United State*
Environmental Protection
Agency
Environmental Research
Laboratory
Gulf Breeze a 32561
EPA 600'3 80 019
Jdnuary 1980
and Development
Effects of Petroleum
Compounds on
Estuarine Fishes
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal spe-
cies, and materials. Problems are assessed for their long- and short-term influ-
ences. Investigations include formation, transport, and pathway studies to deter-
mine the fate of pollutants and their effects. This work provides the technical basis
for setting standards to minimize undesirable changes in living organisms in the
aquatic, terrestrial, and atmospheric environments.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/3-80-019
January 1980
EFFECTS OF PETROLEUM COMPOUNDS ON ESTUARINE FISHES
by
B. J. Martin
Department of Biology
The University of Southern Mississippi
Hattiesburg, Mississippi 39401
EPA Grant R-804527
Project Officer
John A. Couch
Gulf Breeze Environmental Research Laboratory
Gulf Breeze, Florida 32561
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Gulf Breeze, Florida 32561
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DISCLAIMER
This report has been reviewed by the Gulf Breeze Environmental Research
Laboratory, U.S. Environmental Protection Agency, and approved for publica-
tion. Approval does not signify that the contents necessarily reflect the
views and policies of the U.S. Environmental Protection Agency, nor does
mention of trade names of commercial products constitute endorsement or
recommendation of use.
n
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FOREWORD
The protection of our estuarine and coastal areas from damage caused
by toxic organic pollutants requires that regulations restricting the
introduction of these compounds into the environment be formulated on a
sound scientific basis. Accurate information describing dose-response
relationships for organisms and ecosystems under varying conditions is
required. The EPA Environmental Research Laboratory, Gulf Breeze,
contributes to this information through research programs aimed at
determining:
*the effects of toxic organic pollutants on individual species
and communities of organisms;
"the effects of toxic organics on ecosystem processes and
components;
'the significance of chemical carcinogens in the estuarine
and marine environments.
Considerable interest has focused recently on the fate and possible
effects of carcinogens and mutagens in the aquatic environment which
usually is the ultimate receptacle for pollutants. This report presents
the design and results of use of a closed system carcinogen assay
apparatus long-needed by investigators remote from flowing-water laboratory
facilities. Further, the fate and some possibly long-term effects of
polycyclic aromatic hydrocarbons in the marine estuarine environment and
biota are described. This data may serve to alert us to the role of
certain carcinogens in the environment.
Henry F. Enos
Director
Environmental Research Laboratory
Gulf Breeze, Florida
iii
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ABSTRACT
The overall goal of this project was to study the effects of the carcin-
ogenic polycyclic aromatic hydrocarbons (PAH), benzo(a)pyrene (BaP) and
methylcholanthrene (MCA), on sheepshead minnows (Cyprinodon variegatus) and
channel catfish (Ictalurus punctatus).
A closed-circulating system was designed to maintain up to 100 sheeps-
head minnows in artificial seawater for long-term exposures. Fish were main-
tained in this system for up to 31 weeks with weekly contaminations of PAH.
Due to their chemical properties, significant levels of BaP and MCA remained
in the water column for only ca. 24 hours each week, and no tumors were
observed in the exposed fish during the period of the study.
The incidence and types of lesions in control and exposed fish were
basically similar except in catfish that were fed PAH contaminated food.
High levels of contamination (1 mg/gm food) appeared to be toxic, and lower
levels of contamination (0.1 mg/gm food) produced sufficient stress to make
the catfish susceptible to fatal parasite infestations.
Both species accumulated radioactively labelled PAH at concentrations
much higher than their nominal concentrations in the water. Although the
level of accumulation was extremely variable, in general the accumulation
factors were: ca. SOX in gill and liver, ca. 15X in 61 tract, and ca. 2X
in skeletal muscle.
In summary, these results demonstrate that sheepshead minnows function
well as experimental organisms in artificial seawater in a closed system
maintained at a noncoastal facility. Thus, they provide an excellent model
system for the study of long-term effects of chronic exposure to polluting
agents. The sheepshead minnow, widespread in Gulf estuaries, therefore
provides an excellent indicator organism that can be used to make extra-
polations from the laboratory to the feral population.
Future studies should concentrate on tumor induction tests with known
or suspected carcinogens in the system established in this study.
This report was submitted in fulfillment of Grant No. R804527 by the
University of Southern Mississippi, Hattiesburg, MS, under the sponsorship
of the U.S. Environmental Protection Agency. The report covers the period
10 June 1976 to 31 October 1978.
IV
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CONTENTS
Foreword i i 1
Abstract ; i v
Figures v1
Tables vi
Abbreviations and Symbols vii
Acknowledgment vi i i
1. Introduction 1
2. Conclusions and Recommendations 3
3. Materials and Methods 5
Wet Laboratory 5
Specimen Collection 5
Exposure Systems 5
PAH Contamination of Water 6
Chemical Analysis 6
PAH Feeding Experiments 9
, Bioaccumulation Studies 9
Tissue Culture 9
Histological Preparations 10
4. Results and Discussion 11
Closed-circulating Exposure Systems 11
Chemical Analysis 12
Long-term Exposures 15
Feeding Experiments 17
Bioaccumulation Studies 24
Tissue Culture Studies 25
Histological Studies 26
Spontaneous Tumors 27
References 29
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FIGURES
Number Page
1 Schematic diagram of closed-circulating system 7
2 Detailed structure of closed-circulating system subtank 8
3 Profile of BaP concentration in closed-circulating
system over a six-day period 14
4 Vertebral disorientations in BaP exposed catfish 19
5 Myxosporidian cyst in the sheepshead minnow 28
TABLES
Number Page
1 Long-term exposures of sheepshead minnows 12
2 BaP concentration in closed-circulating system 13
3 BaP concentration in aquaria of closed-circulating system 16
4 MCA concentration in aquaria of closed-circulating system 16
5 Feeding experiments 18
6 Bioaccumulation of 3H-benzo(a)pyrene in sheepshead minnows 20
7 Bioaccumulation of 3H-methylcholanthrene in sheepshead
minnows 21
8 Bioaccumulation of 3H-benzo(a)pyrene in channel catfish 22
9 Bioaccumulation of 3H-methylcholanthrene in channel
catfish 23
VI
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ABBREVIATIONS AND SYMBOLS
BaP — benzo(a)pyrene
BaP-H — 1 milligram benzo(a)pyrene per gram food
BaP-L — 0.1 milligram benzo(a)pyrene per gram food
gm ~ gram or grams
1 — liter or liters
mg — milligram or milligrams
MCA — 20-methylcholanthrene
MCA-H — 1 milligram methylcholanthrene per gram food
MCA-L — 0.1 milligram methylcholanthrene per gram food
ng — nanogram or nanograms
PAH — polycyclic aromatic hydrocarbons
yg — microgram or micrograms
ppb — parts per billion
vii
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ACKNOWLEDGMENT
Appreciation is expressed to Lee Courtney for assistance in the chemical
analyses, Richard Pierce and Peter Schoor for advice concerning procedures
for chemical analysis, and John Couch for many timely and helpful suggestions.
viii
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SECTION 1
INTRODUCTION
Reliable estimates that 75% to 90% of the incidence of human cancer may
be environmentally related increased public awareness of the importance of
environmental pollutants as tumorigenie agents (1,2). This has resulted in
demand for more rigorous environmental safety assessment (3,4). Our current
methods for detecting chemical carcinogens and capability of accurately pre-
dicting the human health hazard resulting from a particular chemical agent do
not provide the degree of safety assessment needed (3,5). The Ames quick
detection technique (6) is an example of an important advance in methods of
assessment; however, long-term bioassays continue to be our most reliable
method of testing a chemical for carcinogen!city (4).
