v>EPA
United States
Environmental Protection
Agency
Environmental Research
Laboratory
(Sulf Breeze, FL 32561
EPA-600/9-79-028
September 1979
Research and Development
Research
Review
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FOREWORD
In December 1970, as Director of a U.S. Fish and Wildlife Service Laboratory on
Sabine Island, I had the gratifying experience of helping integrate the Laboratory's
research program into the newly created Environmental Protection Agency (EPA). It
was a time of change, marking the birth of the first governmental organization charged
with examining pollution problems in terms of the total inter-related environment.
September 1979 also represents a time of change for me. I have requested and
received reassignment to a scientific position at the Environmental Research Labora-
tory, Gulf Breeze (ERL,GB). This assignment will provide me the opportunity to work
more closely on scientific aspects of regulatory actions. I will'begin the reassignment
by working a year at Texas University's Institute of Marine Science in Port Aransas,
Texas, under the Intergovernmental Personnel Act. While at the Institute, I plan to
research the impact of drilling mud on an indigenous benthic community and to
become involved in their academic program. The assignment at Port Aransas will pro-
vide a transition from administrative aspects of a regulatory laboratory to more scien-
tific aspects. My experiences during the past few years have convinced me more than
ever that environmental regulations must be based on a strong scientific data base and
that development and interpretation of the base requires our collective best effort.
In retrospect, I have had a unique opportunity of working with many other scien-
tists (from ERL,GB, EPA, Federal, State and local government agencies, and educa-
tional institutions) in designing research programs and recommending standards for a
healthy, productive environment.
Our research has provided a basis for many EPA regulatory decisions related to
hazards posed by toxicants to estuarine and marine resources. It has also demonstrated
that many important questions remain to be answered if scientists are fully able to
comprehend the total effect of pollutant effects on marine animals and their environ-
ment. Some investigations have uncovered uncertainties that must be resolved. It is
now clear that new initiatives must be forthcoming, and new talents must be recruited.
I look forward to the experiences to come and hope to gain new insights to prob-
lems related to the development of a pertinent data base for environmental regulations.
However, I say goodbye to the dedicated staff at ERL,GB and to the supporters of our
research efforts with deep regret. I have always felt the staff of this Laboratory was
supportive to me as I attempted to direct research efforts. I am especially appreciative
of the understanding and support the staff has given me and my family during the last
few months. I am optimistic about the future of this Laboratory and being a part of
it.
Thomas W. Duke
Director
Environmental Research Laboratory
Gulf Breeze, Florida
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EPA-600/9-79-028
September 1979
United States Environmental Protection Agency
Environmental Research Laboratory
Gulf Breeze, FL 32561
CONTENTS
FOREWORD i i
EXPOSURE ASSESSMENT 1
EFFECTS ASSESSMENT 11
CHLORINATION STUDIES 31
ENVIRONMENTAL PATHOBIOLOGY 43
EFFECTS OF OFFSHORE DRILLING FLUIDS ON
THE MARINE ENVIRONMENT 51
CHESAPEAKE BAY PROGRAM 57
PUBLICATIONS 61
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. Environmental Scientist J.P. Connolly adds an aluminum powder solution to illuminate the
flow field in a turbulence generation apparatus used to investigate the effects of sediment
resuspension on the environmental fate of toxic substances.
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EXPOSURE ASSESSMENT
A dramatic increase in the use and production of
chemicals in the United States has prompted the develop-
ment and manufacture of many new compounds that have
subtle, yet-to-be determined ecological consequences.
EPA legislative mandates require a reliable data base
for the evaluation of human and environmental risks
posed by toxic substances. In 1978, ERL,GB researchers
continued to work on the development of new techniques
and methodology for Exposure Assessment applicable to
the development of criteria for the control of toxic sub-
stances and the registration or reregistration of pesticides.
A primary objective of the Exposure Assessment effort
was to develop a simple screening tool that can be used in
the preliminary characterization of the fate of pollutants
in estuaries. Data from the preliminary tests will form the
basis for subsequent toxicological tests and more complex
determinations of the transfer and fate of pollutants in
the marine or estuarine environment.
Fate Studies
A.W. BOURQUIN, Research Microbiologist;
H.P. PRITCHARD, Environmental Scientist
ERL.GB researchers have developed a three-level (tier)
test to predict the fate of toxic organics in estuarine sys-
tems. Procedures are based on tier-testing and allow
extrapolation to real-life aquatic settings. The system
eventually will be integrated into a larger Laboratory
Hazard Assessment Program that is now under develop-
ment.
The central concepts for the tier-test system are:
Level I-rapid screening tests that provide preliminary
information on detoxification, biodegradation, and trans-
formation processes. These screening tests provide infor-
mation for orientation toward more elaborate testing for
both fate and toxicity effects.
Tests use actual media, sediment, and water, and
require no radiolabeled chemicals. Sterile systems are
employed to determine the chemical hydrolysis contribu-
tion to the transformation process. Loss of toxicity will
be assayed with mysid shrimp and a benthic amphipod.
Level Il-a laboratory sediment-core test system capa-
ble of generating "range finding" data that integrates fate
processes under the complexities of a natural estuarine
environment. Testing protocol includes relative estimates
of routes, rates, and the extent of fate processes relative
to standard test chemicals.
Level Ill-laboratory microcosm tests that simulate
natural estuarine conditions and quantitatively evaluate
rates, extent, and capabilities of degradative processes on
toxic organics leading to predictions of exposure concen-
trations.
Fate Study (Dimilin®)
A.W. BOURQUIN, Research Microbiologist;
H.P. PRITCHARD, Environmental Scientist
Of the new type of insecticides that evoke toxicity
through interference with the formation of the exoskele-
ton, Dimilin® (diflubenzuron) is probably the most
widely used. In order to properly assess Dimilin's poten-
tial toxic effects in marine environments, a fate study has
been conducted by researchers at ERL,GB to compare
and contrast results derived from soil studies conducted
by other scientists with the insecticide.
In screening tests, Dimilin was shown to have a long P
(octanol/water partition coefficient) of 3.02, a sediment/
water partition coefficient of 350, and no substantial vola-
tility. Studies in a static core sediment/water laboratory
test system (Eco-core) and in a continuous-flow micro-
cosm have revealed that Dimilin readily hydrolyzes into
p-chlorophenyl urea and diflurobenzoic acid. In the static
Eco-core, 90% of the original Dimilin hydrolyzed after
24 days, indicating a half-life of 17 days.
The hydrolysis products were shown to be non-toxic
to mysid shrimp and did not degrade even after long-term
incubation. In similar soil studies reported in the litera-
ture, shorter half-lives were observed and the hydrolysis
products were readily biodegraded. Only very small
amounts of Dimilin became bound to the sediment used
in these systems. Thus, there appears to be definite differ-
ences between fate processes in estuarine and terrestrial
environments.
Studies in the continuous-flow microcosm showed simi-
lar results. Tests increasing the number of active growth
vessels or lengthening the exposure to sediment revealed
that Dimilin hydrolysis was largely biological and
sediment-mediated (Fig. 2). Since these studies involve
naturally derived sediment/water systems, results probably
reflect similar processes that occur in estuarine salt-marsh
environments. Dimilin appears to break down about two
times slower than methyl parathion under similar labora-
tory conditions.
Sediment-core (Eco-core)
A.W. BOURQUIN, Research Microbiologist;
H.P. PRITCHARD, Environmental Scientist
ERL,GB researchers have developed a sediment-core
fate test, known as Eco-core, that has been proposed as a
standard degradation test system for marine environments.
The system involves analytical chemical tracing of the fate
of a radiolabeled pesticide or toxic organic added to an
©Registered trademark
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Figure 2. The fate of Dimilin and its hydrolysis products in continuous-flow microcosms shown in three
consecutive growth vessels (GV I, II, III). (•) Dimilin, (•) parachlorophenyl urea, (O) 2,4-
difluorobenzoic acid, (U) parachloroaniline.
undisturbed sediment/water core. The Eco-core system
offers these advantages:
(1) It is simple, inexpensive, and easily assembled.
(2) Replication with the Eco-core is excellent.
(3) Many Eco-cores can be set up at once to permit
the simultaneous testing of several environmental param-
eters.
(4) Experimental protocols allow the fate of a toxi-
cant to be followed with time, and the distribution of the
toxicant in water and sediment (total budget analysis) to
be determined.
(5) Results from an Eco-core test reflect an integrated
picture of all the fate processes occurring in the sediment/
water core. With the proper controls, however, rates and
extents of chemical hydrolysis, sediment binding, micro-
bial degradation, and volatilization can be individually
assessed.
(6) Because a sediment water core is employed, the
sediment/water interface is relatively undisturbed. Natural
environmental conditions and complexities (including
anaerobic conditions in the sediment) are maintained,
thus giving validity to data generated.
(7) Simple operations and analyses permit a large
number of compounds to be tested; a sufficient data base
can be generated to provide fate assessment based on
relative rankings with reference compounds.
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Figure 3 presents the results of an experimental series
of Eco-cores using sediment from Range Point saltmarsh
and three pesticides: methyl parathion, pentachloro-
phenol, and Sevin®. The data qualitatively and quantita-
tively demonstrate that in estuarine saltmarsh samples,
Sevin degrades fater than methyl parathion, which
degrades faster than pentachlorophenol. Table 1 gives a
total budget analysis from the same experiment. Because
of extensive calibration, standardization, and characteriza-
tion, the Eco-core test system can be applied to fate
studies in aquatic systems for many other pesticides and
toxic substances.
Mathematical Modeling of Fate and Transport
Processes
J.P. CONNOLLY, Environmental Scientist
A new program in mathematical modeling of fate and
transport processes in estuarine environments was initiated
at ERL.GB in 1978. The program is divided into four
categories:
(1) The development of mathematical models of sedi-
ment adsorption/desorption processes. Studies focus on
the role of organic matter, sediment concentrations, and
sediment dispersion on these sorption processes.
(2) Development of mathematical models to describe
fate processes studied in laboratory microcosms. Special
emphasis was placed initially on continuous-flow micro-
cosms developed at ERL.GB. Rate constants were sought
for fate processes (i.e., degradation, hydrolysis, sorption,
volatility, photolysis) that simulate natural conditions as
much as possible.
(3) Development of mathematical models for predict-
ing exposure concentration in estuarine environments. In
1978, the James River model developed in conjunction
with Manhattan College was utilized as an example of a
typical East Coast estuary offering extensive background,
ecological, hydrodynamical, and physiographical informa-
tion. Studies with Kepone are being employed for calibra-
tion purposes.
(4) Conceptualization of models for hazard assess-
ments in marine ecosystems. This work is designed to
integrate acute and chronic LCSO's, bioconcentration,
behavior, and fate process information generated at the
ERL.GB into an assessment of specific pesticides and to
identify areas requiring further research (Fig. 1).
Aquatic Bacteria Studies
A.W. BOURQUIN, Research Microbiologist;
P.H. PRITCHARD, Environmental Scientist
Effects of toxicant concentrations on the ability of
microorganisms to degrade toxicants were investigated. In
1978, a variety of aquatic bacteria were isolated and
grown; low concentrations of phenylacetic acid (a base
chemical for the synthesis of certain herbicides) serve as
their sole source of carbon and energy. Evidence from
other sources indicate that these bacteria may be adapted
to growth at such low concentrations because of a unique
physiological condition. In the ERL,GB oxygen uptake
studies, cells originally isolated with phenylacetic acid
(PAA) as the substrate continued to produce the PAA
catabolic (constitutive) enzymes even when grown on
acetic acid (without PAA in the medium). This response
is atypical for most aquatic bacteria obtained by conven-
tional means and seems to correlate with the organism's
ability to grow at low concentrations.
Further studies will be undertaken to more fully char-
acterize this constitutive response. If the response is com-
mon in bacteria growing at low nutrient concentrations,
the chance for the occurrence of certain types of bio-
degradation processes (cometabolism) for pesticides or
toxicants in aquatic systems could be high.
Biodegradation Workshop
The Proceedings of the Workshop on Microbial Degra-
dation of Pollutants in Marine Environments, hosted April
9-14 by ERL,GB, were published in June 1979 in the
EPA Ecological Research Reporting Series (EPA-600/9-79-
012).
The workshop was sponsored by the EPA Office of
Research and Development, Office of Toxic Substances,
and Office of Pesticide Programs, and Georgia State
University to evaluate biodegradation studies in aquatic
environments, to develop protocols for methodology, and
to define research needs and experimental limitation in
this field.
Government agencies, universities, and industrialists
were represented in workshop sessions concerned with
fate studies, regulation, or production of potential aquatic
pollutants. ERL,GB Microbiologists A.W. Bourquin and
P.H. Pritchard co-chaired the workshop.
Determination of the Environmental Impact of
Substitutable Chemical Pesticides in Agriculturally
Affected Wetlands
J.W. DAY, Jr., S.P. MEYERS, and R.P. GAMBRELL,
Principal Investigators; EPA Grant R804976, Louisiana
State University, Baton Rouge, LA; F.G. WILKES,
Project Officer
This research will attempt to determine: (1) the fate
of pesticides, such as Guthion®, in brackish wetlands;
correlating results with past productivity, nutrient flows,
application rates, and runoff patterns; (2) the fate and
effects of toxic substances applied to test plots; (3) the
persistence of pesticides under varying conditions; and (4)
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Figure 3. Eco-core microcosm from Range'Point, FL, salt marsh. The total 14C in the
water column CD , the ethyl acetate-extractable portion 0 , and the residual
parent compound |^ , are shown as percentages of initial concentrations.
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TABLE la. MASS BALANCE OF CARBON-14 IN SALT-MARSH MICROCOSMS RECEIVING
14C-PENTACHLOROPHENOL (% OF CARBON-14 INITIALLY ADDED)
14co2
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BALANCE OF CARBON-14 IN
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Day 1
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BALANCE OF CARBON-14 IN
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Day
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SALT-MARSH MICROCOSMS RECEIVING
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*S = Sterile
**NS = Nonsterile
***ND = Not Determined
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new techniques for correlating laboratory and field
methods (Fig. 4).
The physical/chemical studies focus on the effects of
different Eh and pH conditions on pesticide stability.
Organisms isolated from batch cultures are tested for
their ability to degrade the pesticide in pure culture.
Another microbiological study concerns the effect of
pesticide addition in a chemostat on microbial popula-
tions from field samples. Effects of pesticides on nitrify-
ing bacteria are under investigation.
The effect of redox potential on the levels of Guthion
in swamp soil at pH 7 is presented in Fig. 5. Guthion
essentially disappeared from the most oxidized suspension
(+450 mv) in 6 days. At +250 mv, approximately 6 parts
per million (ppm) Guthion remained at 6 days, disappear-
ing by day 12. Moderate and strong reduction conditions
(+5, -150 mv) decreased the rate of Guthion loss com-
pared to better-oxidized treatments; some Guthion
(approximately 0.5 ppm) was recovered at 20 days.
Several microorganisms in soil-water microcosms that
exhibit an ability to degrade Guthion have been isolated;
studies on rates, utilization, and degradation of Guthion
are in progress. In addition, several organisms have
demonstrated resistance to the pesticide. No significant
change in growth rate as a result of pesticide exposure
has been found.
Guthion was found to be very toxic to benthic inverte-
brates in field studies. At concentrations above 1 ppm,
the community population was reduced at 95% (many
species were reduced by 100%). System alteration
occurred even at 0.01 ppm.
The population of benthic communities was reduced
by 84% as long as 26 days after exposure. The complete
recovery time of swamp communities has not yet been
determined; oligocheates and tubellarians appear to
recover first and to be affected the least.
Fate and Effects of Atrazine in Salt-Marsh
Ecosystems
D.E. DAVIS, Principal Investigator; EPA Grant R803835,
Auburn University, Auburn, AL;
F.G. WILKES, Project Officer
Effects of the herbicide, atrazine, on the marshgrass
(Spartina alternaflora), fiddler crab, periwinkel snail, mus-
sel, and detritus were determined in model ecosystems.
Atrazine stress effects in a natural salt marsh were com-
pared with effects observed in the laboratory.
Atrazine was sprayed at 0.0, 0.5, 5.0, and 50.0 kg/ha
on triplicate plots in a salt marsh on Sapelo Island, GA,
and on microecosystems built to simulate the salt marsh.
Residue levels were determined 3 months after spraying.
The top 25 cm of Sapelo soil treated with 50 kg/ha con-
tained approximately 4% of the atrazine applied; the
microecosystems contained approximately 3%. Atrazine
concentrations in 0 to 1, 1 to 10, and 10 to 25 cm of
Sapelo Island soil averaged 1.18, 0.74, and 0.23 ppm,
respectively. Levels from the microecosystems were lower.
Soils from plots receiving 5.0 or 0.5 kg/ha contained the
lowest atrazine concentrations, which were 0 to 0.04 ppm
below the top 1-cm layer.
Harvested Spartina was divided into: living plants less
than 0.5 m, living plants larger than 0.5 m, and dead
plants. Residues for these portions of plants from Sapelo
Island treated at 50 kg/ha were 2.14, 12.6, and 0.4 ppm,
respectively, and for the microecosystems, 16.8, 21.1, and
25.1, respectively. Plants receiving 5 kg/ha had <0.5 ppm
atrazine; those receiving 0.5 kg/ha had many values at or
near 0.
Periwinkle snails, mussels, and fiddler crabs from
microecosystems treated with 50 kg/ha exhibited atrazine
concentrations of 7.7, 3.4, and 0.28 ppm, respectively. At
Sapelo Island, residue levels in snails and mussels were
0.38 and 0.02 ppm, respectively. There were too few fid-
dler crabs to assay. None of the animals from the lower
treatment rates had greater than 0.2 ppm atrazine, and
most had essentially none. Final report for the project
will be published in the EPA Ecological Research Series
in late 1979.
Fate of 14-C Kepone in Estuarine Microcosms
R.L. GARNAS, Research Chemist
In experiments using static and flowing estuarine
microcosms, 14-C Kepone did not degrade under any
simulated environmental conditions, but adsorbed rapidly
to a variety of sediments.
Adsorption data fitted linear isotherms for a broad
range of water concentrations (Fig. 6). The partition co-
efficient (Kp) of Kepone between sediment and water
increased with increasing sediment organic carbon content
(O.C.) from quartz sand (O.C. <0.01%) to a salt-marsh
sediment (O.C. = 25%).
Field samples (designated with triangles and squares in
Fig. 6) from the James River showed a similar concentra-
tion dependence on the organic carbon content of sedi-
ment. The Kp's for these samples were never below 1000,
because the analytical limits of detection were insufficient
for lesser concentrations.
Although a decomposed seagrass substrate (ground
Thalassia) displayed a greater organic carbon content
(O.C. = 60%) than a local salt-marsh sediment (O.C. =
25%), the Kp of the seagrass was less. In larger flowing
systems, benthic polychaetes (Arenicola cristata) accumu-
lated high residues of Kepone, died, and decomposed;
however, the Kepone residue associated with this sub-
strate did not desorb as compared to the salt-marsh sedi-
ment in the system.
Kepone desorbed readily from salt-marsh sediments
and James River sediments and was independent of typi-
cal environmental temperature, salinity, and pH ranges.
The discrepancies with the decomposed seagrass Kp
and the lack of desorption from decomposed animal tis-
sue would imply that the quality, as well as the quantity,
of organic carbon in sediments influence the partitioning
of Kepone between sediment and earth.
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FIELD STUDIES
General Ecology of Swamp
Pesticide Application
DUCKWEED
Effect of pesticide on
1. fauna
2. metabolism
mud and water samples
MICROBIOLOGICAL
STUDIES
ISOLATION OF ORGANISMS
Chitinoclasts
Heterotrophs
effect of bacteria
on pesticide
Pesticide
CHEMOSTAT
samples
pure culture
with pesticide addition
samples
effect of pesticide
on microbiology
1. Diversity \
2. Numbers ^
Effect of pesticide on pure cultures
of nitrifying bacteria
PHYSICAL/CHEMICAL
STUDIES
. Pesticide Analysis
degradation products
effect of
pH, Eh
on pesticide
stability
samples
Figure 4. Schematic diagram of the organization of the project.
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Figure 5. The effect of oxidation-reduction conditions on Guthion
loss from swamp soil suspensions maintained at pH 7.0.
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Figure 6. Adsorption isotherms for Kepone in sediments. Triangles and boxes represent
data obtained from the James River System.
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Figure 7. Biological Aide J. V. Wheat fills a carrier control reservoir in a toxicant
delivery apparatus for life-cycle toxicity tests using decapod crustaceans.
10
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EFFECTS ASSESSMENT
ERL,GB's Effects Assessment program yields data
applicable to EPA requirements for the registration and
reregistration of pesticides, development of water quality
standards, the issuance of permits for waste disposal in
ocean waters, and effluent limitation hearings.
In 1978, Laboratory personnel attempted to develop
or improve methods to determine acute (short-term) or
chronic (longer-lasting) effects of pesticides and other
toxicants on the aquatic environment.
In acute toxicity tests, organisms are exposed to a
series of concentrations sufficient to establish (with sta-
tistical and chemical precision) the concentrations that
reduce survival, growth, or other biological responses.
Chronic effects of pollutants are determined by tests
with animals in sensitive life stages (particularly the
embryonic and larval stages) or throughout an entire life
stage. ERL.GB scientists monitor the effects of toxicants
on survival, growth, and reproduction by aquatic species.
In addition, effects on the extent and rate of develop-
ment and on species composition are studied in tests with
communities of species exposed to toxicants.
In this reporting period, Laboratory personnel contin-
ued to investigate the bioconcentration of pesticides from
water by estuarine organisms and the accumulation of
these substances in the marine food chain.
The Laboratory's analytical chemistry section in 1978
analyzed approximately 3000 water or tissue samples used
in aquatic bioassays for the following pesticides or other
organic chemicals: EPN, phorate, DBF®, Trithion®,
Guthion®, endosulfan, toxaphene, Aroclor® 1254,
Surflo®, pentachlorophenol (PCP), Dimilin®, methyl
parathion, trifluralin, and carbaryl.
Acute Toxicity Tests (Dynamic)
S.C. SCHIMMEL, Research Aquatic Biologist;
J.M. PATRICK, Jr., Biological Lab Technician
Data from acute toxicity tests are required by EPA for
the development of Water Quality Criteria, evaluations of
new, "substitute chemicals," and for pesticide registration,
commencing with a Rebuttal Presumption Against Regis-
tration (RPAR).
In 1978, acute toxic effects of the defoliant, DEF, and
the insecticides, EPN, methyl parathion, phorate, and
Trithion were determined in tests using estuarine orga-
nisms (Table 2).
In tests with three or more species, the commercially
important pink shrimp (Penaeus duorarum) was the most
sensitive species tested. Its 96-hr LC50 values (concentra-
tion estimated to be lethal to 50% of test organisms) were
computed to be 1.15 yg/£ or less for all five toxicants.
Pink shrimp were found to be less sensitive to DEF, but
ten times more sensitive to the defoliant than the other
estuarine species tested (sheepshead minnows [Cyprinodon
variegatus] , pinfish [Lagodon rhomboides] , and spot
[Leistomus xanthurus} ).
Bioconcentration Tests
A long-term Trithion® bioconcentration study (15-day
uptake, 4-day depuration) in 1978 exposed juvenile fish,
spot (Leiostomus xanthurus) to 50 yg/£ and 5.0 jjg/£ con-
centrations of the insecticide. Mortality of spot was
observed in the 50 yg/£ concentration; therefore, these
data were not used. In the 5.0 yg/£ concentration (3 pg/£
measured concentration), uptake was rapid and 90% of
steady-state accumulation was observed in 4 days (Fig. 8).
After 15 days, the Trithion delivery was discontinued.
Approximately 90% of the accumulation of Trithion at
steady-state was depurated in 4 days. The steady-state
Bioconcentration Factor (BCF) for Trithion was approxi-
mately 600X (based on measured 3.0 pg/£).
Acute Toxicity Tests (Static)
P.W. BORTHWICK, Research Biologist
The lethality of industrial effluents and extracts was
investigated in bioassays using the mysid shrimp
(Mysidopsis bahia) and the sheepshead minnow
(Cyprinodon variegatus). Acute, static toxicity tests (96
hr) were conducted with effluent samples from a gun-
powder, paper products, and creosote plant (Fig. 9).
Sheepshead minnows survived for 96 hr in all three
samples. No fish mortality occurred in concentrations of
100, 56, 32, 18, and 10% effluent, or in controls.
Mysid shrimp, in contrast, appeared to be sensitive to
effluents from the gunpowder and paper manufacturing
plants. Mysid mortality was significant in 96-hr exposures
to 32, 18, 10, 5.6, and 3.2% effluent of paper products
plant. In the gunpowder plant effluent, concentrations of
100, 32, and 10% resulted in 100, 30, and 0% mysid mor-
tality. Laboratory findings correlated with simultaneous
on-site effluent tests conducted in ERL.GB's Mobile Bio-
assay Laboratory by Dr. Alan Auwarter.
Chronic Toxicity Test (Fish)
D.J. HANSEN, Research Aquatic Biologist;
L.R. GOODMAN, Research Biologist
Life-cycle toxicity tests with fish provide important
data necessary to evaluate the environmental hazard of
pesticides and other substances. Such tests may require
several months to complete. Tests of shorter duration,
however, can provide initial estimates of chronically
acceptable concentrations.
©Registered trademark
11
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TABLE 2. ACUTE (96-HR) FLOW-THROUGH TOXICITY TESTS
Pesticide
DEF
EPN
Methyl
Parathion
Phorate
Trithion
Species
Penaeus duorarum
Cyprinodon variegatus
Lagodon rhomboides
Leiostomus xantburus
Penaeus duorarum
Cyprinodon variegatus
Lagodon rhomboides
Leiostomus xanthurus
Penaeus duorarum
Mysidopsis bahia
Penaeus duorarum
Cyprinodon variegatus
Leiostomus xanthurus
Penaeus duorarum
Leiostomus xanthurus
Measured LC50, yg/2,
(95% C.I.)
