NATIONAL CANCER INSTITUTE AND U.S.
ENVIRONMENTAL PROTECTION AGENCT
COLLABORATIVE PROGRAM ON
ENVIRONMENTAL CANCER
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NATIONAL CANCER INSTITUTE AND U.S.
ENVIRONMENTAL PROTECTION AGENCY
COLLABORATIVE PROGRAM ON
ENVIRONMENTAL CANCER
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NATIONAL CANCER INSTITUTE AND U.S. ENVIRONMENTAL PROTECTION AGENCY
COLLABORATIVE PROGRAM ON ENVIRONMENTAL CANCER
PROJECT REPORT TO THE NATIONAL CANCER INSTITUTE
CARCINOGENIC EFFECTS OF BLACK ROCK HARBOR SEDIMENT
ON AMERICAN OYSTERS AND WINTER FLOUNDER
PRINCIPAL INVESTIGATORS
George R. Gardner
Paul P. Yevich
A. Russell Malcolm
Richard J. Pruell
Peter Rogerson
T.C. Lee
Andre Senecal
James Heltshe
Lesley J. Mills
PROJECT OFFICERS
Richard Latimer (ERL/N)
William Farland (EPA) and Thomas P. Cameron (NCI)
TECHNICAL DIRECTOR
A. Russell Malcolm
May 17, 1987
U.S. Environmental Protection Agency
Environmental Research Laboratory
Narragansett, Rhode Island 02882
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EXECUTIVE SUMMARY
The purpose of the NCI/EPA program was to determine the potential
of contaminated Black Rock Harbor (BRH) sediments to experimentally
induce neoplasms in benthic marine organisms. Black Rock Harbor,
Connecticut, sediments are considered typical of urban embayments of the
Northeastern United States in both type and quantity of chemical contam-
inants. Reference sediment was collected from a site in Central Long
Island Sound (CLIS). Test organisms, the American oyster (Crassostrea
virginica) and the winter flounder (Pseudopleuronectes americanus) are
indigenous to that area.
Experimental exposures of oysters to BRH sediments for 30 and 60
days induced multiple types and formations of tumors. The induction of
tumors in oysters resulted from a novel approach to aquatic exposure
systems where contaminated sediments were suspended in the water column
and the molluscs were allowed to filter-feed on the organic particulate
material. Experimental tumor induction rate in laboratory-exposed oysters
was 13.6% overall (49 neoplasms in 40 oysters, n = 295). The highest
tumor prevalence was in renal excretory tissues (28), followed in decreas-
ing order by gill (8), gonad (4), gastrointestinal (3), heart (3) and
neural tissues (3). Latency periods of 30 and 60 days after discontinua-
tion of exposure to the sediment demonstrated the autonomous nature of
oyster neoplasms. Neoplasms were not observed in oysters exposed to an
uncontaminated reference sediment (n = 338). In addition to the labora-
tory exposures, in situ tumor induction occurred in one oyster of 27 trans-
planted to Black Rock Harbor North Dock Station for 30 days and in one
oyster of 25 transplanted 400 m east of the disposal area for dredged
ii
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Black Rock Harbor sediment in Central Long Island Sound. Both oysters
had gill neoplasms, the later was more advanced with multiple foci.
In the laboratory, neoplastic lesions developed in kidney, pancreas,
external and oral epithelial surfaces of experimental winter flounder
during four months of direct contact with sediments from BRH and BRH
sediments mixed equally with reference sediment. Of particular interest
were pancreatic intrainsular neoplasms observed in winter flounder exposed
to BRH sediment in the laboratory or exposed to BRH sediment in conjunc-
tion with a diet of mussels exposed to BRH sediments. The observation
of diffuse pancreatic ductule hyperplasia (nesidioblastosis) in fish
collected from BRH, CLIS and New Bedford Harbor established a strong
laboratory to field comparison linking the condition to contaminated
sediment.
Chemical analyses revealed the types and concentrations of poly-
nuclear aromatic hydrocarbons found in BRH sediment were similar to
those related to neoplasia in fish from other geographic locations.