Murine assay systems, our mainstay for testing carcinogens, are not
particularly amenable to assessment of the aquatic environment. Since the
aquatic environment becomes a "sink" for many potentially dangerous pollut-
ants, it seems imperative that valid test systems be developed.
Teleost fishes, ubiquitous in the aquatic environment, are obvious
candidates for a monitoring role. Changes in incidence of tumors in feral
fish populations could provide a built-in "early warning11 of the presence of
a carcinogen. Evidence already exists that such correlations between level
of pollution and incidence of proliferative diseases may be possible (7-10).
Fish make excellent experimental animals for these tests because they are
immersed in an aquatic environment that can be easily manipulated experi-
mentally. Also, they grow throughout a relatively short life span, and yet
many species remain small in size. This small size and the apparently short
latency period for tumor induction (11) are important factors in reducing
test costs.
As the levels of pollution have increased dramatically in many aquatic
ecosystems throughout the world, there has been an apparent increase in the
detection of proliferative diseases in aquatic organisms (12). Carcinogenic
hydrocarbons are found at high levels in some aquatic systems; however, cur-
rently their levels remain relatively low in many of the estuaries of the Gulf
Region. This condition is likely to change in the near future if predicted
rapid increases in population and industrialization occur. The quality of
this environment is likely to be further threatened due to existing plans to
dramatically increase petroleum imports through these waters.
The major goal of this project was to conduct a comprehensive study of
the effects of two carcinogenic hydrocarbons, benzo(a)pyrene (BaP) and methyl-
cholanthrene (MCA), on the sheepshead minnow (Cyprinodon variegatus) and the
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channel catfish (Ictalurus punctatus), two teleost species indigenous to the
Gulf Region. The project provides important background data supporting the
feasibility of using teleost fishes in the assessment of suspected carcin-
ogens. Furthermore, it represents a contribution to the development of new
methodologies critically needed (3) to facilitate the reliable, rapid, and
inexpensive assessment of the ever-growing avalanche of new and possibly
carcinogenic compounds that enter the aquatic environment (13).
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SECTION 2
CONCLUSIONS AND RECOMMENDATIONS
The closed-circulating system designed for the maintenance of sheepshead
minnows in artificial seawater has proven to be adequate for keeping as many
as 100 fish in good health indefinitely. The system can be easily maintained
and contaminated with minimum risk to laboratory workers. Seasonal mortality
problems experienced during the adaptation period could likely be solved by
raising fish from eggs under laboratory conditions. Large numbers of sheeps-
head minnows can be produced in this manner (14), and the possibility of
bringing in parasites from the feral environment would also be eliminated.
In fact, the increasing use of the sheepshead minnow as a laboratory animal
(15-17) suggests that it might be advisable to develop a laboratory strain as
is the case for rats and mice.
The chemical properties of BaP and MCA made it difficult to maintain a
high level of exposure or to accurately evaluate the concentration of PAH
within the water column. Less than 25% of the concentration of BaP placed in
the system remained in the water column after 24 hr, and less than 5% can be
found shortly thereafter. Although MCA can be found at higher levels
initially, it essentially disappears from the water column in an equally
short time. Consequently, weekly contamination provided only one day in
seven of significant exposure.
These results suggest that feral fish are unlikely to be exposed to high
levels of PAH in the water column except in the immediate area of a constant
effluent high in PAH content. It appears that fish are more likely to be
contaminated with PAH as a result of their feeding habits. The fact that
fish accumulated PAH and/or their metabolities provides a mechanism by which
PAH could get into the aquatic food chain and suggests that fish from areas
of high PAH contamination should be monitored for PAH content prior to human
consumption.
The task of establishing a cause-effect relationship between PAH and the
types and incidence of diseases in these fish is a difficult one, and consid-
erable additional work must be accomplished before valid conclusions can be
reached. The project provides evidence that the ingestion of PAH can produce
enough stress to render fish susceptible to parasites and disease. Thus, in
this indirect way, PAH could have serious effects on production and the bio-
logical success of a species. The data provide some evidence that PAH
exposure may contribute to the conditions of lordosis and/or scoliosis and
possibly nervous disorders in channel catfish. A detailed study of electro-
lyte physiology and the morphology of the tissues important in electrolyte
control (i.e., Ultimobranchial gland and Stannius gland) of both exposed and
control fish could possibly provide some clarification concerning this matter.
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The apparent wide variability in the accumulation of label by fish
exposed to radioactively labelled PAH needs further study. It would be use-
ful to study fish that have been fed labelled PAH. Biochemical studies to
disclose the amount of parent compound and the amounts and chemical nature
of the PAH metabolites accumulated will be necessary to determine the real
significance of these studies. A study of the photochemical reactions occur-
ring in the system would also aid our understanding of the results.
Development of fish tissue culture cell lines as carcinogen assay
systems may seem somewhat aside from the main thrust of the project; however,
they provide an additional system for the study of rveoplastic mechanisms at
the cellular level. The tissue culture exposures are likely to be partic-
ularly applicable to screening for mutagenic effects. The development of
nonfibroblastic cell lines from sheepshead minnow embryos might also provide
a valuable tool for this same purpose. Since an organism should be most
vulnerable to carcinogenic and mutagenic agents at times of rapid cell
division, experiments exposing sheepshead minnows to such agents during
embryonic development should be conducted.
Finally, the results of the project demonstrate that the sheepshead
minnow can be maintained in good health In a closed system for the extended
periods necessary to study the long-term effects of chronic exposure to a
polluting agent. Thus, the sheepshead minnow in this system provides a
suitable model for chronic testing and should be extensively employed for
this purpose. Since the sheepshead minnow is common in the estuaries of the
Gulf and Atlantic, data obtained concerning this fish in the laboratory
should be useful in making reliable extrapolations concerning conditions in
the estuaries by observing samples from the feral population. Therefore, it
is recommended that efforts be continued to develop extensive baseline data
concerning the incidence of neoplastic and other lesions at selected sites
throughout the Gulf Region. These baseline data plus data from laboratory
exposures under controlled conditions should provide a valuable "early
warning mechanism," making possible the discovery and removal of a chemical
insult before it produces serious and long-term effects.
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SECTION 3
MATERIALS AND METHODS
Wet Laboratory
In order to provide safe and adequate facilities for working with car-
cinogens, the University of Southern Mississippi expended approximately
$17,000 to modify an existing brick facility located four miles from the
main campus. This construction provided a quality wet laboratory that is
functional and safe enough for long-term involvement in aquatic research.
