U.S. ENVIRONMENTAL PROTECTION
AGENCY
SEMI-ANNUAL REPORT
JULY- DECEMBER 1975
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DISCLAIMER
This report does not constitute publication but is for information only.
All data must be considered provisional. Mention of trade names or
coim ercial products does not constitute endorsement or recomendations for
use.
PROPER’ry OF
U.S. EPA/Opp/J D
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Sr 4 ,
____ UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
‘ GULF BREEZE ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE, FLORIDA 32561
This report describes the activities of the Environmental Research
Laboratory, Gulf Breeze, during the period July - December, 1975. This
period was an exciting one for the laboratory - a time of change in labora-
tory organization, diversification of research programs, and initiation of
construction of a major new laboratory facility. Throughout these six
months the laboratory staff has been involved in many activities, including
research, conferences, public and adjudicatory hearings, technical
assistance to states and regions and interaction with Federal and local
agencies. All of these activities have been entered into with a spirit of
dedication to the mission of the Envionmental Protection Agency and that of
the Environmental Research Laboratory, Gulf Breeze. This dedication is
reflected in the increasing dependence of the Agency on the expertise within
this laboratory as EPA develops and promulgates effective environmental
management and control programs. As a result, ERL,GB is playing an ever
greater role in the formulation of pollution control measures developed by
the Agency for the protection and improvement of our Nation’s estuarine and
coastal environments.
The outlook is for more hard work ahead but the probability of success
for accomplishing the ERL, GB mission has been dramatically increased during
the past six months as a result of the changes made during this time. For
example, the recent reorganization of the Office of Research and Development
has resulted in a redefined laboratory branch structure. Each branch
conducts research directed at particular aspects of estuarine pollution
problems. The synthesis of the information generated by all the branches
produces a total definition of potential environmental impact which ranges
in focus from microbial populations to higher trophic levels such as fish
and man and which varies in complexity from single species through
populations and coninunities to total estuarine ecosystems. 1 ’lore detailed
descriptions of the branch in—house and extramural research programs are
presented in Section I of this report.
As part of the reorganization, Dr. Tudor Davies has assumed the position
of Deputy Laboratory Director. In another organizational change, three
Associate Director positions were established to provtde for greater manage-
ment capability of our tn—house, extramural and Technical Assistance
programs, a need whtch became evident as the laboratory programs expanded in
size and diversity.
Historically, the research programs of ERL, GB have concentrated on the
effects of pesticides on estuarine and marine organisms. In addition to
this research, several new program areas have been initiated at the
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laboratory during the past six months. The first is a program aimed at
determining the health and environmental effects of pollutants associated
with energy extraction, conversion, transmission and use. The specific
objectives of this program are to determine the health and ecological
effects of energy related pollutants which enter these systems during off-
shore drilling activities and from the increased use of new fuels derived
from shale oil. This information is essential to the integration of
potential environmental impacts into the achievement of energy self-
sufficiency for the Nation.
The second major effort initiated during the past six months is con-
cerned with determining the environmental acceptability of pesticide
manufacturing wastes treated by advanced industrial waste treatment
techniques. The information from these experiments will be used to
establish guidelines for the treatment and disposal of such complex wastes.
A third new program, dealing with the environmental impact of off-shore
oil drilling, has been initiated in response to a need of the Bureau of Land
Management, U.S. Department of the Interior.
Of major import to the successful operation and completion of our
programs is the recently initiated construction of a new laboratory
facility. When completed in the spring of 1977 at a cost of approximately
$1 million, this building will house an analytical chemistry section and a
flowing seawater bioassay laboratory. This new facility will greatly
increase our ability to respond to both short and long term Agency needs
with respect to the effects of specific pollutants on estuarine and marine
organisms.
In the Technical Assistance area, the past six months have seen our
staff members involved in problems of National importance. As you know, the
occurrence of PCB’s in the aquatic environment recently came to the fore-
front. As a result of experience and knowledge gained while conducting
research on these compounds over the past few years, our staff was asked by
the State of New York to provide expert testimony at Judicial hearings
concerning the introduction of PCB s into the Hudson River. The data and
information presented will enable the State of New York to more adequately
control the disposal of this dangerous class of compounds in the future.
Another recent development in which we have been active is the investi-
gation of contamination by Kepone of the James River and estuary below
Hopewell, Virginia, the site of a Kepone manufacturing plant.b A program has
been initiated to analyze Kepone levels in James River oysters, determine
the accumulation and depuration rates involved, and determine the effects of
Kepone on individual species and the estuarine ecosystem. This work is
being conducted in conjunction with the State of Virginia, U.S. Corps of
Engineers, and other EPA offices and Federal Agencies. Our data will be
used to aid in the assessment of the problem and in developing effective
corrective action. Details of these and other areas of Technical Assistance
are presented in Section II of this report.
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In sunuiary, the past six months have been extremely productive and we
look forward with great anticipation to continuing in the months to come our
various activities and laboratory programs, both old and new. Each of you
is invited to visit us or, if that is not possible, write or telephone us if
there are any problems we can help you with.
Thomas W. Duke
Laboratory Director
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Bernard F. a, Program Analyst 1
ASSOCIATE DiR FOR EXTRA$JRAL ACTIN .
Norman L. Richards
ASSOCIATE DIR FOR TECHNICAL ASSISTANCE
Jack I. Lowe
MICROBIAL & PHYSICOLOGICAL NECHAIIiSI BRANCH
Al W. Bourquin, Branch Chief
Nelson R. Cooley. MicrobiologIst
David I.. Coppage, Research Aqu. Biologist
Edward Matth.ws, Biological Tech.
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE, FLORIDA 32561
LABORATORY DIRECTOR
Thomas W. Duke
_ [ thia M HerndOn, Secre J
Deputy Director
Tudor T. Davies
ASSOCIATE DIR FOR RES & PLANNING
Frank 6. Wilkes
ADMINISTRATIVE OFFICER
Anice N. Reynolds
FACILITIES
Lester W. Wolf
Helen B. Nay. Adele. Aist.
Zeulah L. Snider, Proc. Clerk
Ann C. Chalk, Library Tech.
Dorothy J. Delk, Clerk Typist
Susan Sellers, Clerk Typist
BIlly 6. Buchanan, GFEHO, Fpwn
Darryl N. Malone, Main. Mach
‘I-
[ PAThOBIOLOGY. TOXIC INTERACTIONS BRANCH
John A. Couch, Branch Chief
Steven S. Foss, Sd. & Tech. Photog.
! ARS BLUFF FIELD STATION
11lian P. Davis, Station Chief
Douglas P. Middaugh, Research Aqu Biologist
Stanton F. Erickson, Research Aqu Biologist
Aiston C. Badger, Research Aqu Biologist
Allan H. Crane, Chenlist
H wRrd P. Foulk, Biological Tech.
Richard A. Roach, Eng. Tech.
Lolse B. Ritchter, Adele. Asst.
Ruth I.. Yoekue. Biological Tech.
Joseph N. Caddie, Laborer
ESTUARINE SYSTEMS BRANCH
H. Peter Schoor, Branch Chief
Marlin E. Tagatz,, Research Aqu. Biologist
Patric H. Borthw*ck, Biologist
Joel N. Ivey, Biologist Tech.
BIOASSAY BRANCH
David .1. Hansen, Branch Chief
Gerald C. Walsh. Research Ecologist
DelWayne R. Nimilo. Research Ecologist
Steven C. Schinsoel, Research Biologist
Larry R. Goodman, Biologist
Lowell H. Bahner, Biologist
Dana Beth Tyler-Schroeder, Biologist
James N. Patrick, Biological Tech.
Walter Burgřss, Biological Aid
LABORAtORY ANALYTICAL SERVICES BRANCH
Alfred J. Wilson, Branch Chief
Gary K. Cook, Ch.n1 t
James C. Moore, Chemist
Jerold Forester, Physical Science Tech.
- Johnny Knidht. Physical Science Tech .
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MISSION
The Environmental Research Laboratory, Gulf Breeze, is responsible for
the conduct and management of research on ecological systems with emphasis
on the determination of exposure-effects relationships in marine, coastal
and estuarine ecosystems of hazardous organic and inorganic pollutants.
This information is required by the EPA pesticide registration and control
program and for the development of water quality criteria designed to
protect man and aquatic life in marine, coastal and estuarine environments.
Research is also conducted to determine the health and ecological impact of
pollutants, both singly and in combination, derived from energy extraction,
conversion, transmission and use. Baseline information and technical
methodologies are developed in order to assess the potential effects of
energy resource development. This information is essential to the inte-
gration of potential environmental impacts into the achievement of energy
self-sufficiency for the Nation.
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I. BRANCH ACTIVITIES
BIOASSAY BRANCH
Objectives
The Bioassay Branch of the Environmental Research Laboratory, Gulf
Breeze, conducts research on effects of pesticides and other synthetic
organics on individual species and comunities of organisms. Studies
include bioassays on phytoplankton, vascular aquatic plants, arthropods,
mollusks, fishes and comunities of animals from several phyla. Duration of
bioassays ranges from a few days to several months and tests may include an
entire life-cycle of the test animal. Bioassays provide data on effects of
chemicals on survival, growth, reproduction, pathogenesis, and accumulation
of chemicals and localization within organisms. Data provide a basis for
water quality criteria, effluent standards, pesticide registration, and
ocean dumping permits. Methods developed and used are incorporated into
manuals for use by government and private laboratories for producing data
for pesticide registration and testing toxicity of waste materials.
Status of Projects
Chronic Bioassays: Hansen, Goodman and Burgess
Research in the chronic bioassay task is conducted to determine effects
of long-term exposures of sensitive life stages of fishes to pollutants.
Projects in progress or completed during the first six months of the fiscal
year include bioassays on effects of toxaphene on embryos and fry of the
sheepshead minnow ( Cyprinodon variegatus ) and effects of heptachlor and
diazinon on its reproduction.
Embryo-fry Bioassays: The effects of toxaphene on sheepshead minnows
exposed for 28-days to five concentrations ranging from 0.2 to 2.5 ig/2, were
determined. Newly hatched fry were affected by, but survived exposure to,
1.1 ig/L. Fry in 2.5 ig/t died. Number of days before embryos hatched and
growth rates were apparently unaffected by 0.2-2.5 pg/2. of toxaphene,
Concentrations of toxaphene in fry averaged 9,800 (range 6,100 to 14,000)
times the concentration measured in the water.
Life-cycle Bioassays: Effects of heptachior and diazinon on the life
cycle of sheepshead minnows are being studied. Results of 96-hr bioassays
will be compared to results from continuing exposures of juvenile fish
through maturation, spawning and subsequent survival of progeny.
Application factors that can be used with data from acute tests on
sheepshead minnows and other fishes to develop water quality criteria will
be calculated from these data.
Heptachlor: Acute 96-hr bioassays to determine effects of heptachior on
various life-stages were investigated. The 96-hr LC5O, (measured
concentration) for fry was 3.6 pg/i, juveniles 10.5 pg/2. , and adults 16.3
ig/R . Tentative analysis of data from the partial life—cycle tests
indicates that the application factor is between 0A8 and 0.27.
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Diazinon: Results from acute bioassays in which juvenile sheepshead
minnows were exposed to between 10 and 500 g/t of diazinon indicate that
the 96-hr LC5O is greater than 500 pg/t.
A partial life-cycle bioassay similar to that with heptachior was begun.
Five exposure concentrations range from 0.6 to 10 ig diazinon/z
cholinesterase concentrations in fishes from all five concentrations were
inhibited by 17 to 77% of normal on day 7 of the exposure;
acetyicholinesterase levels decreased with increasing diazinon
concentration. Chemical analyses of water and fish tissue samples have not
been completed.
CHRONIC AND ACUTE TOXICITY OF METHOXYCHLOR, MALATHION AND CARBOFURAN TO
SFIEEPSHEAID MINNOWS ( Cyprino n variegatus . Contract 68- 264. Bionomic 7
EG&G , Inc. Principal Investigator : P. R. Parrish . Project 1ficer: David
J. Hansen .
A final report on this contract is being prepaired following the
completion of exposures of sheepshead minnows to methoxychior and malathion.
Exposures began with juvenile fish and lasted through maturation and
spawning of adult fish. Exposures ended after progeny from exposed adults
were exposed for one month. Data on survival, growth and reproc ction were
obtained. Chemical analyses of tissue and water samples have not been
completed.
