United States
Environmental Protection
Agency
Environmental Research
Laboratory
Gulf Breeze FL 32561
'/ r
Research and Development
EPA-600/S3-82-074 Sept. 1982
Project Summary
Cycling of Xenobiotics
Through Marine and
Estuarine Species
C.N. D'Arsaro
Cycling of xenobiotics was studied
using time-lapse photography to
evaluate effects of Kepone and sodium
pentachlorophenate on feeding activity
of the lugworm, Arenicola cristata.
The fate and effects of methyl parathion
in microcosms inhabited by lugworms
were determined. Uptake and depura-
tion of chrysene by lugworms were
evaluated.
A toxic sediment bioassay system
was developed to test effect of
dredged material. The system included
mysid shrimp, Mysidopsis bahia,
oysters; Crassostrea virginica; and
lugworms, Arenicola cristata. Criteria
of effects were survival of mysids,
shell deposition and bioaccumulation
by oysters, substrate reworking and
bioaccumulation by lugworms, and
settlement of zooplankton. Kepone-
sorbed sediment and dredge spoil
from James River and Houston Ship
Channel were tested. Long-term tests
were used to evaluate effects of a
specific drilling mud from an active
exploratory platform.
Predator-prey tests of sublethal
effects of xenobiotics demonstrated
effects in one-prey and two-prey
systems. The effects of methyl para-
thion on predator-prey relationships
between grass shrimp, Palaemonetes
pugio; juvenile sheepshead minnows,
Cryprinodon variegatus; and gulf
killifish, Fundulus grandis, were
demonstrated.
Evaluation of sublethal effects, such
as avoidance of pollution gradients,
was studied in a trough-type avoid-
ance-response system. The system
was tested with pinfish to demon-
strate that they will avoid chlorine-
produced oxidants.
The assessment of the potential
impact of environmental contami-
nants depends on the accurate mea-
surement of the fate and effects of
these pollutants on both field and
laboratory. This project was directed
toward developing methods to provide
more sensitive evaluators, other than
acute and chronic toxicity tests for a
xenobiotic's fate and effect in estuarine
and marine ecosystems. The goals of
the project were to:
(1) evaluate cycling of selected
xenobiotics or uptake and effect of
selected energy related compounds in
experimental systems that include the
lugworm, Arenicola cristata;
(2) develop a toxic sediment assay
system involving the lugworm and
other species;
(3) develop tests involving estuarine
and marine crustaceans and fishes
designed to evaluate how exposure to
xenobiotics can alter predator-prey
relationships;
(4) develop and test behavioral
assays that provide reliable means to
evaluate sublethal effects.
This Project Summary was devel-
oped by EPA's Environmental Research
Laboratory, Gulf Breeze, FL, to an-
nounce key findings of the research
project that is documented in a
separate report of the same title (see
Project Report ordering information at
back).
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Cycling of Xenobiotics by the
Lugworm, Arenicola Cristata
The impetus to design an assay
system involving a lugworm resulted
from development of culture methods
for that species and recognition that
toxicity tests employed by EPA for
estuarine and marine species do not
include an infaunal organism.
Benthic Photo-Bioassay
System
The lugworm, Arenicola cristata is
ideal for this type of test because it is
widely distributed in littoral habitats and
has major ecological impact due to its
ability to recycle sediment and transport
xenobiotics into the substrate. The
photobioassay system constructed was
based on the lugworm's habit of
creating feeding funnels on the surface
of sediment it occupies. Patterns on the
surface of the substrate which indicate
activity of the worm, were monitored
with time-lapse photographs taken at
12-hour intervals for 72 hours. Areas of
feeding funnels in exposed and control
aquaria were calculated and compared.
Results indicated that/4, cristata was
sensitive to Kepone at all concentra-
tions tested. The highest concentration
was acutely toxic. Lugworms appeared
to be more sensitive to Kepone than
many other species normally used in
toxicity tests. It appeared that in
Kepone-contaminated habitats the
ability of lugworms to rework sediment
would be markedly decreased.
Cycling of Methyl Parathion
by Lugworms
A second evaluation of cycling of
xenobiotics by lugworms was directed
toward determining compartmentation
and degradation dynamics of methyl
parathion in a small-scale microcosm
occupied by only the worm and micro-
organisms associated with the organic
material on which they feed. Ninety
percent of radio-labelled methyl para-
thion disappeared from the water
column in aquaria after 14 days.
Movement into the sediment proved to
be the major compartmentation phe-
nomenon, with over half of the total
radioactivity residing in the sediment
after two weeks. The lugworm enhanced
movement of radioactivity into the
sediment and caused dispersion through-
out the sediment. Although volatili-
zation losses were negligible, steadily
decreasing mass balance of radioactivity
in the system suggested accumulation
of unextractable residues in the sedi-
ment. Analysis of extractable radio-
activity in the sediment and water
compartments by thin-layer chromatog-
raphy and autoradiography demon-
strated rapid degradation of methyl
parathion into a number of more polar
products, including p-nitrophenol and
amino-methyl parathion. Although A.
cristata was shown to metabolize
methyl parathion readily to /'-nitro-
phenol, microbial activity accounted for
the majority of biological degradation in
the system.