The laboratory allows control of the ambient environment and provides for
the safe handling and disposal of contaminated water.
Specimen Collection
During the course of the study, approximately 6500 sheepshead minnows
(Cyprinodon variegatus) were collected. Approximately 5000 were seined from
a marshy tidal entrance at Range Point on Santa Rosa Island near the U.S. EPA
Environmental Research Laboratory, Sabine Island, Gulf Breeze, Florida.
Approximately 1500 sheepshead were collected from a similar tidal stream on
the north side and at the east end of Horn Island on the Mississippi Gulf
Coast near Pascagoula, Mississippi. Fish were collected during each month
of the year except January and February. All specimens were examined for
gross lesions and treated for 30 minutes with 1:4000 formalin to remove
parasites. The fish were acclimated to 5 to 15 °/oo artificial seawater
(Rila Mix, Rila Products, Teaneck, NJ) at least 5 days prior to being placed
in an experimental system.
Approximately 1400 Channel catfish (Ictalurus punctatus) were obtained
as 1- to 3-inch fingerlings from local commercial fish hatcheries during the
course of the study. These fish were also formalin-treated prior to their
use in experimental systems.
The sheepshead minnows were fed a diet prepared by mixing 400 grams of
cat food (Kozy Kitten brand) with 250 gm of No. 3 Purina Trout Chow. The
mixture was pelletized by running it through a meat grinder, prepared weekly,
and kept under refrigeration until used. The channel catfish were fed either
the catfood-trout chow mixture or commercial catfish food (Purina). Both
species were fed approximately 3% of their body weight per day.
Exposure Systems
Sheepshead minnows were maintained in closed-circulating systems
5
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consisting of 5 aquaria constructed of double-thick glass or fiberglass-
coated 1/2-inch exterior plywood with a double-thick glass front (Fig. 1).
The aquaria have PVC stand pipes that provide a standing water capacity of
150 liters. The aquaria drain into a common subtank. The subtank is con-
structed so that water is forced up through a filter system into a water
holding compartment (Fig. 2). The holding compartment contains a submersible
pump (Little Giant, 3E-12NDVR) controlled by a float valve system (IP504
Automatic Float Switch, W. H. Grainger, Inc., New Orleans, LA) that inter-
mi ttently pumps the water into a head box placed above the five aquaria. Five
drains in the bottom of this box allow water to gravity flow into the aquaria.
The drains are fitted with rubber stoppers that contain holes. Thus, the
flow rate of the system is a function of total water volume and the size of
the holes in the rubber stoppers.
Most of the experiments with catfish were conducted in 950-liter cylin-
drical fiberglass tanks (Reeves Plastic Engineering, Pascagoula, MS). These
tanks contain Venturi lifters and can be operated with constant or intermit-
tent flow-through to maintain water quality. Some experiments involving both
catfish and sheepshead minnows were conducted in 185-liter capacity rectan-
gular fiberglass-coated plywood tanks equipped with bottom drains and stand
pipes. Both types of tanks were provided compressed air through air stones.
External filters were employed with 185-liter tanks.
PAH Contamination of Water
Acetone was used as a carrier for the introduction of PAH into the
systems. To obtain the 10 ppb exposures, 6 ml of acetone containing 1.5 mg
PAH per ml was added to the systems. The 50-ppb exposures were obtained by
adding 10 ml of acetone containing 4.35 mg PAH per ml to the systems. Equal
volumes of acetone were added to controls. The contaminant was always added
to the holding compartment of the subtank (Fig. 2).
Contaminants were added to the tanks in a similar manner for the catfish
exposure experiments.
Chemical Analysis
The concentrations of BaP and MCA in the exposure system water were
determined by two different methods. Initially, the PAH were extracted from
one-liter water samples with 100 ml of hexane. Interference due to high
boiling aliphatic hydrocarbons was eliminated by evaporating the hexane to a
1 ml volume, adding it to a column of 1 g neutral alumina over 1 g silica gel
and eluting the aliphatic hydrocarbons with 10 ml hexane. The BaP (benzo(a)-
pyrene) and MCA (methylcholanthrene) was then eluted with 10 ml of methylene
chloride/hexane (50/50, V/V), evaporated to 1 ml or less, and analyzed by gas
chromatography with flame ionization detectors (GC-FID). A 3mm x 2m stainless
steel column packed with 3% SP-2100 on Supelcoport 80/100 was utilized with a
temperature programmed from 150 to 250°C at 4°/mm. Two inherent difficulties
were experienced with this procedure. In some cases, the removal of the
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Figure 1. Schematic diagram of the basic components and the waterflow in
the closed-circulating system. The arrows indicate the direction of water
flow. The switch is controlled by a float mechanism as the water level
changes in the sub-tank. The head box, aquaria, and the sub-tank are all
completely covered to minimize air contamination.
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FILTER COMPARTMENT
INLET
COMPARTMENT.
WATER HOLDING
COMPARTMENT
Figure 2. Detailed structure of closed-circulating system sub-tank. The
five holes in the wall of the inlet compartment are the points of attachment
for the hoses from the aquaria. The filter compartment contains a false
bottom not illustrated that supports the filter material 3 inches above the
bottom of the sub-tank. This allows water to flow from the inlet compart-
ment, up through the filter material and into the holding compartment. The
submersible pump is placed in the water holding compartment and the float
controlled switch is mounted on the wall of this compartment.
8
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required number of one-liter samples represented an appreciable amount of
the total volume of water in the system, and more importantly, the procedure,
did not provide the sensitivity needed for the low concentrations of con-
tamination encountered.
A second more sensitive procedure that employs HP liquid chromatography
was used to analyze most of our samples (18). Due to the increased sensi-
tivity of this procedure, 10 to 100 ml water samples were sufficient for
analytical purposes.
PAH Feeding Experiments
For the high concentration of contaminate (1 mg/gnr feed), BaP (BaP-H) or
MCA (MCA-H) was dissolved in 20 ml of acetone, blended with 257 gm of feed,
and then pelletized. The low concentration of contaminate-(BaP-L or MCA-L)
was prepared in a similar manner but with only 0.1 mg PAH/gm feed.
The contaminated food was fed once each day at the rate of approximately
3% of body weight per day.
Bioaccumulation Studies
Fish were placed in 38 liter aquaria and allowed to adapt for two days.
Then 0.08 yg/1 of 3H-benzo(a)pyrene or 0.037 yg/1 of 3H-20 methylcholanthrene
(Amersham Corp.) were added to the system. Gill, liver, G.I. tract, and
skeletal muscle tissues were excised, weighed, and homogenized in chloroform:
methanol (3:1). The filtrate from a glass wool-filled pipette was then
evaporated to dryness: scintillation cocktail (1 liter toluene:4 gPPO) was
added to the vial and the filtrate was counted in a Packard Tri-Carb Liquid
Scintillation Spectrometer.