CHRONIC AND ACUTE TOXICITIES OF CHLORDANE, PENTACHLOROPHENOL, TRIFLURALIN
AND 2,3,7 -TETRACHLORODIBENZ 2 -DIOXIN TO SHEEPSHEAD MINNOWS. Contract 68—
U 2U6 . Bionomics, EG&G, Inc. Principal Invest g tor : P. R. Parrii .
Woject Officer: David J. Hansen .
Sheepshead minnows are being exposed throughout their entire life-cycle
to chiordane, pentachiorophenol and trifluralin. The fish exposed to
chiordane have hatched from eggs and grown to maturity. Reproductive
effects are now being assessed. The fish exposed to pentachiorophenol have
hatched from eggs and are approximately one month old. Acute toxicity tests
with trifluralin are complete and life cycle texts will begin next month.
INSECTICIDE EFFECTS ON LARVAL DEVELOPMENT OF BLUE CRAB. Grant R 801128.
Duke University Mari i Laboratory. Princi ’I Investigator : C. G.Thookhout.
Project Officer: Jack I. Lowe .
The objective of this project is to determine the effects of different
concentrations of Mirex, Methoxychlor, and Malathion on the development of
the blue crab Callinectes sapidus , from the time of hatching to the first
crab stage. No effect, sublethal and lethal concentrations will be deter-
mined. Effects of various pesticide concentrations on zoel and megelopal
development, larval behavior and occurance of structural abnormalities will
be determined. The most sensitive larval development stage will be deter-
mined and related to pesticide exposure concentration.
The final report of this project is in press.
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EFFECTS OF INSECT GROWTH REGULATORS AND JUVENILE HORMONE MIMICS ON
CRUSTACEANThEVELOPMENT. Grant R 803838. 5 ke University Marine Laborator
Principal Investigator: John Cos Tow , Jr. roject Officer: Thomas W. Duke .
Chemicals which mimic juvenile hormones are under development for
potential use as alternatives to synthetic organic pesticides. The effects
of these compounds on non-target, but closely related, organisms is unknown.
The objectives of this research are to determine: (1) the effects of
sublethal concentrations of juvenile hormone mimics and insect growth
regulators on the development of marine crustacea, from hatching to juvenile
stages; (2) the effects of sublethal levels of these compounds on larval
stage behavior; and (3) the effects of sublethal concentrations of these
compounds on the long-term mutagenesis of small marine copepods. The
information produced by this research will be used to develop water quality
criteria applicable to juvenile hormone mimics and guidelines for their
registration and application.
During the reporting period, research concentrated on the deterioration
of Altosid (ZR-515) in aqueous solution, the effects of the insect growth
regulator TH-6040 on the larvel development of the estuarine crabs
Rhlthropanopeus harrisli and Sesarma reticulatum , and the mutagenic effects
of ZR-515 on the harpacticold copepod Tisbe hOlothurtae .
Acute Bloassays: Schimel and Patrick
Studies were condticted to determine effects of the insecticides BHC and
Lindane (-isomer of BHC) on several estuarine animals in 96—hr flow—through
bloassays. Results of these studies are listed in Table 1. Edible tissues
from animals that survived the 96-hr tests are being analysed for pesticide
content.
Two, separate, 28-day bioaccumulation studies on BHC were conducted with
the American oyster ( Crassostrea virginica ) and the pinfish ( Lagadon
rhomboides) . Each test was followed by a 28-day depuration period to
determine the rate of loss of each of BHC t s four isomers. Maximum
concentration factors (the concentration of the various isomers of BHC in
tissues divided by the concentration measured in exposure water) were 218 in
oyster meat and 137 in pinfish muscle. After seven days depuration, no BHC
was detected in oysters or pinfish.
A 96—hr flow—through bioassay was conducted to determine the LC5O of DDT
to juvenile brown shrimp ( Penaeus aztecus) . These data were required to
better evaluate the environmental effects of proposed DOT applications to
crops in Louisiana. The measured LC5O for DOT in this test was O.l4pg/9.
(95% C.I. = 0.10 to 0.24 g/ ).
An apparatus was constructed for the purpose of investigating the
bioconcentration of pesticides by estuarine animals. The device delivers
one liter of water each cycle (400 cycles/day) to two control and four
treated aquaria. One control aquarium provides seawater with carrier (if
necessary) and the other seawater without carrier. The treated aquaria
receive concentrations of carrier identical to the control. Two
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Table 1. 96-hour toxicity of the insecticides lindane and BHC to several estuarine animals in
flow-through bioassays. The 95% confidence intervals are in parentheses. Test
animal size is rostrum-to—telson length for shrimps and standard length for fishes.
SPECIES INSECTICIDE 96—HOUR
SIZE
TEMPERATURE
SALINITY
LC5O
(pg/9 .)
(mm)
(°C)
(°/oo)
Penaeus Lindane 0.13 30-52 24.0-26.0 20-22
duorarum (0.10-0.17)
P. duorarum 0.34 21-27 18-22
(0.21—0.44)
_____________ 4.44
_____ (3.44-6.08)
__________ 103.9
__________ (91.8-130.8)
_______ 31.12
__________ (29.20-33.36)
__________ 86.43
(80.77-96.70)
____________ 6.28
_____ (3.81-14.08)
- Palaemonetes
pugio
Cyprinodon
vari atus
Lagodon
rhomboides
L. rhomboides
Mysidopsis
bahia —
BHC
Lindane
Li ndane
Lindane
BHC
Li ndane
17-21
42-61
55-89
8-9.5
23-27
24-28
22.5-25.0
22.0—26.0
23 .0-25 .0
18-22
10-18
21.0-23
20-23
15-22
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concentrations of a toxicant are possible, with two replicates for each
concentration. The apparatus uses Lamda pumps with a predetermined counter
which is capable of delivering 0.01 ml to 100 ml of toxicant stock solution
each cycle.
Studies are continuing to evaluate the pinfish as a fish suitable for
bioassays involving the embryo and larval stages. Although techniques have
not yet been devised to culture larvae beyond 11 days after hatching, static
96-hr bioassays using newly-hatched pinfish are possible.
Special bioassays are now under way to determine the uptake and
depuration rates of the insecticide Kepone by American oysters. Two studies
are anticipated, each lasting two months. Results of these tests may be
helpful in estimating the recovery of oysters in the James River, Virginia,
system from recent Kepone contamination.
Estuarine Productivity: Walsh
Effects of Pollutants on Marine Unicellular Algae
Estuarine algae are subjected to daily variations in salinity and
nutrient composition of the water and to combinations of pollutants. This
task is designed to study growth responses of estuarine unicellular algae to
heavy metal ions, organometals, and other pollutants in relation to salinity
and nutrient status of growth media. The findings will be used to relate
water quality criteria for these pollutants to the composition of individual
bodies of water.
Growth of algae in the presence of lead (Pb): Concentrations up to 100
ppb Pb (in PbCl ) had no effect on growth of Dunaliella tertiolecta or
Isochrysis galbana9n 30 ppt salinity and three nutrient concentrations of
trace metals, minor salts and vitamins. Concentrations up to 100 ppb had no
effect on growth of Chlorococcum sp. at 30 ppt salinity and high nutrient
concentration. Also, toxicity of herbicide was not enhanced when 0.
tertiolecta and I. galbana were grown in the presence of 100 ppb Pb and tfie
EC5O concentrations of atrazine.
Growth of algae in the presence of cadmium (Cd): Dunaliella tertiolecta
was grown in the presence of 100 and 500 ppb Cd (in CdCI and cadmium
acetate) in four nutrient concentrations. In first tests, Cd had no effect
on growth in the three highest concentrations. Cadmium appeared to
stimulate growth at the lowest nutrient concentration.
The role of chaelators in toxicity of heavy metals to algae in our tests
is being studied and will be described in the next report.
Effects of chlorinated naphthalenes on growth of algae: Studies on
growth of algae in the presence of chlorinated naphthalenes began near the
end of the reporting period. No effect of the compounds at 50, 100 and 500
ppb were found in preliminary tests, but this must be confirmed in future
tests.
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Effects of Heavy Metals on Development of Red Mangrove Seedlings
Mangrove stands are subjected to pollutants that occur inshore,
including heavy metals from dumping of sewage. Tests were performed to
determine effects, if any, of lead, cadmium and mercury on seedlings. Data
were collected to determine safe concentrations of the metals in estuarine
soils that contain mangroves.
Seedings of the red mangrove ( Rhizophora mangle ) were treated twice with
up to 500 ppm of lead, cadmium or mercury. No effects of the first
treatment were found on rate of growth, weights of hypocotyls, stems, leaves
or roots, and total productivity. Second treatments with cadmium or mercury
caused chiorosis of leaves followed by death of the plants. Microscope
slides are now being prepared for pathology associated with treatment, and
plant parts are being analyzed for tissue concentrations of the three heavy
metals.
WATER QUALITY AND MAIIGROVE ECOSYSTEM DYNAMICS, Grant R 803340. University
of Florida, Gainesville, florida. Principal Investigator: Samuel C.
edaker. Project Officer: Gerald E. Walsh .
Progress during this past quarter has focused on the nutrient and heavy
metal concentrations present In the major functional compartments of
intertidal mangroves and in the construction of simulation models at Harvard
University using the facilities of the Department of Engineering and Applied
Physics. Evaluations made thus far suggest that mangroves do not
concentrate heavy metals above levels comonly found in other types of
halophytes. However, variations in metal concentrations (and the major and
minor plant nutrients) are relatable to the type of mangrove forest relative
to local topography, intensity of tidal flushing and proximity to sources of
metals. Compartment turnover times for these forest types are known and it
Is now possible to estimate the maximum loadings relative to the incoming
load of metals.
Entrained metals in litterfall may be released during in situ decom-
position or be exported in the detrital flow to the downstream bay or
estuary. Work is underway to begin an intensive monitoring of the rates of
detrital export (ergo export of metals) for the various forest types and
their ultimate fate in the downstream water body. The successful completion
of this task should allow us to define the intertidal mangrove ecosystem as
either a sink (with time dimensions) or as a conduit for each of the metals
of interest. Also possible is speculation on the role of mangroves in con-
concentrating metals in heterotrophic consumers by virtue of the detrital
flux recognized now as a major food source for estuarine organisms.
HERBICIDE TOXICITY IN MANGROVES. Grant R 801178. University of Miami,
Coral Gables, F’IoFTda. Principal Investigator: Howard J. Teas. Project
Officer : GeraldE. Walsh .
The amine salts of 2,4-D and picloram were applied to the Florida
species of mangroves: red mangrove ( Rhizophora mangle) , white mangrove
( La uncularia racemosa and black mangrove ( Avicennia germinans) . Treatments
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Lethal application rates of picloram for young seedlings were
for white mangrove, 13 kg/ha for red and 13 kg/ha for black;
plants they were 2.7, 13 and greater than 53 kg/ha respectively.
application tolerated by young seedlings were 0.01, 5.3 and 5.3
mature plants they were 0.5, 5,3 and 53 kg/ha. “No effect
seedlings were less than 0.01 kg/ha for all species; for mature
were less than 0.1, 0.5 and 2.7 kg/ha.
Spray applications of 6.3 - 12.2 kg/ha of a commercial mixture of 2, 4-
0, and picloram to the canopy of a mixed-species forest caused partial
defoliation within three weeks. Within 16 months it killed all of the
white, 78 - 100% of the mature red, but none of the mature black mangroves.
Radiocarbon-labelled picloram concentrated in
mangrove and it is concluded that the tree is killed
of its effects on them.
The final report for this project has been received.
EFFECTS AND FATE OF SELECTED WASTE-WATER CHLORINATION PRODUCTS AND
PESTICIDESTFI MARINE At AE. Grant R 803943. Syracuse University Resea EFi
Corporation, Syracuse , N. V. Principal Investigator: Harish C. Sikka.
Project 0fficer: Gerald E. Walsh .
Effects of 4-chlororesorcinol, 3—chiorophenol, 3-chlorobenzoic acid and
5—chlorouracil on growth of the marine unicellular algae Dunaliella
tertiolecta and Skeletonema costatum were studied.
Skeletonema was more sensitive to all compounds than was Dunaliella .
Growth of Dunaliella was not altered significantly by any of the four
compounds at concentrations of 1 and 10 ppm. Growth of Skeletonema was
completely inhibited by 10 ppm of 4—chiororesorcinol and 3-chiorophenol and
reduced 15-20% by the same concentration of 3-chlorobenzoic acid. 5-
chiorouracil had no effect on growth of Skeletonema at 1 and 10 ppm.
