrimp cannot be ascertained without further study.
Determination of the Site(s) of Action of Select-
ed Pesticides by an Enzymatic-Immunobiological
Approach ~~~ "
R.B. KOCH, Principal Investigator. EPA Grant R803458,
Mississippi State University, MS; W.P. SCHOOR, Project
Officer
An antibody was produced to an organochlorine pesti-
cide that inhibits ATPase enzymes, an important mecha-
nism in the generation of energy, to gain insight into the
nature of the mechanism of action of certain pesticides.
The pesticide Kelevan, the condensation product of
ethyl levulinate and Kepone, was used as a hapten for co-
valent conjugation to various protein atigens to produce
an antibody against the pesticide, thereby blocking its ac-
tivity. Kelevan was successfully conjugated to bovine
serum albumin (BSA), fibrinogen (BF), and gamma glob-
ulin (BGG); rabbits and chickens preimmunized with BSA
and then immunized with BSA-Kelevan produced anti-
bodies to both the hapten (Kelevan) and the carrier pro-
tein (BSA).
The antiserum to Kelevan protected ATPase activity
against Kepone and its derivatives. The titer of antibody
to Kelevan was critical because antiserum containing only
trace amounts of Kelevan antibody failed to provide pro-
tection against its toxicity.
The antibody was concentrated by Na2SO4 fractional
precipitation of the antiserum and obtained in pure form
by affinity chromatography by using BGG-Kelevan cova-
lently linked to Sepharose-4B. Pure antibody was obtain-
ed from untreated blood serum or plasma with no prior
pretreatment or fractionation by using the BGG-Kelevan
affinity column.
Complete protection of mitochondrial Mg2+ ATPase
activity from in vitro inhibition of Kepone was obtained
by a 1.2 mg quantity of Na2$O4 fractionated antibody
and 120 ^g of pure antibody; ATPase inhibition was
readily reversed, as predicted, when the antibody was
added prior to addition of substrate to the reaction mix-
ture. This indicates that this procedure blocked the tox-
icity of Kepone to the Mg2+ ATPase system.
The three-year investigation will conclude February 15,
1978, after tests to determine if the antibody prevents in-
hibition by other chlorinated hydrocarbon pesticides. Re-
sults will be published in the next reporting period.
59
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PUBLICATIONS
BAHNER, L.H., A.J. WILSON, Jr., J.M. SHEPPARD, J.M.
PATRICK, Jr., L.R. GOODMAN, and G.E. WALSH.
1977. Kepone bioconcentration, accumulation, loss,
and transfer through estuarine food chains.
Chesapeake Sci. 18- 299-308.
BLOCK, R.M., G.R. HELZ, and W.P. DAVIS. 1977. The
fate and effects of chlorine in coastal waters.
Chesapeake Sci. 18(1): 97-101.
BORTHWICK, P.A., and S.C. SCHIMMEL. 1978. Toxicity
of Pentachlorophenol and related compounds to
early life-stages of selected estuarine animals. In:
Pentachlorophenol, 1978. K. RANGO RAO. Ed.,
Plenum Publishing Corp., N.Y. pp. 141-146.
BOURQUIN, A.W. 1977. Effects of malathion on micro-
organisms of an artificial salt-marsh environment. J.
Environ. Qual. Vol 6,4: 373-378.
BOURQUIN, A.W. 1977. Degradation of malathion by
salt-marsh microorganisms. Appl. & Environ. Micro-
biol. 33: 356-362.
BOURQUIN, A.W., M.A. HOOD, and R.L. GARNAS.
1977. An artificial microbial ecosystem for deter-
mining effects and fate of toxicants in a salt-marsh
environment. Dev. Ind. Microbiol. 18: 185-191.
BOURQUIN, A.W., M.A. HOOD, P.A. PRITCHARD, and
R.L. GARNAS. 1977. Degradation of methyl para-
thion in laboratory simulations of salt-marsh envi-
ronments. Abs. Amer. Soc. Microbiol. Q72. p. 273.
BOURQUIN, A.W., and V.A. PRZYBYZEWSKI. 1977.