13.7
(10.5-18.4)
>438
286.2
(237-374)
127.5
(101.5-165.6)
0.29
(0.1-1.1)
188.9
(150.0-255.2)
183
(14.7-23.5)
25.6
(19.2-34.2)
1.15
(0.91-1.40)
0.31
(0.22-0.43)
0.11
(0.08-0.16)
1.28
(0.97-1.74)
3.91
(3.11-5.62)
0.47
(0.37-0.66)
>178
Mean Bioconcentration
Factor
ND1
220
343
67
ND
774
205
ND
ND
ND
127
Lost sample
260
= nondetectable in tissues
12
-------
10000
1000
O
o.
CO
100
cc
Detection Limit
6 9
- UPTAKE -
12
15 18 21
—DEPURATION
TIME (days)
Figure 8. Bioconcentmtion of the insecticide Trithion by the estuarine fish, spot
(Leiostomus xanthurus) in a 15-day uptake, four-day depuration study.
Trithion exposure concentrations were 50 ng/H and 5.0 \jg/H.
The search for "rapid tests" using saltwater fishes is
complicated. Few life-cycle tests have been completed and
only one saltwater species, the sheepshead minnow
(Cyprinodon variegatus), has been successfully tested. In
1978, life-cycle tests were continued with the sheepshead
minnow to expand the data base for reference to "short-
cut" methods such as embryo/juvenile, behavioral, or
physiological tests. "Short-cut" testing utilized chemicals
previously tested in life-cycle tests. Such tests include:
embryo/juvenile tests lasting 28 days, measurements of
stamina, and measurement of acetylcholinesterase in fishes
exposed to organophosphate pesticides.
Life-cycle Tests
Sheepshead minnows were used in 1978 in partial life-
cycle or entire life-cycle toxicity tests with toxaphene,
EPN, and Guthion. Although data from these tests have
not been analyzed statistically, visual examination of the
data suggests the maximum acceptable toxicant concen-
trations (MATC's) shown in Table 3.
In addition to measuring the effects of Guthion, EPN,
and diazinon on survival, growth, and reproduction in life-
cycle toxicity tests, the enzyme acetycholinesterase
(AChE) was monitored to determine if it can be used as
an indicator of chronic effects of organophosphate or
carbamate pesticides. The diazinon test was completed in
1978, and the Guthion test is in progress. Data for the
EPN test have not been statistically analyzed.
Egg production (basis of the maximum acceptable toxi-
cant concentration [MATC] in the diazinon experiment)
may have been impaired in the EPN experiment, and
apparently was impaired in the Guthion experiment.
These three experiments indicate that the average num-
ber of eggs per female per day may be reduced concur-
rently with AChE activity of about 20% of control activ-
ity. However, egg production in the diazinon experiment
was impaired with AChE activity of 78% of control activ-
ity. MATC's and AChE activity will be compared with
data from EPN and Guthion experiments.
13
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Figure 9. Biological Aide K.J. Butler exposes sheepshead minnows to industrial
effluents in acute static toxicity test.
Embryo/Juvenile vs Life-cycle Tests
Embryo/juvenile toxicity tests with the sheepshead
minnow may provide as reasonable an initial estimate of a
maximum acceptable toxicant concentration (MATC) as
that obtained from life-cycle toxicity tests (Table 3). The
duration of embryo/juvenile tests is usually 4 weeks, as
compared to the 12 to 24 weeks required for a partial or
complete life-cycle test. Tentative results of embryo/
juvenile tests predicted MATC's in life-cycle tests within a
factor of 5 for 75% of 9 chemicals tested. Three chemi-
cals for which embryo/juvenile tests poorly predicted the
MATC's caused adverse effects on reproduction or growth
of progeny in life-cycle tests.
Although tests conducted at other laborateries have'
demonstrated an excellent correlation between "no-effect
concentrations" in embryo/larval tests and MATC's in
life-cycle tests with freshwater fishes, ERL.GB researchers
believe that additional testing on marine fishes is neces-
sary before results from embryo/juvenile or other ''rapid
tests" can be substituted for results from life?cyele tesfli
Future tests have been recommended in these areas: (1)
additional life-cycle exposures using the sheepshead min-
now; (2) development of embryo/juvenile (lairval1) and life-
cycle methods for additional marine fishes? (3) examitte-
tion of additional "rapid tests" as predictors of life-cycle
MATC's.
14
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TABLE 3. ESTIMATED MATC's FOR THE SHEEPSHEAD MINNOW (CYPRINODON VARIEGATUS)
Geometric Mean MATC, yg/£
Chemical
Endrin
Carbofuran
Malathion
Methoxylchlor
Pentachlorophenol
Trifluralin
Chlordane
Kepone
Diazinon
Toxaphene**
Guthion**
EPN**
Embryo/Juvenile
Tests
0.19
18
6
1.7
275
13
7.1
>0.8
>6.5
0.7
0.6
>10
Life-Cycle
Tests
0.19
18*
6*
1.7*
64
2.5
0.6
0.087
<0.47*
0.7
0.3*
5.7
Quotient
1
1
1
1
4.3
5.2
142
>9.2
>13.8
1
2
>1.8
Most Sensitive
Life Stage
All
Fry Survival
Fry Survival
Hatching & Adult
Survival
Parental Survival
Eggs Spawned
Eggs Spawned
Growth
Eggs Spawned
Fry Survival
Eggs Spawned
Survival, growth,
& Eggs Spawned
^Partial life-cycle test
*Data are based on visual inspection and not statistical analyses; therefore, conclusions must be considered provisional.
Community Bioassays
M.E. TAGATZ, Research Aquatic Biologist;
J.M. IVF.Y, Biological Technician
A community bioassay is designed to determine the
effects of a toxicant on many different types of settling
benthic organisms. In 1978, ERL.GB tests sought to assess
the effects of toxicants in flowing seawater on planktonic
larvae that are allowed to colonize in sand-filled aquaria.
Aquaria are arranged in groups of eight (Fig. 10); all
but one group are continuously exposed to different con-
centrations of a toxicant. After 7 or more weeks, the
number and species of animals (macroinvertebrates) that
developed in exposed and non-exposed aquaria are com-
pared statistically.
Sevin®, a widely used carbamate insecticide, altered
development of estuarine communities in ERL.GB tests.
The harvest after 10 weeks exposure yielded 7,844 ani-
mals, representing 29 species of 7 phyla. Average number
of species per aquarium was significantly less (a = 0.05)
in aquaria containing 11.1 or 103 pg/£ than in those con-
taining 1.1 jjg/& or in control aquaria.
The amphipod (Corophium acherusicum) was particu-
larly affected; significantly fewer were found in all con-
centrations than in the control.
A marked increase in the abundance of the annelid
(Polydora ligni) in aquaria containing 103 yg/£, corre-
sponded to a marked decrease in the number of other
annelids and to a significant absence of Nemertea.
The abundant clam (Ensis minor) grew significantly
less in length at the higher concentrations of Sevin.
©Registered trademark
15
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Seowater
Syringe Pump
Splitter Box
Delivery Port
(200ml/min)
Figure 10. One of four identical apparatuses (associated with a common splitter box
and consisting of eight aquaria) used to test effects of toxicants on develop-
ment of macrobenthic communities,
Average lengths in mm were 8.5 (control), 8.0 (1.1 yg/£),
6.7 (11.1 pg/£), and 6.4 (103 pg/£).
Food Chain Bioassay
L.H. BAHNER, Aquatic Research Biologist
Food-chain experiments conducted at ERL,GB
attempted to determine and measure variables that could
affect pesticide concentrations in predatory fish.
Experiments focused on three variables that affect pes-
ticide transfer: (1) pesticide concentration; (2) predator
feeding rates; (3) types of food consumed as a regular
diet.
In the first experiment, 23,400 amphipods were con-
taminated with replicate concentrations of 14-C Kepone
and fed to 75 juvenile spot (Leiostomus xanthurus) that
were maintained in both Kepone-free and Kepone-
eontaminated water.
In the second experiment, 36 spot were fed 720,
2,160, or 3,600 amphipods that contained the same con-
stant residues of Kepone fed to the predatory fish.
In the third experiment, mysids, amphipods, and
sheepshead minnows containing similar concentrations of
Kepone were fed to spot. All experiments lasted for 21
days.
Results of the first study indicate that the pathways of
uptake of Kepone from water and food by spot are inde-
pendent and additive. A pesticide uptake/depuration
model developed at ERL.GB determined that Kepone
uptake by spot from food was not affected by previous
or simultaneous exposure to Kepone in water. Bioaccum-
ulation factors for spot consuming 14-C Kepone-dosed
amphipods ranged from 0.42 to 0.47.
16
-------
In the second test, the equilibrium concentration ratio
increased from 0.5 to 0.9 in spot fed varying amounts of
food containing the same concentrations of Kepone, as
the amount of food consumed increased from Ix to 5x.
In the third test, results indicated that variation in the
type of food consumed did not appear to be as important
a factor in the equilibrium concentration as the quantity
of food consumed.
Thus far, ERL.GB food chain experiments suggest that:
(1) field tests can be closely simulated in the laboratory
if all main variables are tested (water, sediment, food);
(2) fate of pesticides in predatory animals can be pre-
dicted mathematically if the rates of pesticide transfer
from one trophic level to another can be derived from 3
or 4 laboratory experiments.
Predictive Models
Stochastic Uptake/Depuration Model
L.H. EARNER, Aquatic Research Biologist;
J.L. OGLESBY, Statistician
The bioconcentration of pesticides by estuarine animals
from water and their bioaccumulation in food sources are
useful indicators of pesticide movement in the natural
environment. Laboratory and field studies have demon-
strated that food is a prime source of pesticide contami-
nation in certain estuarine species.
Investigators at ERL,GB in 1978 examined the impact
of pesticide transfer to estuarine animals via food, water,
or bottom sediment through: (1) analyses of laboratory
studies and field surveys; (2) models designed to predict
movements of pesticides in ecosystems.
Flow-through laboratory experiments with oysters,
worms, shrimp, crabs, and fish indicated that the food-
chain transfer of the pesticide Kepone was important in
predicting Kepone residues in estuarine organisms. There-
fore, a generalized mathematical equation was developed
to describe the uptake of Kepone by such organisms. The
model describes biological data as a single equation, thus
allowing variations (due to many physical, chemical, bio-
logical, and random-error factors) to be analyzed
simultaneously.
Kepone uptake from water, food, and sediments by
estuarine invertebrates and fishes (Fig. 11) were analyzed
by the nonlinear equation,
Y = P1/(1+P2**(TIME-P3)), which requires estimation of
three parameters (PI, P2, and P3). The maximum pre-
dicted residue concentration (yg/g, ppb) in the exposed
organism is equal to EXP(Pl); the bioconcentration factor
is, therefore, EXP(Pl) exposure concentration. The bene-
fits of using such a model are: (1) more realistic Biocon-
centration Factors (BCF's) can be calculated from the
data; and (2) differences due to exposure method or
media, concentration, or time can be statistically analyzed.
(a) Plot of hypothetical uptake curves.
Model equations:
Y (i) + Pl(i)/(l+P2(i)**(TIME-P3(i)))
= 7.5/(l+.8**(TIME-3))
Y (j) = Pl(j)/(l+P2(j)**(TIME-P3(j)))
= 5.0/(1+.9**(TIME-1.7))
(b) Uptake of Kepone from flowing water by:
Oysters (exposed to 0.39 \ig/H):
Y = 7.88/(l+0.59**(TIME-1.81))
Lugworms (exposed to 0.29 yg/&):
Y = 6.72/(l+0.63**(TIME-3.98))
Lugworms (exposed to 0.039
Y = 5.01/(1+0.90**(TIME-0.45))
(c) Uptake of Kepone from food by:
Spot (exposed to 2.0 yg/g):
Y = 6. 817(1+0. 79**(TIME- (-0.47)))
Blue crabs (exposed to 0.25 yg/g):
Y = 5.97/(l+0.95**(TIME-0.0))
(d) Uptake of Kepone from sediments by:
s*
Fiddler crabs (exposed to 0.25 yg/g):
Y = 5.5/(l+0.27**(TIME-10.6))
Lugworms (exposed to 0.25 yg/g):
Y = 5.5/(l+0.73**(TIME-(-0.46)))
Blue crabs (exposed to 0.25 yg/g):
Too few data; analysis not accomplished.
Algal Stimulation and Inhibition Statistical Model
L.H. EARNER, Aquatic Research Biologist;
G.E. WALSH, Research Ecologist
Growth of estuarine algae grown in flask cultures under
controlled conditions indicates that additions of pollutant
chemicals to algal growth media can cause increased
growth (stimulation), decreased growth (inhibition or
toxicity), or "no effect" when compared to control treat-
ments.
Historically, straightline interpolation or probit analysis
have been applied to such data for estimation of the
EC50 (concentration of toxicant that inhibited growth to
50% of the untreated control); in most studies, stimula-
tion might be noted, but not treated statistically. Since
increases of minor nutrients can cause quite dramatic
eutrophication, it would be beneficial to analyze stimula-
tion data so that projections of environmental hazard can
be implied.
17
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P3(j)
EXPOSURE PERIOD-
OYSTER (0.39jjg'»
, • • +
*_J !
• LUGWORM (029yg/l)
*—*
LUGWORM (0.039 jjg/l)
0 7 14 21 28 35
EXPOSURE PERIOD (days)
10000
10
SPOT (2.0ng/g)
BLUE CRAB (0.25 yg/g)
o
fr
5Y CONCENTI
i
IOOOO
1000
5" 100
10
w
FIDDLER CRAB
-/ ,
. .sf*"^
i •""* 7*~~
• ^ol0**! * BLUE CRAB
i r» y^\ IJQ/Ql
vvJ*bO UQ/QJ
14 21
(025yg/g)
28
EXPOSURE PERIOD
EXPOSURE PERIOD (days)
Figure 11. Uptake of Kepone from water, food, and sediment by marine organisms.
The following statistical model was designed to analyze
algal stimulation and/or inhibition data:
Y = Growth Index = P6/(1+P7**(CONC-P8))
P6/(1+P9**(CONC-P10)).
Growth Index is the dependent variable expressed as
culture density, % of control, or other measure of growth;
parameter P6 is the maximum predicted growth (units are
those of Y);
parameter P7 is the stimulation function slope;
CONG is the toxicant concentration of the test
cultures;
parameter P8 is the concentration of toxicant that is
predicted to cause growth equal to 0.5 * P6;
parameter P9 is the inhibition function slope; and
parameter P10 is the concentration of toxicant that is
predicted to cause growth inhibition equal to 0.5 * P6.
Application of this model to algal bioassay data from
selected industrial effluent tests is graphically illustrated
to show the versatility of this model (Fig. 12) for describ-
ing stimulation or inhibition compared to control growth.
The model for algal growth was applied to data from
14 assays of textile plant effluents. Application of the
model allowed concentrations of effluents that caused
20% stimulation or 50% inhibition of growth to be
calculated.
Thirteen of fourteen data sets were easily modeled.
Analysis could not be completed for one data set.
18
-------
X
Ul
Q
O
P6/2 -/
EFFLUENT CONCENTRATION (%)
10 30 50 70 9O
EFFLUENT CONCENTRATION (%)
200
012345
EFFLUENT CONCENTRATION (%)
EFFLUENT CONCENTRATION (%)
Figure 12. Effects of textile industry wastes on growth of Skeletonema costatum.
(a) Plot of hypothetical algal stimulation
and inhibition of growth curve.
Model equation:
Y = P6/(1+P7**(CONC-P8))
P6/(1+P9**(CONC-P10)).
(b) Growth stimulation.
Y = 238./(l+0.94**(CONC-11.42)).
(c) Inhibition of growth.
Y = 125.-125./(l+0.47**(CONC-2.33)).
(d) Stimulation and inhibition of growth by
a single waste.
Y - 182./(l+0.78**(CONC-3.49)) -
182./(l+0.84**(CONC-65.77)).
Effects of Toxicants on Selected Estuarine Flora
and Fauna
GERALD E. WALSH, Research Ecologist
Industrial Waste
The Industrial Waste Program at ERL.GB is designed to
identify toxic outfalls from energy-producing and other
industries and to characterize the toxic wastes biologically
and chemically. Biologists and chemists in 1978 devised a
scheme for integrated biological and chemical tests that
identify toxic fractions of liquid wastes.
19
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Thirty outfalls from sewage treatment plants and indus-
tries such as textiles, pulp and paper, chemicals, and steel
were investigated in 96-hr tests using the alga (Skeleto-
nema costatum) and the mysid (Mysidopsis bahia). Whole
wastes and their chemical fractions were examined for
acute toxicity to the alga and mysid and for stimulation
of growth of the alga.
Of the 30 tested, only one waste (steel plant effluent)
had no effect on the organisms. Some were toxic to algae
and mysids at relatively low concentrations. The response
of (Skeletonema costatum) to whole wastes from textile
industry plants is shown in Fig. 12.
Growth of Skeletonema costatum in whole waste and
various fractions of waste from a sewage treatment plant
showed that toxicity of waste was due to heavy metals
(Table 4). The non-heavy metal subfraction was highly
stimulatory to algae, a property of the waste that would
not have been found without chemical fractionation.
Some industrial wastes tested in the laboratory were
also tested in field by flow-through bioassays of
Mysidopsis bahia in a mobile laboratory. The data con-
firm that some wastes contain both toxic and stimulatory
substances (Table 5).
ERL.GB researchers found that both toxic and stimu-
latory effects must be considered in assessment of poten-
tial impact 'of an outfall on receiving water before con-
trol technology is applied.
TABLE 4. EFFECTS OF WASTE FROM A SEWAGE TREATMENT PLANT AND ITS CHEMICAL FRACTIONS ON
GROWTH OF SKELETONEMA COSTATUM*
PERCENTAGE WASTE
EC50
SC20
Whole waste
Organic fraction
Inorganic fraction
Heavy metal subfraction
Non-heavy metal subfraction
15.4
NE
15.5
16.5
NE
NE
NE
0.4
NE
0.9
*The EC50 is the calculated concentration that would inhibit growth by 50%.
The SC20 is the calculated concentration that would stimulate growth by 20%.
NE = no effect.
TABLE 5. EFFECTS OF WHOLE WASTE ON MYSIDOPSIS BAHIA AND SKELETONEMA COSTATUM
PERCENTAGE WASTE
INDUSTRY
Kraft Mill
Textiles
Chemicals
Chemicals
MYSID OPS IS BAHIA
LC50
8.7
6.3
13.3
NE
SKELETONEMA COSTATUM
SC20
0.7
NE
2.7
4.2
EC50
NE
0.08
79.0
NE
20
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Pesticides
Life-Cycle Tests (Mysid)
ERL,GB maintains the capability for rapid screening of
the toxicity of pesticides to algae. Skeletonema costatum,
a chain-forming diatom, is usually the test species, but
several other diatoms and green and red algae are main-
tained for use when needed.
Five pesticides were tested under the RPAR program
for toxicity with Skeletonema costatum. Their 96-hr
ECSO's and no-effect concentrations (parentheses) are:
Trithion, 0.12 ppm (0.01 ppm); DEF, 0.36 ppm (0.20
ppm); EPN, 0.37 ppm (0.01 ppm); Phorate, 1.29 ppm
(0.01 ppm); and methyl parathion, 5.00 ppm (1.00 ppm).
Water Quality and Eutrophication Studies in
Santa Rosa Sound
G.A. MOSHIRI, Principal Investigator; EPA Grant
R805366, University of West Florida, Pensacola, FL;
G.E. WALSH, Project Officer
Water samples are collected biweekly from the surface,
mid-depth, and bottom of five stations near the ERL.GB
laboratory. The same stations are also monitored over 48-
hr periods in summer, fall, winter, and spring. Parameters
such as BOD, COD, and concentrations of oxygen, nitro-
gen, phosphorous, and chlorophyll were measured;
detailed description analyses of the phytoplankton assem-
blages are being made on a seasonal basis.
D.R. NIMMOt, Research Ecologist;
T.L. HAMAKER, Biological Technician;
E. MATTHEWS, Biological Laboratory Technician
In 1978, ERL,GB investigators continued to build the
data base of the chronic effects of pesticides on the life-
cycle of mysid shrimp (Mysidopsis babia).
In response to requests from the Office of Pesticide
Programs (OPP), four RPAR pesticides were tested for
chronic toxicity to mysids. The results of these 28-day
tests showed mysids to be highly sensitive to these pesti-
cides (Table 6).
In each test, the number of offspring per female was
used to determine the MATC (estimated maximum accept-
able toxicant concentration) values. As in the past, this
criterion appears to be the most sensitive indicator of
chronic toxicity of pesticides to mysids. A no-effect con-
centration could not be determined for the organophos-
phate defoliant DEF.
Life-cycle tests to examine the effect of turbidity on
mysids in flowing water were undertaken in 1978 in.
response to requests by the Florida Department of
Environmental Regulation. Clean bottom sediments from
East Bay, near Pensacola, FL, were tested. Preliminary
tests indicated that turbidity at approximately 1 g/£ of
sediment affected the reproductive success of mysids.
Further development of this flow-through test method
could be of importance in testing the effects of various
complex effluents and sediments on the life-cycle of
mysids.
TABLE 6. RESULTS OF TOXICITY TESTS WITH MYSIDOPSIS BAH1A
Compound
DEF
EPN
METHYL PARATHION
PHORATE
96-hr LC50*
4.36
3.24
0.78
0.37
MATC*
0.34
0.44-3.24
0.11-0.37
0.09-0.21
AF**
0.08
0.14-1.0
0.13-0.47
0.24-0.57
*Micrograms per liter
* Application factor (AF) limits are derived by dividing the Maximum Acceptable Toxicant Concentration limits
by the 96-hr LC50.
tCurrent address, Environmental Research & Technology, P.O. Box 2105,
1716 Heath Parkway, Fort Collins, CO 80522
21
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Figure 13. Biological Aide R.D. Fink counts grass shrimp larvae to determine chronic
effects of Trithion on survival and length of larval development (time from
hatching to completion of metamorphosis),
Life-cycle Toxicity Tests (Decapod Crustaceans)
D.B. TYLER-SCHROEDER, Research Biologist
The grass shrimp (Palaemonetes pugio) was exposed to
the organophosphate, Trithion, at various life stages to
determine its usefulness in assessing the toxicity of
pollutants.
Prior to the life-cycle experiments, a series of 96-hr
acute exposures were conducted with field-collected and
laboratory-reared juveniles to establish concentrations for
the life-cycle exposures and to contrast sensitivity of the
two groups of shrimp. The 96-hr LCSO's were: 3.4 \ig/H
for field shrimp and 2.8 pg/£ for laboratory-reared shrimp.
In 1978, the flow-through system for life-cycle tests
was improved by the addition of a diluter that allowed
fresh water or high-salinity brine to be added to the
ambient, filtered seawater. The diluter allowed greater
control of salinity during freshets or periods of extreme
fluctuations in salinity. Salinity is viewed as a critical
factor in the Survival of grass shrimp during larval develop-
ment.
Although tests are not complete, preliminary results
indicate that long-term effect concentrations of Trithion
on grass shrimp lie close to the 96-hr LC50 concentration
established in the laboratory (Fig. 13). As in earlier tests,
the shrimp appeared to be most sensitive to the toxicant
during their reproductive cycle. Criteria for effects on
reproduction included: the number of females spawning,
the number of eggs laid per female, and the number of
larvae hatching per female in each concentration.
22
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Figure 14. Research Biologist R.A. Rigby removes oyster from Sediment Bioassay
System to measure growth rate.
Effects of Kepone on Development of the Blue
Crab and Mud Crab
C.G. BOOKHOUT and J.D. COSTLOW, Jr., Principal
Investigators; EPA Grant R803838, Duke University,
Durham, NC;
D.B. TYLER-SCHROEDER, Project Officer
Experiments were conducted to determine the effect
of Kepone on the development of the blue crab
(Callinectes sapidus), from the time of hatching until the
1st crab stage. For comparison, the effects of Kepone on
larval development of the mud crab (Rhithropanopeus
harrisii) also were investigated.
Of the concentrations tested, 35, 50, 65, and 80 parts
per billion (ppb) Kepone were found to be sublethal; 95,
110, and 125 ppb Kepone were acutely toxic to R.
harrisii larvae. In contrast, 0.1, 0.5, and 0.75 were sub-
lethal, and 1.0 ppb Kepone was acutely toxic to C.
sapidus larvae.
The duration of zoeal development in R, harisii and
total time from hatching to 1st crab generally were pro-
longed with concentration. In C. sapidus, no significant
relationship could be detected between Kepone concen-
tration and duration of zoeal development, but there was
a significant relationship to 1st crab. The developmental
stages in which the larvae are particularly sensitive dif-
fered in the two species.
23
-------
EXPOSURE SYSTEM
F
Figure 15. Sediment Bioassay System. Test sediment is introduced into 10-gallon test
aquaria (H) containing bioassay organisms by means of a sediment dosing
apparatus (A). Sediment is suspended by recirculating pumps (E) and is
metered into the individual test aquaria from a constant head box through
glass tubes (D) fitted with a length of silicon hose that is threaded through
two aluminum bars (C) normally crimped shut by spring tension. The alu-
minum bars are attached to timer operated solenoids (B). When energized,
the solenoids retract allowing the sediment -water mixture to flow to the
appropriate aquaria. Ambient unfiltered seawater enters the bioassay sys-
tem from a head box (F) and flows to a splitter box (G) where flow rates
are adjusted prior to entering the test aquaria. Effluent water is routed
through a drain (I) to a holding pond.
Toxic Sediment Bioassay System
N.I. RUBINSTEIN, Research Biologist; EPA Grant
R804458, University of West Florida, Pensacola, FL;
C.N. D'ASARO, Principal Investigator;
F.G. WILKES, Project Officer
Dredged material proposed for disposal into the marine
environment must be evaluated by criteria established and
published by the EPA (Federal Register Vol. 42, No. 7,
January 11, 1977) and by the U.S. Army Corps of
Engineers (USCE) and EPA (Implementation Manual for
Section 103, PL92-532, Marine Protection Research and
Sanctuaries Act of 1972).