STUDY OF EFFECTS OF
Vir 9 ini State Co11 ge
Project Officer: Gerald
PESTICIDES
ON DEVELOPMENT OF
p T Investijator:
ALGAE.
Bernard
Grant
R.
R 802388.
Woodson.
.
E.
Princi
Walsh.
The purpose of this study is to investigate effects of selected
pesticides on growth, respiration, photosynthesis, cellular organization and
motility on freshwater algae. The pesticides are dephenamid, Baygon,
atrazine, diuron, malathion, seven and simizine. The study also includes
determination of uptake of pesticides by selected algal species.
______ ___ ______ _____ ___________ AND 2,4-D ON ULVA SP.,
____________ ___ __________ Syracuse University Research
were to soil or water, by aerial spray and to single leaves
The effects on radiochioride uptake and on localization
labelled picloram after leaf application were studied in red
as droplets.
of radiocarbon-
mangrove.
2.7 kg/ha
for mature
Rates of
kg/ha; for
doses” for
plants they
dormant buds
by the mixture
of red
because
UPTAKE AND EFFECTS OF
ENTEkOMORPHA SP. A
MIREX, METHOXYCHLOR
RHODYMENIA SP.
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Corporation. Principal Investigator: Harish C. Sikka. Project Officer:
Gerald E. Walsh .
This was a study concerning effects, uptake, and metabolism of mirex,
methoxychior, and 2,4—D in the seaweeds Ulva sp., Enteromorpha sp. and
Rhodymenia sp. None of the pesticides, at concentrations corresponding to
their maximum solubility in seawater, had any significant effect on photo-
synthesis, protein, carbohydrate, lipid, chlorophyll, carotenoid or trace
metal content of the algae. All three algae removed substantial amounts of
mirex and methoxychior from the medium, but uptake of 2,4-D was extremely
low. The rate of uptake of methoxychlor was considerably greater than that
of mirex. Enteromorpha accumulated considerably more mirex and methoxychior
than Ulva or Rhodyinenia . Both Ulva and Enteromorpha failed to metabolize
either mirex or 2,4-D. Enteromorpha metabolizedniethoxychlor to a limited
extent. After 7 days of incubation with carbon labelled methoxychior, a
major portion of the label in the tissue and medium was present in unchanged
methoxychior. A small amount of radioactive metabolite, 2,2—bis (2.-
methoxyphenyl)-l,l-dichloroethylene, was detected in both the tissue and
medium. In addition, medium contained 2,2-bis ( -hydroxyphenyl)-l,l,l-
trichioroethane and four unidentified minor radioactive metabolites. Unlike
Enteromorpha, Ulva did not metabolize methoxychior.
The final report for this project has been received.
DYNAMICS OF AN ESTUARY AS A NATURAL ECOSYSTEM. Grant R 802928. University
of South Caro’TTna. PrinEfpal Investigatory : F. John Vernberg. Project
Yficer: Gerald E. Walsh .
This study consisted of two separate but interrelated substudies: 1) a
macroecosystem study and 2) a microecosystem study.
The macroecosystem study, designed to continue work on the dynamics of a
relatively undistrubed marsh-estuarine ecosystem, had two objectives: 1) to
establish baseline data on an undisturbed estuary to provide a scientific
basis for comparative studies on the effects of various pollutants, and 2)
to develop models of an estuarine ecosystem that would predict the probable
effects of environmental perturbation. The final report includes sections
on the data retrieval system, radiation balance, hydrography, microbiology,
composition and seasonality of the zooplankton, vertical migration of
crustacean larvae, metabolism of crustacean larvae, and abundance, diversity
and respiration of the macrobenthic fauna.
The objectives of the microecosystem study were: 1) to develop and test
replicate experimental salt marsh units at the microecosystem level as
diagnostic tools for the assessment of both long and short term pollution
effects on the Spartina alterniflora salt marsh comunity, and 2) to utilize
these simulated marshes to test and to provide data to the overall ecosystem
model being constructed. The final report describes comunity metabolism
and benthic coninunity analyses in the system.
The final report for this project has been received.
8
-------
Physiology: Nimmo and Bahner
These studies are divided into four categories: (1) effects of toxicant
combinations on shrimp, (2) methods development for assessing the uptake of
toxicants from sediments by marine organism, (3) methods development of
bioassay procedures, and (4) effects of cadmium on shrimp.
Species used for bioassay in the laboratory are usually exposed to toxi-
cants singly, but they are often exposed to combinations of toxicants under
varying environmental regimes. Consequently, an experimental flowing-water
bioassay system was developed that controls salinity and temperature while
testing toxicants either singly or in combination. Obvious advantages of
this control are that rates of toxicant accumulation, translocation, and
loss, and acute and chronic toxicty to animals can be better assessed and
repeated. Our bioassays were conducted with pink shrimp ( Penaeus duorarum )
exposed to the following toxicant combinations: cadmium-malathion, cadmium-
methoxychior, cadmium_methoxychlor_AroclorR 1254, and a complex industrial
waste which contained both inorganic and organic constituents. The
toxicities of the pesticide-metal combinations, when compared to that of
each constituent singly, appeared to be independent or additive, and varied
with the method(s) of bioassay.
Accumulation of cadmium from natural (contaminated) sediments was tested
using 5 genera from 3 phyla. Sediments containing less than 100 ppm Cd were
collected from Corpus Christi Bay, Texas and placed to a depth of 4 cm in 30
glass aquaria. Seawater, from Santa Rosa Sound at ambient temperature and
salinity flowed (60 l/hr) through each aquarium for 60 days. About 30
animals of each species, Palaemonetes vulgaris, Penaeus duorarum,
Crassostrea virginica , Uca pugilator and Cyprinodon variegatus were placed
in each aquarium. Concentrations of cadmium in water, sediment and tissues
were determined at regular intervals using atomic absorption
spectrophotometry.
A chronic bioassay that induced exposure of the entire life-cycle of an
estuarine/marine crustacean has been completed. In flowing seawater, the
mysid Mysidopsis bahia was affected by exposure to 10 ig/9. cadmium. The 16-
17 day life-cycle exposure showed that survival as well as length of time to
formation of brood pouch and length of time to brood release all appear as
indicators of deleterious effects. This mysid has optimum growth at 20 0/00
salinity, 25°C, and can complete a life cycle in static or flowing water.
Mr. Bill Peltier (EPA Region IV) has recently completed a successful flow—
through bioassay with this species using dilutions of an industrial
effluent.
Cadmium was more toxic to marine decapod crustaceans in 96-hr bioassays
than to marine gastropods, bivalves and teleosts (Eisler, R. 1971. J. Fish.
Res. Bd. Canada 28:1225-1234). Therefore, some of the more subtle effects
which might be apparent in long-term bioassays (up to 30 days) were
investigated using estuarine and marine shrimps. Studies included (1) rates
of accumulation and localization in various tissues, (2) histological
effects of acute (96-hr) and chronic (24-days) exposures, and (3) a
9
-------
comparison of accumulation of cadmium from food with cadmium administered in
water.
Cadmium was accumulated by shrimp from water that contained as little as
8 pg/L. All tissues had detectable concentrations, but muscle cadmium
concentration increased after shrimp were transferred to cadmium-free water.
Background concentrations of the metal were eliminated by holding feral
animals in flowing water in the laboratory.
Shrimp exposed to cadmium consistently developed blackened foci or
lamellae in the branchia. Occasional blackened cuticular lesions on the
appendages and body surfaces were also observed. Often whole branchial
processes were necrotic and congested with hemocytes. Shrimp that survived
these exposures sloughed the blackened portions of the branchial processes
and appeared normal within 14-days after transfer to cadmium-free water.
Accumulation of cadmium from food by Palemonetes pugio appears to be
negligible compared to accumulation from water. In 30-day bioassays, the
toxicity of cadmium to grass shrimp, P. vulgaris , was approximately an order
of magnitude lower than the toxicity to pink shrimp, P. duorarum .
Bioassay-Crustaceans: Tyler-Schroeder
The past six months were spent gaining experience in handling grass
shrimp Palaemonetes pugio larvae, in a flow—through exposure apparatus using
salinity-controlled, temperature-controlled, filtered seawater.
In 96-hr acute tests using this system with Altosid, a synthetic
juvenile horiiione, observations were made on mortality and molting of grass
shrimp exposed to concentrations from 400 to 1000 pg/t . Rostrum-telson
lengths of surviving larvae showed growth of larvae exposed to 400 i.ig/2. of
the chemical was significantly less than larvae reared in the seawater
control (X=0.05). Lengths of larvae from higher concentrations were not
measured.
A 21-day test with Altosid was performed on P. pugio larvae to determine
if the chemical accelerated or retarded the onset of metmorphosis to the
postlarval stage. Exposure to concentrations of 400 to 1000 g/L of the
chemical was begun on the 12th day of larval development. Salinity was 15-
20 0/00 and temperature was held at approximately 25°C. Larvae were fed
Artemia nauplii throughout the test.
The observed success of metamorphosis for the experiment is shown in
Fig. 1. Total percentage metamorphosis and survival at the completion of
the test is given in Table 2.
10
-------
400
542
dU9/I
a
736
ugh
I
/
I
I
I
I
I
I
/
II
I i
/
I
/
/
/
/
—D
A
— —fr- #••
—--0
—-€--
—
4 16
8 20
22 24
26 28
30 32
48
42
36
30
-I
24
IAJ
4
>
4
-J
I-
(1)
0
a-
LL
0
a
z
18
6
0
AGE (days)
-------
Table 2
Percentage survival
Altosid.
and metamorphosis
of grass shrimp exposed
to
Altosid, pg/L
Survival .
Metamorphosis ,
Acetone Control
400 iig/2.
542 ug/2.
736 pg/2
1000 ug/2.
Research concerning effects of endrin, a chlorinated hydrocarbon insec-
ticide, on the life-cycle of the grass shrimp was begun. The 96-hr LC5O of
Endrin to 1st day P. pugio larvae was 1.0 pg/9. in first tests. Exposure to
concentrations from 0.34 to 3.4 ig/L caused a significant reduction in
growth of the larvae from the controls (a=0.01). These tests were perfoniied
in the same flow-through system as were the Altosid tests.
A 500
being readi
Completion
be used in
ml. diluter with exposure aquaria has been constructed and is
ed for uptake and spawning studies on P. pugio , using Endrin.
of these studies should yield an application factor for Endrin to
establishing water quality criteria for marine invertebrates.
Meetings, Conferences, and Workshops
Hansen, D.J.
Presented a paper at the AIBS meeting at Corvallis, Oregon. The
presentation was part of a suninary of research conducted by NERC Corvallis
research laboratories.
Participated in preparing a manuscript at a meeting of the Working Group
on Principles for Developing Coastal Water Quality Criteria in Dubrovnik,
Yugoslavia.
Presented a paper on the effects of PCB’s on estuarine animals at
National Conference on Polychlorinated Biphenyls at Chicago, Illinois.
EPA ’s
Testified in Albany, New York on the matter of PCB’s in the Hudson
River.
Discussed the use of application factors for setting marine water
quality criteria at the ERL, Duluth and the occurrence of PCB’s in marine
waters at an OTS meeting in Washington, D.C.
Bahner, L.H.
Attended Symposium on
Mil’lford, CT. November
laboratory at Miliford and
Pollution and Physiology of Marine Organisms,
3-6, 1975. Symposium sponsored by the NOAA NMFS
the Belle Baruch Institute, SC.
65
48
53
56
32
77
73
73
76
63
12
-------
Nimmo, D.R.
Attended Symposium on Pollution and Physiology of Marine Organisms,
Miliford, CT. November 3-6, 1975. Presented paper entitled “Effects of
cadmium on the shrimps, Penaeus duorarum, Palaemonetes pu io and
Palaemonetes vulgaris (Nimmo, D.R., Lightner, D.W. and Bahner, L.H.).
Schirmiel, S.C.
Attended meeting of American Society for Testing Materials at St.
Louis, MO. October, 1975. Discussed acute bioassay and bioaccumulation
methods and hazard evaluation.
13
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ESTUARINE ECOSYSTEMS BRANCH
Objectives
The objectives of the Estuarine Ecosystems Branch are to deterr ine the
pathways, biological effects and fate of hazardous organic and inorganic
pollutants in estuarine ecosystems, simulated as well as in natura .