Distribution of bacteria with nitrilotriacetate-
degrading potential in an estuarine environment.
Appl. Environ. Microbiol. Vol. 34(4): 411-418,
October 1977.
BRANNON, A.C., and P.J. CONKLIN. 1978. Effect of
sodium pentachlorophenate on exoskeletal calcium
in the grass shrimp, Palaemonetes pugio. In: Penta-
chlorophenol. K. RANGA RAO, Ed. Plenum Pub-
lishing Corp., N.Y. pp. 205-212.
CANTELMO, F.R., and K.R. RAO. 1978. Effects of
pentachlorophenol on the meiobenthic nematodes
in an experimental system. In: Pentachlorophenol.
K. RANGA RAO, Ed. Plenum Publishing Corp.,
N.Y. pp. 165-174.
CANTELMO, A.C., P.J. CONKLIN, F.R. FOX, and K.R.
RAO. 1978. Effects of sodium pentachlorophenate
and 2,4-dinitrophenol on respiration in crustaceans.
In: Pentachlorophenol. K. RANGA RAO, Ed.
Plenum Publishing Corp., N.Y. pp. 251-265.
CARPENTER, J.H. 1977. Problems in measuring residuals
in chlorinated seawater. Chesapeake Sci. 18(1): 112.
CARPENTER, J.H., D.L. MACALADY, and C.A.
MOORE. 1977. Sunlight-induced bromate forma-
tion in chlorinated seawater. Science 195: 1335-
1337.
CARPENTER, J.H., C.A. MOORE, and D.J. MACALADY.
1977. Errors in determination of residual oxidants
in chlorinated seawater. Environ. Sci. Tech. 11(10):
992-994.
CONKLIN, P.J., and K.R. RAO. 1978. Toxicity of sodi-
um pentachlorophenate to the grass shrimp,
Palaemonetes pugio, in relation to the molt cycle.
In: Pentachlorophenol, K. RANGA RAO, Ed.
Plenum Publishing Corp., N.Y. pp. 181-192.
COUCH, J.A. Diseases, parasites, and toxic responses in
penaeid shrimps of the Southeastern and Gulf
Coasts of the U.S. Fish. Wildl. Serv. Bull. 76: 1-44.
COUCH, J.A. Ultrastructural study of lesions in gills of a
marine shrimp exposed to cadmium. J. Invert. Path.
29: 267-288.
COUCH, J.A., and L. COURTNEY. 1977. Interaction of
chemical pollutants and virus in a crustacean: a
novel bioassay system. Ann. N.Y. Acad. Science
298: 497-504.
COUCH, J.A., J. WINSTEAD, and L. GOODMAN. 1977.
Kepone-induced scoliosis and its histological conse-
quences in fish. Science 197: 585-587.
DAVIS, W.P., D.P. MIDDAUGH, J.H. CARPENTER, G.R.
HELZ, and M.H. ROBERTS, Jr. 1977. The chemis-
try and ecological effects of chlorination of sea-
water-a summary of EPA research projects. (EPA/
OEMI Interagency Energy-Environment Program
Report).
60
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DAVIS, W.P., D.P. MIDDAUGH, J.H. CARPENTER, G.R.
HELZ and M.H. ROBERTS, Jr. 1977. The chemis-
try and ecological effects of chlorination of sea-
water: a summary of EPA research projects. Pro-
gram Review Proceedings of Environmental Effects
of Energy Related Activities on Marine/Estuarine
Ecosystems. EPA 660/7-77-111: 175-186.
DOUGHTIE, D.G., and K.R. RAO. 1978. Effects of sod-
ium pentachlorophenate and 2,4-dinitrophenol on
hepatopancreatic enzymes in the blue crab,
(Callinectes sapidus). In: Pentachlorophenol. K.
RANG A RAO, Ed. Plenum Publishing Corp., N.Y.
pp. 213-250.