An ERL,GB research team in 1978 developed a flow-
through toxicity test (bioassay) (Fig. 14) to determine
biological effects of contaminated sediments on represent-
ative estuarine organisms and developing benthic com-
munities. The objective was to provide a screening tool
that would detect potential hazards of dredge spoils (con-
taminated sediments) prior to their disposal in the marine
environment.
Ten-gallon glass aquaria containing silica sand and flow-
ing unfiltered seawater were utilized as test habitats
(Fig. 15). A number of these aquaria received different
concentrations of test sediments (dredge spoils); other
unperturbed aquaria served as controls. Control and experi-
experimental aquaria were compared to identify acute
24
-------
and sublethal effects caused by exposure to test
sediments.
Test organisms were selected from the three environ-
mental compartments affected by dredging activities:
mysid shrimp (Mysidopsis bahia), a water-column crusta-
cean that often scavenges on or near bottom sediments;
the oyster (Crassostrea virginica), an epibenthic mollusk
that filters surrounding water for its nourishment; and a
deposit-feeding polychaete (Arenicola cristata, commonly
called the lugworm) that lives directly within bottom sedi-
ments and feeds primarily on the overlying detrital layer.
Test criteria were developed to identify effects of toxic
sediments on: (1) survival of mysids; (2) shell deposition
and bioaccumulation of known contaminants by oysters;
(3) substrate-reworking and bioaccumulation by lugworms;
and (4) resilience of the benthic community in terms of
numbers and variety of macrofaunal organisms that
settled onto test sediments from planktonic larvae within
28 days.
To date, tests have been conducted with sediment arti-
ficially spiked with the organochlorine pesticide Kepone
at concentrations of 10.0, 1.0, and 0.1 ppb, and with
dredged materials from the James River and the Houston
Ship Channel.
Mysids, oysters, and lugworms were significantly
affected by Kepone sediments. Significant reductions were
detected in colonizing polychaetes exposed to the highest
Kepone concentration. The pesticide was found to bio-
accumulate in oysters and lugworms.
James River sediment, although not acutely toxic to
test organisms, reduced oyster growth and lugworm re-
working activity. These sublethal responses reflect physio-
logical effects directly related to the metabolism of the
animals. Bioaccumulation of Kepone from James River
sediment was detected in lugworms and oysters at con-
centrations that approximated uptake from 1.0 ppm
Kepone-sorbed sediment. (Subsequent analysis indicated
mat James River sediment contained approximately 0.5
to 1.0 ppm Kepone.) Houston Ship Channel sediment did
not significantly affect test organisms in terms of the bio-
assay response criteria.
At present, dredge material bioassays are not con-
sidered precise predictors of environmental impact due to
the number of variables that cannot be addressed in the
laboratory. However, they are quantative estimators of
environmental effects.
The bioassay developed at ERL,GB provides several
distinct advantages over existing dredged material tests.
Standard bioassay procedures are used to compare test
results with an extensive data base compiled on a wide
variety of marine organisms and toxicants. The test spe-
cies are culturable, thus eliminating conditioning and the
cost and time required for field collection of bioassay
animals. The flow-through seawater design excludes many
artifacts inherent to static bioassay systems and more
realistically simulates actual conditions at the disposal
site. Further, the use of benthic community recruitment
as a test criterion provides a long-term measure of the
impact of dredged material on the marine environment.
Method for Determining PCP in Marine Biota and
Seawater
L.F. FAAS, Chemist;
J.C. MOORE, Chemist
A method for measuring pentachlorophenol (PCP) in
die estuarine environment was described in a paper pre-
sented by two ERL.GB chemists at the 176th National
Meeting of the American Chemical Society in 1978 at
Miami Beach, FL.
The method uses gas-liquid chromatography (GLC) to
determine PCP residues in tissues as low as 0.01 ppm by
die formation of the ethyl derivative, followed by Florisil
cleanup.
Seawater concentrations as low as 0.0002 ppb can be
measured by formation of the amyl derivative. Formation
of the amyl derivatives of PCP gives a GLC separation not
possible with methyl or ethyl derivatives (Table 7). Tests
using the method indicate that PCP accumulates in fish
(Mugil cephalus), shrimp (Palaemonetes pugio), and
oysters (Crassostrea virginica).
Protozoan Studies
N.R. COOLEY, Research Microbiologist
Protozoa, algae, and bacteria form the basis of aquatic
food chains. Of the three, ciliated protozoa are the most
numerous animals in the estuarine benthos and may be
more important than bacteria as nutrient regenerators,
particularly of nitrogen and phosphorus. Previous work
at ERL,GB and elsewhere has demonstrated that some
ciliates can bioaccumulate certain persistent pesticides,
thereby aiding their translocation and possible toxic effect
at higher trophic levels.
ERL,GB researchers in 1978 examined the effects of
toxicants, singly and in combination, on population
growth of ciliate protozoa. Toxicity of nickelous chloride
to Tetrahymena pyriformis W (grown axenically in Tetra-
hymena medium in flask cultures) was investigated alone
and in combination with the carbamate insecticide, car-
baryl (Sevin®).
When tested alone, EC50 for reduction of the. 24-hr
growth rate was 13.09 mg Ni++/£. EC50 for reduction of
population size at 96 hr was 19.74 mg Ni++/£. When
tested in combination with carbaryl, the greatest observed
reduction of 24-hr population growth rate (62.35%)
occurred at 15.79 mg Ni++/£ and 20 mg carbaryl/£. All
combinations of carbaryl and 15.79 mg Ni++/£ reduced
the 24-hr population growth rate more than 50%. Great-
est observed reduction of population size at 96 hr was
36.23% in the combination 15.79 mg Ni++/£ and 30 mg
carbaryl/£.
In another investigation, the ciliate Uronema nigricans
(strain PC) was grown axenically at 27° C in 20 m£ of
Soldo and Merlin's M medium in 250-m£ Erlenmeyer
25
-------
TABLE 7. RETENTION TIMES OF ETHYL AND AMYL DERIVATIVES OF SEVERAL PHENOLS AND ACIDS
RELATIVE TO ALDRIN ON THREE DIFFERENT GLC COLUMNS.
Compound
2,4,6-Trichlorophenol
p-Nitrophenol
2,4,5-Trichlorophenol
Dicamba
2,3,4,6-Tetrachlorophenol
2,4-D
Silvex
Pentachlorophenol
2,4,5-T
Tetrachlorohydroquinone
Aldrin
2%
Ethyl
0.14
0.17
0.20
0.27
0.28
0.38
0.57
0.55
0.62
0.65
1.00
SP2100
Amyl
0.40
0.52
0.54
0.72
0.79
0.98
1.41
1.54
1.64
5.37
1.00
0.75% SP2250:
Ethyl
0.13
0.25
0.20
0.32
0.27
0.48
0.63
0.56
0.80
0.68
1.00
0.97% SP2401
Amyl
0.34
0.65
0.51
0.85
0.73
1.24
1.58
1.52
2.11
5.30
1.00
Ethyl
0.17
0.55
0.28
0.51
0.34
0.79
0.96
0.64
1.20
0.83
1.00
5% QF-1
Amyl
0.40
1.38
0.64
1.19
0.79
1.82
2.06
1.47
2.70
4.45
1.00
flasks and studied as a possible test organism. The tests
used sodium lauryl sulfate as a possible reference chemi-
cal for future toxicity tests and as a means of determin-
ing whether response of this ciliate strain to toxicants will
change during prolonged maintenance in stock cultures.
The EC50 for reduction of 24-hr population growth rate
was 1.78 mg/&, and the EC50 for reduction of the 96-hr
population size was 7.94 mg/£.
Cyclic Burrowing Behavior of Pink Shrimp
C.R. GRIPE, Research Biologist; EPA Grant R804458,
University of West Florida, Pensacola, FL;
C.N. D'ASARO, Principal Investigator;
F.G. WILKES, Project Officer
Pink shrimp (Penaeus duorarum) are among the most
sensitive organisms available for aquatic toxicity testing.
P. duorarum normally remain buried in the substrate dur-
ing the day and emerge at night. Exposure to sublethal
pesticide levels in ERL.GB laboratory tests have caused
this aquatic species to alter its burrowing pattern by
remaining continuously above the substrate. Such behav-
ior in the natural environment would render pink shrimp
more susceptible to predators.
An automated apparatus used earlier at ERL,GB to
monitor the avoidance of a toxicant gradient by a test
animal was adapted in 1978 to studies of abberant behav-
ior in burrowing animals exposed to the pesticide, methyl
parathion. Infrared light sources, sensors, and a micro-
processor were used to monitor elapsed time of light
beam interruption.
Both halves of two troughs were filled with sand and
compartmentalized into four areas by plastic screens. A
shrimp was placed in each area and monitored by two
light beams for 6 days. A regime of 12 hr darkness/12 hr
light was maintained throughout the test.
One trough was exposed to 2 ppb methyl parathion on
days 3 and 4 of a 6-day preliminary test. The other
trough containing four shrimp served as a control. Two of
the four exposed shrimp died. The remaining two were
observed above the sand during the daylight period. The
activity of one surviving exposed shrimp is shown in
Fig. 16.
If normal burrowing behavior is altered in an aquatic
environment, the surviving shrimp would be more vulner-
able to predators that feed in daylight. A reduction in
shrimp population through increased predation might
diminish this important commercial seafood.
Preliminary testing demonstrated that the automated
apparatus developed at ERL.GB is useful in quantifying
aberrant behavior in pink shrimp (Fig. 17).
26
-------
EXPOSURE
A
Figure 16. Activity graph indicating time and light beams (.33 sec/hr) during each hr
of a 6-day test. On days 3 and 4, shrimp were exposed to an average 2.0
ppb methyl parathion.
-------
Overflow
drain
Sand
Phototransistor
17. Diagram of one of two replicate troughs used to study pink shrimp burrow-
ing behavior. The trough is partitioned with barriers of Plexiglas and plastic
screen. The presence of each shrimp above the sand is monitored by two
pairs of pbototransistors and infra-red LED's.
Predator-Prey Bioassay System
C.R. GRIPE, C.E. ASHTON, Research Biologists; EPA
Grant R804458, University of West Florida, Pensacola,
FL; C.N. D'ASARO, Principal Investigator;
F.G. WILKES, Project Officer
Earlier ERL,GB studies on effects of toxicants on an
estuarine predator-prey relationship have indicated that
exposure to certain sublethal pesticide concentrations
increase vulnerability of the prey. Test systems were
modified in 1978, focusing on the effect of the toxicant
on: (1) prey only and (2) exposed and control prey in
the same tank.
Three pinfish (Lagodon rbomboides) and equal num-
bers of toxicant-exposed and control grass shrimp
(Palaemonetes pugio) were placed in two replicate tanks
(2082,) containing removable dividers. Approximately 20
min after the dividers were removed, the surviving shrimp
were counted to determine differential predation between
exposed and control prey. Preliminary tests indicate that
marking the prey by removal of the first right or left
pleopod produces no significant effect on predation.
Significantly fewer (a<0.01) shrimp survived predation
after exposure for 24 hr to 1.2 ppb methyl parathion.
Exposure to 1.3 ppb Trithion for 24 or 72 hr produced
no significant difference in predation.
Role of Benthic Invertebrates on Fate and Trans-
port of Xenobiotics
C.R. GRIPE, C.E. ASHTON, Research Biologists; EPA
Grant R804458, University of West Florida, Pensacola,
FL; C.N. D'ASARO, Principal Investigator;
F.G. WILKES, Project Officer
28
-------
The fate of xenobiotics in estuarine systems is deter-
mined by the quantity added and amount of dilution,
degradative processes (i.e., photolysis, hydrolysis, and
metabolism), sorptive processes, biological accumulation,
and transport phenomena.
Little information exists on the relative importance of
benthic invertebrates on the fate of xenobiotics as com-
pared to other processes.
Previous studies at ERL.GB indicate that benthic inver-
tebrates can have a significant effect on pollutant mobili-
zation into the sediment in relatively large microcosms.
The role of these invertebrates is under study to deter-
mine if the same process could be demonstrated in
smaller, more experimentally manageable systems. Prelimi-
nary tests used small, static estuarine water/sediment
systems to which 14C-methyl parathion was added as the
model xenobiotic.
Replicate systems either with or without a juvenile
polychaete (Arenicola cristata) are being compared to a
formalin control to determine the relative impact of the
invertebrates, microbes, or physical processes (Fig. 18).
Initial data indicate that radioactivity in the water
seems to disappear more rapidly in the polychaete sys-
tems during the first week of exposure than in the other
systems. These differences between the systems, however,
were greatly reduced with continued incubation. Further
analysis of extractable and nonextractable radioactivity
may indicate whether this difference is due to degradation
or transport, or both.
Figure 18. C.E. Ashton and C.R. Cripe remove samples from sediment systems.
29
-------
Figure 19. The American oyster is measured prior to exposure to chlorinated seawater
in a study of its potential to bio accumulate chlorination by-products.
30
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CHLORINATION STUDIES
Large-scale use of chlorine as a disinfectant and an oxi-
dant for drinking water and wastewaters has created con-
cern about the toxicity of chlorination by-products to
aquatic life, the persistence of chlorinated organic com-
pounds in the environment, and their accumulation in the
marine food chain.
In 1978, scientists at the Bears Bluff Field Station,
Johns Island, SC, continued investigation of effects of
chlorination by-products on marine/estuarine organisms,
communities, and food webs.
Projects focused on effects of such compounds on the
physiology and growth of oysters, interactions between
low levels of chlorination and the community structure of
bottom-dwelling animals, and the effects of halogenated
compounds on marine phytoplankton.
Oysters
G.I. SCOTT, Biologist;
S. KLINGENSMITH, Biologist;
D.P. MIDDAUGH, Research Aquatic Biologist
American oysters (Crassostrea virginica) were tested to
determine potential to bioaccumulate chlorination by-
products (Fig. 19).
Adult oysters, 6 to 10 cm in height, were exposed in
replicate exposure tanks to a nominal concentration of
1.0 mg chlorine/£ added as Ca(OCl)2- A second group
was exposed to seawater that had been chlorinated at a
nominal rate of 1.0 mg chlorine/i, then dechlorinated
with sodium thiosulphate (Na2S2Oj).
Two other sets of replicate tanks were maintained as
^28203 and seaw'ater controls. Tests were conducted in
flow-through tanks that received seawater of ambient tem-
perature (26° to 30°C) and salinity (22.5 to 30 parts per
thousand [°/oo] ) at 5 £/hr/oyster for 30 days, followed
by depuration for 16 days.
Mortality was 10% in the study for oysters exposed to
chlorinated seawater; measured chlorine-produced oxidant
(CPO) was approximately 0.20 mg/£. Oysters in the
chlorinated-dechlorinated tanks (measured CPO = 0.0)
and controls had a 2% mortality.
Condition and gonadal indices of oysters showed
changes, depending on the exposure regime (Table 8).
After 16 days of exposure, the condition index of oysters
maintained in the chlorinated-dechlorinated tanks was sig-
nificantly lower than that of the seawater control group.
Gonadal indices of oysters maintained in the chlorinated,
chlorinated-dechlorinated, and ^28203 control tanks
were all significantly lower than those of the seawater
control group.
Chemical analyses revealed generation of chlorination
by-products (predominantly bromoform, CHBrj) in the
chlorination exposure tanks (5.5-54.3 yg bromoform/£)
and in the chlorination-dechlorination tanks (4.2-38.3 yg
bromoform/£). A two- to three-fold biological magnifica-
tion of bromoform occurred in oyster tissues, compared
to the levels measured in water samples taken from the
treatment tanks within a week. Oysters depurated bromo-
form after chlorination, and chlorination-dechlorination
exposures were discontinued.
Marine Ecosystem Testing Units (METU)
P.P. SHERIDAN, Marine Ecologist;
W.P. DAVIS, Supervisory Aquatic Biologist;
R.L. YOAKUM, Biological Technician
Since 1975, Bears Bluff investigators have used the
Marine Ecosystem Testing Units (METU) to observe the
complex interactions between low concentrations of
chlorination and community structure of benthic taxa.
METU consists of 96 outdoor tanks that receive a con-
tinuous flow of estuarine water containing eggs and larvae
of marine organisms. The system serves as a habitat for
developing communities (algae, amphipods, gastropods,
barnacles, tunicates, polychaetes, mollusks, and other
groups). Communities are exposed to continuous chlori-
nation at nominal concentrations of 0.125, 0.250, and
0.500 mg sodium hypochlorite (NaOCl) per liter (£) and
are compared with non-chlorinated control communities
(Fig. 20).
Two series of community tests have been conducted.
In the first series (28 months), communities were allowed
to develop for 30, 60, or 120 days. The flow rate of sea-
water entering the tanks was 40 £/hr.
Although the numbers of organisms in the communi-
ties increased as development time increased (from 30 to
120 days), the community composition, in terms of dis-
tribution of organisms among the various taxa, was simi-
lar under the influence of a given chlorination concentra-
tion. Intermediate concentrations of chlorination (0.125
and 0.250 mg NaOCl/£) generally produced larger com-
munities than those found in the control tanks or at the
highest nominal concentration of NaOCl (0.500 mg/£) in
which community densities were similar to controls.
Amphipods were the most abundant organisms:
Gammarus mucronatus and Corophium acherusicum pre-
dominated.
All chlorination concentrations stimulated amphipods,
decapods, and bivalves. Variable responses were observed
in gastropods, polychaetes, and isopods, whereas occur-
rence of insects, barnacles, and tunicates was depressed.
In a second series of experiments (now in progress),
effects of increased flow rates and increased surface area
are being investigated. Communities were allowed to
develop for 60 days in the chlorination concentrations
tested earlier. Seawater flow rates were doubled from 40
£/hr to 80 £/hr in half of the test tanks.
Preliminary results indicated increased occurrence of
amphipods, polychaetes, shrimp, and tunicates, and
decreased occurrence of other species, including bivalves
31
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Figure 20. Biological Aides Joe Caddes, Elizabeth Bee, and Ann Hart (right) assist in
harvest of aquatic species in Marine Ecosystem Testing Units (METU).
32
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TABLE 8. CONDITION AND GONADAL INDICES FOR OYSTERS EXPOSED TO
VARIOUS CHLORINATION REGIMES.
Type of Exposure* Condition Index
N
A 16
B 16
C 16
D 15
X
3.55
3.18**
3.75
4.10
S
0.82
1.06
0.70
0.67
Gonadal Index
N
16
16
16
16
X
10.1**
8.8**
11.6**
16.0
S
5.32
6.22
2.85
3.68
*A, chlorination without dechlorination; B, chlorinated seawater dechlorinated with
entering treatment tanks; C, Na2§2O3 and seawater control; D, seawater control.
before
*Significantly different from seawater controls. P< 0.05.
and barnacles. Cages for sedentary organisms (i.e., mol-
lusks) were installed in half of the test tanks to deter-
mine the effect of increased surface area. Ten times more
tunicates occurred in tanks with cages than in tanks with-
out cages; other community components showed variable
responses. Effects of chlorination continued to show
trends similar to those observed in the first experimental
series.
Mini-METU
A.C. BADGER, Research Aquatic Biologist;
I.B. JOHNSON, Biologist
Organisms from a miniaturized experimental commun-
ity configuration are being sorted and identified. Initial
review of these data indicates that Mini-METU is a less
sensitive toll for assessing effects of chlorination of
marine communities than METU.
Fewer organisms colonized the much smaller, indoor
Mini-METU tanks. Although Mini-METU tests have been
halted, the system may be useful in the future for pre-
liminary testing prior to definitive tests with the more
complex METU system.
Analytical Chemistry and Productivity Studies
A.M. CRANE, Chemist;
S.J. ERICKSON, Research Aquatic Biologist
Many chlorination experiments use aqueous solutions
of NaOCl or Ca(OCl>2 as a source of oxidative chlorine
(HOC1 + OC1"). "Residual oxidants," produced by chlori-
nation of the various experimental media, are determined
via amperometric titration.
The theoretical chemical composition of the measured
residuals in chlorinated saline water is a mixture of many
oxidative species of which approximately 99.8 mole % is
bromine (HOBr and OBr). Chlorine comprises less than
0.2 mole % (Table 9). However, the relationship between
volume of the sample (200 m£) and concentration of
phenylarsine oxide titrant used (0.0056N) is such that 1
mil of titrant is equivalent to 1 ppm (by weight) of ele-
mental chlorine (Cl~).
Therefore, measured residuals have been given as total
residual oxidant and are expressed as mg chlorine/£. In
converting to mg bromine/£, the reported values must be
multiplied by 2.25.
Production of trihalomethanes (THM) during chlorina-
tion of North Edisto River estuarine water (salinity =
23 °/oo) with NaOCl was investigated in three experi-
ments that addressed: (1) the capacity of filtered estua-
rine water to produce THM, (2) the statistical correlation
between the chlorophyll a content of individual species of
marine algae exposed to NaOCl and the concentration of
THM produced, and (3) THM production rate and chlo-
rine demand of algal cell populations.
Chlorination of 0.22 ym Millipore-filtered estuarine
water (salinity = 23 °/oo) from the North Edisto River
caused rapid formation of THM, comprised mainly of
tribromomethane (bromoform) and chlorodibromometh-
ane. At nominal concentrations of 10 mg chlorine/£
(added as NaOCl) or greater, the trihalomethane yield
after 24 hr exposure remained nearly constant at 211 ±8
33
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TABLE 9. CHLORINE AND BROMINE COMPOUNDS EXPECTED IN SEAWATER AFTER
CHLORINATION AT DIFFERENT pH's*
MOLE PERCENTAGE OF TOTAL ADDED CHLORINE
pH
6
7
8
9
10
CLO~
8xlO'3
9xlO"2
0.07
3
3
HCLO
0.09
0.1
0.08
0.03
0.003
CL2
9x1 0~5
IxlO"5
8xlO'7
3xlO"8
3xlO-10
Br2
7
0.7
0.06
2xlO"3
2xlO'5
Br2CL"
10
1
0.09
3xlO'3
3xlO'5
HBrO
83
95
74
20
3
BrO"
0.3
3
26
77
94
E.M.F.
+ 1.06 v.
+ 1.03 v.
+ 1.00 v.
+ 0.95 v.
+ 0.89 v.
*From: Dove, R. A., Reaction of Small Dosages of Chlorine in Seawater, Research Report 42/70,
Central Electricity Operating Board, Southeastern Region, Scientific Services Department,
Southhampton, England, p. 124.
. Within the chlorine concentrations tested, chlorodi-
bromomethane production increased with increasing chlo-
rine concentration; the bromoform concentration
decreased (Table 10) and exhibited a significant linear
correlation with chlorodibromomethane (r=0.94,
p<0.001).
In a second series of experiments, three species of
marine algae (Isocbrysis galbana), (Carteria sp.), and
(Thalassiosira pseudonana) were studied to determine if
their chlorophyll a content and their individual roles in
the generation of THM during chlorination of marine
waters were related (Table 11). In the presence of 10
Isocbrysis galbana cells/m£, the total THM produced by
chlorination with NaOCl to a nominal 10 mg/£ chlorine
averaged 41% greater than THM production in filtered
saline waters (Table 12). The effect of Carteria sp. on
THM production was statistically insignificant, but chlo-
rination of seawater that contained Thalassiosira
pseudonana decreased the total THM production by 24%.
Regression analysis of data for each- algal species revealed
no significant correlation between THM production and
chlorophyll a concentrations.
Determination of residual oxidants in estuarine water
with known algal cell volumes and controlled chlorination
concentrations and contact times revealed that THM pro-
duction and the chlorine demand of the algal culture were
correlated. Rapid reduction in residual oxidant level was
accompanied by the production of THM at nominal chlo-
rine concentrations of 5, 10, and 20 mg/£. Fig. 21 sum-
marizes the relationship at the 20 mg/£ in the presence
of /. galbana.
Single Species Studies (Phytoplankton)
SJ. ERICKSON, Research Aquatic Biologist;
C.E. HAWKINS, Biologist
Fifteen chlorinated and brominated compounds identi-
fied as by-products of chlorination were screened against
four species of phytoplankton (Skeletonema costatum),
(Isocbrysis galbana), (Thalassiosira pseudonana), and
(Glenodinium halli) in seawater of 25 °/oo salinity at
20 ±2 C. Growth in exposed cultures was compared with
that in unexposed controls after 7 days. Concentrations
of the compound that inhibited cell division (50% and
25% of control) or stimulated cell division (110% of con-
trol) are shown in Table 13. The values expressed as
greater than (>) were the highest concentrations tested.
The four species of algae responded in a like manner and
were either stimulated, inhibited, or not affected by the
same compounds.
Monochloramine was the most inhibitory compound
tested. Algal response to the halogenated phenols varied.
Some compounds were stimulatory. At the concentrations
tested, pentachlorophenol and 2,4,6-tribromoanisole were
not inhibitory.
The other five compounds inhibited growth in this
order of increasing inhibition: penta-bromophenol, 2,4,6-
tribromoanisole, 2,4,6-trichlorophenol, pentachlorophenol,
and pentabromophenol. The two most inhibitory com-
pounds, pentachlorophenol and pentabromophenol, con-
tained the greatest numbers of halogen atoms.
34
-------
TABLE 10. AVERAGE TRIHALOMETHANE CONCENTRATIONS 24-HR AFTER CHLORINATION
OF FILTERED ESTUARINE WATER WITH NaOCl.
Nominal Chlorine
Concentration
mg/£
10
20
30
40
50
100
Trihalomethanes Produced
CHBrj
yg/£
180
188
177
146
136
119
CHBr2Cl
yg/£
26
33
38
70
65
91
Total
Mg/£
206
221
215
216
201
210
TABLE 11. EFFECT OF THREE ALGAL SPECIES ON CONCENTRATION OF TRIHALOMETHANES,
PRODUCED AFTER 1 HR IN CULTURE MEDIUM THAT CONTAINED 10 mg NaOCl/£.
