CYCLING OF XENOBIOTICS THROUGH MARINE AND
ESTUARINE SEDIMENTS
Extracted from cited papers by
Charles N. .D'Asaro
Department of Biology
University of West Florida
GRANT NO. R804458
Project Officer
Frank G. Hi Ikes
Environmental Research Laboratory
U.S. Environmental Protection Agency
Gulf Breeze, Florida"32561
ENVIRONMENTAL RESEARCH LABORATORY
OfFICE-OF RESEARCH AND DEVELOPMENT
II.S. -ENVIRONMENTAL PROTECTION'AGENCY
SZ 561
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CONTENTS
Abstract
Figures
1. Introduction
2. Cycling of Xenobiotics by the Lugworm, Arenicola cristata .
a. Bentic Photo-Bioassay System
b. Cycling of Methyl Parathion by Lugworms
c. Effect of sodium Pentachlorophenate on Lugworm Activity
d. Uptake and Depuration of Chrysene by Lugworms
3. Toxic Sediment Bioassay System
a. Methods Development v/ith Kepone-Sorbed Sediment . . . .
b. Tests with Dredge Spoil
c. Tests with Drilling Mud
4. Predator-Prey Tests
a. One and Two-Prey Tests with Separate Controls
b. Exposed and Control Prey in the Same System ,
c. Cryptic Shading and Predation .
5. Evaluation of Sublethal Effects in Special Test Systems. . ,
a. Avoidance of Pollution Gradients
b. Toxicant Induced Changes in Cyclic Burrowing Patterns
6. Biology of Species to Be Use in Small Scale Microcosms . .
References
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ACKNOWLEDGMENT
The cooperation and help of the staff at the Gulf Breeze Environmental
Research Laboratory is gratefully acknowledged. Facilities in which
experiments were completed were made available to the University of West
Florida through the kind assistance of the Laboratory Director, Dr. Thomas
W. Duke, and the Project Officer, Dr. Frank G. Wilkes.
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FIGURES
Number Page
1. Photo-Bioassay System (A - 24-hour timer; B - 35 mm camera with
automatic advance; D, E - aquaria with 35 cm of sand and 75 1 of
seawater).
2. Comparison of the rates of sediment turned under by group of similar
size. A different group of lugworms was used for the six replicate
tests.
3. Comparison of the rates of sediment turned under by lugworms.
C-control: E-experimental group exposed to Kepone. Each group
consisted of six lugworms.
4. Comparison of the rates of sediment turned under by the lugworm,
Arenicola cristata, C-controls; E-experiment group exposed to sodium
pentachlorophenate. Each group consisted of six lugworms.
5. One tank in the exposure system for chrysene.
6. Accumulation of chrysene by lugworms. Each data point is based on
average accumulation of five worms.
7. Exposure system. A-suspended sediment dosing apparatus; B-timer;
C-crimping bar and solenoids; D-delivery tubes; E-submersible
pumps to recirculate suspended sediment to the delivery box (not
labeled); F-seawater headbox; G-splitter box stand pipes; H-mysids,
oysters and lugworms in exposure tank; 1-exposure tanks on a water
table.
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Number Page
8. Percent mortality of mysids exposed to control and Kepone-
sorbed sediments for 10 days.
9. Effect of Kepone-sorbed sediments on oyster shell deposition
(percent growth is relative to controls).
10. Number of individuals and species collected from exposed and
and control aquaria after 28 days.
11. Effect of James River sediment on oyster shell deposition.
12. Kepone residues in oysters and lugworms following 28 days of
exposure to James River sediments.
13. Percent mortality of mysids exposed to control and three con-
centrations of drilling muds for 10 days.
14. Weekly average oyster growth (N=15).
15. Oyster shell deposition relative to controls.
16. Percent mortality of lugworms in control and exposed aquaria.
17. Number of individuals and species collected from exposed and
control aquaria after 100 days.
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Number Page
18. Ba, Cr, and Pb concentrations in oysters exposed to control and
three concentrations drilling muds for 100 days.
19. The ratio of Palaemonetes pugio to Cyprinodon variegatus surviving
after each of five days of predation by Fundulus grandis in con-
trol and 0.457 mg/1 methyl parathion exposed aquaria. The lower
ratio in the exposed aquaria indicated greater predation on P_. pugio.
20. Preference coefficients for £_. grandis predation on P. pugio and
juvenile C_. variegatus during five test days in control and 0.475
mg/1 methyl parathion-exposed aquaria. Higher coefficients
indicate greater predation on P_. pugio. See Farr (1978) for
method of calculation.
21. The ratio of P_. pugio to C_. variegatus surviving after each of five
days of predation by £. grandis in control and acetone control
aquaria.
22. The ratio of P_. pugio to C^. variegatus surviving after each of
five days of predation of F_. grandis in control aquaria and
in three concentrations of methyl parathion.
23. Test apparatus used to observe pinfish reactions to flounder
models: A-fluorescent lights; B-automatic advance camera;
C-realease chamber; D-circular test tank.
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Number Pag
24. Diagrams of pinfish positions for combined trials 1-10 for each
time interval of control, light flounder model, and dark flounder
model. Each point represents the mean position of 10 pinfish for
each trial, and the X represents the mean of all 10 points. The
F values shown below the diagram were calculated by Hotelling's one-
sample test. P values correspond to the probability at the tiven
F value.