ERICKSON, S.J., and A.E. FREEMAN, 1978. Toxicity
screening of fifteen chlorinated and bromtnated
compounds to four species of marine phytoplank-
ton. In: Water Chlorination: Environmental Impact
and Health Effects, Volume II, R.L. JOLLEY, et
al., editor. Ann Arbor Sci. Publ., Inc., Ann Arbor,
Michigan.
FARR, J.A. 1977. Impairment of antipredators' behavior
in Palemonetes pugio by exposure to sublethal doses
of parathion. Trans. Amer. Fish Soc. Vol. 106, No.
3. pp. 287-290.
HANSEN, D.J., S.C. SCHIMMEL, and J. FORESTER.
1977. Endrin. effects on the entire life-cycle of a
saltwater fish Cyprinodon variegatus. J. Toxicol.
Environ. Health, Volume 3, pp. 721-733.
HANSEN, D.J., and P.R. PARRISH. 1977. Suitability of
sheepshead minnows (Cyprinodon variegatus) for
life-cycle toxicity tests. In: Aquatic Toxicology and
Hazard Evaluation, ASTM STP 634, pp. 117-126.
HANSEN, D.J., AJ. WILSON, D.R. NIMMO, S.C.
SCHIMMEL, L.H. BAHNER, and R. HUGGETT.
1976. Keponse: hazard to aquatic organisms.
Science. 193-528.
HANSEN, D.J., L.R. GOODMAN, and A.J. WILSON, Jr.
1977. KeponeR-. chronic effects on embryo, fry,
juvenile, and adult sheepshead minnows,
(Cyprinodon variegatus). Chesapeake Sci. 18(2):
226-232.
M1DDAUGH, D.P., A.M. CRANE, and J.A. COUCH. 1977.
Toxicity of chlorine to juvenile spot, Leiostromus
xanthurus. Water Research, Volume 11, pp. 1089-
1096.1977.
MIDDAUGH, D.P., and J.M. DEAN. 1977. The toxicity
of cadmium to eggs, larvae, and adults of the fishes:
mummichog, Fundulus heteroclitus, and silverside,
Menidia menidia. Bull. Environ. Contam. Toxicol.
17(6).
MIDDAUGH, D.P., and F.W. LEMPESIS. 1976. Labora-
tory spawning and rearing of a marine fish, the
silverside Menidia menidia. Mar. Biol. 35 (4):
295-300.
MIDDAUGH, D.P., J.A. COUCH, and A.M. CRANE.
1977. Chlorine toxicity to early-life history stages
of the striped bass, Morone saxatilis. Chesapeake
Sci. 18 (1) 141-153.
NIMMO, D.R., L.H. BAHNER, R.A. R1GBY, J.M.
SHEPPARD, and A.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, F.L. MAYER and J.L. HAMEUND,
Eds., ASTM STP 634 pp. 109-116.
NIMMO, D.R., R.A. RIGBY, L.H. BAHNER, and J.M.
SHEPPARD. 1977. The acute and chronic effects of
cadmium on the estuarine mysid, Mysidopsis bahia.
Bull. Environ. Contam. Toxicol., 0007-4861/78/
0019-0080.
NIMMO, D.R., D.V. LIGHTNER, and L.H. BAHNER.
1977. Effects of cadmium on the shrimps, Penaeus
duorarum, Palaemonetes pugio, and Palaemonetes
vulgaris. In: Physiological Responses of Marine Biota
to Pollutants, F.J. VERNBERG et al., Ed. Academic
Press, New York. pp. 131-183.
RAO, K.R., P.J. CONKLIN, and A.C. BRANNON. 1978.
Inhibition of limb regeneration in the grass shrimp,
Palaemonetes pugio, by sodium pentachlorophenate.
In: Pentachlorophenol. K. RANGA RAO, Ed.
Plenum Publishing Corp., N.Y. pp. 193-204.
RUBINSTEIN, N.I. 1978. Effect of sodium pentachloro-
phenate on the feeding activity of a lugworm,
Arenicola cristata. In: Pentachlorophenol. K.
RANGA RAO, Ed. pp. 175-180.
SCHIMMEL, S.C., and A.J. WILSON, Jr. 1977. Acute
toxicity of Kepone to four estuarine animals.