Consistent with those analyses, mutagenic activity was demonstrated with
unfractionated solvent extracts of BRH sediment, but not the reference
sediment. Using the standard Ames assay with preincubation and an exoge-
nous S-9 metabolizing system, a strong, concentration dependent, mutagenic
response was obtained with strain TA100, indicating the presence of
mutagenic agents causing base-pair substitutions. A weak but positive
response was also obtained with strain TA104 under the same test condi-
tions. The results of spiking experiments where standard mutagens were
added to unfractionated extracts suggested the presence of metabolic inhib-
itors in the sediments, especially those inhibiting oxidative metabolism
and probably a low concentration of direct-acting mutagens.
iii
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Evidence for the presence of tumor-promoting agents was obtained
with the Chinese hamster V79 cell metabolic cooperation assay in tests
with BRH sediment, but not with the reference sediment.
In addition to activity detected with short-term assays, exposure
to BRH sediment induced sister chromatid exchange in the marine polychaete,
Nepthys incisa in both laboratory and field studies.
Our experience with multiple species studies suggests to us that
the oyster is a sensitive biological model for study of the neoplastic
process in marine invertebrates. The results of the current NCI/EPA
study have demonstrated that it is now technically possible to advance
invertebrate oncology, especially in relation to potential carcinogens
that are sequestered in bottom sediments. The American oyster showed
excellent survival in laboratory studies and can be easily deployed for
field comparison studies. The winter flounder offers similar character-
istics for comparative laboratory and field studies, especially for its
potential as a model in the development of nesidioblastosis.
IV
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CONTENTS
Page
EXECUTIVE SUMMARY ii
LIST OF FIGURES vii
LIST OF TABLES viii
PART I. INTRODUCTION 1
PART II. METHODOLOGY 5
Sediment Collection and Preparation 5
Laboratory Studies of Oysters 6
- Experimental Design 6
Collection and Holding 7
Exposure System 7
Tissue Preparation and Histological Examination 10
Data Collection and Analysis 11
Laboratory Studies of Flounder , 12
Experimental Design 12
Collection and Holding 15
Exposure System 16
Tissue Preparation and Histological Examination 17
Data Collection and Analysis 19
Field Studies of Oysters 20
Experimental Design 20
Indigenous Oyster Studies 20
Caged Oyster Studies 20
Field Studies of Flounder 23
Chemical Analyses 23
Experimental Design 23
Organic Analyses 25
Inorganic Analyses 31
Short-Term Biological Tests 33
Sample Preparation 34
Salmonella/Microsome Assay 34
Chinese Hamster V79 Metabolic Cooperation Assay 35
PART III. RESULTS 38
Sediment Chemistry 38
Laboratory Studies of Oysters 39
Test I 39
Test II 42
Histopathology of Oysters Exposed to BRH Sediment 42
Noninfectious Lesions 42
Degenerative Lesions 49
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CONTENTS (Continued)
Laboratory Studies of Flounder 49
Test I 49
Test II 50
Histopathology of Flounder Exposed to BRH Sediment 52
Noninfectious Lesions 53
Degenerative and Necrotic Lesions 57
Infectious Lesions 60
Field Studies of Oysters 60
Oysters Indigenous to BRH 60
Oysters Caged In BRH 61
Oysters Caged in CLIS 61
Field Studies of Flounder 62
Central Long Island Sound (CLIS) 62
Black Rock Harbor (BRH) 62
Oyster Tissue Chemistry 63
Flounder Tissue Chemistry 63
Short-Term Biological Tests 63
Salmonella/Microsome Mutagenicity Test 63
V79/Metabolic Cooperation Assay 68
Preliminary Ames - V79/MC Results With
Fractionated Solvent Extracts 71
PART IV. DISCUSSION 72
American Oyster 73
Winter Flounder 80
Field Studies 83
Human Health 85
Future Studies 86
PART V. CONCLUSIONS 89
ACKNOWLEDGMENTS 91
REFERENCES 92
PLATE LEGENDS 100
APPENDIX 1. Oyster Tumor Data 104
APPENDIX 2. Winter Flounder Tumor Data - Test I 109
APPENDIX 3. Sediment Chemistry Data Ill
APPENDIX 4. Pathology Data Base 116
APPENDIX 5. Immune Repression Test Summary 127
vi
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LIST OF FIGURES
No. Page