25. Diagram of AGARS trough. Trough is 125 x 17 x 15 cm high and
constructed of 6 mm clear plexiglas. "M" denotes mixing boxes where
1.5 1 min clean water and test compounds or carriers are combined.
The flow is divided evenly between pairs of small chambers on each
side of the trough. Water exits from the chambers through a row
of 7 mm holes. Water flow maintains a gradient of a control zone
and three increasing toxicant concentrations (areas 1-4). Organism
position can be monitored in both the upper and lower half of each
area by pairs of infrared light emitting diodes and photo-transistors
26. Results of a 9-day AGARS test with a group of 4 pinfish. Mean of
24 hourly totals of time spent in each of 4 areas of trough versus
elapsed time of test. Chlorine produced oxidants were present on
days 4 and 7 only.
27- Diagram of one of two replicate troughs used to study pink shrimp
behavior. The trough was modified from that shown in Figure 26 in
that it is partitioned with barriers of plexiglas and plastic screen
and contains sand. The presence of each shrimp above the sand
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Number Page
is monitored by two pairs of photo-transistors and infrared light
emitting diodes.
28. An example of an activity graph indicating time in light beams
(0.33 sec/h) during each hour of the 6-day test. On days 3 and 4
shrimp were exposed to 2.0 ppb methyl parathion and carrier. Light
and dark bars indicate photo-period.
29. Mean increase in length by cultured Arenicola cristata during spring
and early summer; 95% confidence belts are indicated.
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ABSTRACT
The results of five broadly defined projects are reported.
Cycling of xenobiotics was studied with a photo-bioassay system, which used
time-lapse photography to evaluate effects of Kepone and sodium pentachlorophenate
on feeding activity of the lugworm, Arenicola cristata. Radio-labeled methyl
parathion was used to demonstrate fate and effect in microcosms inhabited by
lugworms. Uptake and depuration of chrysene by lugworms was evaluated in a
flow-through system.
A toxic sediment bioassay system was developed to provide a means to test
effect of dredge spoil. The system included microcosms that held mysid shrimp,
Mysidopsis bahia; oysters, Crassostrea virginica; and lugworms, Arenicola cristata.
Effect was tested by using survival of mysids, shell deposition and bioaccumu-
lation by oysters, substrate reworking and bioaccumulation by lugworms, and
settlement of zooplankton as criteria. Kepone-sorbed sediment and dredge spoil
from James River and Houston Ship Channel were tested for 28 days. Long term
tests (100 days), with the same systems, were used to evaluate effect of a
specific drilling mud from an active exploratory platform.
Predator-prey tests of sublethal effects of xenobiotics demonstrated effect
in one prey and two prey systems. The effects of methyl parathion on predator-
prey relationships between grass shrimp, Palaemonestes pugio; juvenile sheeps-
head minnows, Cyprinodon varieqatus; and gulf killifish, Fundulus grandis were
demonstrated. The relationship between Palaemonetes pugio and pinfish, Lagodon
rhomoboides was also demonstrated. A method that could be used to evaluate
effect of xenobiotics on predator-prey relationships between cryptically shaded
flounder and pinfish prey was developed.
Evaluation of sublethal effects, such as avoidance of pollution gradients
was studied in a trough-type avoidance response system. The system was developed
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to be independent of an observer. It was tested with pinfish to demonstrate
that they will avoid chlorine-produced oxidants. The system was modified to
demonstrate toxicant induced changes in cyclic burrowing activity by pink
shrimp, Penaeus duorarum, exposed to methyl parathion.
Usefulness of small scale microcosms was evaluated by developing methods
to culture polychates and crustaceans. Various aspects of the biology of selected
species were studied.
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INTRODUCTION
Five broadly defined projects were included in the project goals of
Grant R804458, "Cycling of Xenobiotics through Marine and Estuarine Sediments."
Two addressed primary goals that were:
(1) to evaluate cycling of selected xenobiotics or uptake and effect of
selected energy related compounds in experimental systems that
included the lugworm, Arenicola cristata; and
(2) to develop a toxic sediment assay system involving the lugworm and
other species.
The remaining projects were directed toward developing methods to provide
more realistic evaluators, other than acute and chronic toxicity tests, for a
xenobiotic's fate and effect in estuarine and marine ecosystems. Specifically
these were:
(1) development of tests involving estuarine and marine crustaceans
and fishes designed to evaluate how exposure to xenobiotics can alter
predator-prey relationships
(2) development and testing of behavioral assays that would provide
reliable means to evaluate sublethal effects such as avoidance; and
(3) establishment of small scale microcosms that could be used to test
fate and effect.
Although the five broadly defined projects are divergent, commonalities
included either use of systems dominated by lugworms or evaluation of sublethal
effects in small scale microcosms.
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.
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BETHIC PHOTO-BIOASSAY SYSTEM
The first test to include an infaunal representative was Rubinstein's
(1979) benthic bioassay which uses time-lapse photography to measure effect of
toxicants on feeding behavior of lugworms (Fig. 1). That organism is ideal for
this type of test because it is widely distributed in littoral habitats and has
major ecological impace due to its ability to recycle sediment and transport
xenobiotics into the substrate. The photo-bioassay system was constructed
based on the lugworm's habit of creating feeding funnels on the surface of sed-
iment it occupies. Under normal circumstances, active worms, unperturbed by
xenobiotics in the water column or sorbed on the sediment, create enlarge, and
recreate obvious funnels. This pattern on the surface of the substrate, which
indicates activity of the worm, was monitored by Rubinstein with time-lapse
photographs taken at 12-hour intervals for 72 hours. Areas of feeding funnels in
exposed and control aquaria calculated and compared initial trials demonstrated
that there was no significant difference in reworking activity between replicates
under control conditions (Fig. 2). As worms were influenced by xenobiotics,
their activity, when compared to controls kept under the same environmental
conditions, decreased. For these experiments the xenobiotic tested was Kepone
at measured concentrations of 2.8, 4.5, 6.6, 7.4, and 29.5 ug/1.