Chesapeake Sci. 18: 224-227.
SCHIMMEL, S.C., J.M. PATRICK, Jr., and J.
FORESTER. 1977. Uptake and toxicity of toxa-
phene in several estuarine organisms. Arc. Environ.
Contam. Toxicol. 5: 353-367.
SCHIMMEL, S.C., J.M. PATRICK, Jr., and L.F. FAAS.
1978. Effects of sodium pentachlorophenate on
several estuarine animals: toxicity, uptake, and
depuration. In: Pentachlorophenol. K. RANGA
RAO, Ed. Plenum Publishing Corp., N.Y. pp. 147-
155.
61
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SCHIMMEL, S.C., J.M. PATRICK, Jr., and J. FORESTER.
1977. Toxicity and bioconcentration of BHC and
Lindane in selected estuarine animals. Arch. Environ.
Contam. Toxicol. 6: 355-363.
TAGATZ, M.E., J.M. IVEY, J.C. MOORE, and M.
TOBIA. 1977. Effects of pentachlorophenol on the
development of estuarine communities. Toxicol.
Environ. Health. 3: 501-506.
TAGATZ, M.E., J.M. IVEY, and M. TOBIA. 1978.
Effects of DowicideR G-ST on development of ex-
perimental estuarine macrobenthic communities. In:
Pentachlorophenol. K. RANGA RAO, Ed. Plenum
Publishing Corp., N.Y. pp. 157-163.
TAGATZ, M.E., P.W. BORTHWICK, J.M. IVEY, and J.
KNIGHT. 1976. Effects of leached mirex on ex-
perimental communities of estuarine animals. Arch.
Environ. Contam. Toxicol. 4: 435-442.
TYLER-SCHROEDER, D.B. 1976. Effects of two poly-
chlorinated biphenyls, Aroclor^ 1016 and 1242, on
the grass shrimp Palaemonetes pugio. MS Thesis.
Univ. of West Florida, Pensacola, EL. 128 pp.
WALSH, G.E. 1977. Exploitation of mangal. In: Ecosys-
tems of the World, V.J. CHAPMAN, Ed. Elsevier
Pub. Co., Amsterdam, pp. 347-362.
WALSH, G.E., K.A. AINSWORTH, and A.J. WILSON.
1977. Toxicity and uptake of Kepone in marine
unicellular algae. Chesapeake Sci. 18(2): 222-223.
Water Quality Committee, American Fisheries Society
(D.J. HANSEN, member). 1976. A survey of efforts
to abate water pollution in fiscal year 1973 by
North American Agencies. Fisheries (1(1): 15-21.
PRESENTATIONS
BAHNER, L.H., and J.L. OGLESBY. 1977. Test of model
for predicting KeponeR accumulation in selected
estuarine species. Symp. American Society for Test-
ing and Materials (ASTM), Cleveland, OH, Oct 10-
14, 1977.
BAHNER, L.H., and D.R. NIMMO. 1977. Uptake of
Kepone from sediments by estuarine organisms.
Kepone Seminar II, EPA Chesapeake Bay Program
and National Marine Fisheries Service, NOAA. Sept
19-21, 1977.
BLOCK, R.M., G.R. HELZ, and W.P. DAVIS. 1977. The
fate and effects of chlorine in coastal waters. In:
Proc. Chlorination Workshop, University of
Maryland, Center for Environmental and Estuarine
Studies, Chesapeake Biological Laboratory, March
15-18, 1976, and Chesapeake Sci., Vol. 18(1):
97-101. 1977.
CARPENTER, J.H., and C.A. SMITH. 1977. Reactions in
chlorinated seawater. Second Conf. on Water Chlo-
rination: Environmental Impact and Health Effects.
Gatlinburg, TN. Nov. 1, 1977.
COUCH, J.A. 1977. Interactions of three pathogens in
mass mortality of penaeid shrimp. Crustacean
Health Workshop. NOAA, Galveston Coastal
Fisheries Laboratory, Galveston, TX. Chaired ses-
sion: Infectious Diseases: Parasitic and Viral Infec-
tions in Crustaceans. April 20, 1977.