1 Central Long Island Sound Reference (REF) Sediment,
BRH Dredge, and BRH Disposal Sites 2
2a Oyster Dosing System 8
2b Oyster Dosing System • 9
3 Uptake of PAHs and PCBs From BRH Sediment by Mussels 13
4 Flounder Exposure System 18
5 Black Rock Harbor Field Stations 21
6 Central Long Island Sound Disposal Site and Field Station
Locations 24
7 Results of Ames Testing with Salmonella Strain TA98 and
S-9 Metabolic Activation 64
8 Results of Ames Testing with Salmonella Strain TA100 and
S-9 Metabolic Activation 65
9 Results of Ames Testing with Salmonella Strain TA102 and
S-9 Metabolic Activation 66
10 Results of Ames Testing with Salmonella Strain TA104 and
S-9 Metabolic Activation 67
11 Effects of Extracts of Reference Sediment on Cell Survival
and Mutant Recovery in Two Independent V79/MC Assays 69
12 Effects of Extracts of Black Rock Harbor Sediment on
Cell Survival and Mutant Recovery in Two Independent
V79/MC Assays 70
vii
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LIST OF TABLES
No. Page
1 Winter Flounder Exposure Regimens 14
2 Oyster Tumor Induction Summary Test 1 40
3 Winter Flounder Tumor Induction Summary Test 1 51
Appendix 1. Oyster Tumor Data
BRH 30 Days Test I 104
BRH 60 Days Test I 104
BRH 60/30 Days Test I 105
BRH 30/60 Days Test I 105
BRH 30 Days Exposure Field Test I 106
BRH 60 Days Exposure Field Test I 106
BRH 30 Days Exposure Field Test II 107
Tumor Occurrence in CLIS-Deployed Oysters 107
Percent Survival 30 and 60 Day Caged Oysters 108
Appendix 2. Winter Flounder Tumor Data - Test I
Percent Tumors for Young-of-Year Flounder 109
Percent Tumors for One-Year-Old Flounder 109
Winter Flounder Survival Test I 110
Appendix 3. Sediment Chemistry
Contaminant Concentrations in Black Rock Harbor (BRH)
and Reference (REF) Sediments Ill
viii
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PART I. INTRODUCTION
The National Cancer Institute (NCI), through a collaborative program
on environmental cancer with the U.S. Environmental Protection Agency (EPA),
awarded the U.S. EPA Environmental Research Laboratory in Narragansett,
Rhode Island (ERL/N) a two-year research grant to study carcinogenic
effects of Black Rock Harbor (BRH) sediment on molluscs and fish. The
NCI/EPA project was based on preliminary studies conducted under an ERL/N-
U.S. Army Corps of Engineers "Field Verification Program" (FVP) (Gentile
and Pesch, 1987; Gentile and Scott, 1986). Results of those studies sug-
gested a relationship between tumor occurrence in the American oyster
(Crassostrea virginica, a marine filter-feeding bivalve), and the winter
flounder (Pseudopleuronectes americanus, a demersal fish species), and
exposure to contaminated BRH sediment.
Black Rock Harbor is a polluted harbor with sediment contamination
considered typical of industrial cities along the Northeast Atlantic
coast. Sediment dredged from BRH has been deposited in a disposal area
in Central Long Island Sound (Figure 1).
Preliminary exposure tests with oysters demonstrated the presence of
kidney tumors originating in epithelial cells in two of ten animals exposed
for 30 days to BRH sediment suspended in the water column. A reproductive
tract tumor was also found in BRH-exposed oysters. In similar tests with
winter flounder, pituitary alterations were observed that included a pre-
sumptive cellular proliferation in the anterior glandular area of one animal.
Necrotic, acidophilic-cell foci were observed in pituitary gland of all fish
(n = 6) examined. In addition, effects on thymus tissue and the central
nervous system were observed, as were symptoms typical of fin erosion.
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BRIDGEPORT
FVP STUDY
REACH
i1
n
I'
• I
//MAINTENANCE
; i /"/DREDGING
N
t
NEW
HAVEN
BRIDGEPORT
BLACK ROCK
FVP
DISPOSAL
SITE
SOUTH REFERENCE
• SITE
Figure 1. Central Long Island Sound Reference (REF) Sediment, BRH Dredge, and BRH Disposal Sites
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The capacity of BRH-dredged sediment to induce malignant tumors in aquatic
species has important implications for comparative oncology and public
health. Accordingly, the NCI/EPA collaborative project was proposed to
provide a more rigorous evaluation of the tumorigenic properties of BRH
sediment.