Results indicated that A_. cristata was sensitive to Kepone at all concentrations
tested (Fig. 3). 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 effected habitats the ability of lugowrms to
reqork sediment would be markedly decreased.
CYCLING OF METHYL PARATHION BY LUGWORMS
A second evaluation of cycling of xenobiotics by lugworms (Garnas, et al. 1977)
was directed toward determing compartmentation and degradation dynamics of methyl
parathion in a small scale microcosm occupied only by the wor, and microorganisms
associated with the organic material on which it feeds. Ninety percent of radio-
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labelled methyl parathion disappeared from the water column in aquaria after
14 days. Movement into the sediment proved to be the major compartmentation
phenonomon, with over half of the total radioactivity residing in the
sediment after two weeks. The lugworm inhanced movement of radioactivity into the
sediment and caused dispersion throughout the sediment. While volatilization
losses were negligible, steadily decreasing mass balance of radioactivity in the
system suggested accumulation of unextractable residues in the sediment.
Analysis of extractable radioactivity in the sediment and water compartments
by thin layer chromotography and autoradiography demonstrated rapid degradation
of methyl parathion into a number of more polar products, including P-nitrophenol
and amino-methyl parathion. While A_. cristata was shown to metabolize methyl
parathion readily to P-nitrophenol, 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 xenobiotics on activities of the lugworm
was Rubinstein's (1978) evaluation of effect of sodium pentachlorophenate 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. Photo-bioassay methods
developed by Rubinstein (Fig. 1) were used in this study. Stock solutions of
Na-PCP were prepared from a commercial bactericide and introduced into experimental
aquaria at 45, 80, 156, and 276 ug/1. 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
concentrations there was significant decrease in activity (Fig. 4). Some mortality
occurred at the higher concentration.
UPTAKE AND DEPURATION OF CHRYSENE BY LUGWORMS
The final analysis of cycling of xenobiotics by lugworms evaluated uptake 1980a
and depuration of chrysene, another energy related compound (Rubinstein et al. 1980a>
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Worms were exposed to chrysene at measured concentrations of 0.07, 0.69, and
2.76 ug/1 in large wooden tanks in an open system that simulated ambient
conditions and the natural habitat (Fig. 5). After 14 days, exposed worms were
moved to uncontaminated systems and allowed to depurate for 14 days (considerable
mortality was encountered due to handling in cold weather.) From lowest to highest
exposure, lugworms accumulated 65, 516, and 682 ug/1 in 14 days (Fig. 6). There
was a continued increase in accumulation during that period so it is probable
that had exposure time been increased, higher levels of chrysene would have been
encountered before equilibrium was reached. Little depuration was observed.
This suggested that lugworms do not have the ability to degrade chrysene; thus
there is a good possibility that they have the potential to introduce chrysene
in various food chains utilized by man.
TOXIC SEDIMENT BIOASSAY SYSTEM
Many xenobiotics in marine environments have a high affinity for particulate
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
related activities were directed toward developing a flow-through toxicity test
that could be used to determine biological effects of contaminated sediments
on representative estuarine organisms and to evaluate resiliency of benthic
communities exposed to contaminated sediments. The test developed (Rubinstein
et al. 1980b) incorporated 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.
METHODS DEVELOPMENT WITH KEPONE-SORBED SEDIMENT
The approach was to simulate and then compare certain aspects of the marine
environment before and after deposition of spoil material. Small scale estuarine
microcosms were assembled in ID-gallon aqauria receiving flowing, unfiltered
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seawater. Three aquaria received different concentrations of test sediments,
while three others remained unperturbed and served as controls. Comparisons
were made after 28 days. Organisms included in the test are 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 bioaccumulation by lugworms; and (4)
resiliency of the benthic community in terms of numbers and variety of
macrofaunal organisms that settled onto test sediments as planktonic larve
within 28 days.
The exposure system employed is shown in Figure 7- Sediments exposed to
Kepone at 0.1, 1.0, and 10.0 ug/1 were used during evaluation of the method.
This was followed by tests with actual dredge spoil material from the James
River and Houston Ship Channel.
Effect of Kepone-sorbed sediment and mysid survival was time and dose
dependent (Fig. 8). Oyster shell growth was significantly inhibited (Fig. 9).
Lugworms had an increasing dose-dependent relationship in concentration of Kepone.
Whole-body residues were 0.043, 0.46, and 1.1 ug/1 19 macrofaunal species were
found (Fig. 10). In terms of test criteria, only polychaetes were effected
at the highest exposure.
TESTS WITH DREDGE SPOIL
James River sediment did not affect mysids significantly although there was
some effect on oysters (Fig. 11). Lugworm substrate reworking was reduced in
experimental aquaria. Oysters and lugworms concentrated Kepone (Fig. 12).
Little difference was seen in survival of recruited larvae perhaps because few
larvae entered the system during the winter when it was operational.
Houston Ship Channel sediment did not significantly affect mysid survival
or oyster shell deposition; nor did lugworm activity or macrofaunal composition
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vary significantly between control and experimental units.