Tissue Culture
A sheepshead cell line (SHF) developed from cultured fin fibroblaats of
a male Archosargus probatocephalus was employed for these studies (19). C-
band staining was accomplished by modifying the procedure described by Howard
et al. (20) for human leukocyte cultures. The incubation time was increased
from 24 to 48 hours and the staining time for the slides in Gierasa stain was
increased to 15 minutes. This modified staining procedure was also applied
successfully to preparations of cell lines from spleen fibroblasts of the
silver perch, Bairdiella chrysura (21),and fin fibroblasts of the salt-water
blue striped grunt, Haemulon sciurns (22).
Chromosomes of the SHF cells were prepared for karyotyping by a modifi-
cation of the method of Sumner et al. (23). Colcemid was added to the;
cultures to a final concentration of 0.25 ug/ml for 2-3 hours. The medium
was then removed and the flasks rinsed with trypsin-versene^ '(ATV) (24).
The ATV treatment was repeated until all the cells were removed. The pooled
ce-11 suspension (original medium and all rinses) was- centrifuged at 1000 rpm
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for 10 minutes, the supernatant was aspirated off, and the cellular pellet
was resuspended and treated for 20 minutes at room temperature with a hypo-
tonic solution of 0.075 M KC1. Fixative (3 parts absolute methanol: 1 part
glacial acetic acid) was added and the cells were pelleted again by centri-
fugation. The fixed cells were dropped on clean dry slides and allowed to
air dry prior to staining in a standard Giemsa stain.
Histological Preparations
Tissues prepared for light microscopy were fixed in Davidson's fixative
(25) in the cold for 24 to 96 hours. Fixed tissues were embedded in paraf-
fin, sectioned, and stained with hematoxylin and eosin according to routine
procedures.
10
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SECTION 4
RESULTS AND DISCUSSION
Cl osed-circulati ng Exposure Systems
Increasing utilization of marine organisms such as the sheepshead minnow
in bioassays and laboratory studies of the effects of pollutants (14-17)
creates a need for closed systems in which reasonably large numbers of
animals can be maintained in good health. In addition to the need to study
marine organisms in inland laboratories, closed systems are needed at marine
laboratories to avoid releasing large amounts of dangerous and sometimes
costly chemicals into the estuarine environment.
The closed-circulating systems designed for this project provide a
relatively inexpensive solution to this problem. They are constructed of
low-cost, easily available materials and designed to be easy and safe to
operate. The system is essentially enclosed yet needs no external aeration,
thus reducing the risk of aerosol contamination. The design of the subtank
provides both mechanical and biological filtration by a filter system that
can be charged with oyster shell, charcoal, or any other commonly used fil-
tration material (Fig. 2). Overflow water from the aquaria flows up through
the filter from the bottom of the subtank into the water holding compartment,
causing debris to accumulate in the bottom of the subtank. Consequently, at
the time periodic water changes are made by draining the subtank, this debris
can be easily washed out through the drains. Also, water can be run "back-
wards" through the filter material for additional cleaning with little risk
of operator contamination. In our experience 75 to 125 adult sheepshead
minnows can be maintained indefinitely with good water quality by draining
the subtank once each week. When the system is stocked and functioning
properly, nitrites have averaged 0.123 ppm, nitrates 18.8 ppm. '• Although we
have experienced problems obtaining reliable readings with our ammonia test,
ammonia seems to stay reasonably low. The pH averages 8.5 and rarely drops
below pH 8.3. These values are within the quantitative ranges considered
ideal for marine aquaria (26).
Our experience indicates that it is extremely important that the fish
be healthy, stress free, and relatively clear of parasites when they are
placed in the system. If these conditions are met, an acceptable survival
rate can be expected over an extended period (Table 1).
The importance of the state of health of the feral fish may be illus-
trated by the fact that during each year of the study sheepshead minnows
collected in the early spring died in large numbers before they could be
adapted to laboratory conditions. Experiment 5 in Table 1, which is the
11
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lowest rate of survival experienced, were survivors of a group of fish caught
at this time of year.
A caution concerning the operation of the system is that care must be
taken to see that an adequate water level is maintained at all times since
circulation will stop when the water falls below a critical level. Addi-
tionally, the brass rod on the float valve switching device must be cleaned
and freed of corrosion periodically if the switch is to function properly. (An
effort is currently underway to redesign the switching system to alleviate
this problem.)
TABLE 1. LONG-TERM EXPOSURES OF SHEEPSHEAD MINNOWS
Experiment
Number
1
2
3
42
5
Number
of Fish
100
125
125
125
45
Contaminant1
Control
1 ug/1 PaP
10 ug/1 MCA
50 ug/1 MCA
50 ug/1 MCA
Length of
Exposure (wks)
10
25
13
31
15
Percent
Survival
70
66
100
86
44
Contaminated once each week.
Continuation of experiment No. 3 with an increase in level of contamination.
Chemical Analysis
Before relationships can be established between tumor incidence and the
concentration and mode of exposure to a tumorigenie agent, it is necessary to
establish a profile of the actual concentrations of the agent in the water
column throughout the time course of the experiment. Table 2 provides data
concerning the levels of BaP at different points in the closed-circulating
system for a six-day period after the system was contaminated with 10 ug/1.
Since much of the BaP precipitates and floats on the water surface, the values
determined shortly after contamination will vary greatly according to the
method of sampling. Consequently, samples were taken with a pipette ca. 27
cm below the water surface. The data in Table 2 indicate that BaP is in the
water column of the subtank at a level of about 80% of the theoretical level
of contamination. The concentration in the different aquaria is somewhat
varied at one hour; however, the mean value for the aquaria at this time is
about 35% of the theoretical concentration (Fig. 3). Although we had ex-
pected large amounts of PAH to adhere to the filter surfaces, the concen-
tration of BaP in the effluent from the filter one hour after exposure is
actually higher than the mean for the five aquaria,suggesting that the filter
12
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did not significantly lower the concentration. After 24 hours the level of
BaP at all sampling points had dropped rapidly to about 20% of the theoret-
ical concentration. From the 4th to the 6th day, the concentration dropped
to about 5% (Fig. 3). These data indicate that weekly contamination of the
system with BaP at a level of 10 yg/1 provides a significant amount of BaP
in the water column for only the first one or two days of the exposure
period.
TABLE 2. BaP CONCENTRATION IN CLOSED-CIRCULATING SYSTEM1
Time of Sampling
1 hr 24 hr 96 hr 144 hr
Sampling Location
Holding Compartment of Sub tank 8.02
Filter Effluent
Aquarium -
Aquarium -
Aquarium -
Aquarium -
Aquarium -
1
2
3
4
5
4.5
2.6
3.0
5.7
3.1
3.2
2.2
2.1
1.6
1.6
2.6
2.0
1.8
0.4
0.5
0.4
0.5
0.5
0.3
0.5
0.3
0.5
0.3
0.3
0.5
0.3
0.4
System contaminated at the beginning of the experiment with 10 yg BaP per
liter of water.
2All values in yg/1 (ppb).
Somewhat similar results were obtained when the system was contaminated
on a weekly basis with BaP at a concentration of 50 yg/1 for a three week
period (Table 3). These data provided no evidence for week-to-week accumu-
lation of BaP in the system over the three-week period since the concen-
trations for the second and third weeks were not significantly higher than
those observed for the first week. Also, the profile of concentration during
each week was similar to that observed at the 10 yg/1 contamination. For
example, one hour after contamination the mean concentration was about 45%
and by 24 hours it had dropped to 22%. By the 4th day, the concentration had
leveled off at 2-3%.