The objective of the laboratory studies was to replicate previous
experimental evidence that prolonged exposure to contaminated BRH-dredged
sediments caused tumors in oysters and winter flounder and to analyze
the data statistically. Effects of contaminant uptake and accumulation
in oysters as a result of water-column filter-feeding were assessed after
30- and 60-day periods of continuous exposure and post-exposure. Exposure
studies with fish investigated long-term effects of direct contact with
BRH-dredged material and consequences of trophic transfer by feeding BRH-
sediment-contaminated blue mussels (Mytilus edulis) to the experimental fish.
Field studies were conducted to determine tumor prevalence in indig-
enous oysters and flounder, on site at BRH and at the disposal area in
CLIS, for compariston with laboratory results. Also, oysters were deployed
at stategic locations in BRH, CLIS, and a reference area and analyzed to
determine occurrence of tumor induction under natural environmental
conditions.
Although ERL/N FVP studies had failed to detect a positive contami-
nant signal one meter above the disposal mound one year post-disposal, an
on-site oyster study was conducted because (1) commercial oyster beds in
Long Island Sound are commonly at 40 and 60 feet in depth, but also extend
to depths of 80 feet, the depth at the disposal mound, and (2) commercial
oyster fisherman routinely deposit oysters from polluted areas on the
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bottom of Long Island Sound at depths of 80 feet to depurate them and
then recover them to sell for public consumption (Personal communication,
Rhoades, NOAA, Milford Laboratory; William Nelson, SAIC, ERL/N).
Chemical analyses were intended to support histopathological evalua-
tions by providing information on the presence and levels of known carcin-
ogens and their metabolites in various biological tissues and/or carcinogens
and mutagens present in sediment. Data are to be assessed for significant
correlations between concentrations of tissue contaminants and tumor induc-
tion. Chemical analyses are to follow a "broad brush" approach, but will
focus on promising leads derived from histopathological and biological
testing.
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PART II. METHODOLOGY
Laboratory research methodology followed the recommended ERL/N
Quality Assurance procedures and American Society for Testing Materials
(ASTM) "Standard Practice for Conducting Bioconcentration Tests with
Fishes and Saltwater Bivalve Molluscs" (ASTM, 1984), where practical.
Sediment Collection and Preparation
Sediment was collected from two locations using a Smith-Mclntyre
grab sampler (area sampled is 0.1 m^). Reference (REF) sediment was
collected in Central Long Island Sound (CLIS) at the south reference
site (Figure 1) on April 29, 1985. The reference site, also used in
ERL/N's Field Verification Program (FVP), is located approximately 700 m
south of the southern perimeter of the FVP disposal area. Contaminated
sediment was obtained from the channel of Black Rock Harbor (BRH) near
navigation marker buoy 12 on April 7, 1985.
Sediment collected on each date was brought to the laboratory, press
sieved wet through a 2-mm mesh stainless steel screen, homogenized and
stored at 4°C until used. REF and BRH sediment to be used in oyster
tests was stored in one-gallon, wide-mouth glass containers. Sediment
for the flounder tests was stored in the barrels used during collection,
and homogenized at the time of testing. For one of three fish exposure
treatments, sediment from the CLIS REF site and from BRH was mixed 1:1
(50% REF to 50% BRH).
Each type of sediment was analyzed chemically, the results are
given in Appendix 2 (Rogerson et al., 1985).
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Laboratory Studies of Oysters
Experimental Design
The laboratory studies of oysters assessed four regimens of exposure
to each type of sediment. The replicate 30- and 60-day REF and BRH
exposure treatments assessed the biological response up to and beyond
the 31 days required for tumor induction reported in preliminary tests
(Gardner and Yevich, 1986). The replicate treatments of 30- and 60-day
continuous exposures to REF and BRH suspended particulate, followed by
60 and 30 days of post-exposure holding, respectively, assessed latent
pathological effects and/or trends in neoplastic lesion development.
Each of the four exposure regimens consisted of three replicates.
A minimal sample size of n = 150 (n = 50/replicate) was used for each
continuous exposure regimen (30 and 60 days) and each exposure/post-
exposure regimen (60/30 and 30/60 days). The total number of oysters
exposed to each type of sediment was 600, for a combined total of 1200
animals. Upon termination of each test, oysters were sampled for histo-
pathological and chemical evaluations.
The number of oysters collected for histology was dependent upon
the test organism survival rates. Thirty-five oysters from each of
three REF (n = 105) and three BRH (n = 105) replicates in four treatments
(REF n = 420; BRH n = 420) were allocated for histology (total n = 840),
while 15 animals from each replicate (n = 45/treatment or total n = 360)
were archived for chemical analyses, assuming 100% survival. Twenty-five
oysters were processed for histology and ten for chemistry as unexposed
controls.