TESTS WITH DRILLING MUDS
Since the toxic sediment assay system was proven to be effective, a long
term (100 day) toxicity test was conducted to determine effect of a specific
drilling mud (Rubinstein et al. 1980c). Drilling muds were obtained weekly
from an active exploratory platform and tested within one week of collection in
the system, previously described (Fig. 7). Three dilutions were tested: 10, 30,
and 100 ml/1 by volume. These concentrations represented those expected
at intervals of 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 only
10 days.
Mysids exposed in the system were not acutely affected (Fig. 12). Oyster
shell growth was significantly inhibited at concentrations of 30 and 100 ml/1
(Fig. 14 & 15), but there was no mortality. Lugworms were severly effected
by exposure to the mud (Fig. 16). Mortalities observed were 75% at 100 ml/1,
64% at 30 ml/I, and 33% at 10 ml/I. Twenty recruited species were present
after 100 days (Fig. 17). There was no significant difference between popu-
lations in the aquaria. Ba, Cr, and Pb were found to have accumulated
significantly in oyster tissue (Fig. 18).
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 in highly
variable; thus impact should be considered on a case by case basis.
PREDATOR-PREY TESTS
Sublethal concentrations of xenobiotics, expecially pesticides, may be
expected to affect various aspects of behavior. This was demonstrated by Farr
(1977) (partly funded by this grant) who demonstrated that methyl parathion
impairs the ability of grass shrimp, Palamonetes pugio, to escape predation by
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the gulf killifish, Fundulus grandis. Although £_. grandis ate no more exposed
than unexposed shrimp, the predators took less time to capture exposed prey. If
pesticides have different effects on species in a multiprey systems, predators
may be expected to consume a higher than normal proportion of affected species.
The result would be more rapid accumulation of a xenobiotic.
TWO PREY SYSTEM
Palaemonetes pugio and juvenile sheepshead minnow, Cyprinodon variegatus
were exposed to methyl parathion for 24 hours before introduction of Fundulus
grandis, the predator (Farr 1978). Two experiments were run for five days:
a preliminary experiment at 0.475 ug/1, and a definitive experiment which included
a carrier control and methyl parathion concentrations of 0.024, 0.119, and 0.475
ug/1.
In the first experiment when the prey were exposed to the pesticide, gulf
killifish consumed a greater proprotion of grass shrimp relative to sheepshead
minnows (Fig. 19). Predation was also relatively greater on P_. pugio than on
C_. variegatus as compared with controls (Fig. 20). The second experiment tested
effect of a range of methyl parathion concentrations and of acetone (carrier)
on prey consumption. In both control and acetone-control aquaria, the ratio of
shrimp to fish increased rapidly during the test and did not differ, indicating
strong predator preferences for sheepshead minnows and no acetone-related
effect (Fig. 21). As the concentration of pesticide was increased in test
aquaria, the ratio of grass shrimp to sheepshead minnows decreased with time
(Fig. 22). Increasing the concentration resulted in increased consumption of
grass shrimp relative to fish prey, an obvious example of how a pesticide can
alter relative proportions of prey in a predator's diet.
EXPOSED AND CONTROL PREY IN THE SAME SYSTEM
Test systems were modified from the work of Farr (1978) and focused on the
effect of xenobiotics on exposed and control prey in the same systems (cripe 1979)
Equal numbers of pinfish, Lagodon rhomboides, and toxicant exposed and control
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grass shrimp. Palaemonetes pugio, were placed in two replicate tanks containing
removable dividers. Approximately 20 minutes after the dividers were removed,
surviving shrimp were counted to determine differential predation between exposed
and control prey. Prey were pleopod clipped for identification. Clipping was
emonstrated to have no significant effect on predation.
Significantly fewer shrimp survived predation after exposure for 24 hours
to 1.2 ppb methyl parathion. Exposure to 1.3 ppb Trithion for 24 and 72
hours produced to significant difference in predation.
CRYPTIC SHADING AND PREDATION
The behavior between a bothid flounder, Paralichthys albigutta, and its
prey the pinfish, Lagodon rhomboides, can be exploited to evaluate how a
xenobiotic could alter predatory strategy of flounder or avoidance response
of pinfish. Before the relationship could be tested it was necessary to
determine what the prey's normal response is to flounder exhibiting various
degrees of cryptic coloration.
For these experiemtns, models of flounder prey were used (Ashton 1980).
These were black and white photographs laminated between plastic and attached
to a plastic outline of a flounder. A circular tank was used as the arena
(Fig. 23). Ten prey were released from a central holding chamber and photographs
were taken at 0.5, 1.0, and 1.5 minutes to record response to the flounder
model. Ten trials were completed for control, dark model, and light model
treatments. Position of the school of prey relative to the predator model was
calculated. The group response for ten trials at each time interval was com-
bined for each treatment group and random versus non-random distribution was
tested.
Control data indicated that pinfish were randomly distributed in the absence
of a model (Fig. 24). In the case of the light model that represented a cyptically
shaded flounder there was no significant avoidance (Fig. 24). Pinfish swam
directly over the model. In the case of the dark model, the pinfish preferred
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the opposite side of the tank from the model (Fig. 24). Pinfish did not swim
around the tank as they did in control and light model experiments.
This system is now ready to be tested to determine how sublethal exposure
to xenobiotics can modify the antipredator response of the pinfish.