COUCH, J.A. 1977. The American oyster as an indicator
of carcinogens in the environment. Symp. Patho-
biology of Environmental Pollutants, Storrs, CT.
National Academy of Science. June 1, 1977.
COUCH, J.A., and J.T. WINSTEAD. 1977. Concurrent
neoplastic and protestin disorders in the American
oyster (Crassostrea virginica). First Colloquim on
Molluscan Pathology, French Academy of Science,
Perpignon, France. Sept. 1977.
DAVIS, W.P., B.S. HESTER, R.L. YOAKUM, and R.C.
DOMEY. 1977. Marine ecosystem testing units: de-
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low-level pollutants. International Symposium;
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Meeresunters. 30: 673-681.
DAVIS, W.P., J.E. RANDALL, and D.O. FRENCH. 1977.
The systematics, biology, and zoogeography of
Ptereleotris heteropterus (Pisces: Gobiidae). Proc.
Third Int. Coral Reef Symp, Coral Gables, FL. May
23-27, 1977.
GARNAS, R.L. 1977. Fate of Kepone in estuarine micro-
cosms. Kepone Seminar II. Easton, MD. Sept. 19-21,
1977.
GARNAS, R.L. 1977. Interdependent microcosms for the
assessment of pollutants in the marine environment.
Symp. on the Role of Microcosms in Ecological Re-
search. American Institute for Biological Sciences,
Lansing, MI. August 1977.
GARNAS, R.L. 1977. The fate of methyl parathion in a
marine benthic microcosm. No. 44 Pesticide Chem-
istry Division. 173rd Meeting American Chemical
Society, New Orleans, LA. March 1977.
62
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HANSEN, D.J. 1977. Bioconcentration of Kepone from
water by estuarine organisms and the effects on the
estuarine organisms. Kepone Seminar II. Easton,
MD. Sept. 20-21, 1977.
MIDDAUGH, D.P., and W.P. DAVIS. 1976. Impact of
chlorination processes on marine ecosystems. Proc.
EPA/AIBS Symposium. (EPA 600/3-76-079) pp.
46-62.
NIMMO, D.R. 1977. Effects of Kepone on marine biota.
42nd Conf. North American Wildlife and National
Resources. Atlanta, GA. March 8, 1977.
NIMMO, D.R. 1977. Kepone: its effects on mysids and
movement through a laboratory food chain. Work-
shop on the Fate and Movement of Kepone in the
James River and Chesapeake Bay Systems. Virginia
Institute of Marine Science, Gloucester Point, VA.
October 12-13, 1976.
NIMMO, D.R. Kepone, PCB effects on marine biota.
Central Atlantic States Association of Food and
Drug Officials, Gettysburg, PA. May 24-27, 1977.
NIMMO, D.R. 1977. Mysids: guinea pigs of the environ-
ment? Crustacean Health Workshop. Galveston, TX.
April 20-22, 1977.
NIMMO, D.R. 1977. Pesticides and the estuarine environ-
ment. Seminar Duke Univ. Marine Laboratory,
Beaufort, NC. March 25, 1977.
RICHARDS, N.L. Accumulation and transport of shale-
oil-derived compounds in the marine environment.
National Institute of Environmental Health
Sciences/USSR Conference on Shale Oil. Denver,
CO. May 17-21, 1977.
RICHARDS, N.L. 1977. Effects of chemicals used in off-
shore oil and gas drilling operations. Second National
Conference on the Interagency Energy/Environment
R&D Program, Washington, DC. June 7-8, 1977.
RICHARDS, N.L. 1977. Marine applications. Symp. on
Application of Short-term Bioassays in the Frac-
tionation and Analysis of Complex Environmental
Mixtures, Williamsburg, VA. Feb. 21-23, 1977.
TYLER-SCHROEDER, D.B. 1977. Effects of endrin on
grass shrimp, Palaemonetes pugio, in a long-term,
entire life-cycle toxicity test. Galveston, TX.
April 20-22, 1977.