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Collection and Holding
Adult oysters were obtained from the Cotuit Oyster Company (Cotuit,
Massachusetts). All the oysters used in laboratory testing were approx-
imately four years in age. Oysters used in these studies were removed
from Cotuit oyster bed 6 on August 15, 1985. Testing began on September 10,
1985, following a short holding period for laboratory temperature acclimation.
Exposure System
Dosing. In the oyster tests, two identical dosing systems were
constructed to deliver REF and BRH sediment to three replicate chambers
(Figure 2a). Each chamber utilized a transmissometer (SeaTech, 10 cm)
coupled with a microprocessor feed-back device to control a dosing airlift
(developed by Sinnett and Davis, 1983; Figure 2b). The dosing apparatus
consisted of a modified 6-L separatory funnel with an airlift and an 18-L
polycarbonate reservoir. Sediment slurry was constantly circulated
between the dosing funnel and the reservoir with the airlift system.
Seawater and sediment slurry were mixed in a common chamber and then
distributed to the three replicate chambers using a 3-way splitter to
deliver equal amounts of sediment slurry to each replicate. Using the
above technology, along with modifications for the present tests, the
REF and BRH suspended sediment concentration in the water column was
maintained at 20 mg/1.
Transmissometers were placed in one REF and one BRH replicate chamber
(Figure 2b) to monitor sediment delivery to the three replicate chambers.
Sediment loading of each replicate was confimed by dry weight measurements
of suspended sediment. Approximately 90 kg of BRH sediment was required
for an oyster exposure test.
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oo
vent hole
resuspension
tube
resuspension
tube
dosing airlift
reservoir
/airlift
seawater
pre-mix chamber
18 L reservoir
6L sep. funnel
dosing funnel
3-way splitter
to tanks
Figure 2a. Oyster Dosing System
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microprocessor
solenoid
valve
low pressure
air supply
1
chart
recorder
dosing
system
152 L aquarium
transmissometer
Figure 2b. Oyster Dosing System
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Exposure chambers. Oysters were exposed to doses of sediments in 6
all-glass, 152-L (50-gallon) aquaria (91 L x 44 W x 38 H cm) (Figure 2b).
Three replicate chambers were used for REF sediment and 3 for BRH sediment.
Seawater flowed through each chamber at a rate of 0.65 L/min; that rate
yielded six daily turnovers of seawater. For the exposure tests, the
seawater was filtered in the laboratory and for the post-exposure periods,
the seawater was unfiltered. The ambient seawater salinity was 32 °/oo
and the temperature was 20°C.
A uniform suspension of test sediments was achieved by aligning 12-
inch-long airstones near one side wall of each test container. Air
bubbles provided a continuous lifting, circular motion that resulted in
constant water movement, sediment suspension and aeration without disrupting
oysters. Suspended organic detritus and living bacteria contained in BRH
and in reference sediments served as the nutritional source during the
exposure experiments. Nutrition during post-exposure holding was derived
from natural food items (i.e., phytoplankton and bacteria) contained in
unfiltered seawater.
Oysters were held at three levels within each chamber during exposure
using a specially designed tier (Figure 2b). The tier was constructed of
nine polyethylene grids (34 cm x 26 cm; with 1/4-inch-square openings)
held in position by 1/2-inch-diameter threaded polyethylene rods with
hexnuts. Horizontally, the three grids at each level were spaced 9 cm
apart and approximately 3 cm from the inside walls of the chamber.
Tissue Preparation and Histological Examination
Upon termination of experimental exposures, the oysters were opened
and fixed according to procedures established at ERL/N (Yevich and Barszcz,
10
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1981). Briefly, those procedures included fixation of tissues in Kelly's
fixative for 16 hours followed by washing overnight in a water bath
tissue washer. Oysters were sagittally sectioned through the visceral
mass during the first one-half hour of the fixation process. Adductor
muscle was cut on the same longitudinal plane, although severence of the
primary sagittal section was finalized only after complete fixation and
during final trimming. A centrally located transection was then made,
as well as parasagittal sections when tissue mass was large. Sections
for embedding were trimmed to a thickness of approximately 3 mm. Thus,
a minimum of four tissue blocks were generated for each oyster. Tissues
were then trimmed, paraffin embedded, cut at 6 y and stained with Harris
Hematoxylin and Eosin for examination. Heidenhain's stain was used in
special cases for observation of connective tissue continuity.