When the systems were contaminated each week with 50 yg MCA/1 for five
weeks, a similar profile of concentrations resulted; however, the absolute
13
-------
u
e
o
CD
10 30 80 70
TIME (hr«)
90
110
130
150
Figure 3. Profile of BaP concentration 1n closed-circulating system over a
six-day period. Initially, the system was contaminated with 10 yg of BaP
per liter of water. The graph Indicates meanst SE of the values from aquaria
1 through 5 of the system.
14
-------
values were higher (Table 4). As 1n the case of BaP contamination the
evidence does not Indicate an accumulation of MCA 1n the system over the
five-week period. The concentration one hour after contamination was 65.3
ug/1, a value which 1s higher than the 50 ug/1 theoretical level of con-
tamlnation. This may seem surprising; however, 1t 1s a common occurrence
Immediately after contamination before the contaminant has become dispersed
throughout the whole system. At 24 hours after contamination, the concen-
tration 1s 47.7 ug/1 which 1s 95% of the theoretical concentration, and by
the 4th and 6th days, the values had dropped to 12% and 10%, respectively.
These data suggest that MCA 1s more soluble 1n aqueous solutions than
BaP. Thus, when 50 ug/1 contamination of BaP and MCA are compared one hour
after contamination, the concentration of BaP 1n the water column 1s less
than 25% of that observed for MCA, and this relative difference remains
essentially the same even after 4 to 6 days. Perhaps this higher solubility
which makes MCA more accessible to organisms and cells In an aqueous environ-
ment Is one reason why MCA 1s sometimes listed as a more potent carcinogen
than BaP (27).
Because of the low solubility properties of both BaP and MCA, 1t seems
that a significant portion of these PAH would remain 1n the water column for
only a transient period. Thus, weekly contaminations produce "spike" type
exposures typical of what might occur when PAH-containing wastes are repeat-
edly released Into the aquatic environment 1n discrete amounts (28). This
method of laboratory exposure 1s therefore a good model for the type of pol-
lution that occurs 1n ocean dumping of petroleum and 1n some types of Indus-
trial effluents.
The relative Insolubility of these PAH suggests that they and similar
PAH may not cause serious and widespread problems for fish due to direct
contact with the compound 1n the water column. However, since PAH may accum-
ulate In somewhat higher concentrations 1n some marine Invertebrates and
bottom sediments (27), 1t 1s feasible that some fish, as a result of their
feeding habits, may be exposed to high levels of PAH through Ingestlon. Even
1f 1n the final analysis PAH does not prove to be a serious carcinogenic
agent 1n marine fish and Invertebrates, 1t 1s of obvious significance that
when exposed to PAH, fish and invertebrates such as the American oyster
(Crassostrea vlrglntca) (28), an Important human food Item, 1s known to
accumulate these potent mammalian carcinogens.
Long-term Exposures
During the course of the study, we have maintained a full complement of
Cyprlnodon 1n our systems for a total of 94 weeks with the same group of fish
In the system for as long as 25 weeks (Table 1). During this time a 73%
overall survival rate occurred. No obvious lesions were observed 1n about
20% of the sheepshead minnows sacrificed and examined because they showed
signs of Illness or were moribund. However, of the remainder that presented
pathologies, 60% were gill-related. Of these, about 25% appeared to have
fungal diseases and another 15% had regions of gill lamellae that were eroded
and necrotlc. Other pathologies observed were: hemorrhaglc areas on the
15
-------
TABLE 3. BaP CONCENTRATION IN AQUARIA OF CLOSED-CIRCULATING SYSTEM1
Week of
Exposure
1
2
3
Mean ± S.E.
1 hr
21 .92
18.8
24.6
21.8±4.5
Time of
24 hr
10.0
15.9
7.0
11.0±4.5
Sampl Ing
96 hr
1.0
1.8
1.7
1.5 ±0.44
144 hr
1.0
1.4
0.9
1.1 ±0.26
'System contaminated at the beginning of each week with 50 ug BaP per liter
of water.
2A11 values In yg/1 (ppb).
TABLE 4. MCA CONCENTRATION IN AQUARIA OF CLOSED-CIRCULATING SYSTEM1
Week of Exposure
1
2
3
4
5
Mean ± S.E.
1 hr
61. 22
65.5
71.4
62.2
66.4
65.3±4.0
Time of
24 hr
42.8
42.4
55.6
41.9
52.0
47.7±5.9
Sampling
96 hr
3.6
7.4
7.4
6.9
5.0
6.1±1.7
144 hr
3.5
6.0
5.8
5.3
5.1
5.1 ±1.0
xSystem contaminated at the beginning of each week with 50 yg MCA per liter
of water.
2A11 values in yg/1 (ppb).
16
-------
body surface 12%, hetnorrhagic gills 8%, and lordosis and/or scoliosis 8%.
Pathological conditions observed in channel catfish that became ill or
moribund while in the laboratory were similar to those observed in the
sheepshead minnows. For example, .about 77% of the abnormalities in catfish
were gill-related. In contrast to the sheepshead minnows, 47% of the cat-
fish cases that we classified under gill .abnormalities were severe gill
infestations of the monogenetic trematode Cleidodiscus. An apparent expla-
nation for this occurrence is that 33% of these infestations of Cleidodiscus
occurred on fish that were stressed because of ingesting high levels of PAH
in their food regime. About 20% of the catfish sacrificed had hemorrhagic
and/or aneuritic lesions on the gills.
These data concerning the types and incidence of lesions of sheepshead
minnows and channel catfish maintained in the laboratory are important since
they serve as baseline information required to accurately assess the effects
of contaminates introduced into the systems. The question regarding whether
the lesions observed may result from maintaining the fish in the laboratory
is problematic since extensive data on the survival rates, types, and inci-
dence of lesions in the fish in their normal environments are not available.
In an experiment in which 10 catfish were maintained in water contami-
nated on a weekly basis with 1.0 yg/1 of BaP the fish remained healthy for
approximately 7 months. During the next three month period, however, 4 of
the fish presented with a similar pathology. In each case they became very
scoliotic and/or lordotic and exhibited nervous disorders in their manner of
movement. Most of the affected individuals also displayed abnormal melano-
cytic control and were much darker in color than normal fish. Radiographs
(Fig. 4) illustrate the vertebral disorientations that occurred. Lesions of
this type can result from inadequate nutrition (30) and we have on occasion
observed scoliosis in catfish maintained as controls; however, the high inci-
dence of these phenomena in the exposed fish that continued to feed normally,
and had normal growth rates, suggests that a cause-effect relationship may
exist between the lesions and the BaP exposure. These lesions, though not
identical, are quite similar to those that appear to occur in teleosts as a
result of exposure to Kepone (29).
Feeding Experiments
The tendency of PAH to adsorb to particulate matter makes it likely that
scavenger feeding fish such as the channel catfish could be exposed to large
amounts of PAH by ingestion. In an effort to reproduce this type exposure,
channel catfish were fed diets containing known concentrations of BaP and
MCA (Table 5).