Algal Species
Isocbrysis galbana
Carteria sp.
Tbalassiosira pseudonana
N*
6
6
6
Cells/m£
1.16xl06
7.22xl04
9.21xl05
Cell Volume
7.48xl06
1.22xl06
4.77xl06
Chlorophyll a
yg/£ ~~
192 ± 10
146 + 27
148 ± 9
THM Range**
yg/£
+35 to +76
-11 to +22
-76 to -41
*N-number of replicates.
**THM Range = concentration in medium with cells - concentration in medium without cells.
TABLE 12. TRIHALOMETHANES PRODUCED IN 1 HR BY CHLORINATION AT 10 mg NaOCl/£
IN CULTURE MEDIUM WITH AND WITHOUT ALGAL CELLS.
Algal Species N Average total trihalomethanes Average total trihalomethanes
produced with 10 cells/m produced in absence of
present Control algae Control
yg/£ % yg/£ %
Isocbrysis galbana
Carteria sp.
Thalassiosira pseudonana
6
6
6
181 +
225 ±
160 +
19
13
16
141
104
76
125
215
165
±_
+_
+_
25
25
35
107
104
81
N-number of replicates.
35
-------
60
120 180
Time (min)
0
240
300
Figure 21. Total trihalometbanes and total residual oxidant as a function of time for
estuarine water containing 10& cells/mH of Isochrysis galbana exposed to a
nominal concentration of 20 mg/i chlorine at time zero; r is the correla-
tion coefficient.
All halogenated aliphatic hydrocarbons tested produced
stimulatory algal growth responses; none inhibited algal
cell division at the highest concentrations tested (16 or
32 mg/£). The stimulatory responses observed could be
the result of utilization of the compounds by the algae,
or they may reflect metabolism of contaminants.
After screening tests were completed, compounds listed
in Table 13 were tested in running seawater experiments
to determine their effect on photosynthesis, as measured
by uptake of *-^C, in natural assemblages of estuarine
phytoplankton. Nominal exposure concentrations ranged
from 0.125 to 2.0 mg/&. In general, halogenated aliphatic
compounds neither stimulated nor inhibited the algae, but
exposure to trichloroethylene increased 14C uptake to
127% of the control value. Phenol and other halogenated
phenolic compounds were inhibitory (14c uptake<25% of
controls). Pentabromophenol and pentachlorophenol
caused greatest photosynthetic inhibition: 14C uptake
was 0.0 and 1.5% of controls. Haloamines formed by
combining NaOCl and ammonia in situ caused similar
inhibition.
In another study to determine the effects of contin-
uous chlorination on entrained estuarine plankton, adeno-
sine triphosphate (ATP) content in plankton was
examined in two running seawater aquarium systems for
1 year. System A (METU) consisted of 96 37-£ aquaria
operated outdoors and System B (Mini-METU) had 40
5.5-£ aquaria operated indoors. Salinities ranged from 21
to 29 °/oo, water temperature from 10° to 31°C, and the
pH from 7.8 to 8.2 units during the study.
In System A, aquaria treated with a nominal concen-
tration of 0.125 mg NaOCl/2, had an ATP content of 87%
of the control value, 0.250 mg/m£, 78%; and 0.5 mg/£
67%.
36
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TABLE 13. CONCENTRATION (mg/2,) OF HALOGENATED COMPOUNDS AFFECTING CELL DIVISION
OF MARINE PHYTOPLANKTON*
Stimulation by
110% of Control
Inhibition by
25% of Control
Inhibition by
50% of Control
Gh Sc
Tp Ig
Gh
Sc
Tp
Ig
Gh
Sc .
Tp
Ig
Chloramine T
Sodium
bromate
Chloroform
Bromoform
Trichloro-
ethylene
Tetrachloro-
ethylene
Ethylene
bromide
2,4,6-Tribromo-
anisole
p-Bromophenol
p-Chlorophenol
2,4,6-Tribromo-
phenol
2,4,6-Trichloro-
phenol
Pentachloro-
phenol
Pentabromo-
phenol
Phenol
Monochlora-
mine
2 8 > 8
16 0.125 16 8 >16
32 8 32 0.5 >32
2 1 8 >32
16 4 >16
16 0.5 4 >16
0.5 1 0.25 2 >16
0.5 0.125 4
0.5 2 > 8
12 1 8 > 8
16
4
0.5
1
16 16
0.125
>8 > 8 4 >8 >8 >8
>16 >16 >16 >16 >16 >16
>32 >32 >32 >32 >32 >32
>32 >32 >32 >32 >32 >32
^ lo ^ lo ^ lo ^ lo ^ lo ^ lo
'
> Xo "^ lo ^ lo ^ lo ^ lo ^ lo
4 > 16 > 16 > 16 4 > 16
2 2 1 4 4-4
4>8 1 >8 4 >8
>8 >8 >8 >8 >8 >8
>16 16 16 >16 >16 16
8 2 0.25 484
1 0.5 0.25 1 2 0.5
1 1 0.063 1 1 1
2 > 16 > 16 > 16 > 16 > 16
0.125 0.125 0.125 .0.125 0.125 0.125
8
>16
>32
>32
>16
> lo
> lo
1
1
> 8
>16
0.5
0.25
0.063
>16
0.125
*Ig = Isochrysis galbana; Sc = Skeletonema costatum-, Gh = Glenodinium halli; Tp = Thalassiosira pseudoncma
Fishes, Crustaceans, and Mollusks
D.P. MIDDAUGH, Research Aquatic Biologist;
A.C. BADGER, Research Aquatic Biologist;
G.I. SCOTT, Biologist;
I.B. JOHNSON, Biologist
A research project initiated in the spring of 1976 to
delineate factors controlling natural spawning in the
Atlantic silverside (Menidia menidia) was completed. Field
observations showed that sexually mature adults in the
North Edisto River estuary, SC, began to spawn as sea-
sonal water temperatures rose to 16 C or above in March.
Spawning continued until June or July when water tem-
peratures were 29° to 30°C (Fig. 22).
Silversides spawned only during daylight hours, and
spawning runs were precisely correlated with time of high
tide (Fig. 23). Also, the intensity of spawning runs peri-
odically increased and decreased; maximum intensity
occurred near the time of new and full moons.
37
-------
Figure 22. Spawning Atlantic silverside photographed in the North Edisto River
estuary.
Extensive examination of gonadal material from field-
collected adults indicated that spermatogenesis and oogen-
esis also were cyclic and that the immature and inter-
mediate egg stages apparently serve as a source of eggs
which pass through a maturing and a final hydrated stage
before spawning (Fig. 24). Females spawned once every
13 to 16 days, releasing approximately 500 eggs per
spawning.
Data from the study are being used to model periodic
variables that cue spawning in the silverside Menidia in
laboratory culture. Laboratory spawning will provide a
method to assess the effects of toxic substances on the
reproductive potential of the silverside, an estuarine fish
that serves as a food source for many commercially
important fishes.
In another study, groups of embryonic grass shrimp
(Palaemonetes pugio) were exposed to 0.1 and 0.3 mg
cadmium/2, for 8 days prior to hatching. Other groups of
embryos were cultured in uncontaminated seawater.
Prehatch exposure to cadmium had no additive effect
on sensitivity of larvae to cadmium or salinity stress for
14 days after hatching (Fig. 25). Only one group of
larvae, exposed to 0.1 mg/£ cadmium for 4 days before
hatching and transferred in 0.1 mg cadmium in 10 °/oo
salinity water after hatching, showed a significant decrease
in survival (X^, P<0.05) compared to control survival. No
significant decreases in survival were observed for any
larvae transferred to 15 and 30 °/oo salinity at a pre- and
posthatch cadmium concentration of 0.1 mg/L
Pre- and posthatch exposure to 0.3 mg cadmium/)!,
caused significant decreases in survival of all larvae trans-
ferred to 10 and 15 °/oo salinity after hatching. Signifi-
cant decreases in survival were observed for only two
groups exposed before hatching and transferred to 30
°/oo salinity and 0.3 mg cadmium/2, after hatching.
Rivulus marmoratus: An Investigation of its Poten-
tial as a Cancer Research and Chemical Carcino-
gen Screening Organism
C.C. KOENIG, Principal Investigator; EPA Grant
R805469, Grice Marine Biological Laboratory, College of
Charleston, Charleston, SC;
W.P. DAVIS, Project Officer
The self-fertilizing hermaphroditic marine fish (Rivulus
marmoratus) is being studied as a potential environmental
monitor of teratogenetic and mutageliic effects of chemi-
cals, chlorination by-products, or effluents. During 1978,
over 1000 individuals have been raised, important data on
their natural history has been obtained and a substantial
wild stock population has been located.
38
-------
O oo
2'5
2.0
1.5
LU
t 4
CO
O 9
CL ^
> 2
t R o
CO
2 o2
C3 51
0
< 1 4
Q- CQ
M co p
or ^
LU 0
CO
O • O • O • O
I III li i
1 15 31 15 30 15 31 15 30 15 31
MARCH APRIL MAY JUNE JULY
1977
A.
en
DEX
B.I
<
2
O
(D
in
|o 100
u-x 50
° 0
^ 100
£< 50
C 5 °
0K100
O? 50
TAGE
^
O
oo
PERCEN
IM
U1
o
o
.1 ii"i on iiiii—i]
- ._>•
v.
\
1 15 31 15 30 15 31 15 30 15 31
MARCH APRIL MAY JUNE JULY
1977
Figure 23. Daily spa-wning-run-intensity values at Bears
Bluff and Point of Pines study sites. Compos-
ite data represent the daily mean at each site.
Predicted high tide elevations in meters above
mean low water (MLW) are for daytime high
tides. Filled circles represent new moons and
open circles represent full moons.
Figure 24. A. Mean daily spawning-run-intensity values
for M. menidia during 1977. Time of new
moons (filled circles and full moons [open
circles] are also shown; B. Mean daily gonadal
index values for males and females collected
during 1977; C. Percentage occurrence of im-
mature (IMM), intermediate (INT), maturing
(MAT) and hydrated (HYD) eggs in ovaries.
-------
6 2 4 fc 6 ' *> 12 VI 6 1466 01214
0 24 6 8 10 12 14
30ppi
: : : : i :
6 2 4 4 6 W 12 H
30ppi
: :;:::::
0 2 4 6 8 10 12 14
Days after Hatching
o 2 4 6 a 10 12 u
Days after Hatching
4 £ 8 O l'2 14
30ppt
5 1 lei Vo 12 M 6 s i6 a b 12 u
Days after Hatching Days after Hatching
Figure 25. Survival of larval grass shrimp exposed to cadmium. A - no prehatcb expo-
sure (0 days); B - 1 day; C 4 days; D 8 days of prehatch exposure. Key-.
Control -+ ,0.1 mg/H Cd - • , 0.3 mg/l Cd - •
Sister chromatid exchange (SCE) is a technique suc-
cessfully used in mammalian cell cultures to analyze muta-
genic characteristics of chemicals or processes. Experi-
mentation was initiated to adopt this technique to marine
fishes and a method has successfully evolved. During
1979, investigations will continue to field test the method
to derive a laboratory screening phase with a field verifica-
tion approach.
In other activities, the culture, life-cycle, and critical
life stages were examined with the bivalve mollusk
(Mulinia lateralis). Induced spawning was successful but
larval culture under laboratory conditions has not yet
been accomplished.
In addition, experiments were conducted to seek
methods of laboratory maintenance of the mysid shrimp
(Neomysis americana). Culture of larvae to reproductive
adults and production of a second filial generation were
accomplished. In testing toxicity of chlorination/chlorina-
tion by-products, these organisms proved to be highly
sensitive to low chlorination levels.
Spawning, egg placement, development, and matura-
tion of the striped killifish (Fundulus majalis) are also
under investigation. This species inhabits coastal lagoons
of the southeastern Atlantic Coast and is an important
food of fish-eating seabirds.
Isolation and Study of Halo-organics
J.H. CARPENTER, Principal Investigator; EPA Grant
R803893, Rosenstiel School of Marine and Atmospheric
Sciences, Miami, FL;
W.P. DAVIS, Project Officer
Techniques for analyzing the larger, lipid-soluble halo-
organics produced by chlorination were developed and
improved in 1978. Results have shown that amino acids
become halo-organic by-products (Fig. 26).
Important research and regulatory questions concern-
ing the complex process of seawater chlorination remain
unanswered: (1) What is the marine environment's assimi-
latory capacity for exotic halo-organic by-products, and
(2) What biological effects result from contact or uptake
of such compounds? These answers would be applicable
to decisions regarding hyperchlorination in sewage sludge
stabilization, the technical management of biocide proc-
esses in electrical power generation using marine cooling
water, and the disposal of municipal and industrial
effluents.
40
-------
Nntur.il orjvm ic
nitrof.cn compounds,
e.g. coll;ir,cn, amino
acids, porphyrins, etc.
M-nr
1
Cci)
rc.ir ran qcrenr.,
bond clo.nv.ii:,->.
re.ict ion with
other spec ies, etc.
t
I
HOBr,
HOC1
wide ranRe of
products including
CH Br,
26. A possible scenario for halogenation of nitrogen containing organics in
marine waters.
An Investigation of the Ecological Effects of
Residual Ozone to Selected Marine Species
D.T. BURTON, Principal Investigator; EPA Grant
R804683, Benedict Research Laboratory, The Academy
of Natural Sciences of Philadelphia, Benedict, MD;
W.P. DAVIS, Project Officer
Effects of ozonation upon selected estuarine organisms
were compared with those of simultaneous chlorination.
Similar by-products of the oxidation processes and effects
of oxidative compounds on invertebrates and fishes were
demonstrated. Ozone has been suggested as a potential
replacement for chlorination, but in terms of marine
waters and production of similar THM by-products no
advantage has been shown. A final report is in prepara-
tion for publication during 1979.
Food Webs, Populations, and Productivity in a
Southeastern Coastal Marine Marsh
N.W. CHAMBERLAIN, Principal Investigator; EPA Grant
R8044688, Grice Marine Biological Laboratory, College of
Charleston, Charleston, SC;
W.P. DAVIS, Project Officer
Aquatic species in marsh ecosystems adjacent to the
Bears Bluff Field Station were monitored in 1978 to pro-
vide information on their life histories, food and trophic
relationships, and parasitism. Data will be used in eco-
system analysis and to validate field tests with selected
pollutants or pesticides. Findings will be reported in 1979.
Ecological Response Team
W.P. DAVIS, Supervisory Aquatic Biologist;
G.I. SCOTT, Biologist;
W.P. LEMPESIS, Environmental Protection Specialist
Personnel of the Bears Bluff Field Station assisted in
the National Response Team (NRT) response to the need
to assess biological impacts of oil and other hazardous
chemical spills in 1978 and 1979.
The NRT has coordinated emergency assistance by the
Federal Government in accidental spills occurring in the
U.S. and foreign countries.
The Bears Bluff scientists participated in the Federal
Government's response to accidental spills in Padre Island
(Texas), Brittany (France), Hackberry (Louisiana),
Savannah (Georgia), Cooper River (South Carolina),
Tampa (Florida), and Fajardo (Puerto Rico). A number
of reports on the biological effects of these spills have
been published, and a method has been defined for an
integrated zonal assessment of ecological damage. The
staff also assisted in designing followup research and
monitoring efforts to be undertaken by appropriate
regional, state, and local governments.
41
-------
Figure 27. Arrows indicate tumors found on fish collected from Gulf Coast estuarine
waters.
42
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ENVIRONMENTAL PATHOBIOL06Y
In 1978, the Environmental Pathobiology Unit at ERL,
GB initiated a new research project, "Carcinogens in the
Aquatic Environment," supported by the National Cancer
Institute. Three disciplines-pathobiology, biochemistry,
and molecular biology-will be applied in the investigation
of carcinogenic pollutants in Gulf coastal waters.
The primary mission of the research is to elucidate the
first series of critical steps in the production of active
carcinogens by pre-carcinogenic chemicals released in
aquatic systems. Selected estuarine and marine species
will be exposed to suspected carcinogens, teratogens, and
mutagens.
Epizootiological Study of Tumors and Carcinogens
J.A. COUCH, Coordinator
ERL,GB scientists are evaluating the role of fish and
shellfish as indicators of carcinogenic pollution in a proj-
ect cosponsored by the National Cancer Institute.
Since field collections began in August 1978, several
tumors and possibly neoplastic lesions have been found in
fish and oysters collected in Gulf coastal waters of
Florida, Alabama, and Mississippi. The types of tumors
are under study to determine if specific cancer-causing
pollutants can be related to tumor prevalence or cellular
diseases in aquatic species (Fig. 27).
Suspected cancer-causing agents are tested in long-term
exposures in a special assay system designed at ERL,GB.
The system, constructed initially to test the carcinogenicy
of the herbicide trifluralin, provides for the control of
light, water temperature, water flow rates, and the nutri-
tional status of test animals (Fig. 28).
Vertebral Dysplasia in Fish Exposed to Trifluralin
J.A. COUCH, Coordinator
Sheepshead minnows (Cyprinodon variegatus), exposed
to 5.5 to 31 pg/iof the herbicide trifluralin throughout
their first 28 days of life, developed a heretofore unde-
scribed vertebral dysplasia.
This dysplasia consisted of semi-symmetrical hypertro-
phy of vertebrae (3 to 20 times normal), characterized by
foci of osteoblast and fibroblasts actively laying down
bone and bone precursors. Effects of the abnormal verte-
bral development were dorsal vertebral growth into the
neural canal, ventral compression of renal ducts, and lon-
gitudinal fusion of vertebrae.
Fish, exposed for 51 days to 16.6 yg/£ trifluralin and
thereafter depurated for 41 days, showed no increase in
vertebral dysplasia during depuration; however, residual
spinal column damage was evident. Serum calcium con-
centrations were elevated in adult fish exposed for 4 days
to 16.6 yg/& trifluralin. Fluorosis or mimicry of hyper-
vitaminosis A are considered possible mechanisms for the
osseous effect, but are not considered to be the only pos-
sible causes.
The highly predictable nature of this disorder in experi-
mental exposures strengthens the probability that young
fish may serve as experimental models for determining
effects of chemicals on early vertebrate ontogeny, partic-
ularly in regard to skeletal development.
Metabolism of Polyaromatic Hydrocarbons by
Mixed Function Qxidase (MFO)
P. MELIUS, Principal Investigator; EPA Grant R806213,
Auburn University, Auburn, AL;
W.P. SCHOOR, Project Officer
The metabolism of polynuclear aromatic hydrocarbons
(PAH) by normal and induced mullet and killifish liver
homogenates and microsomal preparations was measured.
'The Ames test was used to identify the mutagenic metab-
olites of 3-MC.
Rats, mullet, and killifish were induced with either
Aroclor 1254 or 3-MC; 3-MC was used as substrate. In
addition, NADH-ferricyanide reductase activity was meas-
ured. Sodium dodecyl sulfate (SDS) electrophoresis was
carried out in order to measure the appearance of heme-
containing proteins.
In the experiment using the TA 98 and TA 100
mutants, mullet S-9 preparations, and 3-MC (25 yg) sub-
strate, a significant increase in revertants occurred at the
100 mg Aroclor/kg dose level. For no known reason, no
increase was found at the 200 mg Aroclor/kg level. Possi-
bly the 200 mg level of Aroclor was toxic to the cells.
Under these conditions, chrysene apparently was not
mutagenic. The 3-M had a very slight but erratic muta-
genic effect with killifish S-9 preparations. The TA 1535
and TA 1538 organisms gave no significant mutagenesis
in any of these experiments. Problems were encountered
in maintaining the Salmonella TA 98 and TA 100 mutants
without spontaneous reversion.
The NADH-ferricyanide reductase activities were meas-
ured in controls and Aroclor-treated rats, mullet, and killi-
fish. These activities slightly increased in adult rats, as
compared to neonatal rats. The 250-mg Aroclor/kg dose
level in the adult rat caused a three-fold increase in
enzyme activity, whereas the 200-mg Aroclor/kg dose
level in the mullet caused a 30% increase in enzyme activ-
ity. These results appear to agree with those reported for
earlier experiments which indicated that induced enzyme
levels were significant in the rainbow-trout, but lower
dian in the rat.
43
-------
Figure 28. Visiting Investigator L.A. Courtney checks aquaria designed to test carcin-
ogencity of the herbicide trifluralin in aquatic species.
Oxidation and Conjugation of Carcinogenic
Hydrocarbons in Marine Animals
D.R. STRENGTH, Principal Investigator; EPA Grant
R806368, Auburn University, Auburn, AL;
W.P. SCHOOR, Project Officer
Six enzymes were studies in the investigation of oxida-
tion and conjugation of carcinogenic hydrocarbons: (1)
UDP-glucuronosyl transferase, (2) 3-phosphoadenosine-5-
phosphosulfate sulfotransferase, (3) UDP-glucose dehydro-
genase, (4) glucose-1-phosphate uridyltransferase, (5) b-
glucuronidase, and (6) aryl sulfatase. The criterion used
for validation was a direct proportionality between reac-
tion rate and enzyme concentration.
Systematic procedures have been developed for han-
dling tissue samples, including the selection of appropriate
buffers, proper homogenization, centrifugation for optical
clarification, and use of Sephadex G-25 to remove inter-
fering low molecular weight substances. The assay will be
validated in tests with rat and marine animal tissue.
Carcinogen Assay System for Estuarine Fishes
B.J. MARTIN, Principal Investigator; EPA Grant
R804527, University of Southern Mississippi,
Hattiesburg, MS;
J.A. COUCH, Project Officer
A closed-circulating assay system was designed to study
the effects of the carcinogenic polycyclic aromatic hydro-
carbons (PAH), benzo[a] pyrene (BaP), and methylcholan-
threne on sheepshead minnows and channel catfish.
Fish were maintained in the system for up to 31 weeks
in weekly contaminations of PAH. Significant levels of
BaP and methylcholanthrene remained in the water col-
umn for only ca. 24 hr each week. No tumors were
observed in the exposed fish during the study.
The incidence and types of lesions in control and
exposed fish were basically similar except in catfish that
were fed PAH-contaminated food. High levels of contami-
nation (1 mg/gm food) appeared to be toxic and lower
levels of contamination (0.1 mg/gm food) produced suffi-
cient stress to make the catfish susceptible to fatal para-
site infestations.
44
-------
Both species accumulated radioactively labelled PAH at
concentrations much higher than their nominal concentra-
tions in the water. Although the level of accumulation
was extremely variable, the accumulation factors, in gen-
eral, were: ca. SOX in gill and liver, ca. 15X in GI tract,
and ca. 2X in skeletal muscle.
In an experiment in which 10 catfish were maintained
in water contaminated on a weekly basis with 1.0 yg/£
BaP, the fish remained healthy for approximately 7
months. In the next 3 months, four of the fish became
very scoliotic and lordotic and exhibited nervous disorders
in their manner of movement.
Most of the affected fish also displayed abnormal
melanocytic control and were much darker in color than
normal fish. Radiographs (Fig. 29) illustrate the vertebral
disorientations that occurred. The high incidence of this
phenomenon in exposed fish that continued to feed nor-
mally and had normal growth rates suggests that a cause-
effect relationship may exist between the lesion and BaP
exposure.
Chemical Carcinogens in Bivalve MoHusks from
Oregon Estuaries
M.C. MIX, Principal Investigator; EPA Grant R806224010,
Oregon State University, Corvallis, OR;
J.A. COUCH, Project Officer
Indigenous populations of bivalve mollusks were used
as monitors for detecting and quantifying environmental
BaP in Oregon estuaries. Short-term and long-term studies
were conducted to establish base-line levels of BaP and to
identify seasonal variations in BaP concentrations in shell-
fish. A presumptive cellular proliferative disorder (thought
possibly to be neoplastic) was also studied in mussels
(Mytilus edulis) from Yaquina Bay.
Histological studies revealed that mussels inhabiting
polluted environments had an average 6 to 8% prevalence
of the cellular proliferative disorder not observed in mus-
sels maintained in clean environments. The cellular condi-
tion followed a seasonal pattern: a low prevalence in the
summer and fall was followed by an increase in early
winter; prevalence peaked in January-February. The atypi-
cal, large cells that characterize the disorder in M. edulis
possess many ultrastructural properties in common with
malignant vertebrate cells.
Findings were published in March 1979 in the EPA
Ecological Research Series.
Separation of Compounds with Carcinogenic
Properties Found in Marine Invertebrates
C.W. CHANG, Principal Investigator; EPA Grant R806108,
The University of West Florida, Pensacola, FL;
N.L. RICHARDS, Project Officer
Analytical methods will be developed for the charac-
terization of genotoxic compounds bioaccumulated by
marine organisms from the water column. A concentra-
tion and separation scheme will be designed and validated.
Investigation of Chemical Mutagen Accumulation
in the Tissues of Marine Organisms
J.R. BAYLIS, Jr., Principal Investigator; EPA Grant
R806339, The University of West Florida, Pensacola, FL;
N.L. RICHARDS, Project Officer
Activation and detection methods were developed and
validated with diverse reference mutagens/carcinogens.
These techniques will be used to screen fractions of oyster
tissue extracts taken from clean and polluted waters.
Enzymatic Screening Tests for Mutagens
J.J. SCHMIDT-COLLERUS, Principal Investigator; EPA
Grant R805671, University of Denver, CO;
N.L. RICHARDS, Project Officer
An enzymatic screen developed for chemical carcino-
gens was based on the selective in vitro stimulation of
microsomal biphenyl-2-hydroxylase by known chemical
carcinogens.
An attempt was made to repeat published work, using
a spectrophotofluorometric assay for biphenyl metabo-
lites. The assay system, however, was not found to be
valid for use with complex mixtures. Tests showed that
metabolites must be separated from interfering com-
pounds prior to quantitation.