Specific responsibilities include the design of laboratory microcosms, and a
conceptual estuarine mathematical model for assessment of routes, rates,
effects, sources and sinks of pollutants as integrated into the overall
model . The approach in the design of these systems is to establish a simple
and basic system for the screening of pollutants. A secondary objective is
to develop a system to the point where it can be used as a rigorous
scientific tool in establishing the effects of stress applied to an estuary.
Status of Projects
Effect of Mirex on Predator-Prey Interaction in an Experimental
Estuarine Ecosystem: Schoor, Tagatz, Ivey and Borthwick
Tests of 14- to 16-days duration were conducted to determine the distri-
bution and sublethal effects of mirex in an experimental estuarine
ecosystem. Observations of the system and water data indicated a healthy
community that appeared to be of sufficient size not to be stressed by
removal of replicate samples. The water remained clear, turbidity averaging
0.6 nephelometric unit (range 0.2 to 1.8). No algal growth was observed in
the tanks during Experiment 1, but small amounts were visible near the end
of Experiment 2. Dissolved oxygen ranged from 7.2 to 8.5 ppm, but was
usually at the saturation level (8.1 ppm). Overall pH of the water averaged
7.2 (range 7.0 to 7.4). All plants survived, some showed new growth, and
most of their leaves remained green. Shrimp were closely associated with
the plants, eating leaf detritus or epiphytic material on the leaves or
both.
Concentration of mirex in the water averaged 0.025 pg/P. (range 0.015-
0.050 pg/P.) for Experiment 1, 0.046 pg/2. (0.017-0.11 pg/P.) for Experiment 2
(one day predation), and 0.044 pg/P. (0.011-0.13 pg/P.) for Experiment 2 (two
days predation).
Mirex was translocated from water to sand and biota. All components
sampled during 16-days of exposure in Experiment 1 contained mirex. Only
trace amounts (< 0.02 mg/kg) occurred in sand. Mirex was not detected in
Thalassia at day 1, but subsequent concentrations in leaves (trace to 0.033
mg/kg) were as great as l,300X the average concentration in the water.
Roots had less mirex (not-detected, less than 0.020 mg/kg, to 0.024 mg/kg)
than did leaves (trace to 0.033 mg/kg). Mirex in shrimp ranged from trace
amounts to 0.20 mg/kg. Concentrations in shrimp increased with time and
were as great as 8,000X the average concentration in the water. After three
days exposure, pinfish contained 0.050 to 0.063 mg/kg mirex, up to 3,800X
that in the water.
14
-------
In both experiments, an alteration of predator-prey interaction due to
the effect of mirex was evident. There was no significant difference ( ix =
0.05) survival of grass shrimp in control and treated tanks after 13-days
exposure in the absence of predation by pinfish. However, there was a
significant difference ( = 0.01) in survival after one, two, or three days
of predation by pinfish (Table 3). Survival of shrimp after three days of
predation was based on two, instead of three, treated tanks due to death of
a pinfish in the third tank.
That more deaths due to predation occurred in the treated tanks than in
the control tanks could be interpreted as an alteration of the normal
behavior of either shrimp or pinfish by mirex; It is believed, however, that
the behavior of only the grass shrimp was altered. This is supported by the
experiments of Lowe et al. (1971), who found that mirex was acutely toxic to
grass shrimp, but on the basis of mortality, pathology, and observations for
symptoms of pesticide poisoning, had no apparent affect on pinfish.
Thus, while the criterion of death may be useful in single-species
bioassay, in an ecosystem bioassay an effect on the predator-prey
interactions may have far greater implications regarding the total welfare
of the system. In addition, concentrations at which this behavioral effect
occurs are quite low and come closer to the environmental levels.
Effect of Mirex on Lugworm Ecosystem: Schoor and Borthwick
An inexpensive bioassay system has been developed to estimate pollutant
effects on the benthic comunity. The system is composed of washed beach
sand, turtlegrass and periphyton on the sand surface, artificial seawater
and the lugworm ( Arenicola cristata) . Observations are made of surface
phenomena, indicative of fugworm activity , including feeding funnels (head
shafts), respiratory tubes (tail shafts), and egg masses. Preliminary
experiments have demonstrated that the pesticide Mirex taken into the
substrate by the burrowing and feeding activity of the lugworm seemed to
affect the behavior of this species.
Environmental Chambers: Schoor and Borthwick
Two fiberglass equipment shelters have been purchased and adapted to
provide self—contained facilities for static estuarine system bioassays.
Artificial light within 91% of the natural daylight spectrum can be provided
for any pre-selected photoperiod. A wide range of temperatures may also be
selected. These facilities provide space for simultaneous experiments in up
to thirty-six 180 liter aquaria under controlled conditions.
DETERMINATION OF THE SITE(S ) OF ACTION OF SELECTED PESTICIDES BY AN
ENZYMATICI UN JOLO ICAL APPROA H. Grant R 803458. Mississippi State
University. Principal Invei Igator: Robert B. Kock. Project Officer : W.
Peter Schoor .
The objective of this project is to develop antibodies to Kelevan, an
“active” derivative of Mirex which inhibits the ATP’ase system of fire ants.
These antibodies will be used to determine if the inhibited enzyme system
15
-------
Table 3 - Survival of grass shrimp after 13 days exposure to mirex in the absence of predation and after
an additional 1 to 3 days of exposure and predation by two pinfish per tank. (Numbers in
parentheses indicate numbers of shrimp in tanks before deaths occurred)
No. tanks
(Control!
Treated)
Avg. concn.
mirex
(i.ig/l)
Survival
after 13 days
Chi-
squarea
No.
days of
predation
Survival
after predation
Chi-
squarea
Control Treated
Control
Treated
1/1
0.046
82% 68%
(63) (63)
NS
1
44%
(52)
23%
(43)
4•57*
1/1
0.044
78% 81%
(63) (63)
NS
2
16%
(49)
0%
(51)
9 .05**
3/3
0.025
94% 88%
(189) (189)
MS
3
24%
(177)
4 %b
(115)
19.39**
aNS = non—significant; *sjgnjficant at 5% level (x 2 1 d.f. = 3.84); **significant at 1% level
(x 2 1 d.f. = 6.63).
bBased on two, instead of three, treated tanks due to death of a pinfish in the third tank.
-------
can be reactivated in vitro by the removal of Kelevan through competitive
binding of the highly specific antibodies.
The initial objective of this project was to reduce the ketone func-
tional group of Kelevan by the t4olff-Kishner method to the methylene unit
and then to attach the reduced product through its N-hydroxysuccinimide
derivative to bovine serum albumin (hereafter called BSA). Although several
attempts were made with the Woiff-Kishner method, reduction of the ketone
function has not been achieved.
Fortunately, Dr. R. 0. Hutchins of Drexel University has developed an
alternate method for reducing ketone to methylene units. His method, which
involves the formation of the ketone tosyihydrozone followed by reduction of
the hydrazone with the highly selective reagent sodium cyanoborohydride, can
be used with esters present and, therefore, will be used on Kelevan
directly.
The whole sequence of attachment reactions on Kelevan itself (ketone
group present) has been studied in order to gain some familarity with the
reactions. Kelevan has been hydrolzyed under acid conditions to the free
acid. Treatment of the free acid with N-hydroxysuccinimide resulted in the
formation of a colorless crystalline compound with a m.p. of 245° C and an
infrared spectrum compatable with the desired adduct. Other spectral data
(ms and nmr) and elemental analysis data are being collected now. Most
bondings to BSA seem to result in about 5 moles of nonprotein material being
bonded per mole of BSA. Therefore a 25 fold excess of the succimide
derivative (149 mg) was added to a 100 ml aqueous solution of 100 mg of BSA
which had been buffered to about pH 7.5 with bicarbonate and HC1. The
succinimide derivative was dissolved in 0.5 ml of tetrahydrofuran (THE) and
added dropwise to the BSA solution. Preliminary checks indicated this
amount of THF would not denature BSA. The suspension was allowed to stand
overnight in the refrigerator to complete the formation of the BSA-Kelevan
(BSA-K) adduct. The volume of the solvent was reduced by freeze-drying, and
the residue was then subjected to gel-filtration.
Preliminary experiments have been conducted on the effect of the
Kelevan- Nhydroxysuccinimide derivative (KS), prepared as described above,
on the ATP’ase activities from catfish brain homogenate fraction. The data
(Table 4) showed that the KS inhibited the three ATP’ase activjties with
pronounced effect on Na+ _K+ and oligomycin-sensitive Mg + ATP’ase
activities. A freeze-dried sample of the clear portion (a small sediment
was discarded) of the reaction mixture in which the BSA-K adduct was formed
on the ATP’ase activities was also tested (see Table 4). At 1.0 and 4.0 uM
concentrations in th reaction mixture, BSA-K inhibited Na -K and
oligomycin-sensitive Mg + ATP’ase activities.
The 1.0 to 4.0 urn Kelevan concentrations (Table 4) were determined
assuming that 5 moles of KS interacted with one mole of BSA. However, it is
possible that additional KS was absorbed onto the BSA by charge-transfer
complex formation, hydrophobic interaction, and unreacted KS not sedimented.
The latter concentrations were ignored because there is no way of estimating
their values at this time.
17
-------
Table 4. Effects of Kelevan** and BSA Derivatives on the ATPase activities from a Catfish Brain
Homogenate Fraction
Kelevan
Derivatives
Concentration
( 3 jM)
Na+ -K
ATPase
Specific
Sensitive
Activity
Mg 2 ATPase
Oligomycin
Insensitive
Control
none
17.5
6.1
8.7
KS**
20
4.3
1.1
6.9
BSA-K
‘ 1
15.7
4.2
9.2
BSA-K
4*
15.3
4.3
7.6
BSA-K
(Sephadex effluent)
l*
17.7
6.4
9.9
BSA-K
(Sephadex effluent)
l0*
13.7
5.2
12.0
** KS — Kelevan N-hydroxysuccinimide
Kelevan was tested in earlier studies and found to be nearly as inhibitory to the ATPase activities
from Catfish brain homogenates as was the reduction product of Kepone 5 .
03
* pM concentrations are estimates of Kelevan added to the reaction mixture in the various forms
listed.
-------
In order to remove unreacted KS from the protein solution, a portion of
the freeze-dried BSA-K derivative was dissolved in buffer and passed through
a Sephadex G-75 column using 0.02 M imidazole (pH 7.5) buffer as the mobile
phase. Most of the 280 nm absorbing material was collected in the void
volume effluent. The effluent sample was freeze-dried and tested for
ATP’ase inhibition. The amount of BSA-K added to the reaction mixture was
again calculated assuming 5 moles of KS was covalently bound to BSA.
However, it is assumed that most, or at least a significant portion, of the
non-covalently bound KS was removed by the Sephadex treatment.
The BSA-K sample from the Sephadex column showed less effect on the
ATP’ase system compared to the BSA-K sample before it was passed through the
Sephadex column (Table 4). Of possible interest is the stimulation observed
of the oligomycin-inserisitive Mg 2 ATP’ase (Table 4, 10 pM BSA-K, Sephadex-
effluent). No explanation for this observation is available at present.
The above results indicate that the Kelevan inhibition of ATP’ase activity
is maintained even after forming an adduct with BSA.
The above results are very promising in indicating that Kelevan, a
derivative of kepone from which a mono-hydrogen rnirex derivative may be
prepared, can be covalently bound to BSA. The observation that the BSA-K
adduct was still somewhat inhibitory to ATP’ase activity after passing
through a Sephadex G-75 column was not necessarily expected because of the
size of the molecule. However, if this preliminary observation provides to
be correct, it could indicate that antibodies to Kelevan used as a hapten
would be able to interact with and remove Kelevan from the inhibited enz ie.
The following paper describing this research is currently in press:
Desaiah, D. and R. B. Koch. Preliminary investigation on the effects of
Mirex and its derivitives on AlP t ase activities on Fire Ants. J. Agr.
Food Chem.
FATE AND EFFECTS OFATRAZINE IN SALT MARSH ECOSYSTEMS. Grant R 803835.
Auburn University. Principil Investigator: Donald E. Davis. Project
Officer : W. Peter Sch’ .
The objective of this research is to investigate the accumulation,
degradation, and effects of atrazine on a model micro-ecosystem consisting
of organisms from a salt marsh representing four trophic levels. During the
first 6 months investigations were initiated aimed at determining the level
oof C14 atrazine and its metabolites in individual components of the
ecosystem, beginning with the primary autotroph Spartina alterniflora .