In an experiment in which the fish were fed 1 mg BaP/gm food,56% of the
fish were dead in 24 hr and all but one had died by the 4th day of the exper-
iment. Most of the fish exhibited a "spiraling" behavior just before becoming
moribund. In a second experiment in which catfish were fed 1 mg BaP/gm food,
all the fish lived until the 6th day and 60% had died by the 7th day. The
fish continued to die until the remaining 5 were sacrificed on the 16th day.
17
-------
TABLE 5. FEEDING EXPERIMENTS
EXPT. FOOD
NO. REGIME1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
1 BaP-H -14 -1 -9 -1
2 BaP-H -2 -13 -1 -2 -2 *
3 MCA-H -1 -1 -1 -1 -1 -1 -1 -1 -6 -5 -2 *
4 BaP-L -8 -14 t
5 MCA-L -3 -2 -3 -4 -1 §
twenty-five fingerling channel catfish were used in each of these experiments. The food regime in
_ experiments 1 and 2 (BaP-H) contained 1 mg BaP/g of food. In experiment 3 (MCA-H) the food was con-
oo taminated with 1 mg MCA/g of food. The contamination in experiment 4 (BaP-L) was 0.1 mg BaP/g food,
and 0.1 mg MCA/g in experiment 5 (MCA-L).
*Experiment terminated and surviving fish were sacrificed.
tSurviving fish were fed uncontaminated food for 10 additional days and then sacrificed.
§2 additional fish died on day 27 and 1 on day 35. The experiment was terminated on the 36th day and
the surviving fish were sacrificed.
-------
4a
r - •
Figure 4. Vertebral disorientations in BaP exposed catfish. Images are
positive prints of radiographs.
4a) Dorsal view. The vertebral column appears essentially normal. Mag. 2X,
4b) Lateral view. The disorientation of vertebra in the anterior half of
the vertebral column is evident. Note the twisting of some of the vertebrae
to an extent that the neural spines are out of the focal plane (arrowhead).
Mag. 2X.
4c) Dorsal view. A higher magnification of the anterior region of the
vertebral column. Mag. 5X.
4d) Lateral view. The disarticulation of many of the vertebrae is evident.
Note that some vertebrae extend above adjacent vertebra and others have ab-
normally large spacing (arrows). The rotation of some of the vertebrae re-
moves their neural spines from the plane of the image (arrowhead). Mag. 5X.
19
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TABLE 6. BIOACCUMULATION OF 3H-BENZO(A)PYRENE IN SHEEPSHEAD MINNOWS1
ro
o
Time
Exposed
6 hr
12 hr
24 hr
48 hr
96 hr
Gills
ng/gm^
tissue
0.06-0.42
0.08-0.21
0.12-0.27
0.20-0.26
0.04-0.13
BF3
2.3
1.8
2.5
2.9
1.1
Liver
ng/gm
tissue
Q. 15-0. 59
0.2 -1.6
0.29-0.4,4
0.34-0.60
w
0.04-0.60
BF
4.9
1-Q
4-4
6.4
5.0
RI
ng/gm
tissue
0'. 58-0. 73
0.89-1.9
0.19-0.75
1.41-1.60
0.53-0.75
Tract
BF
8.2
18
5.9
19
8
Muscle
ng/gm
tissue
Q. 01-0.06
0.02-0. 05
0.03-0.12
0.04-0.08
0.02-0.11
BF
Q.5
0-6
P.p
0.7
P.6
theoretical concentration of 3H-BaP, 0.08 ng/ml water.
2Range of values determined from 3 experiments.
3BF = Bioaccumulation Factor: mean cqj|cenjiration °f Bap in tissue divided by theoretical con-
centration of BaP in water.
-------
TABLE 7. BIOACCUMULATION OF 3H-METHYLCHOLANTHRENE IN SHEEPSHEAD MINNOWS3
ro
Time
Exposed
24 hr
48 hr
72 hr
96 hr
120 hr
144 hr
168 hr
Gill
ng/gmz
tissue
1.6 -5.6
0.2 -9.6
1.0 -4.0
0.65-3.0
3.3 -6.9
3.6 -4.0
0.56-12.7
s
BF3
97
97
57
49
124
86
189
Liver
ng/gm
tissue
0.4 -8.2
0.19-2.5
0.10-6.0
2.9 -7.5
1.4 -8.5
1.3 -7.5
0.46-0.5
BF
132
35
65
140
135
135
13
GI
ng/gm
tissue
6.4 -11.3
0.26-3.8
0.48-1.46
0.25-1.02
0.12-0.68
0.16-1.03
0.09-0.26
Tract
BF
227
54
25
16
10
12
5
Muscle
ng/gm
tissue
0.11-0.30
0.11-0.20
0.08-0.18
0.03-0.26
0.01-0.04
0.03-0.43
0.02-0.04
BF
6
4
3
4
1
4
1
'•Theoretical concentration of 3H-MCA, 0.037 ng/ml water.
2Range of values determined from 3 experiments.
3BF = Bioaccumulation Factor: mean concentration of MCA in tissue divided by theoretical
concentration of MCA in water.
-------
TABLE 8. BIOACCUMULATION OF 3H-BENZO(A)PYRENE IN CHANNEL CATFISH1
ro
r\>
Gills
Time
Exposed
6 hr
12 hr
24 hr
48 hr
72 hr
96 hr
120 hr
ng/gm2
tissue
0.12-0.62
0.25-0.71
0.17-0.53
0.04-0.19
0.07-0.16
0.08-0.20
0.04-0.46
BF3
4.7
5.5
4.1
1.4
1.5
1.6
3.4
Liver
ng/gm
tissue
0.60-3.0
1.7 -2.7
0.5 -2.8
0.5 -3.5
0.17-2.0
0.04-3.2
0.04-0.12
BF -
21
4.4
25
26
14
18
1
GI Tract
ng/gm
tissue
0.13-1.3
0.53-2.3
0.09-1.6
0.3 -2.6
0.25-1.3
0.08-2.6
0.03-0.85
BF
7
14
11
11
10
17
6
Muscle
ng/gm
tissue
0.14-0.30
0.23-0.26
0.26-0.43
0.14-0.42
0.19-0.66
0.06-0.51
0.11
BF
2
3
4
4
5
2
1
theoretical concentration of 3H-BaP, 0.08 ng/ml water.
2Range of values determined from either 3 or 5 experiments.
3BF=Bioaccumulation Factor: mean concentration of BaP in tissue divided by theoretical con-
centration of BaP in water.
-------
TABLE 9. BIOACCUMULATION OF 3H-METHYLCHOLANTHRENE IN CHANNEL CATFISH1
ro
CO
Gills
Time
Exposed
24 hr
48 hr
72 hr
96 hr
120 hr
144 hr
168 hr
ng/gmz
tissue
0.03-1.8
0.07-0.16
0.014-0.02
0.01-0.06
0.02
0.07
0.06
BF3
25
3
0.5
1
0.5
2
1.5
Liver
ng/gm
tissue
0.02-0.59
0.01-0.20
0.03-0.08
0.05-1.20
0.04-0.7
0.20-0.40
0.14-0.6
BF
9
14
2
14
8
8
10
GI Tract Muscle
ng/gm
tissue
0.01-0.91
0.04-1.32
0.02-0.54
0.01-0.10
0.01-0.06
0.02-0.04
0.02-0.08
ng/gm
BF tissue
9 0.10-0.13
14 0.05-0.10
8 0.06-0.10
2 0.06-0.2
1 0.03-0.1
1
1
BF
3
2
2
3.5
2
theoretical concentration of 3H-MCA, 0.037 ng/ml of water.