A high pressure liquid chromatography method was
developed to permit rapid separation of metabolites.
Nanogram quantities of metabolites were detectable by
chromatographic separation in conjunction with a spectro-
photofluorometric detector. It was not possible to demon-
strate in vitro stimulation of biphenyl-2-hydroxylase by
chemical carcinogens with this method.
In studies with alternative assays, terphenyl was metab-
olized to at least three different compounds by hamster
microsomes. Further work is necessary to validate the
utility of this substrate in an enzymatic screen for
carcinogens.
A marine protozoan (Parauronema acutum) has been
shown to metabolize biphenyl in vivo to 2- and 4-
hydroxybiphenyl. This organism may provide a reliable,
inexpensive source of biphenyl hydroxylase for an in vitro
enzymatic assay system.
The effect of the addition of biphenyl on growth of P.
acutum was examined at both 22 and 25 C. In addition,
the effect of the carrier (dimethylsulfide [DMSO] or
Tween 80) on the ability of the organism to respond to
biphenyl was investigated.
At 22 C, biphenyl dissolved in DMSO at final concen-
trations in the culture of above 0.2 mM caused immediate
death and lysis of the cells (Fig. 30). The lower concen-
tration allowed normal growth of the culture in terms of
cell counts. The loss in viability of the cultures was
45
-------
4a
4d
Figure 29. Vertebral disorientations in BaP-exposed catfish: (4a) dorsal view, Mag.
2X, (4b) lateral view, Mag. 2X; (4c) dorsal view, Mag. 5X; (4d) lateral view,
Mag. 5X. Some vertebrae extend above adjacent vertebrae; others have
abnormally large spacing (arrows). The rotation of some vertebrae removed
their neural spines from the plane of the image (arrowhead).
caused by the biphenyl and not the DMSO carrier. How-
ever, when Tween 80 was the carrier, the lethal effect at
higher concentrations of biphenyl was decreased.
At 25 C, there appeared to be no difference between
biphenyl dissolved in DMSO or Tween 80, with cultures
being unaffected by 0.2 mM concentrations of biphenyl
(Figs. 31 and 32). Extracts of these cultures were
examined by HPLC-SPF to quantitate the metabolites
produced (Table 14). (Numbers are provided for those
extracts in which metabolites were detected.)
It can be seen that both 2- and 4-hydroxybiphenyl
were produced, and that neither carrier nor medium pro-
duced material which interfered with metabolite determi-
nation. When Tween 80 was used as a carrier, the results
obtained seemed to indicate that the 4-hydroxybiphenyl
metabolite may be located intracellularly in a form which
is released by freezing and thawing the cells.
46
-------
TABLE 14. QUANTITIES OF 2- AND 4-HYDROXYBIPHENYL PRESENT IN EXTRACTS OF
Parauronema acutum CULTURES*
Additions to
Incubation
Mixture
cells + medium**
0.2 mM biphenyl
in DMSO (.01/10)***
1.0 mM biphenyl
in DMSO (.05/10)
DMSO (.01/10)
DMSO (.05/10)
0.2 mM biphenyl
in 1.5 x 10'2%
Tween2
1.0 mM biphenyl
in 7.5 x 10'2%
Tween2
1.5 x 10-2%
Tween
7.5 x 10-2%
Tween
Treatment
Before
Extraction
None
Frozen
None
Frozen
None
Frozen
None
Frozen
None
Frozen
None
Frozen
None
Frozen
None
Frozen
None
Frozen
r
2-hydroxybiphenyl
-0-
-0-
0.43
0.44
0.43
0.40
-0-
-0-
-0-
0.10
0.75
0.80
0.34
0.20
-0-
-0-
-0-
0.35
'g
4-hydroxybiphenyl
-0-
-0-
1.26
0.98
0.60
3.24
1.06
-0-
-0-
-0-
1.38
3.12
0.40
1.05
-0-
-0-
-0-
0.45
Ratio
4-OH/
2-OH
2.9
2.2
1.4
8.1
- -
. .
1.8
3.9
1.2
5.2
- -
. .
*Cultures were grown at 25°C. Growth data are presented in Figures 17 and 18.
**Numbers are the average obtained from two different culture flasks. All others represent one flask.
***Numbers in parentheses indicate the volume (ml) of biphenyl in DMSO or DMSO alone added to
the 10 ml of medium.
47
-------
o NO ADDITIONS
• 0.2 mM biphanyl
m DMSOI 01/lOml I
A DMSOl.OI/IOml I
A 0.5mM biphanyl in
DMSOI 025/IOml)
£ DMSO 1.025/IOml)
• I.OmM biphanyl in
OMSOl.OS/IOml)
o DMSOI.OVOml)
A0.2mM btpnanytin
I.5«IO"*% Twaan
Q I.5«IO'Z% T*e«n
• 0.4 mM blphinyl In
3 « 10"* % T*««n
o 3 » I0"z % Twe«n
4 6 6 IO 12
DAYS
Figure 30. Growth of Parauronema acutum in the presence of biphenyl at 22 C. The
arrows indicate time of addition of biphenyl or carrier (7 days), and each
point is the mean of the cell counts from at least two different cultures
in one experiment.
48
-------
O NO ADDITIONS
• OZmM tipfctnyl In
I.S.K>-«%Tw.n
'
• 1.0 ml* Wphinyl In
7.5«IO-I%T.it»
O 7.91 «J'%T...f,
A 2 OnH WrMKl III O.I3%T.t,n
O NO ADDITIONS
& 0 Z mM ofpnnnvl In
DUSOI.OI/IOmlt
• OUSOt.OI/IOnl)
A 1.0 mH Mrntnyl In
DMSO(OVIOml)
A DM 901.0V 10 in 11
• Z.OmH hlthflMl In
DUSOI.OI/IOml)
O DHSOl.OI/IOml)
I 2343 «
MYS
Figure 31. Growth of Parauronema acutum at 25 C in the
presence of biphenyl dissolved in DMSO. The
arrows indicate time of addition of biphenyl
or carrier (2 days), and each point is the mean
of the cell counts from at least two different
cultures in one experiment.
Figure 32. Growth of Parauronema acutum at 25°C in the
presence of biphenyl dissolved in Tween 80.
The arrows indicate time of addition of
biphenyl or carrier (2 days), and each point is
the mean of the cell counts from at least two
different cultures in one experiment.
49
-------
Figure 33. Biological Technician J.M. Patrick is assisted in securing full-face mask
prior to diving for test samples from ERL.GB's offshore laboratory in
the Gulf of Mexico.
50
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EFFECTS OF OFFSHORE DRILLING
FLUIDS ON THE MARINE ENVIRONMENT
The urgent demand for new sources of domestic oil
and gas has brought about an intensified search for off-
shore petroleum. The number of "wildcat" or exploratory
wells required to locate petroleum reserves has increased
as the more accessible energy sources have become
exhausted. Further, improved drilling technology has
introduced a capability for drilling deeper wells.
Accelerated drilling activity in marine waters and
deeper drilling practices have resulted in an increase in the
quantity of chemicals being discharged into the marine
environment. Many new chemicals have been developed
for use in drilling fluids; some have not been tested for
environmental effects.
ERL,GB's assessment of the potential impact of drill-
ing fluids on the marine environment seeks to provide a
data base for mitigating decisions regarding adverse
effects. Drilling muds composed of diverse chemicals and
released at varying underwater depths are evaluated on the
basis of their effects on marine organisms and communi-
ties. Selected drilling fluid components are screened to
determine their relative toxicity.
Data required for environmental impact statements,
lease stipulations, discharge permits, and monitoring pro-
grams will be compiled from ERL.GB's investigation of:
(1) ecological impact of different classes of drilling
fluids, cuttings, and packer fluids, including biocides
(Fig. 33);
(2) advisability of banning or restricting the use of
specific chemical additives;
(3) impact of drilling near areas of high biological pro-
ductivity, such as coral reefs and bathometric highs.
Community Bioassays
M.E. TAGATZ, Research Aquatic Biologist;
J.M. IVEY, Biological Technician
Knowledge of the toxicity of biocides is required to
assess their potential impact on the estuarine and marine
environment. Communities of bottom-dwelling animals
were used to investigate two biocides, Aldacide® (active
component, paraformaldehyde) and Surflo® B3 3 (active
component, sodium salt of 2, 2'-methylenebis), in labora-
tory tests at ERL.GB.
After analysis, results will be compared with data from
earlier experiments using another biocide, pentachloro-
phenol (PCP). Initial findings indicate that paraformalde-
hyde was less toxic to macrobenthic communities used in
experiments conducted at ERL,GB and in the field. The
chlorophenols were found to be particularly toxic to
mollusks.
Effects of Drilling Fluids and Oil on Corals
J.H. THOMPSON, Research Biologist; TJ. BRIGHT,
Principal Investigators; EPA Grant R805441, Texas A&M
Research Foundation, College Station, TX;
N.L. RICHARDS, Project Officer
Field experiments were conducted on Stage I, an off-
shore research platform leased from the U.S. Naval
Coastal Systems Laboratory (Fig. 34), to determine the
direct and indirect effects of drilling fluids on corals,
coral communities, and coral reef processes. The platform,
located 19 km south of Panama City, FL, in the Gulf of
Mexico, provided field validations for laboratory tests to
assess the potential of environmental hazards to coral
reefs adjacent to offshore oil and gas drilling sites. Data
relevant to the impact of chemicals discharged in offshore
oil and gas drilling operations are required by EPA regula-
tory offices for decisions on applications for the National
Pollutant Discharge Elimination System (N.P.D.E.S.) Per-
mits.
The potential impact of effluents released near coral
reefs during normal drilling activities has created concern
for the biological, commercial, and aesthetic aspects of
the reefs and the communities they shelter. In the 1978
experiments, Madracis mirabilis was selected as the test
organism because of its significance to the Flower Gardens
off the coast of Galveston, TX, and its behavioral patterns
(expansion of polyps during the day and night that per-
mit observation by time-lapse photography).
Coral samples were collected from the East Flower
Garden banks in the western Gulf of Mexico and trans-
ported to the Stage I laboratory in tanks containing sea-
water (Fig. 35).
Previous work by the research team has indicated that
the individual major sedimentary components of typical
drilling fluids can be less harmful to coral colonies than
the whole drilling mud taken from an actual drilling oper-
ation. Therefore, the hermatypic coral (M, mirabilis) was
exposed to various lignosulfonates and chrome-modified
lignosulfonates used as thinners in most drilling fluids.
Chronic behavioral bioassays also were conducted in flow-
ing seawater at Stage I. The coral also was exposed to
various concentrations of ferrochrome lignosulfonate.
In separate experiments, colonies of M. mirabilis were
subjected to similar concentrations of a whole drilling
mud. Specially designed coral exposure chambers main-
tained a constant suspension of the drilling fluid in sea-
water. In both experiments, the behavior of the corals
was measured with time-lapse movie cameras. Analysis of
the movies permitted calculation of degree of polyp
retraction: 100 ppm ferrochrome lignosulfonate (FCL)
was 100% fatal to the coral after 5 days; 10 ppm FCL
caused significant negative behavioral reaction as measured
51
-------
Figure 34. ERL,GB researchers head ashore from Stage I, a U.S. Navy platform that
serves as an offshore laboratory to test effects of compounds used in oil-
drilling activities.
by the R factor. Increased sedimentation and turbidity
were observed. In the natural environment, these condi-
tions are known to be deleterious to growth and recruit-
ment rates of corals.
The results of chronic bioassays performed in flowing
seawater on M. mirabilis indicate that some coral reef
organisms were much more sensitive to FCL than were
fish and other organisms used in earlier acute bioassays.
Under some circumstances, concentrations of FCL capable
of stressing M. mirabilis may be found more than 2 miles
(3.2 km) from a drilling rig. Further dilution of an order
of magnitude may not occur for 20 miles (32 km).
The effect of Q-Broxin on M. mirabilis was dramatic.
Most corals treated with 100 ppm FCL retracted their
polyps in about 1.6 hr. Although partial re-expansion
occurred in some cases, all polyps (22 colonies) failed to
re-expand after 19 hr exposure. After 120 hr (5 days), all
corals in the 100 ppm treatment tanks went into the
"shutdown reaction." (When this phenomenon occurs, the
corals produce large amounts of mucus, often expel their
zoozanthellae, and, within a few hours, their tissue disin-
tegrates and decays, leaving a bare carbonate skeleton.)
As a result of preliminary observations, the ERL.GB
coral effects program will be expanded. Drilling fluid com-
ponents and whole drilling fluids will be tested at concen-
trations normally found near offshore oil and gas drilling
operations to determine if these operations may pose a
hazard to coral reefs.
Negative Ion Screening for Marine Xenobiotic
Chemicals
R.C. DOUGHERTY, Principal Investigator; EPA Grant
R806334, Florida State University, Tallahassee, FL;
N.L. RICHARDS, Project Officer
Negative chemical ionization mass spectrometry (NCI-
MS) is uniquely suited to the detection of toxic sub-
stances in the environment. This suitability stems from
the fact that toxic substances generally have significant
electron affinities and anion affinities. In contrast, bio-
molecules generally have negative electron affinities and
attach anions only weakly in the gas phase.
Under an EPA grant, scientists examined a series of
reagent gases to obtain a negative chemical ionization
spectra of polynuclear aromatic hydrocarbons. With
hydrocarbon or halocarbon reagent gases (i.e., isobutane
or methylene chloride), the NCI mass spectrum is gen-
erally dominated by the molecular anion of the arene.
Sensitivity for many polynuclear aromatics increases
significantly by the addition of 10% oxygen to a hydro-
carbon reagent gas. The major ions in the spectra include
52
-------
Figure 35. Stage I Biological Aides CM. Teaf and W.S. Ravenel steady tanks being
lifted by crane into a landing barge for delivery to the offshore laboratory.
the molecular anion, an oxygen exchange ion that corre-
sponds to a deprotonated arene-ol, and oxygen aduct
anions.
NCI mass spectra have been described for a series of
polynuclear aromatic hydrocarbons. Sensitivity data and
detection limits have been established for selected com-
pounds in water.
Toxic, Sublethal, and Latent Effects of Selected
Petroleum Hydrocarbons on Grass Shrimp
F.R. FOX, Research Biologist; EPA Grant R8044541,
University of West Florida, Pensacola, FL;
K.R. RAO, Principal Investigator;
N.L. RICHARDS, Project Officer
The short-term uptake, tissue distribution and depura-
tion of two radio-labeled polycyclic aromatic hydro-
carbons, benzol a] pyrene (BaP) and benzanthracene (BA),
were studied with the grass shrimp (Palaemonetes pugio)
at known stages of the intermolt cycle.
Premolt shrimp accumulated less BaP and BA than
intermolt shrimp. The newly molted shrimp accumulated
more BaP and BA than intermolt shrimp. The relative
increase in uptake by newly molted shrimp was more pro-
nounced for BA than for BaP.
When exposed to 1.25, 2.5, 5, and 10 ppb BaP or BA,
the intermolt shrimp accumulated BA to a greater extent
than BaP at each of the concentrations tested.
The level of BA or BaP accumulated by shrimp
increased in relation to the environmental levels of these
compounds. The relative accumulation of BA and BaP in
the tissues examined was in the following order: digestive
tract (stomach + intestine) hepatopancreas thorax abdo-
men. Each tissue accumulated more BA than BaP.
A rapid uptake was observed in shrimp exposed to
media containing 2.5 ppb BA or BaP during the first 6
hr; subsequently uptake was somewhat reduced. However
53
-------
0.4
c
•;= 0.3
+">
o
O)
j:
CL
w
• Control
DNP
6543
— log inhibitor concn. (M)
Figure 36. In vitro effects of sodium pentachlorophenate and 2,4-dinitrophenol on a
calcium-activated ATPase in the microsomal fraction of the hepatopancreas
from the blue crab (Callinectes sapidus). The values are mean ± 5. D, of
five experiments. (Reprinted by permission of Plenum Publishing Corp.,
New York.)
even at the end of 96 hr exposure, the shrimp exhibited a
trend of continual accumulation of BA and BaP.
When transferred to seawater, the shrimp appeared to
depurate BA more rapidly than BaP. The level of radio-
activity in the shrimp exposed to BA declined by 80% at
the end of a 7-day period of depuration; under similar
conditions the BaP level (radioactivity) declined by only
35%.
Toxicity of Pentachlorophenol to Crustaceans
K.R. RAO, Principal Investigator; EPA Grant R8044541,
University of West Florida, Pensacola, FL;
N.L. RICHARDS, Project Officer
Crustaceans are capable of regenerating lost limbs. The
incidence of limb regeneration, trre rate of growth of the
limb bud and the relative size of the new limb after
ecdysis depend on (1) the stage of the molt cycle at
which limb removal occurs and (2) the interval between
limb removal and ecdysis.
Exposure to media containing sodium pentachloro-
phenate (Na-PCP) caused a dose-related inhibition of limb
regeneration in grass shrimp (Palaemonetes pugio). The
early phases of regeneration (wound healing, cell division,
and dcdifferentiation) appeared to be more sensitive to
Na-PCP than later phases of regenerations.
The effects of Na-PCP on oxygen consumption by the
grass shrimp varied depending on the stage of the molt
cycle and the concentration of Na-PCP. Intermolt shrimp
exposed to high concentrations of Na-PCP (10 or 20 ppm)
exhibited an initial increase in oxygen consumption fol-
lowed by a rapid decline, leading to death.
A decline in oxygen consumption followed by death
can be induced in newly molted shrimp by a lower con-
centration (5 ppm) of Na-PCP. Tests on isolated tissues
(muscle, gill, and hepatopancreas) from the blue crab
(Callinectes sapidus) revealed that Na-PCP and 2,4-dini-
trophenol (DNP) inhibit oxygen consumption' in vitro.
Na-PCP and DNP had inhibitory effects on several
hepatopancreatic enzymes in blue crabs. The enzymes
affected were: fumarase, succinate dehydrogenase, malate
dehydrogenase, glucose-6-phosphate dehydrogenase, pyru-
vate kinase, lactic dehydrogenase, glutamate-pyruvate
transaminase and a microsomal calcium-activated ATPase.
Under in vitro conditions, isocitrate dehydrogenase was
stimulated by a low concentration of Na-PCP (10~^M)
whereas higher concentrations inhibited it.
These studies on crustaceans and earlier studies on fish,
mollusks, and rats seem to indicate that PCP affects car-
bohydrate metabolism, lipid metabolism, ion transport,
and possibly protein metabolism. The inhibitory effects
on a wide variety of enzymes suggest that the actions
may be due to non-specific interactions of this phenol
with membrane proteins.
In addition to the proven uncoupling effects on oxida-
tive phosphorylation, the overall inhibitory effects of PCP
on a variety of enzymes (Fig. 36) may account for the
broad-spectrum biocidal effects of pentachlorophenol
(PCP) and its salts.
54
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Toxic Photooxygenated Products Generated Under
Environmental Conditions from Phenanthrene
J.L. LASETER, Principal Investigator; EPA Grant
R804647, University of New Orleans, LA;
N.L. RICHARDS, Project Officer
The photooxidation of phenanthrene, a model poly-
cyclic aromatic hydrocarbon (PAH), was accomplished in
a hexane aqueous phase. A light source similar to sunlight
was used to simulate environmental conditions.
The involvement of singlet oxygen also was examined.
Several oxygenated toxic photoproducts were character-
ized by a gas chromatograph-mass spectrometer computer
system. Some products were found to be soluble in water,
suggesting the possibility of oxygenated PAH intrusion
into natural waters.
Generic Variation and Resistance to Carcinogens
in Natural Waters
RJ. SCHULTZ, Principal Investigator; EPA Grant
R805195, University of Connecticut, Storrs, CT;
N.L. RICHARDS, Project Officer
When test species are selected to investigate the toxic
or carcinogenic properties of pollutants, selection criteria
are usually influenced by availability and adaptability to
the laboratory. A third consideration-the genetic back-
ground of the indicator species-is being evaluated in tests
that will compare the resistance of fish of different stocks
to dimethylbenzanthracene and diethylinitrosamine.
Novel Techniques for Concentration and Separa-
tion of Toxic Substances from Estuarine Waters
E. KLEIN, Principal Investigator; EPA Grant R805656,
Gulf South Research Institute, New Orleans, LA;
N.L. RICHARDS, Project Officer.
The organic carcinogens benzo [a] pyrene (BaP), diel-
drin, and N-acetyl-2-amino-fluorene were recovered on
XAD-2 macroreticular resin in yields of 90% or more
from distilled water or seawater and in yields of 40% or
more from Lake Pontchartrain water containing a high
concentration of organic material.
The original solutions contained less than 500 parts per
trillion (ppt) of carcinogen. These results show that XAD-
2 provides an efficient means for recovering nonpolar
organic carcinogens from dilute solutions.
More polar carcinogens such as diethylinitrosamine
were not effectively recovered on XAD-2 columns. There-
fore, the ability of the above carcinogens to bind to
nucleic acid both before and after S9 liver microsomal
activation was investigated.
The DNA-carcinogen interaction systems included
direct binding, equilibrium dialysis, nuclei binding, and
binding to DNA-cellulose. Radiolabeled carcinogens were
used to quantify the amount bound.
Either rat liver nuclei (0.1 mg DNA) or DNA-cellulose
(1 mg DNA) bound 18% of the acetylaminofluorene and
up to 66% of the dieldrin from solutions containing 150
to 280 nmoles of compound. Up to 30% from solutions
containing as much as 320 pmoles was bound. Ten-fold
or lower recoveries were found when direct-binding or
equilibrium-binding methods were used.
Less carcinogen was recovered with the DNA fraction
when liver microsomes were used to activate die binding
systems. In these cases, the microsomal protein can
decrease the net DNA binding by competing with the
DNA for carcinogen or by converting the carcinogen to
products that do not bind well to DNA.
In the nuclei binding studies, DNA was isolated from
the nuclei by extraction with sodium dodecyl sulfate and
pehnol followed by recovery of the DNA by ethanol pre-
cipitation and spooling. The recovered DNA contained
more than 10% of the input radioactivity, indicating that
a significant portion of the input sample may be tightly
bound to DNA.
The portion that bound after DNA isolation was not
dissociated by further extractions with chloroform-
isoamyl alcohol or 1% sodium dodecyl sulfate solution.
At least some of this material may be covalently asso-
ciated. Whether or not DNA-binding is more specific for
recovery of organic carcinogens than is XAD-2 has not
been determined.
Activation of 2-aminofluorene to Mutagen(s) by
the Marine Ciliate (Paramonema acutum)
D.C. LINDMARK, Principal Investigator; EPA Grant
R805364, Rockefeller University, New York, NY;
N.L. RICHARDS, Project Officer
Living cells of the marine ciliate (Parauronema acutum)
can convert 2-aminofluorene into compounds with muta-
genic activity in the Ames/Salmonella plating test using
tester strains TA 98, 1537, 1538. The ciliate, however,
does not activate benzo[a] pyrene or destroy the muta-
genic properties of N-methyl-N'-nitro-N-nitrosoguanidine,
and does not accumulate any of the tested compounds.
Homogenates of P. acutum, when substituted for a
liver microsomal fraction in the Salmonella/microsome
test, activate 2-aminofluorene and produce revertants at a
high rate. Benzo[a] pyrene is not activated nor is nitro-
soguanidine inactivated.
After differential sedimentation of an homogenate, the
activating ability is equally distributed between a non-
sedimentable fraction and a fraction sedimenting at high
speed. Though the activation of aminofluorene does not
require NADP and may not be due to mixed function
oxidase activity, it does demonstrate that a marine ciliate
can produce mutagens and can^possibly contribute to an
increase of the mutagen load in the marine environment.
The role of marine protozoa in the accumulation of
polynuclear aromatic hydrocarbons (PAH) will be assessed.
Microsomal fractions were prepared in 1978 and the
microsomal activity of marine protozoa will be charac-
terized in 1979.
55
-------
Figure 37. The Cheasapeake Bay (above), a popular site for water recreation, is the
site of an EPA research effort designed to restore and preserve the quality
of natural systems of the Bay estuary.
56
-------
CHESAPEAKE BAY PROGRAM
In the independent Agencies Appropriation Bill of
1976, the 94th Congress directed EPA to assess adverse
environmental impacts on the Chesapeake Bay system and
to coordinate a research/abatement program aimed at
restoring the quality of the estuary.
In response, EPA established the Chesapeake Bay Pro-
gram (CBP) and joined with environmental agencies of the
states within the Bay drainage basin, interested scientists,
and private citizens in an attempt to define and establish
pollution problem priorities and to recommend measures
for the proper management of Bay resources.
ERL, Gulf Breeze Deputy Director T.T. Davies is
director of CBP's varied and interdependent scientific and
management investigations of the Chesapeake Bay System.
Results of the research will establish a framework to iden-
tify problems, describe processes affected, and project
consequences of identified control measures in both the
natural and management systems.
In fostering an atmosphere of partnership in CBP,
funds were awarded to the water pollution control agen-
cies in Maryland and Virginia and to an "umbrella" organ-
ization of citizen interests. The State management grants
allow coordinated channeling of all State concerns through
one designated lead agency and enhance the communica-
tion between the EPA program and each State. The public
particpation program fosters development of communica-
tion channels between citizens and the CBP management
and informs the public about the program and its
objectives.
At the outset, three priority areas were identified for
scientific investigations: (1) accumulation of toxicants in
the food chain, (2) the decline of submerged aquatic vege-
tation, and (3) eutrophication (nutrient enrichment).
An evaluation of the role of toxics in the ecological
health of the Chesapeake Bay system requires a thorough
understanding of the chemical, physical, and biotic
dynamics of the estuarine system. The CBP toxics pro-
gram is concerned with the sources, pathways, and fate of
toxic substances present in the estuary. Initial toxic
studies will develop a baseline inventory of the distribu-
tion of toxicants in sediments, pore water, the water col-
umn, and biota. Results of the toxics program will be
used to delineate management options to control toxi-
cants and minimize their adverse impacts.