EVALUATION OF SUBLETHAL EFFECTS IN SPECIAL TEST SYSTEMS
AVOIDANCE OF POLLUTION GRADIENTS
It has often been observed that fish and invertebrates avoid pollution
gradients. Most apparatus designed to detect avoidance of pollutants by
aquatic organisms require visual observations of the test organisms in steep
pollution gradients. The aquatic Gradient Avoidance Response System (AGARS)
was developed to eliminate these limitations (Cripe, 1979a). This system
(Fig. 25) allows animals to choose between one uncontaminated zone and three
increasingly toxic zones in a gradient trough that is monitored for extended
periods by infrared light sources, sensor, and a microprocessor. Data are
accumulated hourly and processed by a paper tape reader/calculator/plotter system
that records the time test animals remain in each zone and compares behavior
before and during test exposures. Initial tests in AGARS indicated that pinfish,
Lagodon rhomboides will avoid chlorine-produced oxidants at concentrations of
0.02-0.04mg/l (Fig. 26). The system is a prototype that can be enlarged by
using more powerful lights and greater microprocessor memory capacity. In
addition to several species of fish, baseline data have also been obtained
with blue crabs, Callinectes sapidus, and penaeid shrimp. The system could
also be used to test thermal or salinity preferences.
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 criterior 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
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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 (Gripe 1979b) from a modified AGARS system.
Two troughs were employed with sensors only in the upper level. Each trough
was partially filled with sand and compartmentalized into four areas by plastic
screen (Fig. 27). Shrimp were placed in each compartment on a 12L-12D cycle and
monitored for six days. One trough was exposed to 2 ppb methyl parathion on
days 3 and 4 of the test.
The results indicated variability in absolute activity level of a particular
shrimp on different days as well as between shrimp. An activity index was
calculated by dividing the mean of the hourly light beam interruptions for each
dark period into the mean hourly counts for the succeeding light period. The
index was lower for controls that exposed shrimp. On a daily basis there was
significant difference in activity between days when toxicant was added and days
when it was not (Fig. 28).
In conjunction with the development of trough systems a device to detect
potentially dangerous electrical currents in saltwater holding tanks was developed
(Gripe & Stokes 1978).
SMALL SCALE MICROCOSMS
The usefulness of microcosms in evaluating fate and effect of various
xenobiotics is well documented. Several of tests developed under grant auspices
were actually completed in laboratory microcosms. Bourquin et al. (1979)
redescribed these as well as three other types. A part of the grant effort was
directed toward evaluating various aspects of the biology of selected species
that could be used in microcosms.
POLYCHAETES
Pond culture of lugworms (D'Asaro, in manuscript) was directly linked to
development of techniques to use lugworms in small scale systems. The results
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demonstrated rapid growth rates of lugworms over a 90-day period at densities
of 60 or more worms per square meter when ground seagrass (Thalassia) was used
as a food (Fig. 29). These data were used by Rubinstein (1979) to develop various
toxicity tests in which lugworms were used as a primary component in the system.
White (1978) used microcosms to evaluate the impact of three predators
(Neanthes succinea, Glycera americana, and Callinectes sapidus. The blue crab,
C_. sapidus was the most effective predator especially on juvenile lugworms that
do not burrow deeply.
Lasfargues (1980) examined the role of bacteria as a food source for lugworms
in microcosms. Lugworms were allowed to consume composted seagrass; then
comparisons were made between biomass of bacteria and yeast in the grass and
feces. There was a significant reduction in the population of bacteria but not
.in the population of yeast in the feces. There was also evidence of selective
digestion of bacteria. A gram negative bacterium, isolated from composted
seagrass, was cultured and used to enrich compost fed to worms. The enrichment
caused a significant increase in growth.
Redig (1980) examined culture methods that could be used to rear Polydora
ligni, a species that can be easily included in microcosms.
ISOPODS
Ligia exotica is a semi terrestrial isopod restricted to the immediate
supralittoral zone, an area often heavily impacted by oil spills. Orientation
and social behavior in L_. exotica was evaluated by Farr (1978). The tendency
of L_. exotica to aggregate and orient to enviornmental stimuli was examined in
circular outdoor tanks or small aquaria.
In the circular tanks, experimental animals released in the center of the
arena were allowed to aggregate for 24 hours under clay saucers placed around
the periphery of the arena. The resulting aggregations were not random indicating
that \^. exotica actively seeks conspecifics. In tests in aquaria, L_. exotica
significantly selected shelters containing conspecifics or a shelter conditioned
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by previous occupancy by a conspecific. Other experiments demonstrated that
distribution of L^. exotica appears to be influenced initially by raised landmarks
on the shoreline. Observations on social behavior were also reported.
Levy (1980) described the breeding habits and brood pouch development of
Erichsonella altenuata, an isopod ideal for microcosm studies because it is
easy to culture in closed systems, and it is a representative of marine grassflat
communities.
MYSIDS
Plaia (1980) described the complete embryogenesis and organogenesis of
Mysidopsis bahia, a species presently used as a standard organism in toxicity
tests.
ANOSTRACANS
Logue (1980) examined the effects of temperature and diet on growth of a
freshwater anostracan, Streptocephalus seali. This species wer found to be easy
to culture. It matures rapidly in two weeks thus it may be useful for life-
cycle tests.
-------
Fig. 1. Photo-Bioassay System (A - 24-hour timer; B - 35 mm camera with automatic advance; D, E - aquaria
with 25 cm of sand and 75 1 of seawater).