During the first six month study period two experiments were initiated
simultaneously. The first was to determine the tolerance of S. alterniflora
to atrazine and study its effects on growth. The second was to develop
methods for the isolation and identification of atrazine and its metabolites
in S. alterniflora . A similar experiment was conducted with a previously
studied plant, Sorghum vulgare (sorghum), to compare the metabolites formed
in Spartina and to aid in their identification.
Tolerance of S. alterniflora to atrazine and its effects on growth.
19
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S. alterniflora plants were collected from the marsh at Sapelo Island,
GA., and maintained at Auburn Univ. in Hoagland’s nutrient solution in 2—
liter beakers in a growth chamber having a 14 hour photoperiod with 60%
relative humidity, a temperature of 28°C and 30 Klux of light provided by a
mixture of incandescent and fluorescent lamps. The photoperiods were
followed by 10 hour dark periods at 60% relative humidity and 24°C. Healthy
plants were divided into five lots of 16 plants each. The plants were
weighed and the number of leaves per clump of plants, and the height of the
plants were recorded. They were then transferred to 1-liter plastic beakers
containing 900 ml of Hoagland’s solution containing: 0, 5 x 10-8; 5 x
5 x i c r 6 or 5 x i - M atrazine. Each beaker contained four plants and each
treatment was replicated four times. Results of this experiment are given
in Table 5. Generally, the values of all parameters measured decreased with
increasing concentrations of atrazine. The lowest concentration used in
this experiment, 5 x 10-8 M, had little apparent effect on growth. However,
in spite of the above effects on the weight, number, and length of plant
parts even those plants growing in the highest concentration of atrazine (5
x 10-a M) showed no obvious symptoms of unhealthy plants such as chiorosis,
necrosis, wilting, etc.
Isolation and identification of C atrazine metabolites from
alterniflora .
Plants were grown as described above. Leaf sections from S.
alterniflora and Sorghum vulgare were divided into 25 samples each of 20 g
which were placed in 250 ml flasks containing 150 ml 0.1M phosphate buffer
containing 25uCi ring-labeled atrazine plus nonlabeled atrazine to give a
final concentration of 9 x io M. Flasks were incubated for 24-hr under 30
klux of light at 28°C. Tissues were then homogenized in 400 ml of 80%
ethanol, filtered, and the combined extracts of each species were
concentrated under N 2 , diluted to 1000 ml with deionized water, and
partitioned three times with 1000 ml chloroform. The chloroform and aqueous
extracts were concentrated and stored at -5°C. Plant debris collected
during filtration of the original homogenate was re-extracted with 200 ml
portions of chloroform, and the extracts were concentrated and combined with
the original chloroform phase. The plant residue was also stored at -5°C.
Atrazine and any metabolites in the chloroform, aqueous, and insoluble
fractions will be isolated and identified. At this time only the chloroform
fraction has been studied. The chloroform extract was taken to dryness
under N , dissolved in hexane, and placed on top of a silica gel (60-200
mesh) co’umn (25 x 2 cm). The column was washed successively with petroleum
ether (PE), 25% diethyl ether, 50% diethyl ether in PE, 100% diethyl ether,
ethyl acetate, methanol and water. Each fraction was again placed on a
silica gel column and washed with the respective solvent while ca. 5 ml
fractions were collected. Tubes containing radioactivity were combined and
taken to dryness.
Pigments, mostly chlorophyll and its degradation products, were at this
point inseparable from the radioactive materials. Attempts to separate the
pigment from the radioactive substances by thin-layer chromatography were
unsuccessful. First attempts using microparasil columns with a high
20
-------
TABLE 5. Atrazlne tolerance Expt. I
Effect of various concentrations of atrazine on growth of Spartina, A 5 days after treatment
Atrazine
Conc. CM)
Fresh wt. of
root (g)
Fresh wt.
of shoot (g)
No. of
leaves
Total
heiQht
(cm)
Dry wt. of
root (g)
Dry wt. of
shoot (g)
Control
19.65
27.26
15.56
68.25
3.29
7.18
5 x io- 5
7.53
7.85
5.13
45.88
0.67
2.60
5 x 10-6
7.92
13.79
9.88
61.13
1.14
4.64
5 x i0 ’
9.44
16.64
11.19
63.06
1.43
5.54
5 x 10-8
13.70
24.11
15.0
69.71
2.01
7.90
Each value is the average of four replications with four plants in each replication.
-------
pressure liquid chromatograph to separate the pigments and radioactive
substances were also unsuccessful. However, separation was achieved with an
anion ion-exchange column. While the preparative procedure is slow,
isolation of a single radioactive component from the 100% diethyl ether
fraction has been accomplished. This compound has been tentatively
identified as atrazine. Similar results were obtained with the 100% diethyl
fraction of Sorghum vulgare . Radioactive constituents of other fractions
(ethyl acetate, methanol) oUthe chloroform extract are being isolated in a
similar manner. The identity of each radioactive component will be
confirmed by mass spectrometry. Preliminary separations by thin-layer
chromatography and autoradiography suggest that only one radioactive
metabolite is present in each remaining fraction of the chloroform extract;
these are anticipated to be the dealkylated derivatives of atrazine.
Methods that will provide for the separation of plant pigments from
radioactive substances on a preparative scale and thereby speed up the
analyses are being developed. Apparently, such a separation of pigments
from atrazine metabolites in chloroform extracts has not been completely
accomplished in other reported studies. While the study has been delayed by
the difficulty encountered in separating pigments from the radioactive
metabolites, the problem has been resolved for chloroform extracts. Similar
problems are not anticipated with the other, non-pigmented, components of
the model ecosystems.
BIOCIDE RESIDUES IN A MARSHLAND-ESTUARY EC0S’ STEM. Grant R 801185. Rice
University. Princi il Tnvest gator: Frank M. Fisher , Jr. P o1ect Officer:
Thomas W. Duke .
Pesticides used for the control of agricultural pests may ultimately
enter adjacent aquatic ecosystems. The ultimate fate and effects of these
synthetic substances in such systems is basically unknown. Information
about this potential impact is necessary in order to develop and implement
environmental controls and application guidelines. This research project
will determine the fate and effects in wetlands of pesticides used to
control insect pests in agricultural fields. The specific objectives of
this project are to describe and quantify the movement and accumulation of
specific pesticides in a Texas Gulf Coast marshland ecosystem. Residues of
aidrin, dieldrin, endrin, lindane, heptachior epoxide, and DDT and its
metabolites will be examined in representative species of several trophic
levels, including oysters, clams, blue crabs, shrimp and carnivorous fish.
Water and sediment samples will be analysed. Emphasis in the final year of
research will be given to the pesticide carbofuran, a compound of special
interest as it is now being used as a substitute chemical for dieldrin and
DDT.
This project has been completed and the final report is in preparation.
22
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Meetings, Conferences , and Workshops
Borthwick, P.W.
Attended ADP coordinators meeting at Research Triangle Park, N.C.,
August 19-20, 1975, to discuss Agency and ORD policies and procedures.
23
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MICROBIAL AND PHYSIOLOGICAL MECHANISMS BRANCH
O bj e Ct V e S
The Microbial and Physiological Mechanisms Branch conducts research on
the metabolism of toxic organic compounds by marine organisms. Metabolites
are isolated and identified. Possible accumulations of substrates or meta-
bolites in food chains are investigated. Research is currently conducted on
the microbiological metabolism of hydrocarbons and chlorinated pesticides.
The effects of hydrocarbon and pesticide metabolites, including
cholinesterase inhibiting pesticides on marine organisms, is being
investigated.
Status of Projects
Fate of Nitrilotriacetic Acid: Bourquin
Investigations into the fate of nitrilotriacetric acid in estuarine
waters has been completed. Tests on the effects of salinity on NTA
degradation by a soil Pseudomonas sp. showed decreasing degradation with
increasing salinities. Several bacteria were isolated from 11-Mile Creek, a
freshwater stream, which degraded NTA readily after a 2-week induction
period. No microorganisms from a saline environment were capable of
degrading NTA in the laboratory.
Microbiology of Surface Films: Bourquin
Studies on the microbiology of surface films indicate dense populations
of bacteria and fungi in surface microlayers of most estuarine environments
sampled as compared to samples from 10cm depths. Additionally, preliminary
gas chromatographic - mass-spectroscopic data indicates hydrocarbons of C 16
-C 34 carbon chains with alkane/alkenes and some aromatics present in
naturally occurring surface films. Studies are continuing to determine
microbiology and chemistry of surface films which may be useful in
determining water quality through the use of microbial indicies.
Metabolism of Aromatic Hydrocarbons: Bourquin
Laboratory studies on the metabolism of aromatic hydrocarbons have
yielded limited promising results for the co-metabolism of chlorinated
pesticides. Studies on biphenyl metabolism show the compound to be degraded
through the dihydrodiol tocatechol. Thin-layer chromatography has been used
to show the existence of the diol. Additional studies are planned with
biochemical mutants which are blocked for hydrocarbon degradation and
accumulate metabolites. Future studies include both mutant and growth
studies on eucaroytic metabolism of polycyclic aromatic hydrocarbons.
Efforts are being made to determine the rate of degradation of organo-
phosphate pesticides in artificial salt-marsh envirOnments. Data
accumulated on the stability of these compounds under various physico-
chemical parameters by Gulf Coast Research Laboratory (W. W. Walker) is
being incorporated into these studies.
24
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Effects of Carbamate Pesticides: Coppage
Research on short-term effects of carbamate pesticides on cholinesterase
activity in the central nervous system of fishes was completed and a
publication of findings is in press. Tests of effects of 140 day exposure
of sheepshead minnows to malathion on brain cholinesterase was completed in
cooperation with Bionomics/Marine Lab. In cooperation with the Bioassay
Branch, initial tests were completed for brain cholinesterase inhibition
caused by 144-168 hrexposures of sheepshead minnows to 10-560 g diazinon/ .
Techniques are being developed for study of effects of methyl parathion on
cholinesterase in brains of exposed estuarine fish. Findings will be useful
in determining concentrations of pesticides that may be harmful to fishes by
inhibition of enzyme essential to nerve impulse transmission and will aid in
interpreting enzyme inhibition in fishes from the environment.
Effects of Toxicants on Ciliate Protozoa: Cooley
The objectives of this research are to: 1) determine effect of specific
organic and inorganic toxicants, single and in combination, on population
growth of ciliate protozoa; and 2) determine whether and to what extent
these toxicants are accumulated by the test organisms.
Experiments on the effect of methoxychlor and cadmium chloride, singly
and in factorial combinations, on 24-hr growth rate and population size of
the ciliate protozoan, Tetra mena pyriformis , have been completed and
statistical analysis of population growth data is nearly complete. Chemical
analyses of cells and media to determine extent of bioaccumulation of
methoxychior and of cadmium in 7-day cultures are nearly completed. Tests
on toxicity of malathion to I. pyriforinis at constant temperature and
salinity are complete and tests at varying salinity and temperature are
expected to be completed during the First Quarter, 1976.
EC5O’s for cadmium chloride, tested singly, were 11.1 ppm for 24-hr
growth-rate reduction and 12.5 ppm for 96-hr population-size reduction.
EC5O’s for methoxychior, tested singly, were unobtainable at concentrations
up to 5 ppm. The EC5O’s for methoxychior in the presence of 10 ppm cadmium
were 1.02 ppm for 24-hr growth rate reduction and 1.6 ppm for 96-hr
population site reduction. The same concentrations in the presence of 15
ppm cadmium chloride caused more than 99% reduction of both 24-hr growth
rate and 96-hr population size. The interaction of the ions in the presence
of 15 ppm cadmium chloride caused more than 99% reduction two toxicants thus
produced EC5O’s at lower concentrations than did either toxicant alone. It
is unlikely that such concentrations of cadmium and methoxychlor would be
encountered in estuarine waters, but in exceptional situations, they might
occur in sediments.
INSECTICIDE PERSISTENCE IN NATURAL SEAWATER AS EFFECTED BY SALINITY
TEMPERATURE, AND STERItTTY. Grant R 80394T. Gulf Co t ResearcI
Laboratory. Principal Investigator: William W. Walker. Project Officer :
MW. Bourguin .