2Range of values determined from 3 experiments.
3BF=Bioaccumulation Factor: mean concentration of MCA in tissue divided by theoretical con-
centration of MCA in water.
-------
These fish were sluggish, not feeding, and their gills were infested with the
monogenetic trematode Cleidodiscus. In a similar experiment in which catfish
were fed 1 mg MCA/gm food, a slow mortality begain to occur on the 8th day
and continued until the 18th day when 6 of the remaining fish died. On the
20th day an additional 5 of the survivors died. In this experiment also, the
fish became sluggish and heavily infested with trematodes simultaneously with
the high mortality. When catfish were fed 0.1 mg BaP/gm food, they eventually
became sluggish and fed poorly, but no mortality occurred until the 13th day
when within 2 days 88% of the fish died. Somewhat similarly, when catfish
were fed 0.1 mg MCA/gm food,the fish began to show the same signs after about
a week. The first mortalities occurred on the 10th day, and by the 14th day
52% of the fish had died. In a control experiment in which catfish were
maintained on a regime to which acetone had been added as in the PAH con-
taminated food, the fish continued to feed normally during a two-month period.
They were thereafter maintained on a regular diet for an additional seven
months with no visible effects.
These results seem to indicate that BaP fed at the level of 1 mg/gm food
is toxic to the channel catfish and that MCA fed at the same level is toxic
to a somewhat lesser degree. Further, either of these compounds fed at the
lower level (0.1 mg/gm food) seem to be less toxic but still stressful enough
to allow parasitic infestation and eventual death.
These results, although preliminary, suggest a need for more work to
clarify the toxic effects of PAH on channel catfish. Of particular interest
is the indication that ingestion of nontoxic amounts of PAH may cause enough
stress to lower resistance sufficiently to allow secondary infestations by
viruses, bacteria, or parasites.
Bioaccumulation Studies
The range of variability in the quantities of PAH at each time period
makes interpretation of these data problematic (Tables 6-9). The technique
employed in the studies allows for some amount of experimental error (e.g.,
wet weights of tissues in milligram quantities must be determined); however,
previous experience with these techniques suggests that this could not account
for all the variability observed. Consequently, some of the experiments were
carefully repeated to provide as many as six data points at one exposure
time. The variability persisted, suggesting that rather large and capricious
variabilities in bioaccumulation actually occur.
When the bioaccumulation of BaP and MCA are considered in both sheeps-
head minnows and catfish, the values for the liver and 61 tract are consis-
tently the highest. The result in the liver could be anticipated because of
the major role this organ plays in the metabolism, detoxification, and storage
of exogenous compounds. The relatively high levels of PAH in GI tract per-
haps would not have been predicted. The tendency for PAH to adhere to
surfaces rather than remain in solution in the water column may be signi-
ficant to this result. For example, this may mean that the major route of
exposure to the labelled compound was via the GI tract as a result of the
24
-------
MATERIAL BELONGS . o
US EPA TOXICS LI3RARY
fish ingesting debris from the aquaria with adhe
--
The level of label in the gills was intermediate, Tower than the liver
and 61 tract but consistently higher than the levels observed in skeletal
muscle. This likely results from the fact that the gills are highly vascu-
lar! zed and have an excretory role.
In each case the level of PAH in the skeletal muscle was significantly
lower and less variable than that observed in the other tissues. Since
muscle tissue is not directly involved in the metabolism of exogenous com-
pounds such as PAH, a lower level of label could be expected.
No consistent trend toward either an increase or decrease in the level
of the label was observed during the time the fish were exposed to the la-
beled compound. This remained true even in some experiments in which fish
were exposed for as long as 12 days.
The basic pattern and the absolute values of label in the tissue were
essentially similar for both species of fish with respect to BaP. In the
catfish, the MCA experiments were also comparable to the BaP results; how-
ever, in the MCA experiments with sheepshead minnows, the levels were signi-
ficantly higher. It is possibly important in this respect that MCA seems to
remain in the water column of the closed-circulating systems at higher levels
than BaP (Tables 3-4). It is our casual observation that MCA is also more
soluble in the acetone carrier used to contaminate the systems. This obser-
vation does not, however, explain why a similar increase in label was not
observed in the catfish experiments with MCA.
When the bioaccumulation experiments are considered in toto, one reason-
able explanation for the results is that the major route of contamination is
by ingestion rather than uptake through contact with the labeled compound
suspended in the water column. In this method of exposure, one would predict
that the level of radioactivity in the tissues of the fish would be related
more to whether a particular fish had recently ingested significant amounts
of the contaminant than how long it had been exposed to the contaminated
water. Experiments in which fish will be fed food containing labeled con-
taminate are planned in an attempt to clarify this point. Also, autoradio-
graphic studies of tissues from these experiments are currently being con-
ducted to elucidate the route of exposure.
Due to the techniques used in these experiments, the results indicate
only the tissue location of the radioactive label and provide no indication
of the biochemical changes that may have occurred in the parent compound due
to metabolic processes. Valuable information of this type must await more
sophisticated biochemical studies.
Tissue Culture Studies
The basic mechanisms of chemical carcinogenesis function at the cellular
level and remain incompletely understood. With the long term goal of studying
these phenomena in fish tissue culture cells, we have karyotyped an
25
-------
established fin fibroblastic cell line designated SHF (19). Since many
teleosts possess large groups of morphologically similar chromosomes, it
was necessary to modify existing techniques for C-banding mammalian cells
so that C-bands could be observed on SHF cells before they could be ade-
quately karyotyped for use in an experimental system. A publication con-
cerning the development of this technique and its successful application to
three different species of fishes is currently in press (31).
A complete karyotyping of SHF cells has been completed and the results
have been submitted for publication (32). It appears from this study that
the SHF culture has a relatively stable karyotype with a modal chromosome
number of 48. Essentially no morphological changes were observed in the SHF
cell chromosomes, so this cell line seems to provide an excellent model for
in vitro studies for carcinogenicity and mutagenicity. Currently, prelim-
inary experiments are in progress to determine the toxicity level of BaP with
respect to these cells so chronic exposures can be accomplished.
Histological Studies
To date, histological examinations have been conducted on 250 (3%) of
the approximately 8,000 specimen collected. About 70% of these specimen were
sheepshead minnows, and the tissues examined most extensively were gills,
liver, 61 tract, and skeletal muscle. To a lesser extent gonads, kidneys,
and heart tissues were also examined. Some of the fish represent random
sampling of the feral population, and others were taken from either control
or contaminated exposure systems. However, a majority were from moribund
fish or fish that appeared to be ill. Most of the histopathic lesions
observed were in the gills. These included hyperplasia, aneurisms, necrosis,
and parasitic infestations. These data are not sufficient to determine
whether the incidence of any of the lesions could be consistently associated
with PAH exposure. This determination will have to await collection of
additional data concerning the incidence of the various lesions in feral
laboratory maintained controls, and experimentally exposed sheepshead minnows
and channel catfish.