Data Collection and Analysis
Observations of tumorigenic activity and other characteristics of
each oyster were recorded in a logbook and in a data base developed at
ERL/N, especially for the NCI/EPA collaborative effort. (A description
of the National Cancer Institute data base design developed by J. Rosen
and D. Sheehan is contained in Appendix 4.)
The experimental design used (i.e., three replicate aquaria contain-
ing fifty animals for each exposure) allowed testing of tank-to-tank
variability and accounting for such variability in statistical analyses.
In the absence of tank-to-tank variability, the sample size of n = 150
animals per exposure was adequate to detect an absolute increase of 20%
over control if the control incidence was 10% or less. If tank varia-
bility was apparent, then the sample size was adjusted accordingly to
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incorporate tank-to-tank variability (Feder and Collins, 1980).
Laboratory Studies of Flounder
Experimental Design
Winter flounder were exposed to REF and BRH sediments in two types of
treatments: the flounder were in direct contact with both REF and BRH
bedded sediments and were fed with mussels (Mytilus edulis) that had
been exposed to both REF and BRH sediments. Blue mussels were selected
as a vector of BRH contaminants via trophic transfer because of their
demonstrated rapid uptake and bioaccumulation of BRH materials. Based
on the FVP chemical characterizations (Rogerson et al., 1985), blue
mussels reach steady-state bioaccumulation of most inorganic and organic
materials contained in BRH-dredged material in eight days (Figure 3).
The experiment had a 2 x 2 x 3 factorial design, summarized in Table 1.
There were three levels of direct exposure to bedded sediment: 100% REF sed-
iment, a 50%/50% mixture of REF and BRH sediment, and 100% BRH sediment as
bedded sediment. Two regimes of feeding sediment-exposed mussels to winter
flounder were used: feeding mussels exposed to REF sediment (C = Clean) and
feeding mussels exposed to BRH sediment (CT = Contaminated). For each of
these six feeding and exposure conditions, flounder of two age classes (0-1
year and 1-2 year) were exposed. There were four replicate exposure chambers
(n = 4) for each treatment of 1-2 year condition (n = 3 for 0-1 year). For
the 0-1 year class of winter flounder, each replicate chamber contained 15
individuals (g = 15). Each replicate chamber contained 3 individuals (g = 3)
of the 1-2 year class. The feeding and exposure treatments are hereafter
referred to as REF/C, 50/C, BRH/C for REF-exposed mussels used as food
and REF/CT, 50/CT and BRH/CT for BRH-exposed mussels used as food.
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10 000 -i
1000 -
100 -
10 -
1.0
Uptake of Parent PAH & PCBs from
Black Rock Harbor Sediment by Mussels
0 Day
Control
7 Dag
Exposed
14 Dag
Exposed
28 Day
Exposed
166 176 202 228 252 PCBs
PAH Mol. Wt. & PCBs
Figure 3. Uptake of PAHs and PCBs From BRH Sediment by Mussels
13
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Table 1.
Winter Flounder Exposure Regimens *
AGE FEED
0-1 YR C
CT
1-2 YR. C
Bedded Sediment
REF 50/50 BRH
n=4 n=4 n=4
g = 15 g = 15 g = 15
n=4 n=4 n=4
g = 15 g = 15 g = 15
n=4 n=4 n=4
g=3 g=3 g=3
CT n=4 n=4 n=4
g = 3 g = 3 g = 3
* There were n = 3 replicate test chambers, each containing g - 15
fish aged 0-1 year and n = 4, g = 3 fish aged 1-2 years. C = Clean
(exposed to REF sediment) and CT = Contaminated (exposed to BRH sediment)
mussels were used as feed in the laboratory studies of flounder.
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Each of the six treatments in the 0-1 year class of winter flounder
had a total of n = 60 (15/replicate) fish per treatment, or n = 360 total
of 0-1 year winter flounder. Each of six treatments in the 1-2 year
class of fish had three individuals in four replicates for n = 12 per
treatment. Each treatment was replicated six times to provide a total of
n = 72 animals. The total number of winter flounder in six treatments of
the experimental design was n = 432. Winter flounder testing presently
ongoing is expected to be continued for 8 months, or until approximately
July 1, 1987.