Effect of Sodium
Pentachlorophenate on
Lugworm Activity
The third analysis of effect of xeno-
biotics on activities of the lugworm was
the evaluation of effect of sodium
pentachlorophenate (Na-PCP) on feeding
activity. Na-PCP was used because it is
an energy-related compound (oil-well
drilling fluids) and because it enters
estuarine and marine systems occupied
by lugworms from numerous non-point
sources. The photo-bioassay methods
described previously were used. Stock
solutions of Na-PCP prepared from a
commercial bactericide were introduced
into experimental aquaria at 45, 80,
156, and 276 /ug/l. Comparisons were
made between the areas of feeding
funnels in exposed and control aquaria.
Na-PCP had no marked effect on feeding
activity at the lowest concentration
tested; however, at the other concentra-
tions, there was significant decrease in
activity. Some death occurred at the
higher concentration.
Uptake and Depuration of
Chrysene by Lugworms
The final analysis of cycling of
xenobiotics by lugworms involved
uptake and depuration of chrysene,
another energy-related compound.
Worms were exposed to chrysene at
measured concentrations of 0.07,0.69,
and 2.76 /ug/l large wooden tanks in an
open system that simulated ambient
conditions and the natural habitat. After
14 days, exposed worms were moved to
uncontaminated systems and allowed
to depurate for 14 days. From lowest to
highest exposure, lugworms accumu-
lated 65, 516, and 682/ug/l in 14 days.
There was a continued increase in
accumulation during that period, so it is
probable that had exposure time been
increased, higher concentrations of
chrysene would have been encountered
before equilibrium was reached. Little
depuration was observed. This suggested
that lugworms are unable to degrade
chrysene; thus their potential to intro-
duce chrysene into various food chains
utilized by man is high.
Toxic Sediment Bioassay
System
Many xenobiotics in marine environ-
ments have a high affinity for particu-
late material (especially organics) and
thus become sequestered in bottom
sediments. Due to increased dredging
and maintenance of navigable water,
there is a greater need to evaluate
impact of toxic sediments on the biota.
For that reason, grant activities were
directed toward developing a flow-
through toxicity test that could be used
to determine biological effects of
contaminated sediments on represent-
ative estuarine organisms and to
evaluate resiliency of benthic com-
munities exposed to contaminated
sediments. The test developed incor-
porated several established toxicity
tests that were modified to examine
acute and sublethal effects of dredged
sediments on the biota. It was designed
to serve as a screening tool to detect
potential hazards of dredge spoils prior
to disposal in the marine environment.
Kepone-Sorbed Sediment
Small scale estuarine microcosms
were assembled using 10-gallon aquar-
ia that received flowing, unfiltered sea-
water. Artificially prepared sediments
containing Kepone at 0.1,1.0, and 10.0
fjg/\ were used. Three aquaria received
different concentrations of test sedi-
ments, while three others remained
unperturbed and served as controls.
Comparisons were made after 28 days.
Organisms included in the test were
representative of three environmental
compartments affected by dredging
activities. Included were mysid shrimp,
Mysidopsis bahia; oysters, Crassostrea
virginica; and lugworms, Arenicola
cristata. Test criteria used to identify
effect were: (1) survival of mysids; (2)
shell deposition and bioaccumulation of
known contaminants by oysters; (3)
substrate reworking and bioaccumula-
tion by lugworms; and (4) resiliency of
the benthic community in terms of
numbers and variety of macrofaunal
organisms that settled onto test sedi-
ments as planktonic larvae within 28
days.
Effect of Kepone-sorbed sediment on
mysid survival was time- and dose-
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dependent. Oyster shell growth was
significantly inhibited. Lugworms had
an increasing dose-dependent relation-
ship in concentration of Kepone. Whole-
body residues were 0.043,0.46 and 1.1
/ug/l. Nineteen macrofaunal species
from four major taxa were identifred.
Using test criteria, only polychaetes
were affected at the highest exposure.
Dredge Material
Tests with actual dredged material
from the James River and Houston Ship
Channel were conducted. James River
sediment did not affect mysids signifi-
cantly although there was some effect
on oysters. Lugworms substrate re-
working was reduced in experimental
aquaria. Oysters and lugworms concen-
trated Kepone. Little difference was
seen in survival of recruited larvae,
perhaps because few larvae entered the
system during the winter when it was in
operation. Houston Ship Channel sedi-
ment did not significantly affect mysid
survival or oyster shell deposition; nor
did lugworm activity or macrofaunal
composition vary significantly between
control and experimental units.