Submerged Aquatic Vegetation (SAV) Program will
examine the cause-and-effect relationships potentially
responsible for the decline of bottom grasses in the Bay
system. Studies are designed to identify, and, when possi-
ble, to quantify important functions performed by SAV
as a source for food, shelter, and habitats and breeding
areas for finfish and shellfish, water fowl, and species of
the lower trophic levels. Research results will provide data
for a management plan aimed at protecting and enhancing
the growth and propagation of the Bay's submerged
plants.
Eutrophication studies traditionally focus on the super-
abundant nutrients that promote excessive algal growth,
subsequent depletion of dissolved oxygen, and an eventual
imbalance of the ecosystem. To date, very little scientific
data are available on the liabilities and benefits of nutrient
enrichment in an estuarine environment. The Eutrophica-
tion Program will address the question of nutrient enrich-
ment in the estuary. Additionally, intensive watershed and
modeling studies will investigate in-stream and ecosystem
responses to non-point source loadings. An understanding
of the eutrophication and enrichment processes will aid
Bay managers in evaluating the consequences of possible
control alternatives and developing control priorities.
The scientific and technical investigations undertaken
by the Chesapeake Bay Program are designed to provide
products and data targeted toward improved management
of the Bay and its resources. An Environmental Quality
Management Study (EQMS) will survey and describe cur-
rent Bay management institutions and agencies, review
available water resource management alternatives, and
define institutional strategies to implement improved Bay
management. Socio-economic costs and benefits related to
various levels of water quality and abatement control
alternatives also will be identified within the EQMS effort.
Chesapeake Bay research projects are listed below:
TOXICS
Chesapeake Bay Earth Science Study-Sedimen-
tology of the Chesapeake Bay
R.T. KERHIN, Princiapl Investigator;
EPA Grant R805965, Maryland Geological Survey,
Baltimore, MD;
L.H. BAHNER, Project Officer
Baseline Sediment Studies to Determine Distribu-
tion, Physical Properties, Sedimentation Budget,
and Rates
J.M. ZIEGLER, Principal Investigator;
EPA Grant R806001, Virginia Institute of Marine
Sciences, Gloucester Point, VA;
L.H. BAHNER, Project Officer
Investigation of Organic Pollutants in the
Chesapeake Bay
R.J. HUGGETT, Principal Investigator;
EPA Grant R806012, Virginia Institute of Marine
Sciences, Gloucester Point, VA;
L.H. BAHNER, Project Officer
Chesapeake Bay Earth Science Study
Water Chemistry
Interstitial
O.P. BRICKER, Principal Investigator;
EPA Grant R805963, Maryland Geological Survey,
Baltimore, MD;
L.H. BAHNER, Project Officer
57
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The Biogenic Structure of Chesapeake Bay
Sediments
D.F. BOESCH, Principal Investigator;
EPA Grant R805982, Virginia Institute of Marine
Sciences, Gloucester Point, VA;
L.H. BAHNER, Project Officer
Chesapeake Bay Sediment Trace Metals
G.R. HELZ, Principal Investigator;
EPA Grant R805954, University of Maryland, College
Park, MD;
L.H. BAHNER, Project Officer
Monitoring Particle-associated Toxic Substances
and Suspended Sediment in the Chesapeake Bay
W. TAYLOR, Principal Investigator;
EPA Grant R805959, The Johns Hopkins University
Chesapeake Bay Institute, Baltimore, MD;
L.H. BAHNER, Project Officer
Fate, Transport, and Transformation of Toxics:
Significance of Suspended Sediment and Fluid
Mud
M. NICHOLS, Principal Investigator;
EPA Grant R806002, Virginia Institute of Marine
Sciences, Gloucester Point, VA;
L.H. BAHNER, Project Officer
Chesapeake Bay Earth Science Study Animal
Sediment Relationship
O.P. BRICKER, Principal Investigator;
EPA Grant R805964, Maryland Geological Survey,
Baltimore, MD;
L.H. BAHNER, Project Officer
Sediment and Pore Water Chemistry
S.Y. TYREE, Jr., Principal Investigator;
EPA Grant R805966, College of William and Mary,
Williamsburg, VA;
L.H. BAHNER, Project Officer
The Characterization of the Chesapeake Bay: A
Systematic Analysis of Toxic Trace Elements
C.C. GRAVETT, Principal Investigator; Interagency
Agreement EPA-79-D-X0717, National Bureau of
Standards, Washington, DC;
L.H. BAHNER, Project Officer
Investigation of the Chester River Oyster
Mortality
H. WILSON, Principal Investigator;
EPA Grant R805976, Water Resources Administration,
Maryland Department of Natural Resources,
Annapolis, MD;
L.H. BAHNER, Project Officer
SUBMERGED AQUATIC VEGETATION (SAV)
Distribution of Submerged Vascular Plants in the
Chesapeake Bay, Maryland--1978
R.R. ANDERSON, Principal Investigator;
EPA Grant R805977, The American University,
Washington, DC;
W. COOK, Project Officer
Distribution and Abundance of SAV in the
Chesapeake Bay--1978
RJ. ORTH, Principal Investigator;
EPA Grant R805951, Virginia Institute of Marine
Sciences, Gloucester Point, VA;
W. COOK, Project Officer
Distribution and Abundance of SAV in the
Lower Chesapeake Bay-1979
RJ. ORTH, Principal Investigator;
Project X-003201-01, Virginia Institute of Marine
Sciences, Gloucester Point, VA;
T. NUGENT, Project Officer
Distribution of SAV in Chesapeake Bay,
Maryland--1979
R.J. MACOMBER, Principal Investigator;
Project X-003202-01, Chesapeake Bay Foundation,
Annapolis, MD;
T. NUGENT, Project Officer
Biostratigraphy of the Chesapeake Bay: A
Feasibility Study
G.S. BRUSH, Principal Investigator;
EPA Grant R805962, Department of Geography and
Environmental Engineering, The Johns Hopkins
University, Baltimore, MD;
T. NUGENT, Project Officer
Biostratigraphy of the Chesapeake Bay and its
Tributaries
G.S. BRUSH, Principal Investigator;
EPA Grant R806680, Department of Geography and
Environmental Engineering, The Johns Hopkins
University, Baltimore, MD;
T. NUGENT, Project Officer
58
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The Functional Ecology of Submerged Aquatic
Vegetation in the Lower Chesapeake Bay
R.L. WETZEL, Principal Investigator;
EPA Grant R805974, Virginia Institute of Marine
Sciences, Gloucester Point, VA;
T. NUGENT, Project Officer
Submerged Aquatic Vegetation in the Chesapeake
Bay: Its Role in the Bay Ecosystem and Factors
Leading to its Decline
J.C. STEVENSON, Principal Investigator;
EPA Grant R805932, University of Maryland, Center for
Environmental and Estuarine Studies, Horn Point
Environmental Laboratories, Cambridge, MD;
T. NUGENT, Project Officer
Relationship Between Submerged Aquatic Vegeta-
tion and Waterfowl: Criteria and Techniques
F. MARTIN, Principal Investigator;
Interagency Agreement EPA-78-D-X-391, Migration Bird
and Habitat Research Laboratory, U.S. Fish and Wildlife
Service, Laurel, MD;
T. NUGENT, Project Officer
Studies on the Value of Vegetated Habitats and
their Roles as Nursery Areas and Shelter from
Predation with Emphasis on Utilization by Com-
mercially Exploited Species
K.L. HECK, Jr., Principal Investigator;
EPA Grant R806151, Benedict Estuarine Research
Laboratory, Benedict, MD;
T. NUGENT, Project Officer
Assessment of the Potential Impact of Industrial
Effluents on Submerged Aquatic Vegetation
G.E. WALSH, Principal Investigator;
U.S. Environmental Protection Agency, Environmental
Research Laboratory, Gulf Breeze, FL;
T. NUGENT, Project Coordinator
Zostera Marina: Biology, Propagation, and Impact
of Herbicides
RJ. ORTH, Principal Investigator;
EPA Grant R805953, Virginia Institute of Marine
Sciences, Gloucester Point, VA;
T. NUGENT, Project Officer
Effects of Recreational Boating on Turbidity and
Sedimentation Rates in Relationship to Sub-
merged Aquatic Vegetation
J. WILLIAMS, H. GUCINSKI, Principal Investigators-;
Interagency Agreement EPA-78-D-X-426, Department of
Oceanography, U.S. Naval Academy, Annapolis, MD;
T. NUGENT, Project Officer
EUTROPHICATION
Definition of Chesapeake Bay Problems of Exces-
sive Enrichment or Eutrophication
L.E. CRONIN, Principal Investigator;
EPA Grant R806189, Chesapeake Research Consortium,
Annapolis, MD;
T. PHEIFFER, Project Officer
An Assessment of IMonpoint Source Discharge,
Pequea Creek Basin, Lancaster County,
Pennsylvania
R.J. BIELO, Principal Investigator;
Project X-003146-02, Susquehanna River Basin Com-
mission, Harrisburg, PA;
T. PHEIFFER, Project Officer
Fall Line Monitoring of the Potomac,
Susquehanna, and James Rivers
F. WHITE, Principal Investigator;
Interagency Agreement EPA-78-D-X-420, U.S. Geological
Survey, Water Resources Division, Towson, MD;
T. PHEIFFER, Project Officer
Chesapeake Bay Circulation Model
R. SHUBINSKI, Principal Investigator;
Project 68-01-5125, Water Resources Engineers, Inc.,
Springfield, VA;
T. PHEIFFER, Project Officer
Evaluation of Water Quality Management Tools
in the Chester River Basin
H. WILSON, Principal Investigator;
EPA Grant R806343, Water Resources Administration,
Maryland Department of Natural Resources,
Annapolis, MD;
T. PHEIFFER, Project Officer
Intensive Watershed Study (Patuxent River Basin)
H. WILSON, Principal Investigator;
EPA Grant R806306, Water Resources Administration,
Maryland Department of Natural Resources,
Annapolis, MD;
T. PHEIFFER, Project Officer
59
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Evaluation of Management Tools in Two
Chesapeake Bay Watersheds in Virginia
R.B. DAVIS, Principal Investigator;
EPA Grant R806310, Virginia State Water Control Board,
Richmond, VA;
T. PHEIFFER, Project Officer
Assessment of Nutrients from Various Sources
G. LANICK, Principal Investigator;
EPA Grant R804917, School of Public Health, University
of North Carolina at Chapel Hill, Chapel Hill, NC;
N. JAWORSKI, Project Officer
Modeling Philosophy and Approach for
Chesapeake Bay Program Watershed Studies
R. AMBROSE, Principal Investigator;
U.S. Environmental Protection Agency, Environmental
Research Laboratory, College Station Road, Athens, GA;
T. PHEIFFER, Project Coordinator
Water Quality Laboratory for Chesapeake Bay
and Its Subestuaries at Hampton Institute
W. BOWIE, Principal Investigator;
EPA Grant R806229, Department of Chemistry and
Physics, Hampton Institute, Hampton, VA;
T. NUGENT, Project Officer
Land Use and Point Source Nutrient Loading in
the Chesapeake Bay Region
B.J. MASON, Principal Investigator;
Project 68-01-4144, GEOMET, INc., Gaithersburg, MD;
T. PHEIFFER, Project Officer
ENVIRONMENTAL QUALITY MANAGEMENT
Preparation of a Strategy and Plan of Action for
Designing the Research of Management Resources
for the Chesapeake Bay Area
J. KEENE, Principal Investigator;
Project X-003149-01, Department of City and Regional
Planning, University of Pennsylvania, Philadelphia, PA;
G. MCGINTY, Project Officer
Environmental Management in the Chesapeake Bay
R. HARRISON, Principal Investigator;
Project X-003200-01, Environmental Law Institute,
Washington, DC;
G. MCGINTY, Project Officer
PUBLIC PARTICIPATION
Chesapeake Bay Program's Public Participation
Program
F. FLANAGAN, G.M. HAGERMAN, Principal
Investigators;
Project T-9008748-01 and T-9008790-01, Citizens Pro-
gram for the Chesapeake Bay, Inc., Baltimore, MD;
W. COOK, Project Officer
STATE PARTICIPATION
Development and Coordination of Technical
Assessments, Scientific Planning, and Data
Organization for the Chesapeake Bay Program
H. WILSON, Principal Investigator;
EPA Grant R805874, Water Resources Administration,
Maryland Department of Natural Resources, Annapolis,
MD;
W. COOK, Project Officer
Data Organization, Technical Support, and Co-
ordination for the Environmental Protection
Agency's Chesapeake Bay Program
R. DAVIS, Principal Investigator;
EPA Grant R805859, Virginia State Water Control Board,
Richmond, VA;
G. MCGINTY, Project Officer
60
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PUBLICATIONS
Recent publications by the ERL.GB and Bears Bluff Field
Station personnel and by researchers supported by EPA
grants or contracts are listed below. Limited reprints are
available and may be obtained from Betty Jackson, Tech-
nical Information Coordinator, Environmental Research
Laboratory, Gulf Breeze, FL 32561, or through the
National Technical Information Service, Springfield, VA
22161.
Research Reports
Anderson, Robert S. 1978. BENZO[a] PYRENE METAB-
OLISM IN THE AMERICAN OYSTER CRASSOSTREA
VIRGINICA. Environmental Research Laboratory, Gulf
Breeze, FL. U.S. EPA Ecological Research Series, EPA-
600/3-78-009. 19 p.
This research program focuses on the role of NADPH-
dependent microsomal mono-oxygenase in the metab-
olism of the widespread environmental carcinogen
benzo [a] pyrene (BP) by the oyster (Cmssostrea
virgimca). The enzyme system is important in detox-
ifying various xenobiotics and in activating poly-
cyclic aromatic hydrocarbon oncogens as BP.
A sensitive radioisotopic system developed to permit
quantification of alkalid-soluble and water-soluble BP
metabolites produced by oyster mono-oxygenase is
described. An NADPH- and O2-dependent aryl hydro-
carbon hydroxylase (AHH) is shown to be located in
the digestive glands of bivalves associated with the
microsomal subcellular fraction. Some indication that
oyster AHH is induced by chronic exposure of the
animals to the environmental carcinogens BP and 3-
methyl-cholanthrene is reported. Experimental evi-
dence indicates that exposure to polychlorinated
biphenyls (PCB) caused AHH induction. The gen-
eration of various dihydrodiol, quinone, and hydroxy
BP derivates is shown.
Bierman, Victor, William Richardson, and Tudor T.
Davies. 1978. MATHEMATICAL MODELING STRATE-
GIES APPLIED TO SAGINAW BAY, LAKE HURON. In:
American-Soviet Symposium on Use of Mathematical
Models to Optimize Water Quality Management, T.T.
Davies and V.R. Lozanskiy, editors. Environmental
Research Laboratory, Gulf Breeze, FL. EPA Ecological
Research Series, EPA-600/9-78-024. pp. 397-432.
This research is directed toward water quality problems
of international waters of the North American Great
Lakes. The prime objective is to develop quantitative
tools to supplement intuition and scientific judgment
in policy decisions related to water quality. Transport
models and algal growth modeling concepts are applied
to Saginaw Bay to describe prevailing conditions.
Borthwick, Patrick W. 1978. METHODS FOR ACUTE
STATIC TOXICITY TESTS WITH MYSID SHRIMP
(MYSIDOPSIS BAHIA), In: Bioassay Procedures for
Ocean Disposal Permit Program, Environmental Research
Laboratory, Gulf Breeze, FL. U.S. EPA Ecological
Research Series, EPA-600/9-78-010. pp. 61-63.
Methods are described for using the bay mysid
(Mysidopsis bahia) in acute toxicity tests of complex
wastes. M. bahia is recommended as a test species due
to its sensitivity, short life cycle, small size, and adapt-
ability to laboratory conditions. Results of these tox-
icity tests can be used to estimate the impact of ocean-
dumped materials on other saltwater crustaceans.
Bourquin, A.W., and P.H. Pritchard. 1979. PROCEED-
INGS OF THE WORKSHOP: MICROBIAL DEGRADA-
TION OF POLLUTANTS IN MARINE ENVIRONMENTS.
Environmental Research Laboratory, Gulf Breeze, FL.
U.S. EPA Ecological Research Series, EPA-600/9-79-012.
551 p.
This international workshop, held April 10-14, 1978,
at Pensacola Beach, Florida, focuses on pertinent issues
related to the scientific investigation of microbial
degradation of organic chemicals in aquatic environ-
ments. Participants discuss methodological criteria for
these investigations and the need for biodegradation
studies. Speakers and contributed papers for open ses-
sions explore these topics: (1) biochemistry of micro-
bial degradation; (2) transformation in aquatic environ-
ments; (3) compartmentalization in aquatic environ-
ments; (4) biodegradation in microcosms; (5) degrada-
tion methodology; and (6) persistence and extrapola-
tion. Discussions within each session are presented.
These proceedings conclude with a summary report
and workshop consensus reports drafted by special
task groups with recommendations concerning the
research, production, and regulation of potential
aquatic pollutants.
Butler, P.A., and J.I. Lowe. 1978. FLOWING SEA-
WATER TOXICITY TEST USING OYSTERS (CRASSOS-
TREA VIRGINICA). In: Bioassay Procedures for Ocean
Disposal Permit Program, Environmental Research Labora-
tory, Gulf Breeze, FL. U.S. EPA Ecological Research
Series, EPA-600/9-78-010. pp. 25-27.
A "special bioassay" for evaluating short-term effects
of specific wastes on marine mollusks is described. The
procedure is recommended only for use with the com-
mercial Eastern oyster (Crassostrea virginica) and
requires flowing, unfiltered seawater. The test is used
at the Environmental Research Laboratory, Gulf
Breeze, to evaluate the effects of insecticides, herbi-
cides, and other toxic organics on oysters.
Cross, F.A., W.P. Davis, D.E. Hoss, and D.A. Wolfe. 1978.
BIOLOGICAL OBSERVATIONS. In: The Amoco Cadiz
Oil Spill: A Preliminary Scientific Report, Wilmot N.
Hess, editor. U.S. Department of Commerce National
61
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Oceanic and Atmospheric Administration (NOAA)/
Environmental Protection Agency (EPA) Special Report,
Government Printing Office (GPO), Washington, DC. pp.
197-215.
This report is a compilation of observations and data
gathered along the Brittany Coast of France after the
Amoco Cadiz oil spill. The information does not reflect
results of a pre-planned biological study, but rather the
qualitative observations by NOAA/EPA biologists from
late March to May 1978. The material is described as
preliminary; final assessment of the full extent of the
impact is expected to require several years.
Davies, T.T., and V.R. Lozanskiy, editors. 1978.
AMERICAN-SOVIET SYMPOSIUM ON USE OF MATHE-
MATICAL MODELS TO OPTIMIZE WATER QUALITY
MANAGEMENT. Environmental Research Laboratory,
Gulf Breeze, FL. U.S. EPA Ecological Research Series,
EPA-600/9-78-024. 453 p.
The American-Soviet Symposium on Use of Mathe-
matical Models to Optimize Water Quality Management
examines methodological questions related to simula-
tion and optimization modeling of processes that deter-
mine water quality of river basins. Participants describe
the general state of development and application of
mathematical models designed to predict and optimize
water quality management in the USA and USSR.
American and Soviet specialists discuss graphic-
economic aspects of pollution control systems; identifi-
cation of ecosystem models by field data; management
decisions for lake systems on a survey of trophic
status, limiting nutrients, and nutrient loadings; and a
descriptive simulation model for forecasting the condi-
tion of a water system. Publication of the proceedings
held December 9-16, 1975, in Kharkov and Rostov-on-
Don, USSR, is in compliance with the Memorandum
from the Fourth Session of the Joint American-Soviet
Committee on Cooperation in the Field of Environ-
mental Research.
Duke, Thomas W., and Anatoliy I. Simonov, editors.
1978. FIRST AMERICAN-SOVIET SYMPOSIUM ON
THE BIOLOGICAL EFFECTS OF POLLUTION ON
MARINE ORGANISMS. Environmental Research Labora-
tory, Gulf Breeze, FL. U.S. EPA Ecological Research
Series, EPA-600/9-78-007. 166 p.
American and Soviet specialists discuss state-of-the-art
for hydrobiological analysis of basic structural compo-
nents of marine ecosystems and the influence of vari-
ous pollutants on these components. Participants define
problems related to methods for modeling the influ-
ence of pollutants on the marine environment, long-
term forecasting and determination of permissible loads
of pollutants, and the unification and intercalibration
of methods for determining production of microorga-
nisms of ocean bacterioplankton and phytoplankton.
Results of laboratory research on the influence of pol-
lution on the marine environment are presented. Pro-
ceedings held September 20-24, 1976, in Gulf Breeze,
FL, were published in English and Russian in compli-
ance with the Memorandum from the Fourth Session
of the Joint American-Soviet Committee on Coopera-
tion in the Field of Environmental Research.
EPA Ocean Disposal Bioassay Working Group. 1978. BIO-
ASSAY PROCEDURES FOR THE OCEAN DISPOSAL
PERMIT PROGRAM. Environmental Research Labora-
tory, Gulf Breeze, FL. U.S. EPA Ecological Research
Series, EPA-600/9-78-010. 121 p.
Bioassay procedures are described for toxicity evalua-
tions of waste materials being considered for ocean dis-
posal under EPA's Ocean Disposal Permit Program.
Procedures specify use of various organisms represent-
ing several trophic levels. Flow-through and static tests
are included; methods vary in their utility and com-
plexity. These procedures are not considered "standard
methods," but as reference methods or official
methods to be used as specified by the EPA Regional
Administrator responsible for the permit program. This
manual is a revision of EPA-600/9-76-010 published in
May 1976.
Evans, John E. 1978. FEASIBILITY OF USING BACTE-
RIAL STRAINS (MUTAGENESIS) TO TEST FOR
ENVIRONMENTAL CARCINOGENS. Environmental
Research Laboratory, Gulf Breeze, FL. U.S. EPA Ecologi-
cal Research Series, EPA-600/3-78-042. 118 p.
This literature review includes published data on the
feasibility of using bacteria as screening agents to
detect environmental carcinogens. Mutagenicity data
are included because growing experimental evidence
indicates that most chemical carcinogens are mutagens,
and many mutagens may be carcinogens. This report
indicates that bacterial mutagenesis can be used to
initiate studies designed to screen for potential muta-
gens and carcinogens in mixed chemical wastes.
Hansen, D.J. 1978. LABORATORY CULTURE OF
SHEEPSHEAD MINNOWS (CYPRINODON
VARIEGATUS). In: Bioassay Procedures for Ocean Dis-
posal Permit Program, Environmental Research Labora-
tory, Gulf Breeze, FL. U.S. EPA Ecological Research
Series, EPA-600/9-78-010. pp. 107-108.
Techniques used at the U.S. EPA Environmental
Research Laboratory in Gulf Breeze for the culture of
sheepshead minnows in aquaria with under-substrate
filters or in aquaria supplied with saltwater are
described. The procedure accommodates planning for
tests to assure availability of required embryos for life-
cycle tests, as well as sufficient juveniles for acute
static or flow-through tests after acclimation for 2
weeks.
Hansen, David J. 1978. IMPACT OF PESTICIDES ON
THE MARINE ENVIRONMENT. In: First American-
Soviet Symposium on the Biological Effects of Pollution
on Marine Organisms, Thomas W. Duke and Anatoliy I.
Simonov, editors. Environmental Research Laboratory,
62
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Gulf Breeze, FL. U.S. EPA Ecological Research Series,
EPA-600/9-78-007. pp. 126-137.
Effects of toxicants on the entire life cycle of an ovip-
arous estuarine fish, Cyprinodon variegatus, can now be
studied; preliminary experiments reveal that this fish
typically develops from an embryo to maturity in 10
to 14 weeks, with about 70% survival in the labora-
tory. Females produce an average of eight eggs per day
and fertilization success exceeds 90%. Effects of poly-
chlorinated biphenyl, Aroclor® 1254, and of a pesti-
cide, toxaphene, on developing communities of estua-
rine animals have been investigated. These studies pro-
vide data for predicting pollution-induced shifts in
composition of estuarine and animal communities.
Hansen, D.J., P.R. Parrish, S.C. Schimmel, and L.R.
Goodman. 1978. LIFE-CYCLE TOXICITY TEST USING
SHEEPSHEAD MINNOWS (CYPRINODON VARIE-
GATUS). In: Bioassay Procedures for Ocean Disposal Per-
mit Program, Environmental Research Laboratory, Gulf
Breeze, FL. U.S. EPA Ecological Research Series, EPA-
600/9-78-010. pp. 109-117.
The method described determines effects of continuous
exposure of a toxic material on sheepshead minnow
embryos and fry; their survival and growth to adult-
hood, and spawning success. Spawning success is meas-
ured by the ability of the fish to spawn naturally,
number of eggs spawned, fertilization success, and sur-
vival of embryos and fry. Experiment requires 4 to 6
months.
Hansen, David J., Steven C. Schimmel, Del Wayne Nimmo,
Jack I. Lowe, Patrick R. Parrish, and William H. Peltier.
1978. STATIC METHOD FOR ACUTE TOXICITY
TESTS USING FISH AND MACROINVERTEBRATES.
In; Bioassay Procedures for Ocean Disposal Permit Pro-
gram, Environmental Research Laboratory, Gulf Breeze,
FL. U.S. EPA Ecological Research Series, EPA-600-9-78-
010. pp. 89-96.
Procedures are described for acute toxicity tests with
fish in containers 15 to 20 cm in depth. Tests require
saltwater in which healthy animals can survive through-
out acclimation and testing without stress as evidenced
by unusual behavior or discoloration. Appropriate test
animals and test materials are specified.
Hansen, David J., Steven C. Schimmel, Del Wayne Nimmo,
Jack I. Lowe, Patrick R. Parrish, and William H. Peltier.