Collection and Holding
Winter flounder were collected between the months of April and
November, 1985, in the Narrow River, South Kingstown Salt Pond, and
lower Narragansett Bay with a beach seine and skiff Otter Trawl. Beach
seine (a nylon 1/4-inch-mesh 100 ft. seine) was used primarily for col-
lecting young-of-the-year (0-1 years; 2-9.5 cm) winter flounder. The
beach seine and a 20 ft. x 22 ft. Otter Trawl with a one-inch cod end
were used to collect 1-2 year class (10-14 cm) winter flounder. All
specimens collected were brought to the laboratory in containers of
aerated seawater and then acclimated for at least two weeks prior to
testing. After a minimum holding period of one week, the winter flounder
were separated according to sizes representative of 0-1 class and 1-2
class animals.
Blue mussel (Mytilus edulis) shucked and chopped served as their
maintenence diet during laboratory holding. Mussels used in flounder
tests were collected or purchased from a routinely used ERL/N source.
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Exposure System
Bedded sediment. Homogenized REF and BRH sediment as 100% REF, 100%
BRH and a 50%/50% REF/BRH mixture were bedded in identical glass test
compartments for continuous exposure of winter flounder. A test sediment
depth of approximately 7 cm was maintained during exposure. Sediment
was added as required to maintain that depth. (Fish activity caused
sediment resuspension and loss via flow-thru seawater turnover.)
Mussels. A system was designed for dosing mussels that incorporated
the same technological features utilized for dosing oysters. Blue mussels
exposures to REF and BRH suspended sediment were continuous for seven days
to provide a contaminated food source for the winter flounder. After
seven days, REF- and BRH-sediment-exposed mussels were placed into frozen
storage (-20°C) until required. The uptake of BRH contaminants by blue
mussel was monitored periodically.
Diets of REF- and BRH-mussels were prepared by removal of byssal
threads, shucking from the shell and then chopping with a stainless
steel blender. Once mussels were chopped to the desired consistency,
the preparation was poured into polyethylene freezer icecube trays and
placed in frozen storage (-20°C) until needed. Consistent cube size
helped to assure known feeding quantities for individual fish in each
exposure chamber. Fish were fed a daily diet of 20% of body wet weight.
Ten kilograms of BRH sediment was required for the mussel exposures
before the mussels were fed to winter flounder. The quantity of BRH-
dredged material needed in these tests was based on the mussel wet weight
needed to provide a minimum diet that was equal to 20% of fish wet weight.
A minimum of 24 cubic feet (0.68 cubic meters) of BRH sediment was used
for these tests.
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Exposure chambers. Test chambers for bedded sediment fish exposures
were constructed of glass (Figure 4). For young-of-the-year winter floun-
der, those chambers had outside dimensions of 42L x 23W x 21H cm. The
bottom was 6-mm plate glass while the sides and ends measured 3 mm thick.
The dimensions of the chambers for 1-2 year winter flounder were
28.5L x 28.5W x 21H cm; they were constructed of 3 mm glass. A 2.5-cm-
diameter hole was cut in one side of each chamber at a height of 16 mm,
providing a water level at the 15.3 mm mark. Four supporting legs
13 x 13 mm were placed at each outside corner on the bottom plate.
Chambers were assembled using a clear silicone cement (Dow Corning General
Purpose No.8641).
Seawater was gravity-fed to each test chamber through a 2-mm I.D.
polyethyelene tube connected to a central supply at a flow rate of 180
ml/min. The distribution container was constructed of a 3-inch-diameter
schedule 40 PVC pipe with a 1-3/4-inch-wide longitudinal section removed.
Holes 3.1 mm in diameter drilled into the PVC pipe at the bottom received
3.2-mm O.D. polyethylene tubing and were the sole seawater supply to
each individual chamber. The seawater was filtered twice, first in the
central laboratory filter station and secondly prior to entering the PVC
pipe distribution chamber with a standard Aquapure filter.
Tissue Preparation and Histological Examination
The requirement of long-term exposures imposed severe restraints on
a sub-sampling routine in favor of a single eight-month sample, although
moribund animals were sub-sampled in the interim. Major tissues and
organs were examined grossly and microscopically for lesions.
17
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oo
Figure 4. Flounder Exposure System
-------
Winter flounder being tested were examined daily for visible signs
of distress, abnormal behavior, and atypical coloration. Flounder were
fixed in Dietrich's Fixative when found moribund or at the termination
of the test. Fixation procedures established at ERL/N were followed.