25
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The objective of this research effort is to determine the effect of
salinity, temperature, light, and sterility on th persistence and
degradation of representative organophosphorus and organochiorine
insecticides in natural seawater. Surface water samples varying in salinity
from zero to 30 ppt will be amended with insecticide chemicals and incubated
for varying lengths of time under varying conditions of light, temperature
and sterility. Insecticide residues and degradation products will be sought
by gas-liquid and thin-layer chromatographic methods. Malathion, parathion,
methyl parathion, diazinon and methoxychior are currently being
investigated.
MICROBIAL INTERACTIONS WITH PESTICIDES IN ESTUARINE SURFACE SLICKS. Grant R
803141. Georgia State University. rincipal Investigator: Donald G
Ahearn. Project Officer : MW. BourQuin .
Indegenous microorganisms of natural and man-medicated surface slicks
are being investigated for their interactions with selected organochiorine
pesticides. The effects of heptachior and other selected pesticides on the
metabolism of hydrocarbons and fatty acids are being evaluated using
labelled (C14) heptachior and hexadecane in a model system. The
accumulation and/or biodegradation of pesticides by bacteria, yeasts and
molds is being considered in regard to their primary role in the food chain.
Bacteria and fungi from North Sea surface slicks have been identified.
Thirty-six representative isolates have been tested for growth on
hydrocarbon substrates. All isolates grew well on alkanes and are being
tested for growth on a mixed hydrocarbon substrate using both axenic and
mixed culture systems. Predominate numbers of bacteria were in agreement
with previous results. Studies have been initiated on extracellular enzymes
to help determine emulsification factors which aid in formation of surface
films.
New Methology Developed: A rapid qualitative screening test for determining
the range of concentrations of heavy metals to use to determine EC5O s in
protozoan population-growth studies. The test requires only 24 hrs, exposes
the protozoa to graded concentrations of the toxicant in La Motte cells held
in a Petri dish moist chamber, and uses death and loss of ability to swim as
criteria of effect.
Meetings, Conferences, and Workshoffis
Coppage, D.L.
Attended and gave paper at the 105th Annual Meeting of the American
Fisheries Society, Sept. 10-13, 1975 Las Vegas, Nevada.
Attended and gave paper at Symposium on Pollution and Physiology of
Marine Organisms, Nov. 3—6, 1975, Milford, Connecticut. Co-sponsored by
MACFC, National Marine Fisheries Service and Belle W. Baruch Institute for
Marine Research, Univ. N. Carolina.
26
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Bourquin, A.W.
Attended and gave paper at Society for Industrial Microbiology Meeting,
Aug. 1975, Kingston, R.I.
Attended and gave invited presentation at the 3rd International
Biodegradation Symposium, Kingston, R.I., Aug. 1975.
Invited presentation at Gulf Coast Research Laboratory, December, 1975,
Ocean Springs, MS.
Attended Estuarine Research Federation Meeting, Galveston, TX, Oct.
1975.
Technical Assistance
Coppage, D.L.
Data, review material and coninentary were provided to the Office of
Water and Hazardous Materials, Office of Pesticide Programs, Criteria and
Evaluation Division on “Fate and Significance in the Environment” of several
pesticides for their “Initial Scientific and Mini—Economic Reviews”. Data
and comentary on pesticides were also provided to the Office of Water and
Hazardous Materials to write “Quality Criteria for Water” as required by the
Federal Water Pollution Control Acts Amendments of 1972.
Bourquin, A.W.
Served on hearing panel for Adjudicatory Hearing for City of
Philadelphia dumping of sewage sludge off Atlantic coast.
Completed study on fate of nitrilotriacetic acid in estuarine waters for
OR&D Headquarters.
27
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PATHOBIOLOGY, TOXIC INTERACTIONS BRANCH
Objectives
The Pathobiology, Toxic Interactions Branch plans, conducts and manages
research concerned with the effects of toxicants on marine and estuarine
species at the subcellular, cellular, tissue and organ levels. Use of
contemporary tools, instruments, and techniques of pathology such as light
and electron microscopy, histochemistry, tissue culture and microtechniques
(Table 6) provide data and knowledge necessary to understand how pollutants
damage aquatic organisms. This information is used to help assess the
significance of numbers and figures that go into establishing water quality
criteria (Table 7). Pathobiology is further concerned with understanding
interactions between natural diseases and man—made disease agents such as
pollutants. Research is conducted on the use of aquatic species as
indicators of carcinogens, mutagens, and teratogens in the environment.
Development of methods that utilize aquatic species as early—warning
indicators for human health and ecosystem damage has high priority in
pathobiology and GBERL.
The philosophy of the pathobiology branch is based on the tenet that an
understanding of mechanism(s) of toxicity and damage in aquatic species is
the best background upon which to predict an manage the impact of toxicants
in aquatic ecosystems.
Status of Projects
Structural and functional changes in the gills of crustacea after
exposure to heavy metals and organics: Couch
It has been found that cadmium exposure is associated with death of gill
epithelial cells. Electronmicroscopy has revealed that osmoregulatory, ion
regulatory, and respiratory cells die and become necrotic resulting in
blackened gills. Levels of 718 ppb CdC1 9 in water for 30 days and less
produces gill destruction and death. This information will be used by the
Office of Water and Hazardous Materials ocean dumping program and the Office
of Pesticide Programs in setting Standards, particularly for heavy metals.
Effects of interactions of biocontrol and chemical agents: Couch
Using the shrimp—virus system in interactive bioassays (see Figure 2)
with polychiorinated biphenyals, it has been found that exposure to PCB’s of
groups of shrimp with only a few infected individuals results in up to 70%
increase in virus infected individuals. These results will be used by the
Office of Pesticides in registration of biocontrol agents (i.e. viruses) as
pesticides.
The use of aquatic species as indicators of the Environmental
Carci nogens: Couch
Research on this project is just beginning. Oysters will be used as
test animals for select suspect carcinogen exposure. A conceptual paper on
28
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TABLE 6
PATHOB I OLOGY
FOUR GENERAL, COMPLEMENTARY LEVELS
EPIZOOTIOLOGY GROSS MICROSCOPIC ULTRASTRUCTURAL
PATHOLOGIC EFFECTS DISSECTION HISTOLOGY SUBCELLULAR CHANGES,
OF POLLUTANTS ON CASE HISTORY CYTOLOGY CYTOCHEMISTRY
POPULATIONS HISTOCHEMISTRY FUNCTIONAL LESIONS
INVOLVES
MORBIDITY INDIVIDUALS INVOLVES SYSTEMS, EFFECTS ON STRUCTURE
MORTALITY ORGANS, CELLS AND FUNCTIONS IN
LIVING SYSTEMS
MERGE
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TABLE 7
APPLICATION OF RESULTS
1. USE REPRODUCIBLE PATHOLOGIC SIGNS * CHARACTERISTIC OF SPECIFIC TOXICANT CONCENTRATIONS TO DEMONSTRATE
SUBLETHAL INJURY.
* DAMAGE SIGNS OR SYNDROMES MAY BE AT ANY ONE (OR MORE) OF THE LEVELS:
EPIZOOTIC,
GROSS,
MICROSCOPIC
ULTRASTRUCTURAL
2. APPLY SUBLETHAL DAMAGE LEVELS IN ESTABLISHING MAXIMUM “SAFE LEVELS” FOR TOXICANTS, OR (AFTER THE FACT)
TO SHOW INJURY FOR ENFORCEMENT PURPOSES.
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SHRIMP-VIRUS MODEL
for
INTERACTIVE BIOASSAY
CHRONIC, SUBLETHAL EFFECTS
RUSIN I
2a. CELL CULTURES
1
.
DISCOVERY, IDENTIFICATION AND CHARACTERIZATION
OF VIRUS IN PENAEID SHRIMPS
2
S
ABILITY TO RECOVER, AT WILL, INFECTED SHRIMP
AND VIRUS IN QUANTITY FROM NATURE
-+
UNDERSTANDING OF NATURAL FACTORS THAT AFFECT
3,
SHRIMP-VIRUS SYSTEM IN EXPERIMENTS,
CROWDING. SALINITY, TEMPERATURE
*
EFFECTS OF CHEMICALS (MUTAGENS, CARCINOGENS), HEAVY
4,
PETALS . HORMONES ETC. ON SHRIMP-VIRUS INTERACTION.
-EXPOSURE EXPERIMENTS-
+
RESULTS
1. INCREASE IN VIRAL PREVALENCE
2. INCREASE IN INFECTION INTENSITY
3. INCREASE IN HOST MORTALITY
4. ENHANCED CYTOPA11IIC EFFECTS
NO EFFECT
1. DECREASE IN VIRAL PREVALENCE
2. DECREASE IN INFECTION INTENSITY
3. REDUCTION IN HOST MORTALITY
4. REDUCED CYTOPATHIC EFFECTS
31
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use of aquatic species for carcinogen indicators has been written (Joint
US/Soviet Journal - in press). The Office of Health and Ecological Effects
will use these results for determining threats of specific pollutants as
carcinogens.
Pathology service: Couch
This is a continuous project providing diagnostic, descriptive and
advisory service to projects of other branches in the interpretation of
pathological responses of test organisms. Gill and pseudo-branch damage in
‘fish exposed to chlorine at the Bears Bluff EPA laboratory has been
described. Levels of chlorine sufficient to damage gills of estuarine
fishes was determined.
BIOCHEMICAL CHARACTERIZATION, INSECT CELL LINE CULTURE , AND DETERMINATION OF
HOST SPECIFICITY OF PINK SHRIMP NUCLEAR POLYHEDROSIS VIRtJ T Grant R 8O339
University of Tex . Principal Investig tor : M. A. Summers . — Project
Officer: o1 A. touch . — — ______ ______
Shrimp baculovirus proteins and DNA have been characterized (DNA mol.
wt. @ 50 x 106) and Kleinschmidt preparations of virus DNA molecules have
been photographed.
This information and these data will permit comparisons and separations
of the shrimp virus and similar insect viruses that are being developed as
viral insecticides. Biochemical characterization of the shrimp virus is
necessary for future correct identification of the virus in field studies
and laboratory studies in which the virus is used as part of experimental
models for study of effects of pollutants on host-pathogen relationships.
New Methodologies: Pathobiology has outlined the potential use of the
shrimp-virus system for bioassays of interactive effects of chemicals and
pollutants in estuarine animals (Figure 2). This outline is suggested as a
model for understanding how low level pollutant exposure might enhance or
increase the effects of natural pathogens in estuarine species, or how a
pollutant might upset the host-parasite relationships in estuarine
ecosystems.
Meetings, Conferences, and Workshops
John A. Couch
Attended and presented an invited paper to the III International
Congress of Virology in Madrid, Spain during September. The paper and a
data demonstration was about the shrimp baculovirus .
Represented Dr. Duke and GBERL at Conference on the Review of EPA
Estuarine and Marine Research in Washington, D.C. in June.
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BEARS BLUFF FIELD STATION
Objectives
Bears Bluff Field Station is uniquely situated among pristine salt
marshes to conduct experiments on estuarine and marine organisms in the
South East Atlantic coast. In this regard an overall research objective is
the scientific assessment of impact of man’s activities on estuarine/marine
coastal ecosystems. Within this broad area the laboratory specifically
coordinates EPA marine research in ecological effects of the biocides used
in power generation plants to control marine growth and disinfectants
applied in municipal sewage treatment to control pathogens. These efforts
include development of bioassays, assessment of estuarine habitats, marine
chemical analytical techniques, and specific studies in the areas of marine
organism development, reproduction, toxicology, chronic effects, food webs,
nutrient and pollutant assimilation, physiological and biochemical
measurements, data analysis, etc. The field station facilities consist of
outdoor ponds, a high capacity flowing sea water system, physiological labs,
marine productivity analysis equipment, chemical analysis laboratory, a
reference library, small boats and marine sampling equipment, temperature
control sea water systems, and telecomunications equipment.
Status of Projects
During the period July through December, 1975, research activities at
Bears Bluff Field Station were directed towards scientific evaluation of
effects of chlorination processes to marine ecosystems. This effort
involved both investigations conducted at the field station, and initiation
of three grant-supported projects.
Marine Ecosystem Test Unit Experiments: Davis, Erikson, Crane, Yoakum
and Foulk
Mixed species experiments (Marine Ecosystem Test Units - METU), consist
of an array of ninety-six, 38-liter flowing water units, each receiving 40
liters/hr of unfiltered marine water from the tidal North Edisto River,
containing entrained natural planktonic organisms and suspended particles.