-------
40'
IOC
uJ
-oo-
3OC-
Trial
Trial 4
Trial 2
Trial 3
Trial 5
Trial 6
12 24 36 48 60 72 0 12 24
36 48 60 72
TIME (hours)
Fig. 2. Comparison of the rates of sediment turned under by group of similar size. A different group
of lugworms was used for the six replicate tests.
-------
cvJ
Q
LU
GQ
~r"_
ID
Q
800f
600h
Non- Detectable
144
800
600
400
200
0
2.8 jjg /I Kepone
•
P-- —-"" c
96 120 144
LJ
QL
LJ
O
£
(T
ID
CO
soor
6.6 jjg/l Kepone
800
600
400
200
72 96 120 144
0
29.5 ;jg/l Kepone
0 24 48 72 96 120 144
Fig. 3.
TIME (hours)
Comparison of the rates of sediment turned under by lugworms,
exposed to Kepone. Each group consisted of six lugworms.
C-control; E-experimental group
-------
IIMF. (hour',)
Fig. 4. Comparison of the rates of sediment turned under by the lugworm,
Arenicola cristata, C-controls; E-experiment group exposed to sodium
pentachlorophenate. Each group consisted of six lugworms.
-------
CHRYSENE
HEAD30X
SYRINGE PUMP
EPOXY COATED
WOOD TANK
EFFLUENT
POND
Fig. 5. One tank in the exposure system for chrysene.
-------
1000^
800
600
LU
LJ
cr:
o
400
200
0
5
6 8
TIME (doys)
10
12
2.76
0.035 pa/I
.CONTROL
14
Fig. 6. Accumulation of chrysene by lugworms. Each data point is based on average accumulation of
five worms.
-------
EXPOSURE SYSTEM
. F
exposure tanks on a water table.
oysters and lugworms in
-------
OO
>>
CO
50
40
h-
cc
O
^>
D
CO
f-
30
20
UJ
0 10
en u
LU
£X
SEDI MENT KEPONE
CONCENTRATION
(ppm)
10.0
0 ^
0
OJ
a>
to
-a
ai
.a
S-
o
to
I
(U
c:
o
Q.
O)
sz
fO
o
S-
4->
C
o
o
-a
cu
CO
o
Q.
X
OJ
to
cu
u
-------
LU
> 100
\-
-J n: 80
LU \—
CC ?>
h- 6 GO
^_
LU
o
cr
LU
o_
LU
X
CO
40
20
2.0
0
SEDIMENT KEPONE CONCENTRATION (ppm)
10.0
— o
14
21
0
28
Q
d
p o
— o o
0 — 0
SEDIMENT KEPONE CONCENTRATION,
14 21
DAYS
28
Fig. 9. Effect of Kepone-sorbed sediments on oyster shell deposition (percent
growth is relative to controls).
-------
ANNELIDA MQLLUSCA ARTHROPODA CHORDATA
ouu
250
_i
§ 200
•=i
~2L
<
o 15°
cc
UJ
| 100
1
50
n
-
—
-
4
-j
6
// ' •
//v.
// ' ' '
j%.
'///.
%
fv
^
v> •
•'/ '
P
:::::::
x£.
•'-•X
-•-•.'.•
x:;::
I-.':X;
«:•>
H
K"-X:
?-:•:•:
,* • • •
:>'x:
T
NUMBERS OVER BARS REPRESENT
THE NUMBER OF SPECIES
1
1 i 1 r^i
7 fi -rr-C-II--'^,'.'--1
^_ r-"«*«*» ' ! ' J r • i ^ 1 ^ /x^«"»" ""• "* • ^
g 0.1 10.0 cc 0.1 10.0 cc 0.1 10.0 cc 0.1 10.0
I 1.0 § 1.0 § 1.0 I 1.0
cj o o u
NOMINAL KEPONE SEDIMENT CONCENTRATION, ppm
Fig. 10. Number of individuals and species collected from exposed and
control aquaria after 23 days.
-------
SHELL GROWTH, PERCENT RELATIVE
mm GROWTH
N ^ M -^ C7) CO O
ooo oooooo
CONTROL
EXPOSED \''S*\\n
- -r 9.*"•'*"> t^-:*
CONTROL
O
ro
CONTROL
EXPOSED^
NJ
CO
CONTROL
EXPOSED]
CO
Fig. 11. Effect of James River sediment on oyster shell deposition.
-------
en
3.
Q
co
I_U
cr
1.25
CO
w 1.00
0.75
Q
O 0.50
CO
UJ
_J
g 0.25
0
OYSTERS
0.51
N.D.
CON- EX-
TROL POSED
LUG won MS
0.75
Aig/g
N.D.
CON- EX-
TROL POSED
N.D., NON-DETECTABLE «0.02/K)/g).
Fig. 12. Kepone residues in oysters and lugworms following 28 days of
exposure to James River sediments.
-------
£
cr
o
a
CO
40
30
20
0
0
to
1_
o
CD
cr>
DRILL MUD
CONCENTRATION
(m I /I)
4 6
TIME (days)
8
100
30
10
10
12
Fig. 13. Percent mortality of mysids exposed to control and three concentrations of drilling muds for
10 days.
-------
£
E
h-
O
cr
LJ
cr
LJ
LJ
0
DRILL MUD CONCENTRATION
(ml/1)
CONTROL
I s
0
468
TIME (weeks)
10 12
Fig. 14. Weekly average oyster growth (N=15).