1978. FLOW-THROUGH METHODS FOR ACUTE TOX-
ICITY TESTS USING FISH AND MACROINVERTE-
BRATES. In: Bioassay Procedures for Ocean Disposal Per-
mit Program, Environmental Research Laboratory, Gulf
Breeze, FL. U.S. EPA Ecological Research Series, EPA-
600/9-78-010. pp. 97-106.
Continuous-flow (often referred to as flow-through)
bioassays are preferred over static tests in evaluating
certain types of wastes to be disposed of at sea, partic-
ularly those with high biochemical oxygen demands
and those that are unstable or volatile. Many test spe-
cies of fish and macroinvertebrates have high metabo-
lic rates and are difficult to maintain in jars or tanks
of standing seawater. A method is described for a 96-
hr, flow-through bioassay on marine fish and macro-
invertebrates appropriate for the evaluation of wastes.
Jackson, Betty P., editor. 1978. RESEARCH REVIEW.
1977. Environmental Research Laboratory, Gulf Breeze,
FL. U.S. EPA Ecological Research Series, EPA-600/9-78-
014, 64 p.
This report summarizes results of aquatic research con-
ducted by the Environmental Research Laboratory,
Gulf Breeze, FL, office of Research and Development,
U.S. Environmental Protection Agency, from January
1 to December 30, 1977. The research program exam-
ines the impact of pesticides and other organic com-
pounds on marine species and communities, and seeks
to develop new methodology for determining ecologi-
cal hazards of chemical substances under simulated
natural conditions. Projects are outlined under four
categories: research related to lexicological testing;
biological processes and effects; development of off-
shore oil resources; and Kepone in the marine environ-
ment. Investigations conducted at the laboratory's
Atlantic Coast field station at Bears Bluff, SC, also are
reviewed for the year 1977.
Jackson, Betty P., editor. 1978. SYMPOSIUM ON PRO-
TECTING THE MARINE ENVIRONMENT: RESEARCH
AND REGULATION. Environmental Research Labora-
tory, Gulf Breeze, FL. U.S. EPA Ecological Reporting
Series, EPA 600/9-78-006. 38 p.
This symposium focuses on the essential role of
research and regulator agencies in protecting marine
ecosystems. Purpose of the symposium is to commem-
orate dedication of a new toxicological test facility at
the U.S. Environmental Protection Agency's Environ-
mental Research Laboratory in Gulf Breeze, FL, on
October 7, 1977. Participants define the special func-
tion of the federal agency scientist, the social responsi-
bility of the scientist, and the need for research in
support of environmental regulation. Historical and
future objectives of the Gulf Breeze Laboratory are
also reviewed.
Jackson, Betty P. and Andree F. Lowry, editors. 1979.
PUBLICATIONS GULF BREEZE LABORATORY.
Environmental Research Laboratory, Gulf Breeze, FL.
U.S. EPA Ecological Research Series, EPA-600/3-79-036.
115 p.
This bibliography, inclusive from 1971 through 1978,
lists all publications authored by researchers employed
by the Environmental Research Laboratory, Gulf
Breeze, and its field station on St. Johns Island, SC, or
by researchers conducting studies under funding or
direction of the laboratory.
63
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Koch, Robert B. 1978. DETERMINATION OF THE
SITE(S) OF ACTION OF SELECTED PESTICIDES BY
AN ENZYMATIC-IMMUNOBIOLOGICAL APPROACH.
Environmental Research Laboratory, Gulf Breeze, FL.
U.S. EPA Ecological Research Series, EPA-600/3-78-093.
29 p.
This report describes development of an antibody to
an organochlorine pesticide to be used in studies
related to its inhibition of the ATPase system. Kelevan,
the condensation product of ethyl levulinate and
Kepone, was successfully conjugated to bovine serum
albumin (BSA), fibrinogen (BF), and gamma globulin
(BGG). Rabbits and chickens preimmunized with BSA
and then immunized with BSA-Kelevan produced anti-
bodies to both the hapten, Kelevan, and the carrier
protein BSA. Antiserum to Kelevan protected ATPase
activity against Kepone and its derivatives. The titer of
antibody to Kelevan was critical since antiserum with
only trace amounts of Kelevan antibody failed to pro-
tect the ATPase activity against Kepone inhibition.
Antibody was concentrated by Na2SO4 fractional pre-
cipitation of the antiserum and obtained in pure form
by affinity chromatography with BGG-Kel covalently
linked to Sepharose 4B. Pure antibody was obtained
from untreated blood serum or plasma with no prior
pretreatment or fractionation with the BGG-Kel affin-
ity column. Complete protection of mitochondrial
mg2+ATPase activity from in vitro inhibition of
Kepone was obtained with a 1.2 mg quantity of
Na2SO4 fractionated antibody and only 120 yg of
pure antibody. Reversal of ATPase inhibition was
readily obtained by addition of antibody prior to
addition of substrate to the reaction mixture.
Mix, Michael C. 1979. CHEMICAL CARCINOGENS IN
BIVALVE MOLLUSKS FROM OREGON ESTUARIES.
Environmental Research Laboratory, Gulf Breeze, FL.
U.S. EPA Ecological Research Series, EPA-600/3-79-034.
The research undertaken involved the use of indigenous
populations of bivalve mollusks as monitors for detect-
ing and quantifying environmental benzo[a] pyrene
(BaP) in Oregon estuaries. Short-term and long-term
studies were conducted in order to establish baseline
levels of BaP and to identify seasonal variations in
BaP concentrations in shellfish. A presumptive cellular
proliferative disorder, though possibly to be neoplastic,
was also studied in mussels (Mytilus edulis) from
Yaquina Bay.
Histological studies revealed that mussels inhabiting
polluted environments, and with high BaP body bur-
dens, had an average 6-8% prevalence of the cellular
proliferative disorder while those from clean environ-
ments and with low or undetectable levels, did not
have the disorder. The cellular condition showed a
definite seasonal pattern, there was a low prevalence
during the summer and fall followed by an increase
during the early winter and a peak prevalence occurred
in January-February. The atypical, large cells that
characterize the disorder in M. edulis possess many
ultrastructural properties in common with malignant
vertebrate cells.
Nimmo, D.R., T.L. Hamaker, and C.A. Sommers. 1978.
CULTURING THE MYSID (MYSIDOPSIS BAHIA) IN
FLOWING SEAWATER OR A STATIC SYSTEM. In:
Bioassay Procedures for Ocean Disposal Permit Program.
Environmental Research Laboratory, Gulf Breeze, FL.
U.S. EPA Ecological Research Series, EPA-600/9-78-010.
pp. 59-60.
Methods are, described for the culture of the bay mysid
(Mysidopsis babia) for life-cycle toxicity tests in (1)
flowing seawater and (2) a re-circulating aquarium. The
mysid is considered a practical organism for toxicologi-
cal and physiological studies during sensitive stages of
development.
Nimmo, D.R., T.L. Hamaker, and C.A. Sommers. 1978.
ENTIRE LIFE-CYCLE TOXICITY TEST USING MYSIDS
(MYSIDOPSIS BAHIA) IN FLOWING WATER. In: Bio-
assay Procedures for Ocean Disposal Permit Program,
Environmental Research Laboratory, Gulf Breeze, FL.
U.S. EPA Ecological Research Series, EPA-600/9-78-010.
pp. 64-68.
Procedures of a method are outlined for determining
effects of continuous exposure of a pollutant on the
survival, reproduction, growth, and behavior of a crus-
tacean (Mysidopsis bahia) throughout a life cycle. The
test species can be captured from small shallow ponds
fed by saltwater with a small fish net or a 3- to 4-foot
push net of small mesh.
Parrish, Patrick R., Elizabeth E. Dyar, Joanna M. Enos,
and William G. Wilson. 1978. CHRONIC TOXICITY OF
CHLORDANE, TRIFLURALIN, AND PENTACHLORO-
PHENOL TO SHEEPSHEAD MINNOWS (CYPRINODON
VARIEGATUS). Environmental Research Laboratory,
Gulf Breeze, FL. U.S. EPA Ecological Research Series,
EPA-600/3-78-010. 53 p.
Test results are reported of exposures of sheepshead
minnows (Cyprinodon variegatus) to three chemicals--
chlordane, trifluralin, or pentachlorophenol-in flowing,
natural seawater to determine acute and chronic (full
life-cycle) effects.
Mortality of parental fish exposed to mean measured
chlordane concentrations J> 2.8 yg/£ was significantly
greater than that of control fish. Hatch of juveniles
from embryos of parental fish exposed to _> 0.8 yg/£
was significantly less than hatch of control juveniles.
The estimated maximum acceptable toxicant concen-
tration (MATC) was >0.5<0.8 yg/Ji and the applica-
tion factor (AF) limits, 0.04-0.06.
Exposure to mean measured trifluralin concentrations
^9.6 yg/£ significantly decreased growth of parental
fish. Fecundity of parental fish exposed to concentra-
tions _> 4.8 yg/£ was significantly less than that of con-
trol fish. Survival and growth of second generation fish
-------
were significantly less than the control in concentra-
tions _> 9.6 yg/£. The estimated MATC was >1.3<4.8
yg/£ and the AF limits, 0.007-0.025.
Mortality of parental sheepshead minnows exposed to
mean measured pentachlorophenol concentrations >88
yg/£ was significantly greater than mortality of con-
trol fish. The estimated MATC was >47<88 pg/£ and
the AF limits, 0.11-0.20.
Roberts, Morris H., Jr., Chae E. Laird, and Jerome E.
Illowsky. 1979. EFFECTS OF CHLORINATED SEA-
WATER ON DECAPOD CRUSTACEANS AND MULINIA
LARVAE. Environmental Research Laboratory, Gulf
Breeze, FL. U.S. EPA Ecological Research Series, EPA-
600/3-79-031. 110 p.
Eggs and larvae of decapod crustaceans and embryos of
Mulinia lateralis were exposed to chlorinated seawater
for varying periods in continuous flow systems. Mortal-
ity, developmental rate, and general behavior were
recorded. Pcmopeus herbstii zoeae were more sensitive
to chlorine-induced oxidants (CIO) than eggs or adults
(96-hr LC50 ca. 2.8 yeq/i = 0.1 mg/£). The 96-hr
LC50 for Pagurus longicarpus zoeae was approximately
the same as for Panopeus zoeae. The 120-hr LC50 for
Pagurus zoeae was 1.4 yeq/£ (0.05 mg/£). Development
was slightly delayed for Pagurus zoeae at CIO levels as
low as 0.6 yeq/£ (0.02 mg/£). Mulinia embryos exposed
for 48-hr had an LC50 between 0.3 and 2.8 yeq/£
(0.01 and 0.1 mg/£). Mulinia embryos exposed to chlo-
rinated seawater for 2 hr had an LC50 of about 2.0
yeq/£ (0.072 mg/£); subsequent survival rates for larvae
in unchlorinated seawater were unaffected by prior
exposure to CIO.
The effects of CIO on serum constituents in
Callinectes sapidus occurred sporadically and appeared
unrelated to dose or mortality. Similar effects were
noted for oxygen consumption in whole crabs and
excised gills.
Turekian, Karl K., and Anotoliy I. Simonov, editors.
1978. FIRST AMERICAN-SOVIET SYMPOSIUM ON
CHEMICAL POLLUTION OF THE MARINE ENVIRON-
MENT. Environmental Research Laboratory, Gulf Breeze,
FL. U.S. EPA Ecological Research Series, EPA-600/9-78-
038. 199 p.
This symposium, organized under a U.S.-U.S.S.R.
Environmental Agreement (Project 02.06-21), focuses
on the impact of chemical pollution on the world's
oceans. Soviet and American specialists discuss the fate
of heavy metals in estuaries and the Gulf of Mexico;
transport of natural radionuclides in shelf waters of
the eastern U.S.; the distribution and dynamics of
trace metals in pore water and sediment; biogeo-
chemical research on metals in the world's oceans;
monitoring chemical pollution and forecasting its bio-
logical consequences; arsenic, antimony, and mercury
in seawater; pollution of the Caribbean Basin; oil and
oil products in surface waters of the Atlantic, Pacific,
and Indian Oceans; the forms of heavy metals in sea-
water (e.g. mercury); methods of sampling water from
the ocean surface microlayer and the technical compo-
sition of the microlayer; a method for determining
mercury; scientific aspects of marine pollution prob-
lems; and the management of the quality of the
marine environment.
Tyler-Schroeder, Dana Beth. 1978. CULTURE OF THE
GRASS SHRIMP (PALAEMONETES PUGIO) IN THE
LABORATORY. In: Bioassay Procedures for Ocean Dis-
posal Permit Program, Environmental Research Labora-
tory, Gulf Breeze, FL. U.S. EPA Ecological Research
Series, EPA-600/9-78-010. pp. 69-72.
The grass shrimp (Palaemonetes pugio) is useful in
assessing toxicity of various materials. It is (1) easily
cultured in the laboratory and sensitive to toxicants,
and (2) can be exposed to toxicants in flow-through
aquaria throughout its life cycle. Culture and holding
procedures are described.
Tyler-Schroeder, D.B. 1978. STATIC BIOASSAY PRO-
CEDURE USING GRASS SHRIMP (PALAEMONETES
SP.) LARVAE. In: Bioassay Procedures for Ocean Dis-
posal Permit Program, Environmental Research Labora-
tory, Gulf Breeze, FL. U.S. EPA Ecological Research
Series, EPA-600/9-78-010. pp. 73-82.
Procedures are outlined for static 96-hr bioassays with
the grass shrimp larvae, Palaemonetes sp. Three species
of the genus, P. pugio, vulgaris, and intermedius, are
easily collected in the field and maintained in the
laboratory. Spawning can be induced in the laboratory
by manipulating temperature and light. Developing
larvae have demonstrated a greater susceptibility to
polychlorinated hydrocarbons than observed in adults
or juveniles.
Tyler-Schroeder, Dana Beth. 1978. ENTIRE LIFE-CYCLE
TOXICITY TEST USING GRASS SHRIMP
(PALAEMONETES PUGIO HOLTHUIS). In: Bioassay
Procedures for Ocean Disposal Permit Program, Environ-
mental Research Laboratory, Gulf Breeze, FL. U.S. EPA
Ecological Research Series, EPA-600/9-78-010. pp. 83-88.
A method to assess toxicity of a material to all life
stages of the grass shrimp in flow-through systems is
described. Tests are conducted throughout the life
cycle of the shrimp-from juvenile stage of the parental
generation, sexual maturation and reproduction,
through hatching, larval development and growth of
the Fj generation to juvenile stage. Thereafter, tests
may terminate, or exposures can be continued if a
determination of effects on F^ reproduction and p2
larval development is required.
Vernberg, F.J., W. Kitchens, H. McKellar, K. Summers,
and R. Bonnell. 1978. THE DYNAMICS OF AN ESTU-
ARY AS A NATURAL ECOSYSTEM, VOL. II. Environ-
mental Research Laboratory, Gulf Breeze, FL. U.S. EPA
Ecological Research Series, EPA-600/3-78-092. 29 p.
This report describes two separate but interrelated sub-
studies: an update of the macroecosystem model of
65
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the North Inlet Estuary near Georgetown, SC, and a
continuing study of experimental saltmarsh microeco-
systems. The model is under development to help
understand the interactions of various parts of a natu-
ral ecosystem. The principal objective of the study is
to develop and test replicate experimental salt-marsh
units at the microecosystem level as diagnostic tools
for assessing long- and short-term pollution effects on
the Spartina alterniflora salt-marsh community.
Because of the complexity, this study was conceived as
a five-year work. Two years of study (March 1, 1976,
to February 28, 1978) are reported. A summary of
the first phase of this research is contained in the
Ecological Research Series (EPA-600/3-77-016, January
1977).
Walker, William W. 1978. INSECTICIDE PERSISTENCE
IN NATURAL SEAWATER AS AFFECTED BY SALIN-
ITY, TEMPERATURE, AND STERILITY. Environmental
Research Laboratory, Gulf Breeze, FL. U.S. EPA Ecologi-
cal Research Series, EPA-600/3-78-044. 25 p.
Effects of temperature, salinity, and sterility on the
degradation of malathion, parathion, methyl parathion,
diazinon, and methoxychlor in fresh and estuarine
water under controlled conditions are reported. Surface
water samples of 1, 10, 20, and 28 °/oo salinity were
amended with these insecticides 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 chromatographic techniques.
No significant differences between sterile and non-
sterile treatments were observed for any of the insecti-
cide studies; the effect of increasing temperature was
highly significant with regard to increased degradation
of malathion, parathion, methyl parathion, and diazi-
non. Methoxychlor reflected the recalcitrance charac-
teristic of the chlorinated hydrocarbon insecticides
throughout 84 days of incubation and was not signifi-
cantly affected by salinity, temperature, or sterility.
Salinity effects were varied among the four organo-
phosphates: highly significant for malathion and diazi-
non, significant for methyl parathion, and not signifi-
cant for parathion.
Wilkes, Frank G. 1978. MICROCOSMS AS BIOLOGICAL
INDICATORS OF POLLUTION. In:' First American-
Soviet Symposium on the Biological Effects of Pollution
on Marine Organisms, Thomas W. Duke and Anatoliy I.
Simonov, editors. Environmental Research Laboratory,
Gulf Breeze, FL. U.S. EPA Ecological Research Series,
EPA-600/9-78-007. pp. 155-56.
Research conducted and supported by the Environ-
mental Research Laboratory, Gulf Breeze, to develop
microcosms as a method for investigating pollutant
fate and effects in the environment is described. Eco-
system compartments under investigation include
direct accumulation from water and food by organisms
at all trophic levels, bioaccumulation through food
chains, direct effects of pollutants on organisms, i.e.,
mortality, reproduction and behavior, and indirect
effects of sublethal levels of pollutants, such as changes
in predator-prey relationships. Microbial processes at
both air-water and sediment-water interfaces are investi-
gated as well as physical and chemical transformations.
Journal Articles
Bahner, L.H. and J.L. Oglesby. 1979. TEST OF A
MODEL FOR PREDICTING KEPONE ACCUMULATION
IN SELECTED ESTUARINE SPECIES. Aquatic Toxicol-
ogy, ASTM STP 667, L.L. Marking and R.A. Kimerle,
editors, American Society for Testing and Materials, pp.
221-231.
Bourquin, A.W., P.H. Pritchard, and W.R. Mahaffey. 1978.
EFFECTS OF KEPONE ON ESTUARINE MICROORGA-
NISMS. Dev. Ind. Microbiol., Vol. 19, pp. 489-497. (ERL,
GB Reprint #345).
Bourquin, A.W., R.L. Garnas, P.H. Pritchard, F.G. Wilkes,
C.R. Gripe, and N.I. Rubinstein, 1979. INTERDEPEND-
ENT MICROCOSMS FOR THE ASSESSMENT OF POL-
LUTANTS IN THE MARINE ENVIRONMENT. Intern. J.
Environ. Studies. 13:131-140.
Brannon, Anita C. and K. Ranga Rao. 1979. BARIUM
STRONTIUM AND CALCIUM LEVELS IN THE EXO-
SKELETON, HEP ATOP ANCREAS AND ABDOMINAL
MUSCLE OF THE GRASS SHRIMP, PALAEMONETES
PUGIO; RELATION TO MOLTING AND EXPOSURE
TO BARITE. Comp. Biochem. Physiol. 63A:261-274.
Butler, Philip A., Charles D. Kennedy, and Roy L.
Schutzmann. 1978. PESTICIDE RESIDUES IN ESTUA-
RINE MOLLUSKS, 1977 VERSUS 1972-NATIONAL
PESTICIDE MONITORING PROGRAM. Pestic. Monit. J.
12(3):99-101.
Butler, P.A. and R.L. Schutzmann. 1979. BIOACCUMU-
LATION OF DDT AND PCB IN TISSUES OF MARINE
FISHES. Aquatic Toxicology, ASTM STP 667, L.I.
Marking and R.A. Kimerle, editors, American Society for
Testing and Materials, pp. 212-220.
Caldwell, R.S., D.A. Armstrong, D.V. Buchanan, M.H.
Mallon, and R.E. Millemann, 1978. TOXICITY OF THE
FUNGICIDE CAPTAN TO THE DUNGENESS CRAB,
CANCER MAG1STER. Mar. Biol. 48(1): 11-18.
Cantelmo, F.R. and K.R. Rao. 1978. EFFECT OF
PENTACHLOROPHENOL (PCP) ON MEIOBENTHIC
COMMUNITIES ESTABLISHED IN AN EXPERI-
MENTAL SYSTEM. Mar. Biol. 46:17-22.
66
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Cantelmo, Angela C. and K. Ranga Rao. 1978. THE
EFFECTS OF PENTACHLOROPHENOL (PGP) AND 2,4-
DINITROPHENOL (DNP) ON THE OXYGEN CON-
SUMPTION OF TISSUES FROM THE BLUE CRAB,
CALLINECTES SAPIDUS, UNDER DIFFERENT
OSMOTIC CONDITIONS. Comp. Biochem. Physiol. 60C:
215-219.
Conklin, Philip J. and K. Ranga Rao. 1978. TOXICITY
OF SODIUM PENTACHLOROPHENATE (Na-PCP) TO
THE GRASS SHRIMP, PALAEMONETES PUGIO, AT
DIFFERENT STAGES OF THE MOLT CYCLE. Bull.
Environm. Contam. Toxicol. 20:275-279.
Cooky, Nelson R. 1978. AN INVENTORY OF THE
ESTUARINE FAUNA IN THE VICINITY OF
PENSACOLA, FLORIDA. Florida Dept. of Natural
Resources, St. Petersburg, FL, Florida Marine Research
Publication No. 31. 119 p. (ERL,GB Contribution
#102).
Couch, John A. 1978. DISEASES, PARASITES, AND
TOXIC RESPONSES OF COMMERCIAL PENAEID
SHRIMPS OF THE GULF OF MEXICO AND SOUTH
ATALNTIC COASTS OF NORTH AMERICA. U.S. Natl.
Mar. Fish Serv. Fish. Bull. 76(1): 1-44. (ERL,GB Reprint
#283).
Couch, J.A., J.T. Winstead, D.J. Hansen, and L.R.
Goodman, 1979. VERTEBRAL DYSPLASIA IN YOUNG
FISH EXPOSED TO THE HERBICIDE TRIFLURALIN.
J. Fish Diseases. 2:35-42.
Gripe, C.R. and B. Stokes. 1978. DEVICE TO DETECT
POTENTIALLY DANGEROUS ELECTRICAL CUR-
RENTS IN SALTWATER HOLDING TANKS. Prog. Fish-
Cult. 40(2):74-75. (ERL,GB Reprint #335).
Gripe, C.R. 1979. AN AUTOMATED DEVICE (AGARS)
FOR STUDYING AVOIDANCE OF POLLUTANT GRA-
DIENTS BY AQUATIC ORGANISMS. J. Fish. Res. Board
Can. 36:11-16.
Engler, R.M. and F.G. Wilkes, editors. 1979. ACTIVITY
REPORT: ENVIRONMENTAL PROTECTION AGENCY/
CORPS OF ENGINEERS TECHNICAL COMMITTEE ON
CRITERIA FOR DREDGED AND FILL MATERIAL.
Environmental Laboratory, U.S. Army Engineer Water-
ways Experiment Station, Vicksburg, MS. 31 p.
Farr, James A. 1978. THE EFFECT OF METHYL PARA-
THION ON PREDATOR CHOICE OF TWO ESTUARINE
PREY SPECIES. Trans. Am. Fish. Soc. 107(1):87-91.
Farr, James A. 1978. ORIENTATION AND SOCIAL
BEHAVIOR IN THE SUPRALITTORAL ISOPOD LIGIA
EXOTICA (CRUSTACEA: ONISCOIDEA). Bull. Mar.
Sci. 28(4):659-666.
Pass, Linda F., and James C. Moore. 1979. DETERMI-
NATION OF PENTACHLOROPHENOL IN MARINE
BIOTA AND SEA WATER BY GAS-LIQUID CHROMA-
TOGRAPHY AND HIGH-PRESSURE LIQUID CHROMA-
TOGRAPHY. J. Agri. Food Chem. May-June 1979, pp.
554-557.
Fox, Ferris R. and K. Ranga Rao. 1978. CHARACTER-
ISTICS OF Ca2+-ACTIVATED ATPASE FROM THE
HEPATOPANCREAS OF THE BLUE CRAB,
CALLINECTES SAPIDUS. Comp. Biochem. Physiol.
59B:327-331.
Helz, George R., and Rong Y. Hsu. 1978. VOLATILE
CHLORO- AND BROMOCARBONS IN COASTAL
WATERS. Limnol. Oceanogr. 23(5):858-869.
Johnson, H.E., R. Parrish, G.F. Lee, C.E. Johnson, J.
Sanborn, D. Hansen, and J. Hamelink. 1978. ENVIRON-
MENTAL CONCENTRATION AND FATE-DISCUSSION
SESSION SYNOPSIS. Estimating the Hazard of Chemical
Substances to Aquatic Life, J. Cairns, K.L. Dickson, A.W.
Maki, editors, American Society for Testing and Materials,
STP 657. pp. 71-80.
Laughlin, Roger A., Claude R. Gripe, and Robert J.
Livingston. 1978. FIELD AND LABORATORY AVOID-
ANCE REACTIONS BY BLUE CRABS (CALLINECTES
SAPIDUS) TO STORM WATER RUNOFF. Trans. Am.
Fish. Soc. 107(l):78-86.
Lucyszyn, E., and P.H. Pritchard. 1979. CHARACTER-
ISTICS OF BACTERIA ADAPTED TO LOW NUTRIENT
CONDITIONS IN LAKE ONTARIO. Dev. Ind. Microbiol.
20:579-589.
Mahaffey, W.R., P.H. Pritchard, and A.W. Bourquin. 1979.
PHENYLACETIC ACID METABOLISM BY THREE
AQUATIC BACTERIA ISOLATED FROM CONTIN-
UOUS CULTURE ENRICHMENTS. Dev. Ind. Microbiol.
20:489-495.