A library of slides representing the major organs and tissues from both
sheepshead minnows and channel catfish has been prepared to serve as a base-
line of "normal" tissues for reference when studying tissues from the species
that were maintained under long-term exposures. We have also cataloged
representative slides illustrating the lesions observed in sick and moribund
fish sacrificed from both control and contaminated exposure systems.
Because of the continued need for more sensitive indices of the effects
of pollutants on the aquatic environment, we conducted the following morpho-
logical studies in addition to routine histology. The morphology of the
gills from both healthy and sick or moribund fish are being studied with the
scanning electron microscope, and white blood cells of healthy and diseased
sheepshead minnows are being studied by light and transmission electron
microscopy. The obvious need to understand the effects of pollutants on
fish during the preadult stages of their life cycle has prompted a careful
study of the embryologic development of the sheepshead minnow. The results
26
-------
of these studies will be published subsequent to submission of this report.
Spontaneous Tumors
Two lesions with a tumor-like appearance were observed in the sheepshead
minnow feral population. In the first case, when the specimen was necropsied,
a large mass was observed protruding from the liver. Its color and texture
was obviously different from a normal liver; however, attempts at histolog-
ical analysis disclosed that the tissue had experienced such severe autolytic
damage that its histological nature was completely obscured.
In a second case, a tumor-like mass was observed in a sheepshead minnow
shortly after it was brought into the laboratory. The lesion was a relatively
large mass of soft tissue protruding from the body surface in the isthmus
region between the pectoral fins.
At low magnification, the tissue has the appearance of a tumor in that
it consists of a randomly oriented connective tissue stroma that forms sinus-
like cavities containing a population of loosely oriented and apparently
pleomorphic cells (Fig. 5a). However, at higher magnification, it becomes
clear that the loosely associated cells are myxosporodian spores that appear
pleomorphic because of their various orientations (Fig. 5b). At an even
higher magnification, the morphology of the spore and its two internal polar
capsules may be observed (Fig. 5c). The histozoic and possibly cytozoic
nature of this parasite is illustrated by the close association of spores
with striated muscle fibers that extended in an irregular orientation through
some regions of the cyst (Fig. 5d). Although not prevalent in the fishes of
the Gulf, this type lesion has been observed in the sheepshead minnow and is
likely the myxosporidian Myxobolus lintoni (33). It has been suggested that
the incidence of this parasite may be increased by pollution or other forms
of stress (33). The lesion is illustrated and described in some detail in
this report in order to point out that these type lesions should not be
confused with true neoplasms.
27
-------
Figure 5. Myxosporidian cyst in the sheepshead minnow. H & E stained
sections viewed with bright field optics.
5a) At low magnification the cyst has a tumor-like appearance. There is a
connective stroma with a parenchyma of loosely associated cells that appear
to be pleomorphic. Mag. 160X.
5b) At somewhat higher magnification the vacuolated nature of the structures
within the stroma is evident (arrows). Mag. 634X.
5c) At even higher magnification it is evident that these structures are
flattened ovoidal myxosporidian spores. In properly oriented spores the two
anteriorly located polar capsules are evident (arrows). Mag. 4800X.
5d) Scattered sparsely throughout this lesion are randomly oriented muscle
fibers. Mag. 1585X.
28
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REFERENCES
1. Cairns, J. The cancer problem. Sci. Am. 233:64-80. 1975.
2. Sonstegard, R. A. Environmental carcinogenesis studies in fishes of the
Great Lakes of North America. Ann. N.Y. Acad. Sci. 298:261-269. 1977.
3. Kraybill, H. F. and C. T. Helmes. Biomedical aspects of biorefractories
in water. Second Int. Symp. on Aquatic Pollutants, Noordwijkerhout,
Amsterdam, The Netherlands, Sept. 26-28, 1977.
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29
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31
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TECHNICAL REPORT DATA
(Pltait nod Inttnictlont on the nvmt btjort completing)
1. REPORT NO.
FPAnfinry 3.80.019
a.
3. RECIPIENT'S ACCBS8ION NO.
B. REPORT OAT*
4. TITLE AND!
EFFECTS OF PETROLEUM COMPOUNDS ON ESTUARINE FISHES
B. PERFORMING ORGANIZATION COOI
7. AUTHOR(S)
B.J. MARTIN
I. PSP1PORMINQ ORGANIZATION REPORT NO
B. PERFORMING ORGANIZATION NAM1 AND AOORB88
Department of Biology
The University of Southern Mississippi
Hattiesburg. MS 39401
10 PROGRAM ELEMENT NO.
R804527
12, SPONSORING AQBNCV NAME AND ADDRESS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Gulf Breeze. Florida 32561
13. TYPE Of RtPORT AND PERIOD COVERED
14. SPONSORING AOBNCY CODE
EPA/600/A
IB. SUPPLEMENTARY NOTES
16, AB8TR
Effecta of the carcinogenic polycycllc aromatic hydrocarbons (PAH), benzo[a]-
pyrene (BAP), and methylcholanthrene (MCA) were investigated with sheepshead minnows
(Cyprinodon variegatue) and channel catfish (Ictalurus punctatua). A closed-circulat-
ing system was designed to maintain up to 100 sheepshead minnows in artificial sea-
water for longterm exposures. Fish were maintained in this system for up to 31 weeks
v»th weekly contaminations of PAH. Due to their chemical properties significant lev-
ult» of BaP and MCA remained in the water column for only ca. 24 hours each week and
no tumors were observed in the exposed fish during the period of the study.
The Incidence and types of lesions in control and exposed fish were basically
similar except in catfish that were fed PAH contaminated food. High levels of con-
tamination (Img/gm food) appeared to be toxic and lower levels of contamination
(0.1 mg/gm food) produced sufficient stress to make the catfish susceptible to fatal
parasite infestations. Both species accumulated radioactively labelled PAH at con-
centrations much higher than their nominal concentraions in the wnter.
These results demonstrate that sheepshead minnows function well as experimental
organisms in artificial seawater in a closed system maintained at a noncoastal
facility. Thus, they provide an excellent model system for the study of longterm
effects of chronic exposure to polluting agents.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b. IOENTIPIBRS/OPBN E.NDID TERMS
C. C08ATI I'icld/Uoup
Carcinogens
Aromatic polycycllc hydrocarbons
Bioassay
Pathology
Teleosts
iLongterm exposure
Bloaccumulation
Tissue culture
Histology
06/C
06/F
06/T
It. DISTRIBUTION iTATBMENT
UNLIMITED
19. SECURITY CLASS (Ttlll Rtportl
Unclassified
31
30. SECURITY CLASS (Thupagtl
Unclassified
aa. PRICE
CPA Form 2220-1 (Rtv. 4-77) PWBVIOU* KOITION n O»*OI.ITE
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