-------
X
h-
O
oc
CD
UJ
LxJ
cr
120
I I 0
00
90
80
70
60h
50
0
DRILL MUD CONCENTRATION
(ml /I)
10
468
TIME (weeks)
30
100
10
12
Fig. 15- Oyster shell deposition relative to controls.
-------
H
_J
-------
CO
u_
O
cr
w*-r \J
480
420
360
300
240
1 80
1 20
60
n
-
-
NUMBERS OVER
MOLLUSC A
7
BARS
REPRESENT THE NUMBER
OF SPECIES
-
-
6
CRUSTACEA
POLYCHAETES
8
_7_
V.'.v
8
^
i
^
6
^
5
3
'•;':<•'.
?
y/
//
2
$o
YV
. • "• ' '
6
^
y/
i.
XX
y/
^
y'/
//
ft
y/
y/
//,
//
3
$$
S^
C 10 30 !00
C 10 30 100
DRILL MUD CONCENTRATION, m!/l
Fig. 17. Number of individuals and species collected from exposed and control aquaria after 700 days.
-------
OYSTER TISSUE RESIDUES (jjg/g DRY WT)
o
o
o
2:
o
m
;z
H
O
O -
o
o
o
o
o
ro
—r~
OJ
\ \\
O I\> -k (7) 00 O O
ro
O
O
CD
o
I i r
CD
Fig. 18. Ba, Cr, and Pb concentrations in oysters exposed to control and three concentrations drilling
muds for 100 days.
-------
3.0
o
Q
O
D_
O
^
CO
UJ
I—
Ld
*•" _
O
UJ
.-r
2.0
1.0
C
CONTROL
0
OF PREDATION
Fig. 19.
The ratio of
of predation
lower ratio
Palaemonetes pugio to Cyprinodon van'egatus surviving after each of five days
by Fundul us grandi s i n . control and 0.475 mg/1 methyl oarathion exposed aquaria.
in the exposed aquaria indicated greater predation on P_. pugio.
The
-------
).0
UJ
o
t 2.C
6
o
LJ
UJ
ir
UJ
Q.
i.c
0.475 jjg /I
CONTROL
0
234
DAYS OF PREDATICM
Fig. 20. Preference coefficients for F. grandis predation on P. pugio and juvenile C varieaatus durina
five test days in control and 0.475 mg/1 methyl parathic^posed aquar a Hiaher^coefficients
indicate greater predation on P. ^u^o. See Farr (1978) for method of calculation COefflclents
-------
6.0
DAYS OF PREDATION
Fig. 21. The ratio of P_. pugio
F. qrandis in~"control
to C_. variegatus surviving after each of five days of predation by
aquaria and in three concentrations of methyl parathion.
-------
6.0
-Z. 5-°
b
b
| 4.0
3.0
O
^
co
£
u
•- »
0
< 2.0
1.0
0
0
CONTROL
0.024 ijg/l
234
DA.YS CF PREDATION
Fig. 22. The ratio of £_. pugio to C_. variegatus surviving after each of five days of predation by
£. grandis in concentrations of methyl parathion.
-------
1
Fig. 23. Test apparatus used to observe pinfish reactions to flounder models: A-fluorescent lights;
B-autoniatic advance camera; C-release chamber; D-circular test tank.
-------
elapsed
time
in
minutos
control
light
model
dark
model
0.5
= O.I445
P=0.8677
F=3 9156
P =0.0652
F=I5.0950
P = 0.0020
.0
F=0.2753
P=0.7663
P =0.0652
= 35.7808
P=O.OOOI
1.5
F = 0.207I
P = 0.8I72
F=IJ077
P = 0.2282
F=3b.yb69
P=O.OOOI
Fig. 24. Diagrams of pinfish positions for combined trials 1-10 for each time
interval of control, light flounder model, and dark flounder model. Each
point represents the mean position of 10 pinfish for each trial, and the
X represents the mean of all 10 points. The F values shown below the
diagram were calculated by Hotelling's one-sample test. P values correspond
to the probability at the given F value.
-------
HIGH
MEDIUM
LOW
CONTROL
OVERFLOV/
PHOTOTRANSISTOR
Fig. 25. Diagram of AGARS trough. Trough is 125 x 17 x 15 cm high and constructed of 6 mm clear plexiglas.
"M" denotes mixing boxes where 1.5 1 min clean water and test compounds or carriers are combined.
The flow is divided evenly between pairs of small chambers on each side of the trough. Water exits
from the chambers through a row of 7 mm holes, inlater flow n.aintains a gradient of a control zone
and three increasing toxicant'concentrations (areas 1-4). Organi
the upper and lower half of eacn area by pairs of infrared light
sm position can be monitored in both
emitting diodes and onoto-transistors
-------
o
ro
UJ
cr
UJ
8000
6000
4000
< 2000
0
;x:x;:.v
;:::Jv"
.:'••':'£:'
x:xXv
CIO
PRESENT
(mg/l)
.21
SH
1
•HB~ i^
07
.02
00
•;:SS:
ix^
Cl
PF
0
}E
r
[
[
L.
m
m
•x^xxl
:SENT
ng/D
1
0
.02
04
09
1 234 56
DAY
Area 4
Area 3
Area 2
Area I
iv :.:. ;
-
I
j
i
i
789
Fig. 26.