Middaugh, Douglas P., and Genie Floyd. 1978. THE
EFFECT OF PREHATCH AND POSTHATCH EXPO-
SURE TO CADMIUM ON SALINITY TOLERANCE OF
LARVAL GRASS SHRIMP, PALAEMONETES PUGIO,
Estuaries, 1(2): 123-125. (ERL,GB Reprint #260).
Middaugh, D.P., J.M. Dean, R.G. Domey, and G. Floyd.
1978. EFFECT OF THERMAL STRESS AND TOTAL
RESIDUAL CHLORINATION ON EARLY LIFE
STAGES OF THE MUMMICHOG FUNDULUS
HETEROCLITUS, Mar. Biol. 46(l):l-8. (ERL.GB Reprint
#308).
Nimmo, D.R., R.A. Rigby, L.H. Bahner, and J.M.
Sheppard. 1978. THE ACUTE AND CHRONIC EFFECTS
OF CADMIUM ON THE ESTUARINE MYSID,
MYSIDOPSIS BAHIA. Bull. Environ. Contam. Toxicol
19(l);80-85. (ERL,GB Reprint #287).
67
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Nimmo, D.R., T.L. Hamaker, J.C. Moore, and C.A.
Simmers. 1979. EFFECT OF DIFLUBENZURON ON AN
ESTUARINE CRUSTACEAN. Bull. Environ. Contam.
Toxicol, 22:767-770.
Reish, Donald J., Thomas J. Kauwling, Alan J. Mearns,
Philip S. Oshida, Steven S. Rossi, Frank G. Wilkes, and
Marjorie J. Ray. 1978. MARINE AND ESTUARINE
POLLUTION. J. Water Pollut. Control Fed. 50(6): 1424-
1469.
Reish, Donald, Steven S. Rossi, Alan J. Mearns, Philip S.
Oshida, and Frank G. Wilkes. 1979. MARINE AND
ESTUARINE POLLUTION. J. Water Pollut. Control Fed.
51(6):1477-1516.
Schimmel, Steven C., James M. Patrick, Jr., Linda F.
Faas, Jerry L. Oglesby, and Alfred J. Wilson, Jr. 1979.
KEPONE®: TOXICITY AND BIOACCUMULATION IN
BLUE CRABS. Estuaries. 2(1):9-15.
Sheridan, P.P. 1979. FOOD HABITS OF THE BAY
ANCHOVY, ANCHOA MITCHILLI, IN APALACHICOLA
BAY, FL. Northeast Gulf Science 2(2): 126-132.
Tagatz, Marlin E., and Michael Tobia. 1978. EFFECT OF
BARITE (BaSO4) ON DEVELOPMENT OF ESTUARINE
COMMUNITIES. Estuarine Coastal Mar. Sci. 7(4):401-
407. (ERL,GB Reprint #340).
Tagatz, M.E., J.M. Ivey, H.K. Lehman, and J.L. Oglesby.
1978. EFFECTS OF A LIGNOSULFONATE-TYPE
DRILLING MUD ON DEVELOPMENT OF EXPERI-
MENTAL ESTUARINE MACROBENTHIC COMMUNI-
TIES. Northeast Gulf Science 2(l):35-42. (ERL,GB
Reprint #370).
Tagatz, M.E., J.M. Ivey, H.K. Lehman, and J.L. Oglesby.
1979. EFFECTS OF SEVIN ON DEVELOPMENT OF
EXPERIMENTAL ESTUARINE COMMUNITIES. J.
Toxicol. Environ. Health. 5:643-651.
Tyler-Schroeder, D.B. 1979. USE OF THE GRASS
SHRIMP (PALAEMONETES PUGIO) IN A LIFE-CYCLE
TOXICITY TEST. Aquatic Toxicology, ASTM STP 667,
L.L. Marking and R.A. Kimerle, editors, American Society
for Testing and Materials, pp. 159-170.
Walsh, G.E. 1978. TOXIC EFFECTS OF POLLUTANTS
ON PLANKTON. In: Principles of Ecotoxicology, G.E.
Butler, editor. John Wiley & Sons, New York, NY. pp.
257-274. (ERL,GB Reprint #341).
Walsh, Gerald E., Karen A. Ainsworth, and Rebekah
Rigby. 1979. RESISTANCE OF RED MANGROVE
(RHIZOPHORA MANGLE L.) SEEDLINGS TO LEAD,
CADMIUM, AND MERCURY. Biotropica. 11(1)-.22-27.
Wilson, Alfred J., and Jerrold Forester. 1978. PERSIST-
ENCE OF AROCLOR 1254 IN A CONTAMINATED
ESTUARY. Bull. Environ. Contam. Toxicol. 19(5):637-
640. (ERL.GB Reprint #339).
Presentations
Block, Ronald M., Dennis T. Burton, Steven R. Gullans,
and Leonard B. Richardson. 1978. RESPIRATORY AND
OSMOREGULATORY RESPONSES OF WHITE PERCH
(MORONE AMERICANA) EXPOSED TO CHLORINE
AND OZONE IN ESTUARINE WATERS. In: Water
Chlorination: Environmental Impact and Health Effects,
Vol. 2, Robert L. Jolley, Hend Gorchev, and D. Heyward
Hamilton, Jr., editors. Ann Arbor Science Publishers, Inc.,
Ann Arbor, MI. pp. 351-360.
Borthwick, Patrick W., and Steven C. Schimmel. 1978.
TOXICITY OF PENTACHLOROPHENOL AND
RELATED COMPOUNDS TO EARLY LIFE STAGES OF
SELECTED ESTUARINE ANIMALS. In: Pentachloro-
phenol: Chemistry, Pharmacology, and Environmental
Toxicology, K. Ranga Rao, editor. Plenum Publishing
Corp., New York, NY. pp. 141-146. (ERL.GB Reprint
#343).
Bourquin, A.W., and David T. Gibson. 1978. MICROBIAL
DEGRADATION OF HALOGENATED HYDROCAR-
BONS. In: Water Chlorination, Environmental Impact and
Health Effects, Vol. 2, Robert L. Jolley, Hend Gorchev,
and D. Heyward Hamilton, Jr., editors. Ann Arbor
Science Publishers, Inc., Ann Arbor, MI. pp. 253-258.
(ERL.GB Reprint #361).
Brannon, Anita C., and Philip J. Conklin. 1978. EFFECT
OF SODIUM PENTACHLOROPHENATE ON EXO-
SKELETAL CALCIUM IN THE GRASS SHRIMP,
PALAEMONETES PUGIO. In: Pentachlorophenol:
Chemistry, Pharmacology, and Environmental Toxicology,
K. Ranga Rao, editor. Plenum Publishing Corp., New
York, NY. pp. 205-211.
Caldwell, Richard S., Elaine M. Caldarone, and Barbara A.
Rosene. 1979. FATTY ACID COMPOSITION OF PHOS-
PHOLIPIDS IN THERMALLY ACCLIMATING
SCULPINS (LEPTOCOTTUS ARMATUS) TREATED
WITH POLYCHLORINATED BIPHENYLS (AROCLOR
1254). In: Marine Pollution: Functional Responses, W.B.
Vernberg, A. Calabrese, F. Thurberg, and F.J. Vernberg,
editors. Academic Press, Inc., New York, NY. pp. 271-
290.
Cantelmo, Angela C., Philip J. Conklin, Ferris R. Fox,
and K. Ranga Rao. 1978. EFFECTS OF SODIUM
PENTACHLOROPHENATE AND 2,4-DINITROPHENOL
ON RESPIRATION IN CRUSTACEANS. In: Pentachloro-
phenol: Chemistry, Pharmacology, and Environmental
68
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Toxicology, K. Ranga Rao, editor. Plenum Publishing
Corp., New York, NY. pp. 251-263.
Cantelmo, Frank R., and K. Ranga Rao. 1978. EFFECTS
OF PENTACHLOROPHENOL ON THE MEIOBENTHIC
NEMATODES IN AN EXPERIMENTAL SYSTEM. In:
Pentachlorophenol: Chemistry, Pharmacology, and
Environmental Toxicology, K. Ranga Rao, editor. Plenum
Publishing Corp., New York, NY. pp. 165-174.
Carpenter, James H., and Donald L. Macalady. 1978.
CHEMISTRY OF HALOGENS IN SEAWATER. In:
Water Chlorination: Environmental Impact and Health
Effects, Vol. 1, Robert L. Jolley, editor. Ann Arbor
Science Publishers, Inc., Ann Arbor, MI. pp. 161-179.
Carpenter, James H., and Carroll A. Smith. 1978. REAC-
TIONS IN CHLORINATED SEAWATER. In: Water
Chlorination: Environmental Impact and Health Impacts,
Vol. 2, Robert L. Jolley, Hend Gorchev, and D. Heyward
Hamilton, Jr., editors. Ann Arbor Science Publishers, Inc.,
Ann Arbor, MI. pp. 195-207.
Conklin, Philip J., and Ferris R. Fox. 1978. ENVIRON-
MENTAL IMPACT OF PENTACHLOROPHENOL AND
ITS PRODUCTS - A ROUND TABLE DISCUSSION. In:
Pentachlorophenol, K. Ranga Rao, editor. Plenum Pub-
lishing Corp., New York, NY. pp. 389-394.
Conklin, Philip J., and K. Ranga Rao. 1978. TOXICITY
OF SODIUM PENTACHLOROPHENATE TO THE
GRASS SHRIMP, PALAEMONETES PUGIO, IN RELA-
TION TO THE MOLT CYCLE. In: Pentachlorophenol:
Chemistry, Pharmacology, and Environmental Toxicology,
K. Ranga Rao, editor. Plenum Publishing Corp., New
York, NY. pp. 181-192.
Costlow, John D., Jr. 1979. EFFECT OF DIMILIN®
ON DEVELOPMENT OF LARVAE OF THE STONE
CRAB, MENIPPE MERCENARIA, AND THE BLUE
CRAB, CALLINECTES SAPIDUS. In: Marine Pollution:
Functional Responses, W.B. Vernberg, A. Calabrese, F.
Thurberg, and FJ. Vernberg, editors. Academic Press,
Inc., New York, NY. pp. 355-363.
Couch, J.A. 1978. BIOMONITORING AND CARCINO-
GEN INDICATORS ORGANISM PROGRAM OF ERL,
GB. Biological Monitoring Workshop, Raleigh, NC. March
1978.
Couch, J.A. 1978. Chairman for panel: HISTOPATH-
OLOGY OF OIL SPILLS DOCUMENTS. Gulf Coast Oil
Spill Response Workshop, Tampa, FL. April 1978.
Couch, J.A. 1978. HISTOPATHOLOGY AS A TOOL IN
THE STUDY OF AQUATIC ANIMALS AS INDICATORS
OF CARCINOGENS IN THE ENVIRONMENT. Third
Annual Eastern Fish Health Workshop, Auburn, AL. May
1978.
Couch, J.A., and J. Harshbarger. 1978. Co-chairman of
Session: NEOPLASMS IN INVERTEBRATES. Inter-
national Colloquin for Invertebrate Pathology, Prague,
Czech oslavakia. September 1978.
Couch, J.A. 1978. ELECTRON MICROSCOPY AS A
TOOL IN STUDIES OF AQUATI C ANIMAL DISEASES.
Louisiana Society for Electron Microscopy, Ocean Springs,
MS. October 1978.
Couch, J.A., W.P. Schoor, and N.L. Richards. 1978.
REVIEW OF CARCINOGENIC RESEARCH PROJECT.
National Cancer Institute Committee, Bethesda, MD.
March 1978.
Davis, W.P. 1978. IMPACTS OF OIL SPILL AND
CLEAN-UP ON THE EUROPEAN COAST: AMOCO
CADIZ. Proceedings of U.S./Japanese Experts Meeting on
Management of Bottom Sediments Containing Toxic Sub-
stance, Tokyo, Japan. October 1978.
Davis, William P., and William F. Mcllhenny. 1978.
MARINE WORKSHOP SUMMARY. In: Water Chlorina-
tion: Environmental Impact and Health Effects, Vol. 2,
Robert L. Jolley, Hend Gorchev, and D. Heyward
Hamilton, Jr., editors. Ann Arbor Science Publishers, Inc.,
Ann Arbor, MI. pp. 859-862.
Davis, William P., and Douglas P. Middaugh. 1978. A
REVISED REVIEW OF THE IMPACT OF CHLORINA-
TION PROCESSES UPON MARINE ECOSYSTEMS. In:
Water Chlorination: Environmental Impact and Health
Effects, Vol. 1, Robert L. Jolley, editor. Ann Arbor
Science Publishers, Inc., Ann Arbor, MI. pp. 283-310.
Doughtie, Daniel G., and K. Ranga Rao. 1978. ULTRA-
STRUCTURAL CHANGES INDUCED BY SODIUM
PENTACHLOROPHENATE IN THE GRASS SHRIMP,
PALAEMONETES PUGIO, IN RELATION TO THE
MOLT CYCLE. In: Pentachlorophenol: Chemistry,
Pharmacology, and Environmental Toxicology, K. Ranga
Rao, editor. Plenum Publishing Corp., New York, NY.
pp. 213-250.
Erickson, Stanton J., and Anne E. Freeman. 1978. TOX-
ICITY SCREENING OF FIFTEEN CHLORINATED AND
BROMINATED COMPOUNDS USING FOUR SPECIES
OF MARINE PHYTOPLANKTON. In: Water Chlorina-
tion: Environmental Impact and Health Effects, Vol. 2,
Robert L. Jolley, Hend Gorchev, D. Heyward Hamilton,
Jr., editors. Ann Arbor Science Publishers, Inc., Ann
Arbor, MI. pp. 307-310. (ERL,GB Reprint #359).
69
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Fox, Ferris R., and K. Ranga Rao. 1978. EFFECTS OF
SODIUM PENTACHLOROPHENATE AND 2,4-DINITRO-
PHENOL ON HEP ATOP ANCREATIC ENZYMES IN THE
BLUE CRAB, CALLINECTES SAPIDUS. In: Pentachloro-
phenol: Chemistry, Pharmacology, and Environmental
Toxicology, K. Ranga Rao, editor. Plenum Publishing
Corp., New York, NY. pp. 265-275.
Garnas, R.L., and D.G. Crosby. 1979. COMPARATIVE
METABOLISM OF PARATHION BY INTERTIDAL
INVERTEBRATES. In: Marine Pollution: Functional
Responses, W.B. Vernberg, A. Calabrese, F. Thurberg, and
F.J. Vernberg, editors. Academic Press, Inc., New York,
NY. pp. 291-305.
Goodman, Larry R., David J. Hansen, John A. Couch,
and Jerrold Forester. 1978. EFFECTS OF HEPTACHLOR
AND TOXAPHENE ON LABORATORY-REARED
EMBRYOS AND FRY OF THE SHEEPSHEAD MINNOW.
Proc. 30th Annu. Conf. Southeast. Assoc. Game Fish
Comm., October 24-27, 1976, Jackson, MS, Wilmer
Rogers, editor, pp. 192-202 (ERL,GB Reprint #297).
Hansen, D.J., and M.E. Tagatz. 1978. EVALUATION OF
A LABORATORY TEST FOR ASSESSING IMPACTS
OF SUBSTANCES ON DEVELOPING COMMUNITIES
OF BENTHIC ESTUARINE ORGANISMS. Symposium
American Society for Testing and Materials (ASTM), New
Orleans, LA. November 3, 1978.
Helz, George R., Richard Sugam, and Rong Y. Hsu. 1978.
CHLORINE DEGRADATION AND HALOCARBON PRO-
DUCTION IN ESTUARINE WATERS. In: Water Chlori-
nation: Environmental Impact and Health Effects, Vol. 2,
Robert L. Jolley, Hend Gorchev, and D. Heyward
Hamilton, Jr., editors. Ann Arbor Science Publishers, Inc.,
Ann Arbor, MI. pp. 209-222.
Nimmo, DelWayne R. 1979. PESTICIDES: THEIR
IMPACT ON THE ESTUARINE ENVIRONMENT. In:
Marine Pollution: Functional Responses, W.B. Vernberg,
A. Calabrese, F. Thurberg, and,F.J. Vernberg, editors.
Academic Press, Inc., New York, NY. pp. 259-270.
Rao, K. Ranga, Ferris R. Fox, Philip J. Conklin, Angela
C. Cantelmo, and Anita C. Brannon. 1979. PHYSIOLOG-
ICAL AND BIOCHEMICAL INVESTIGATIONS OF THE
TOXICITY OF PENTACHLOROPHENOL TO CRUSTA-
CEANS. In; Marine Pollution: Functional Responses,
W.B. Vernberg, A. Calabrese, F. Thurberg, and F.J.
Vernberg, editors. Academic Press, Inc., New York, NY.
pp. 307-339.
Rao, K. Ranga, Philip J. Conklin, and Anita C. Brannon.
1978. INHIBITION OF LIMB REGENERATION IN THE
GRASS SHRIMP, PALAEMONETES PUGIO, BY SOD-
IUM PENTACHLOROPHENATE. In: Pentachlorophenol:
Chemistry, Pharmacology, and Environmental Toxicology,
K. Rango Rao, editor. Plenum Publishing Corp., New
York, NY. pp. 193-203.
Roberts, Morris H., Jr. 1978. EFFECTS OF CHLORI-
NATED SEAWATER ON DECAPOD CRUSTACEANS.
In: Water Chlorination: Environmental Impact and
Health Effects, Vol. 2, Robert L. Jolley, Hend Gorchev,
and D. Heyward Hamilton, Jr., editors. Ann Arbor
Science Publishers, Inc., Ann Arbor, MI. pp. 329-334.
Rubinstein, Norman I. 1979. A BENTHIC BIOASSAY
USING TIME-LAPSE PHOTOGRAPHY TO MEASURE ,
THE EFFECT OF TOXICANTS ON THE FEEDING
BEHAVIOR OF LUGWORMS (POLYCHAETA
ARENICOLIDAE). In: Marine Pollution: Functional
Responses, W.B. Vernberg, A. Calabrese. F. Thurberg, and
F.J. Vernberg, editors. Academic Press, Inc., New York,
NY. pp. 341-351.
Rubinstein, Norman I. 1978. EFFECT OF SODIUM
PENTACHLOROPHENATE ON THE FEEDING ACTIV-
ITY OF THE LUGWORM, ARENICOLA CRISTATA
STIMSON. In: Pentachlorophenol: Chemistry, Pharma-
cology, and Environmental Toxicology, K. Ranga Rao,
editor. Plenum Publishing Corp., New York, NY. pp.
175-180.
Schimmel, Steven C., James M. Patrick, Jr., and Linda F.
Faas. 1978. EFFECTS OF SODIUM PENTACHLORO-
PHENATE ON SEVERAL ESTUARINE ANIMALS:
TOXICITY, UPTAKE, AND DEPURATION. In: Penta-
chlorophenol: Chemistry, Pharmacology, and Environ-
mental Toxicology, K. Ranga Rao, editor. Plenum Pub-
lishing Corp., New York, NY. pp. 147-155. (ERL,GB
Reprint #336).
Schoor, W.P. 1978. ACETYL CHOLINESTERASE
ACTIVITY IN SHRIMP. 176th American Chemical
Society National Meeting, Miami Beach, FL. September
17, 1978.
Scott, G.I., and W.B. Vernberg. 1979. SEASONAL
EFFECTS OF CHLORINE PRODUCED OXIDANTS ON
THE GROWTH, SURVIVAL, AND PHYSIOLOGY OF
THE AMERICAN OYSTER, CRASSOSTREA
VIRGINICA (GMELIN). In: Marine Pollution: Func-
tional Responses, W.B. Vernberg, A. Calabrese, F.
Thurberg, and F.J. Vernberg, editors. Academic Press,
Inc., New York, NY. pp. 415-435.
Scott, Geoffrey I., and Douglas P. Middaugh. 1978.
SEASONAL CHRONIC TOXICITY OF CHLORINATION
TO THE AMERICAN OYSTER, CRASSOSTREA
VIRGINICA (G). In: Water Chlorination: Environmental
Impact and Health Effects, Vol. 2, Robert L. Jolley,
Hend Gorchev, and D. Heyward Hamilton, Jr., editors.
Ann Arbor Science Publishers, Inc., Ann Arbor, MI. pp.
311-327. (ERL,GB Reprint #360).
70
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Tagatz, M.E., J.M. Ivey, and M. Tobia. 1978. EFFECTS
OF DOWICIDE® G-ST ON DEVELOPMENT OF EXPER-
IMENTAL ESTUARINE MACRO-BENTHIC COMMUNI-
TIES. In: Pentachlorophenol: Chemistry, Pharmacology,
and Environmental Toxicology, K. Ranga Rao, editor.
Plenum Publishing Corp., New York, NY. pp. 157-163.
(ERL.GB Reprint #352).
Walsh, G.E. 1978. TOXICITY OF TEXTILE INDUSTRY
WASTES TO U.S. MARINE ORGANISMS. Textile Waste
Symposium, Williamsburg, VA. (Sponsored by EPA Indus-
trial Environmental Research Laboratory, Research Tri-
angle Park, NC.) October 1978.
Wilkes, Frank G. 1978. LABORATORY MICROCOSMS
FOR USE IN DETERMINING POLLUTANT STRESS.
In: Aquatic Pollutants: Transformation and Biological
Effects, O. Hutzinger, I.H. Van Lelyveld, and B.C.J.
Zeeteman, editors. Pergamon Press, New York, NY. pp.
309-321. (ERL,GB Reprint #357).
71
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TECHNICAL REPORT
(Please read Instructions on the reverse
DATA
before completing)
. REPORT NO.
EPA-6on/q-79-o?a
2.
3. RECIPIENT'S ACCESSION NO.
4. TITLE ANDSUBTITLE
5. REPORT DATE
September 1979
Research Review
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 & Development
U.S. Environmental Protection Agency
Gulf Breeze, FL 32561
10. PROGRAM ELEMENT NO.
1EA615
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
13. TYPE OF REPORT AND PERIOD COVERED
October 1978-Septemher 1Q7Q
14. SPONSORING AGENCY CODE
EPA/600/4
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This report reviews aquatic research programs conducted or managed
by the Environmental Research Laboratory, Gulf Breeze, Florida, for
the Office of Research and Development, U.S. Environmental Protec-
tion Agency in 1978 and 1979. The research program examines the
impact of pesticides and other organic compounds on marine species
and communities, and seeks to develop new methodology for determin-
ing ecological hazards of chemicals under conditions simulating the
natural environment. Projects are outlined in the areas of:
Exposure Assessment, Effects Assessment, Chlorination Studies,
Offshore Oil Drilling, Environmental Pathobiology, and the
Chesapeake Bay Program.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Marine biology
Toxicology
Carcinogens
Mutagens
Pesticides
Bioassay
Environmental Research
Laboratory, Gulf Breeze,
Florida
Environmental Protection
Agency
Mirrnrnsms
6F
6A
6E
18. DISTRIBUTION STATEMENT
Release to public
19. SECURITY CLASS (This Report)
unclassified
21. NO. OF PAGES
72
20. SECURITY CLASS (This page)
unclassified
22. PRICE
EPA Form 2220-1 (Rev. 4-77)
PREVIOUS EDITION IS OBSOLETE
72
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Additional information on research reviewed in this publication is available from laboratory personnel listed below with
subject area.
Environmental Research Laboratory, Gulf Breeze, FL 32561
Bears Bluff Field Station, P.O. Box 368, Johns Island, SC 29455
(904) 932-5311
FTS 686-9011
Director
Deputy Director
Associate Director for Extramural Activities
Chief, Processes and Effects Branch
Chief, Experimental Environments Branch
Chief, Bears Bluff Field Station
Coordinator, Environmental Pathobiology Unit
Fate Studies
Sediment-core
Modeling of Fate/Transport Processes
Kepone in Estuarine Microcosms
Acute Toxicity Tests (Dynamic)
Acute Toxicity Tests (Static)
Chronic Toxicity Tests (Fish)
Community Bioassays
Food Chain Bioassay
Algal Stimulation/Inhibition Statistical Model
Effects of Toxicants (Flora and Fauna)
Life-cycle Tests (Mysid)
Life-cycle Tests (Decapod Crustaceans)
Protozoan Studies
Toxic Sediment Bioassay
Analytical Chemistry Section
Method for Determining PCP
Burrowing Behavior (Pink Shrimp)
Predator-Prey Bioassay
Effects of Chlorination on Aquatic Organisms
Marine Ecosystem Testing Units (METU)
Mini-METU
Chemistry and Productivity Studies (Chlorination)
Single Species Studies (Phytoplankton)
Carcinogens in the Aquatic Environment
Effects of Drilling Fluids
Chesapeake Bay Program
T.W. Duke
T.T. Davies
N.L. Richards
F.G. Wilkes
J.I. Lowe
W.P. Davis
J.C. Couch
A.W. Bourquin
P.H. Pritchard
A.W. Bourquin
P.H. Pritchard
J.P. Connolly
R.L. Garnas
S.C. Schimmel
J.M. Patrick, Jr.
P.W. Borthwick
DJ. Hansen
L.R. Goodman
M.E. Tagatz
J.M. Ivey
L.H. Bahner
L.H. Bahner
G.E. Walsh
G.E. Walsh
T.L. Hamaker
E. Matthews
D.B. Tyler-Schroeder
N.R. Cooley
N.I. Rubinstein
R.L. Garnas
J.C. Moore
C.R. Gripe
C.R. Gripe
C.E. Ashton
G.I. Scott
S. Klingensmith
D.P. Middaugh
I.E. Johnson
P.P. Sheridan
R.L. Yoakum
A.C. Badger
I.B. Johnson
A.M. Crane
S.J. Erickson
S.J. Erickson
C.E. Hawkins
J.C. Couch
W.P. Schoor
N.L. Richards
T.T. Davies
Credits: B.P. Jackson, editor; S.S. Foss and T.E. Miller, illustrations; F.G. Wilkes, technical advisor; B.A. Maloney and
G.S. Schmitt, production assistants.
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