WILKES, F.G. 1977. Laboratory microcosms for use in
determining pollutant stress. Proc. Second Int.
Symp. on Aquatic Pollutants, Amsterdam, the
Netherlands. Sept. 26-28, 1977.
WILKES, F.G. and C.W. HALL. 1977. Microcosms as
indicators of ecosystem stress. Third Joint U.S.-
USSR Symp. on Comprehensive Analysis of the
Environment. Tashkent, USSR. Oct. 10-14, 1977.
ECOLOGICAL RESEARCH SERIES
VERNBERG, F.J., R. BONNEL, B. COULL, R. DAME,
Jr., P. DE COURSEY, W. KITCHENS, Jr., B.
KJERFVE, H. STEVENSON, W. VERNBERG, and
R. ZINGMARK. 1977. The dynamics of an estuary
as a natural ecosystem. EPA-600/3-77-016.
SIKKA, H.C., and G.L. BUTLER. 1977. Effects of select-
ed wastewater chlorination products and captan on
marine algae. EPA-600/3-77-029.
AHEARN, D.G. S.A. CROW, and W.L. COOK. 1977.
Microbial interactions with pesticides in estuarine
surface slicks. EPA-600/3-77-050.
PARRISH, P.R., E.E. DYAR, M.A. LINDBERG, CM.
SHANIKA, and J.M. ENOS. 1977. Chronic toxicity
of methoxychlor, malathion, and carbofuran to
sheepshead minnows (Cyptinodon varicgatus). EPA-
600/3-77-059.
COLWELL, R.R., and G.S. SAYLER. 1977. Effects and
interactions of polychlorinated biphenyl (PCB) with
estuarine microorganisms and shellfish. EPA-600/
3-77-070.
HANSEN, D.J., D.R. NIMMO, S.C. SCHIMMEL, G.E.
WALSH, and A.J. WILSON, Jr. 1977. Effects of
Kepone on estuarine organisms. Recent advances
in fish toxicology, a symposium. Ecological Re-
search Series, EPA-600/3-77-085, July 1977. pp.
20-30.
CRANE, A.M., and A.E. FREEMAN. 1977. Water soften-
ing and conditioning equipment: a potential source
of water contamination. EPA-600/3-77-107,
September 1977.
SUMMERS, M.D. 1977. Characterization of shrimp
baculovirus. EPA-600/3-77-130, November 1977.
CALDWELL, R.S. 1977. Biological effects of pesticides
on the Dungeness crab. EPA-600/3-77-131,
November 1977.
63
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TECHNICAL REPORT DATA
fPlease read Instructions on the reverse before completing)
1. REPORT NO.
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
Research Review 1977
5. REPORT DATE
June 1978
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO
Betty P. Jackson, editor
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Gulf Breeze, Florida 32561
10. PROGRAM ELEMENT NO.
1EA615
1. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
13. TYPE OF REPORT AND PERIOD COVERED
January - December 1977
14. SPONSORING AGENCY CODE
EPA/600/4
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This report summarizes results of aquatic research conducted by the Environmental
Research Laboratory, Gulf Breeze, Florida, Office of Research and Development,
U.S. Environmental Protection Agency from January 1 to December 30, 1977. The
research program examines the impact of pesticides and other organic compounds
on marine species and communities, and seeks to develop new methodology for
determining ecological hazards of chemical substances under simulated natural
conditions. Projects are outlined under four categories: research related to
toxicological testing; biological processes and effects; development of off-
shore oil resources; and Kepone in the marine environment. Investigations
conducted at the laboratory's Atlantic Coast field station at Bears Bluff,
South Carolina, are also reviewed for the year 1977.
7.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/GlOUp
Marine biology
Toxicology
Carcinogens
Mutagens
Pesticide
Bioassay
Environmental Research
Laboratory, Gulf Breeze
Environmental Protection
Agency
Microcosms
6F
6A
6E
8. DISTRIBUTION STATEMENT
19. SECURITY CLASS (This Report I
Unclassified
21. NO. OF PAGES
Public
20. SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION is OBSOLETE
64
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