United States
 Environmental Protection
Environmental Research
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
  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
  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
  (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

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
  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
  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
  Effect of Kepone-sorbed sediment on
mysid survival  was  time- and  dose-

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

 Avoidance of Pollution
  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

 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.

  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

      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
Center for Environmental Research
Cincinnati OH 45268
Postage and
Fees Paid
EPA 335
Official Business
Penalty for Private Use $300
                                  PS    0000329