Thirty-two of these units are “harvested” each 30-days, another set (32)
each 60—days and a third set (32) each 120—days. Within each harvest set
three levels of continual chlorination are applied using sodium hypochiorite
adjusted to calculated levels of 0.0 (control), 0.125, 0.250, and 0.500 ppm
at the delivery head. The control and each of the chlorination levels is
triplicated for three sets of eight test tanks. Chlorination delivery rates
are maintained by the use of syringe pumps. Ainperometric titration analysis
can detect only traces of total residual oxidants at the highest level
cited.
Harvests are followed by identification and enumeration of specimens in
each unit. The enumeration requires 5-8 people to sort and identify. The
principal question under consideration is: Will continuous low levels of
sodium hyperchiorite affect the settling and development of species in a
benthic estuarine coninunity?
33
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This experiment, conducted in outdoor tanks, fulfills the need for a
bioassessment technique which correlates data from laboratory single-species
bioassays with data from field experiments. Furthermore, it produces data
representing a sumation of all the potential ecological effects of chlorine
introduced into marine waters, regardless of the state of the art of
chemical detection.
In addition, individual trays of oysters (well known bioaccumulators),
have been placed in the water collector systems downstream from the
coimiunity test tanks. These oysters will provide samples for body burdens
of potential halogenated organics formed during degradation of chlorine. No
oyster tissue analyses have been completed at the time of this report.
During the reporting period the species enumeration effort developed a
technique to increase the rate of sorting. Utilizing colloidal sillica and
adjusting specific gravity, small crustaceans such as the very abundant
amphipods are rapidly separated from detritus and other organisms. This has
significantly accelerated the rate of data output.
Preliminary statistical analyses of the first two harvests (June/July
1975) indicate excellent uniformity of data among dosage replicates, and
organisms in comunities in the test units.
Single Species Bioassays: Davis and Middaugh
Single species bioassays are being conducted on such fishes as spot
( Leiostomus xanthurus) ; muninichog ( Fundulus heteroclitus) , silversides
( Menidia menidia) , and striped bass ( Morone saxitilis) .
Some of the results at this time from fish bioassays include:
Short term studies with juvenile spot (10-15 m IL) showed that they
were sensitive to chlorination of flowing sea water. So-called “total
residual chlorine” in these bioassays was determined by amperometric
titrator. Chemically speaking, however, it is not residual chlorine that is
being measured in sea water systems. The estimated incipient LC5O at 10°C
was 0.12 mg/l and at 15°C, 0.06 mg/i instrument readings. Spot also showed
temperature dependent avoidance responses to chlorination in experiments run
simultaneously to the LC5O exposures. Concentrations avoided were in
general similar to the estimated incipient LC5O’s at respective test
temperatures of 10 and 15°C (Middaugh et al., in press).
Histological monitoring of juvenile spot demonstrate damage to gill
tissue in specimens exposed to chlorine at 1.5 mg/i instrument reading.
Histopathological sections of pseudobranchs from control, untreated
specimens show plump epithelial cells surrounding lamellar capillaries.
Histological sections of pseudobranchs from specimens exposed to
chlorination levels yielding instrument readings of 1.5 mg/i for 95 minutes
show separated epithelial membranes, with a scalloped border; this has been
shown not to be a normal artifact.
34
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In work with early life history stages of the striped bass, we found
that the eggs were less sensitive than newly hatched prolarvae and that the
sensitivity of prolarvae and larvae to chlorination decreased with
increasing age.
Through an extramural grant (R 803872) to Dr. Morris Roberts at the
Virginia Institute of Marine Science, bioassays are being conducted with
selected invertebrate species including the oyster crab ( Penopeus herbstii) ,
hermit crab ( Pagurus pj, and blue crab ( Callinectes sapidus) , and coot
clam ( Mulinia lateralis) .
In the area of halogen chemistry in sea water, Dr. James H. Carpenter
has completed a state-of-the-art review paper for the EPA-ERDA sponsored
“Environmental Impact of Water Chlorination” symposium, Oak Ridge, 22-24
October, 1975. Results of preliminary work (Grant R-803893) on the
inorganic chemistry of chlorine added to sea water has underscored the lack
of basic knowledge in the area. Experiments conducted by Dr. George Helz,
University of Maryland (Grant R 803839) have demonstrated that within a few
seconds of addition of chlorine to sea water, bromine complexes are
activated which proceed to oxidize organic compounds present. Additionally,
Dr. Carpenter has demonstrated that the currently accepted analytical
technique of amperometric titration does not represent total residual
chlorine (TRC) in sea water, as has been generally assumed. Chioro-bromo
complexes are demonstrably present and their activity and ecological effects
must be assessed. Carpenter’s work is proceeding into identification of
halogenated organics induced by chlorination processes. These marine
chemical revelations present considerable and important insight into the
extreme difficulty posed to EPA in establishing scientific criteria for
regulation of all oxidation compounds (chlorine, brornochioride, and ozone)
used in marine waters, especially inshore coastal zones.
Students from the College of Charleston Biology Division are conducting
coniiiunity studies on experimental effluent ponds at Bears Bluff. Their
efforts, jointly supported by Bears Bluff Field Station and a “SOS” grant
from National Science Foundation focuses on the species invasion,
recruitment, and development of comunities in marine habitats. One pond
receives effluents from MET1J and laboratory chlorine bloassays. Another
pond receives only “control tt or non-exposed sea water. One year’s effort
has been completed and sumarized and the students are initiating their
second year of sampling.
ISOLATION AND STUDY OF CHLORO-ORGANICS RESULTING FROM CHLORINATION OF
SEAWATER. rant R 3893. UnT irsit of Miami. Principal Investigator:
James 11. Carpenter. Prolect Officer: WiliTam P. Davis .
Identification and quantification of halogenated compounds formed when
chlorine is added to seawater. Initial emphasis will describe the inorganic
and organic reactions which lead to long lasting by-products. Followup
studies will investigate the complex and physical chemical fate of
identified compounds in simulated and natural marine ecosystems, including
complexing with sediments, or uptake and bioaccumulation by organisms.
35
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Analytical capability will
investigations and coninunity
be applied both
studies by the Bears
to on-site experiments
Bluff Field Station.
FIELD INVESTIGATION
IN SALINE POWER PLANT
1 ryland. Principal
William P. DavIs .
OF CHLORINATED AND BROMINATED
TOOLING WATERS. Grant R
Investigator: George R.
ORGANIC
803839.
Helz .
COMPOUNDS FORMED
Universit ’ of
Project Officer :
(1) IdentIfy and quantify the halogenated organic compounds produced by
power plants; (2) Determine chemical factors controlling yields of produced
compounds by study of power plant cooling waters under different conditions
in the Chesapeake Bay Region. Included in the first year’s funding is a
workshop for chlorine/halogen investigators researching chemical and
ecological impacts in marine ecosystems.
Meetings, Conferences, and Workshops
ON MARINE VASCULAR PLANTS AND DECAPOD
Vlrglnia Institute of Marine Science .
H. Roberts. Project 0Tficer: William P.
Davis, W.P.
Substances Workshop:
Effects Testing of
1975.
of Model Systems in
Materials, Annapolis,
EPA-ERDA “Workshop on Use
Criteria”, Pensacola, Florida,
of Microcsms in Settling
16-17 September, 1975.
Water Standard
Ocean Disposal Bioassays, Pensacola, Florida, 18-19 September, 1975.
Estuarine Research Federation Biannual Symposium, Galveston, Texas, 6-10
October, 1975.
EPA-ERDA “Environmental Impact of
National Laboratory, 22-24 October, 1975.
Water Chlorination”,
Oak Ridge,
Presented EPA position to the Conference/Workshop
Program for the South Atlantic Outer Contental Shelf
1975.
Technical Assistance
“Environmental Studies
Area, 14-17 October,
Technical assistance rendered by Bears Bluff Field Station personnel
include coments on a DEIS from Fish and Wildlife Service to reinterpret the
Lacey Act as a regulatory authority to restrict importation and possession
SUBLETHAL EFFECTS OF
CRUSTACENANS . GrantR
Principal Investigatoi :
Davis .
CHLORINE
803872.
Morris
Determine the effects of chlorine (and Induced
organisms including Impact upon decapod crustacean
upon selected mollusks and other marine organisms.
compounds)
development
on marine
and effects
EPA Office
Environmental
Maryland, 3-4
of Toxic
Fate and
September,
Use
Tox i C
36
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of exotic organisms, including tropical fishes. Our comments became
realized when Congressman Leggett later urged U.S.FW.S. to revise their
strategy in this endeavor.
Personnel at Bears Bluff Field Station provide manuscript reviews to
National Marine Fisheries Service Fishery Bulletin, and grant proposals to
EPA, ERDA, and National Science Foundation, on a regular basis.
Further technical assistance to BLM was provided at the HLive Bottom,
Biological Sensitive Areas” Meeting, Charleston, in January 1976. Review of
EPA assistance to OCS Lease sales was given to Region IV representative to
other meetings and reviews.
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LABORATORY ANALYTICAL SERVICES BRANCH
Objectives
This branch provides analytical support to other Research Branches of
GBERL. This support consists of conducting chetnical analyses of pesticides
and other organics in marine water, sediment and biota as required.
1184 samples were analysed for pesticides and related organics
the reporting period.
during
A method for the detection
developed. The technique offers,
this organophosphate pesticide in
to malathion showed detectable
malathion within 30 minutes whereas
never detected.
of the metabolites of malathion has been
for the first time, a means of monitoring
the aquatic environment. Exposure of fish
levels of the monocarboxylic acid of
the parent compound, malathion, was
An analytical adaptation laboratory has been established. In this
laboratory, techniques are developed for analyses of pollutants in the
marine environment. Liquid chromatography is being evaluated as a means of
analyzing for some of the carbamate and organaphosphate pesticides and their
degradation products. A considerable amount of time has been devoted to
adapting methods for the analyses of diazinon, endosulfan, methyl parathion
and kepone in marine water and biota.
Meetings, Conferences , and Workshops
Wilson, A.J.
Attended a workshop on analytical methods for the analyses of kepone
January 14, 1976, at EPA Research Triangle Park, North Carolina.
Cook, G.H.
on
Attended 12th Annual Pesticide Review
July 14, 1975. Presented papers entitled
malaoxon, mono-and Dicarboxylic Acid of
shrimp tissue.”
Moore, J.C.
Conference, Palm Beach, Florida
“Determination of malathion,
Malathion in fish, oysters, and
Attended 12th Annual Pesticide Review Conference, Palm
July 14, 1975. Presented, papers entitled Uptake and
malathion and its hydrolysis products in pinfish and the
residues with acetyicholinesterase inhibition.”
Technical Assistance
Analysis of whale and manatee tissues for pesticides and polychiorinated
biphenyl compounds for EPA Office of Pesticides.
Beach, Florida,
depuration of
correlation of
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Analysis of sea herrings for pesticides for the Organization of Economic
Cooperation and Development.
Analysis of kepone residues as requested by the State of Virginia and
EPA Region III.
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II. TECHNICAL ASSISTANCE
Approximately 200 man-days of technical assistance (TA) was provided to
groups or agencies outside of the laboratory from June through December,
1975. The recipients of this assistance were universities, professional
societies, state agencies, federal agencies (other than EPA), EPA offices
(other than OR&D) and foreign countries or international organizations.
This TA does not include numerous telephone conversations with private
citizens on environmental questions and/or issues.
Some specific examples of Technical Assistance follow:
PCB Hearings - State of New York vs. General Electric
In December, 1975 Del Nirno and David Hansen prepared statements and
testified for the State of New York on the PCB problems in the Hudson River.
GE plants on the upper Hudson were implicated in PCB-contamination of the
river and aquatic life. Since that time, the courts have ruled in favor of
the State of New York.
Bioassay Organisms supplied to EPA, R 1on .L .
Juvenile marine fish and crustaceans were reared in the laboratory by
the Bioassay Branch of ERL, Gulf Breeze and supplied to the Surveillance and
Analysis Division of Region IV. These organisms were used for in situ ,
flowing water bioassays to evaluate toxicity of effluents from various
industrial plants within Region IV. Mobile laboratory trailers are used by
Region IV personnel for these surveillance operations.
Technical Assistance involving foreign travel
Dr. Tudor Davies traveled to Moscow in December to participate in the
joint US-Soviet working group on Water Pollution. This group is developing
and accomplishing a program of joint projects in the Management of Drainage
Basins - Protection of Lakes and Estuaries and Water Quality Criteria. This
specific activity entailed an assessment of the State of River Basin
Management Modelling and Large Lake Management Modelling in both countries
through a symposium and subsequent publication and the development 0 f work
plans for future cooperation.