Drilling Muds
A long-term (100-day) toxicity test
was conducted using the toxic sediment
assay system to determine effects of a
specific drilling mud. Drilling muds
were obtained weekly from an active
exploratory platform and tested within
one week of collection. Three dilutions
were tested: 10, 30, and 100 ml/I.
These concentrations represented
those expected to occur at intervals of
from several meters to several hundred
meters from a point source. Mud was
added to test aquaria to simulate
periodic discharge. The same species
previously employed were included in
this test but mysids were exposed for
only 10 days.
Mysids exposed in the system were
not affected acutely. Oyster shell
growth was inhibited significantly at
concentrations of 30 and 100 ml/l but
there were no deaths. Lugworms were
severely affected by exposure to the
mud. Mortalities observed were 75% at
100 ml/l, 64% at 30 ml/I, and 33% at
10 ml/l. Twenty recruited species were
present after 100 days. There was no
significant difference between popula-
tions in the aquaria. Ba, Cr, and Pb were
found to have accumulated significantly
in oyster tissue.
The results indicate that physical as
well as chemical properties must be
considered before environmental impact
of drilling fluids can adequately be
assessed. It was also recognized that
composition of drilling muds is highly
variable; thus, impact should be consid-
ered on a case-by-case basis.
Predator-Prey Tests
Sublethal concentrations of xenobio-
tics, especially pesticides, may be
expected to affect various aspects of
behavior. If pesticides have different
effects on species in a multiprey system,
predators consume a higher than
normal proportion of affected species.
The result would be more rapid accumu-
lation of a xenobiotic.
Palaemonetes pugio and juvenile
sheepshead minnow, Cyprinodon varie-
gatus, were exposed to methyl para-
thion for 24 hours before introduction of
Fundulus grandis. the predator. The kill-
ifish consumed a greater proportion of
grass shrimp relative to sheepshead
minnows. Increasing the concentration
resulted in increased consumption of
grass shrimp relative to fish prey, an
example of how a pesticide can alter
relative proportions of prey in a preda-
tor's diet.
Evaluation of Sublethal
Effects in Special Test
Systems
Avoidance of Pollution
Gradients
It has often been observed that fish
and invertebrates avoid pollution gra-
dients. Most apparatus designed to
detect avoidance of pollutants by
aquatic organisms require visual obser-
vation of the test organisms in steep
pollution gradients. The Aquatic Gra-
dient Avoidance Response System
(AGARS) was developed to eliminate
these limitations. This system allows
animals to choose among one uncon-
taminated zone and three increasingly
toxic zones in a gradient trough that is
monitored for extended periods by
infrared light sources, sensors, and a
microprocessor. Initial tests in AGARS
indicated that pinfish, Lagodon rhom-
boides, avoid chlorine-produced oxi-
dants at concentrations of 0.02-0.04
mg/l.
Toxicant Induced Changes in
Cyclic Burrowing Patterns
The pink shrimp, Penaeus duorarum,
is a species that is very sensitive to
xenobiotics. Since no life-cycle toxicity
test exists for penaeid shrimp, the only
criterion of effect that has been used for
hazard assessment is death. Pink
shrimp normally remain buried in
substrate during the day and emerge at
night. Stress from both lethal and
sublethal pesticide exposures disrupt
this pattern and may result in the
shrimp's continuous presence above
the substrate. Such activity would
increase predation and cycling of
xenobiotics. To evaluate the effect of
toxicant-induced disruptions in the
cyclic burrowing pattern, an apparatus
was constructed from a modified
AGARS system.
The results indicated variability in
absolute activity level of a particular
shrimp on different days as well as
between shrimp. On a daily basis, there
was significant difference in activity
between days when toxicant was added
and days when it was not.
Conclusions
The toxic sediment bioassay system
developed under this project shows
promise of becoming a useful method in
determining biological effects of poten-
tially toxic sediments on representative
estuarine organisms and benthic com-
munities. It provides several distinct
advantages over existing dredged
material tests. Thus, it may be a suitable
methodology for future inclusion in the
testing manuals used to generate data
in support of permitting programs such
as S. 103 of The Marine Protection,
Research and Santuaries Act of 1972.
The behavorial tests investigated
demonstrate that measurable biological
responses occur at contaminant levels
below those acutely toxic. Further
research is required, however, to
achieve full understanding and inter-
pretability of the information generated
by these tests.
o US GOVERNMENT PRINTING OFFICE.19BZ-559-017/0802
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C. N. D'Arsaro is with the University of West Florida, Pensacola, FL 32506.
Frank G. Wilkes is the EPA Project Officer (see below).
The complete report, entitled "Cycling of Xenobiotics Through Marine and
Estuarine Species, "(Order No. PB 82 -239 252; Cost: $9.00, subject to change)
will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Environmental Research Laboratory
U.S. Environmental Protection Agency
Gulf Breeze, FL 32561
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Postage and
Fees Paid
Environmental
Protection
Agency
EPA 335
Official Business
Penalty for Private Use $300
PS 0000329
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