Results of a 9-day AGARS test with a group of 4 pinfish. Mean of 24 hourly totals of time spent
in each of 4 areas of trough versus elapsed time of test. Chlorine produced oxidants were prese
on days 4 and 7 only.
present
-------
Overflow
drain
Sand
Phototransistor
Fig. 27. Diagram of one of two replicate troughs used to study pink shrimp behavior. The trough was
modified from that shown in Figure 26 in that it is partitioned with barriers of plexiglas and
plastic screen and contains sand. The presence of each shrimp above the sand is monitored by
tow pairs of photo-transistors and infrared light emitting diodes.
-------
6000
o
"
n)
rO
UJ
CD
5000
4000
3000
2000
1000
0
III
EXPOSURE
24
48
72
HOUR
96
120 144
Fig. 28.
An example of an activity graph indicating time in light beams (0.33 sec/h) during each
hour of the 6-day test. On days 3 and 4 shrimp were exposed to 2.0 ppb methyl parathion
and carrier. Light and dark bars indicate photo-period.
-------
IO.O h
20
60
Days
140
Fig. 29. Mean increase in length by cultured Arenicola cristata during spring and
early summer; 95% confidence belts are indicated.
-------
REFERENCES
Ashton, Charles. 1980. The response of pinfish, Lagodon rhomboides, to cryptic
and non-cryptic flounder, Paralichthyes albigutta, an examination of the
value of cryptic shading to a predator. Masters thesis, University of
West Florida, June 1980.
Bourquin, A.W., R.L. Barnas, P.M. Pritchard, F.G. Wilkes, C.R. Cripe, and
N.I. Rubinstein. 1977. Interdependent microcosms for the assessment of
pollutants in the marine environment. Intern. J. Environ. Studies., 13:131-140.
Cripe, C.R. 1979a. An automated devise (AGARS) for studying avoidance of
pollutant gradients by aquatic organisms. J. Fish. Res. Bd. Can., 35(1):11-16.
Cripe, C.R. 1979b. An automated apparatus to record toxicant-induced changes
in cyclic burrowing patterns of pink shrimp (Penaeus duorarum). Research
Review, September 1979, pp 26-27.
Cripe, C.R. and B.E. Stokes. 1978. A device to detect potentially dangerous
electrical currents in saltwater holding tanks. Prog. Fish. Cult., 40(2):74-75.
D'Asaro, C.N. 1980. Pond culture of the lugworm, Arenicola cristata as a source
of bait (manuscript).
Farr, J.A. 1977. Impairment of antipredator behavior in Palaemonetes pugio
by exposure to sublethal doses of parathion. Trans. Am. Fish. Soc. , 106(3):287-
290.
Farr, J.A. 1978. The effect of methyl parathion on predator preference for two
estuarine prey species. Trans. Amer. Fish. Soc., 107(1):87-91.
Farr, J.A. 1978. Orientation and social behavior in the supralittoral isopod
Ligia exotica Bull. Mar. Sci., 28(4):659-666.
Garnas, R.L., C.N. D'Asaro, N.I. Rubinstein, and R.A. Dime. 1977. The fate of
methyl parathion in a marine benthic microcosm. Paper #44 in Pesticide
Chemistry Division, 173 rd ACS meeting, New Orleans, Louisana, March 20-25, 1977
Lasfargues, J.E. 1980. Evaluation of bacteria as a food resource for Arenicola
cristata. Masters thesis, University of West Florida, September 1980.
Levy, I.C. 1980. Breeding habits and ontogeny of Erichsonella attenuata reared
in the laboratory. Masters thesis, University of West Florida, September 1980.
Logue, C.L. 1980. Effect of temperature and diet on growth, longevity, and egg
production of Streptocephalus seali. Masters thesis, University of West
Florida, September 1980.
Plaia, W.C. 1980. Embryogenesis and organogenesis of Mysidopsis bahia. Masters
thesis, University of West Florida, September 1980.
-------
Redig, M.X. 1980. Optimal temperature-haline conditions for development
of Polydora ligni. Masters thesis, Florida State University.
Rubinstein, N.I. 1978. Effect of sodium pentachlorophenate on the feeding activity
of the lugworm, Arenicola cristata. In: K.R. Rao, ed., Chemistry, Pharmacology
and Environmental Toxicology of Pentachlorophenol, Plenum Press, New York,
pp.175-179.
Rubinstein, N.I. 1979. A benthic bioassay using time-lapse photography to
measure effects of toxicants on feeding behavior of lugworms. In: W.B.
Vernberg et al. editor, Marine Pollution: Functional Responses.
Academic Press, New York. pp.341-355.
Rubinstein, N.I., R.A. Dime, and C.E. Ashton. 1980a. Chrysene uptake and
depuration by the lugworm (Arenicola cristata). In: N. Richards, editor,
Proceedings Symposium on Carcinogenic Polynuclear Aromatic Hydrocarbons in
the Marine Environment, Academic Press, New York.
Rubinstein, N.I., F.G. Wilkes, C.N. D'Asaro, and C. Sommers. 1980b. Effects of
contaminated sediments on representative estuarine organisms and developing
benthic communities. In: Robert Baker, editor, Contaminants and Sediments,
Vol. I., Ann Arbor Sciences pp. 445-461.
Rubinstein, N.I., R.A. Rigby, and C.N. D'Asaro. 1980c. Effects of whole used
drilling muds on representative estuarine organisms. In: Proceedings from
a Symposium or Research on Environmental Fate and Effects of Drilling Fluids
and Cutting.
White, C.B. 1978. Predation on cultured lugworms (Arenicola cristata). Masters
thesis, University of West Florida.
------- |