Dave Hansen traveled to Dubrovnik, Yugoslavia to attend a meeting of the
Working Group on the Principles for Developing Coastal Water Quality
Criteria. A manuscript will be published by FAO.
Analyses of sea herring for the Organization of Economic Co-operation and
Development [ OECD )
The Laboratory Analytical Services Branch, headed by Alfred J. Wilson,
is participating in the OECD program of wildlife sampling and analysis over
a three year period to determine the levels of pesticides and related
compounds in the environment. A shipment of sea herring, Clupea harengus ,
collected off the North Atlantic coast is currently being analysed.
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Ocean dumping activities
In July of 1975 Dr. Bourquin and Dr. Davis served on the Adjudicatory
Hearing Panel for the Philadelphia sewage disposal hearings. State and
federal agencies are pursuing alternatives to the present practice of
barging and dumping Philadelphia sewage in the Atlantic Ocean.
Kepone in the James River, Virginia estuary
In December of 1975 the laboratory became involved in the potential
health and ecological effects of Kepone (a chlorinated hydrocarbon
insecticide) contamination in the lower James River. Our involvement with
the Kepone problem during the period covered by this report (July-December
1975) was limited to planning, validation of analytical methods, and initial
analyses of shellfish exposed to Kepone. However, this laboratory in
cooperation with the Virginia Institute of Marine Science (VIMS), has
proposed an action plan for ecological research specifically related to the
James River Estuary and the SiKepone incident”. This research is planned to
answer the following specific questions:
1. Determine the concentration of Kepone in organisms that are
comercially harvested in the James Estuary and are direct routes of the
carcinogen to human food. Determine the effective strategies that can be
used to reduce present levels of Kepone, i.e., transplanting of shellfish,
etc.
2. Determine direct toxic effects of Kepone on the individual organisms
and potential ecological effects on the James River estuary.
3. Determine the fate and persistence of the insecticide as a hazardous
compound in the estuary.
This research will require a considerable amount of technical assistance
during the next six months. Studies are presently underway at Gulf Breeze
to determine uptake, accumulation and loss of Kepone from oysters and at
VIMS to determine in-situ depuration rates by removing oysters with known
levels of Kepone from the James River and placing them in “clean” waters for
subsequent analysis.
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III. LABORATORY PUBLICATIONS
Bahner, L.H. and D.R. Nimmo. 1975. A salinity controller for flow—through
bioassays. Trans. Am. Fish. Soc. 104:388-389.
Bourquin, Al W. 1975. Densities of bacteria and fungi in coastal surface
films as determined by a membrane adsorption procedure. Liminol. and
Oceano . 20:644-645.
Bourquin, Al W. 1975. Microbial-malathion interaction in artificial salt-
marsh ecosystems; effect and degradation. U.S. EPA Ecological Research
Series, EPA-660/3-75-035. 4lpp.
Carpenter, James H. and Donald L. Macalady. Chemistry of halogens in
seawater. EPA- ERDA Conference Environmental Impact of Water
Chlorination, Oak Ridge, Tenn. 22-24 Octo., 1975.
Cook, G.H., J.C. Moore, and D.L. Coppage. 1976. The relationship of
malathion and its metabolites to fish poisoning. Bull. Environ. Contam.
Toxicol. 16(2).
Coppage, David L. 1975. Brain-acetylcholinesterase inhibition in fish as a
diagnosis of environmental poisoning by malathion, 0,0-dimethyl S-(1,2-
dicarbethoxyethyl) phosphorodithioate. Pest. Biochem. Physiol.
5:536-542.
Coppage, David L. 1975. Effects of some “persistent pesticide”
replacements on fishes. (Abstract). Papers presented at the 105th
Annual Meeting of the American Fisheries Society, Sept. 10-13, 1975, Las
Vegas, Nevada. p. 32
Couch, J. 1975. Evaluation of the exposure of fish and wildlife to nuclear
polyhedrosis and Granulosis viruses (Discussion). In: Baculoviruses
for Insect Pest Controls: Safety Considerations. Summers et al. (Ed.)
Amer. Soc. Micro. 1913 I St. N.W. Washington, D.C.
Couch, J.A., Sumers, M.D., and Courtney, L., 1975. Environmental
significance of Baculovirus infections in estuarine and marine shrimp.
Annals Mew York Acad. Sciences. 266:528—536.
Hansen, David J. and Steven C. Schimel. 1975. Entire life-cycle bioassay
using sheepshead minnows ( Cyprinodon variegatus) . Fed. Regist.
40(123) :26904-26905.
Hansen, David J., S. C. Schimel, and J. Forester. 1975. Effects of
Aroclor 1016 on embryo, fry, juvenile and adult sheepshead minnows
( Cyprinodon variegatus) . Trans. Am. Fish. Soc. 104(3):584-588.
Hollister, T. A.., G. E. Walsh and J. Forester. 1975. Mirex and marine
unicellular algae: Accumulation, population growth and oxygen
evolution. Bull. Environ. Contam. Toxicol. 14:753-759.
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Middaugh, D. P., W. R. Davis, and R. L. Yoakum. The response of larval
fish, Leiostomus Xanthurus , to environmental stress following sublethal
cadmium exposure. Contributions in Marine Science, 19: 13-19.
Parrish, P. R., S. C. Schininel, D.J. Hansen, J. ft. Patrick, Jr., and J.
Forester. 1976. Chlordane: Effects on several estuarine organisms.
J. Toxicol. Environ. Health 1:485-494.
Schimel, S. C. and D. J. Hansen. 1975. Sheepshead minnow ( Cyprinodon
variegatus) : An estuarine fish suitable for chronic (entire life-cycle)
bloassays. Proc. 28th Ann. Conf. Southeast Assoc. Game Fish. Con n.
White Sulphur Springs, West Virginia. p. 392-398.
Schimel, S. C., P. R. Parrish, D. J. Hansen, J. M. Patrick, Jr. and J.
Forester. 1975. Endrin: Effects on several estuarine organisms.
Proc. 28th Ann. Conf. Southeast. Assoc. Game Fish. Conin. White Sulfur
Springs, West Virginia. p. 187-194.
Schoor, W.P. 1975. Problems associated with low solubility compounds in
aquatic toxicity tests: Theoretical model and solubility charac-
teristics of Aroclor 1254 in water. Water Research 9:937-944.
Walsh, G.E. 1975. UtilizatIon of energy by primary producers in four ponds
in northwestern Florida. p. 249-274. In Proceedings:
Biostimulation/and/Nutrient Assessment Workshop, T. 1 a1one (Ed) EPA
Ecological Series Report, EPA-660l3-75-034.
Wilson, A.J. 1976. Effects of suspended material on measurement of DDT in
estuarine water. Bull. Environ. Contam. Toxicol. 15 (5).
Manuscripts In Press
Bahner, L.H. and D.R. Nimo. A precision live-feeder for flow-through
larval culture or food chain bioassays. Prog. Fish-Cult.
Bahner, L.H. and D.R. Ninino. Methods to assess effects of combinations of
toxicants, salinity, and temperature on estuarine animals. Proc. 9th
Ann. Conf. on Trace Substances in Env. Health. Univ. of Missouri at
Columbia.
Bahner, LH., C.D. Craft, and D.R. Nimo. A saltwater flow-through bioassay
method with controlled temperature and salinity. Prog. Fish Cult.
Bourquin, Al W. Microbial populations in coastal surface slicks In: j-
deterioration of materials Vol III (Eds: A. Kaplan et al.).
Cook, G.H., and J.C. Moore. The determination of malathion, malaoxon, mono-
and dicarboxylic acids of malathion in fish, oyster, and shrimp tissue.
J. Ag. Food Chem.
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Coppage, D. L. Anticholinesterase action of pesticidal carbamates in the
central nervous system of poisoned fishes. In: Pollution and Physiology
of Marine Organisms. (Al Calabrese and F. P. Thurberg, Eds.) Academic
Press: New York.
Coppage, 0. L. The relationship of malathion and its metabolites to fish
poisoning. Bull, of Environ. Contam. Toxicol. Vol. 16.
Couch, J. A. Aquatic species as possible indicators of environmental
carcinogens. Joint U.S./Soviet Union Journal of Marine Environmental
Quality.
Couch, J. A. Attempts to increase Baculovirus prevalences in shrimp by
chemical exposure. Proc. Symposium on 11 Tumors in Aquatic Animals as
Indicators of Environmental Carcinogens”. Progress in Experimental
Tumor Research, Vol. 20 Karger, Geneva.
Couch, J.A. Parasites, diseases, and toxic responses in penaeid shrimps of
the Southeastern Coasts of the U.S. Invited paper presented at Special
Symposium of The Gulf Estuarine Research Society (May, 1975). 66 pp. 50
figures.
Davis, W. P. and D. P. Middaugh. A review of the impact of chlorination
processes upon marine organisms. EPA-ERDA Conference on Environmental
Impact of Water chlorination, Oak Ridge, Tenn. 22-24 Octo., 1975.
Duke, 1., Couch, J., Herndon, C. Review of EPA Estuarine and Marine
Research. Fed. Work Group on Pest Management. Proc. Special Meet.
Wash. D.C.
Hansen, 0. J. Techniques to assess the effects of toxic organics on marine
organisms. EPA Ecol. Res. Ser.
Hansen, D. J. ‘CBs: Effects on and accumulation by estuarine organisms.
Proc. EPA Conf. on “Polychiorinated Bipheny1s ’.
Hansen, D. J. (As a member of “Working Group on Principles for Developing
Coastal Water Quality Criteria; FAO”). Principles for developing
coastal water quality criteria.
Middaugh, D. P. and G. Floyd. 1976. The effect of prehatch and posthatch
cadmium exposure on salinity tolerance and activity of larval grass
shrimp, Palaemoneter puglo . Chesapeake Science.
Middaugh, D. P. and P. W. Lempesis, 1976. Laboratory spawning and rearing
of the marine fish: Silverside, Menidia menidia . Marine Biology.
Nitmio, DR. and L.H. Bahner. 1976. Metals, pesticides, and PCB’s:
Toxicities to shrimp singly and in combination. Proc. 3rd Ann.
Internat. Estuarine Res. Fed. Conf., Galveston, Texas.
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Nimo, D.R., D.V. Lightner, and L.H. Bahner. 1976. Effects of cadmium on
the shrimps, Penaeus duorarum, Palaemonetes pugio and Palaenionetes
vulgaris . Poll. and Phys. of Mar. Organisms. Vol. 2. Vernberg and
Vernberg Eds. Academic Press.
Schininel, S. C., J. M. Patrick, Jr. and J. Forester. 1976. Heptachior:
Toxicity to and uptake by several estuarine organisms. J. Toxicol.
Environ. Health.
Manuscripts Submitted
Hansen, D. J. Impact of pesticides on the marine environment. Joint US-
USSR Biological Seminar.
Tagatz, M. E. Effect of Mirex on predator-prey interaction In an
experimental estuarine ecosystem. Trans. Mi. Fish. Soc.
Manuscripts In Preparation
Schimmel, S. C., J. M. Patrick, Jr. and J. Forester. Uptake and toxicity of
toxaphene in several estuarine organisms.
Schiimnel, S. C., J. M. Patrick, Jr. and J. Forester. Heptachior: Uptake,
depuration, metabolism and retention by spot, Leiostomus xanthurs
(PISCES: SCIAENIDAE).
Special EPA Publication
The Ocean Dumping Bioassay Comittee, chaired by Dr. Duke, has prepared
a manual entitled “Bioassay Procedures for the Ocean Disposal Permit
Program.” Staff members from the Environmental Research Laboratories at
Gulf Breeze, Narragansett and Corvallis contributed to this manual. The
manual is now in press.
The bioassay procedures given in this manual were developed to provide
tests for conducting toxicity evaluations of waste materials considered for
ocean disposal under EPA s Ocean Disposal Permit Program. Nine bioassay
procedures are described; three of which are considered “special” and are
not recomended for routine use. The procedures specify the use of various
organisms representing several trophic levels. Both flow-through and static
tests are included. Methods given vary in their utility and complexity of
performance. The procedures are not intended to be considered “standard
methods,” but are to be used as reference methods or official methods
depending on the .judgement of the EPA Regional Achninistrator responsible for
the management of the permit program.
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