Specimens were placed in fixative whole, removed after a short period of
fixation (10 min.) to be sagitally and parasagittally sectioned. One
of every ten animals was transectioned into 3-mm wide tissue sections.
Trimmed tissue was decalcified as necessary and then washed in a water
bath overnight, embedded in paraffin, cut at 6 u and stained with Harris
Hematoxylin and Eosin. Special stains used included Heidenhain's and
Brown and Brenn.
Data Collection and Analysis
The duration of the experiment was 131 days for 0-1 year class
flounder and 92 days for 1-2 year class flounder. The number of moribund
or dead fish removed and fixed in Dietrich's before the test's termination
was 153 for 0-1 year animals and 75 for the 1-2 year class.
The experimental design allowed estimation of tumor incidence in
relation to exposure conditions, feeding regimes and age class. Analysis
of variance techniques were used to test for equality of exposure condi-
tions, feeding regimes and age classes. Tests of interactions among the
three factors were performed. Those tests included appropriate pairwise
comparisons and other comparisons of interest.
19
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Field Studies of Oysters
Experimental Design
Oysters indigenous to BRH proper were collected and analyzed histo-
pathologically and chemically as a one-time spatial assessment of tumor
prevalence in animals near the BRH entrance, an area presently closed to
public shellfishing. Oysters were also deployed at two stations in BRH
and a reference station to test effects of 30-day and 60-day exposures.
Indigenous Oyster Studies
Beds of oysters in the BRH area were identified with the assistance
of the Connecticut Department of Marine Resources. The oyster beds began
on the south shore of the harbor at a location 475 m from a fixed navigation
lighthouse marking the entrance to BRH (Figure 5). The oyster bed continued
along the south shore into the harbor entrance approximately 225 m. The
northernmost extreme of those oyster beds was about 1230 m from Buoy 12.
The Connecticut Department of Marine Resources also assisted in the collec-
tion of animals from the selected site; 99 indigenous oysters were col-
lected using an oyster dredge on May 8, 1985. Of those, 84 were processed
for histopathological examination and 15 were archived for chemical
evaluations. Comparisons with laboratory-derived effects were made where
appropriate.
Caged Oyster Studies
Oyster were deployed in field tests inside polypropylene pipet
baskets (either 15cm x 15cm or 23cm x 23cm) as a standard test chamber.
Ten oysters per basket served as replicate tests. For deployment purposes
replicates were placed end-to-end in a custom-made "tube" constructed
20
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BRIDGEPORT
OYSTER
M COLLECTION
SITE
MAINTENANCE
DREDGING
FVP STUDY
REACH
LIGHTHOUSE
SLACK
ROCX
HAR80R
Figure 5. Black Rock Harbor Field Stations
21
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from a 2-inch-mesh fish net. The nets were shaped to the pipet test
chambers at the time of deployment by sewing a seam along the longitudinal
axis and then tying off each end. Each "tube" could then be suspended
from a fixed above-surface object (as was done in BRH) or attached to a
subsurface device for use in deep water (such as in CLIS).
Black Rock Harbor. Two field stations were selected for experiments
in Black Rock Harbor. They were Buoy 12, located at the south side of
the channel, and North Dock, on the inland side of the channel (Figure 5).
North Dock was located 160 m above a sewage outfall near Cove Marina
while Buoy 12 was located 300 m below the outfall. The two stations
were approximately 460 m apart. A reference station was chosen at the
Milford, CT, NOAA National Marine Fisheries Laboratory; it was selected
because its salinity, conductivity, and temperatures were similar to
those at the two BRH stations.
Oysters deployed at the two BRH stations and the Milford reference
station tested effects of 30-day and 60-day exposures on two occasions,
referred to as BRH Caged Oyster Test I and BRH Caged Oyster Test II.
The first deployment began on May 25, 1985, with recovery on June 25 (30
days) and 23 July (60 days). The second deployment began on August 12,
1986, with recovery and termination of the in situ studies on September 12
(30 days). In each test, five replicates (n = 50) per treatment (i.e.,
30-day or 60-day deployment) (total 100 oysters) were deployed at each
station. Three hundred oysters were utilized in each test (i.e., Test I
and Test II).
Central Long Island Sound. Four Central Long Island Sound (CLIS)
field stations were selected for testing based on their location relative
22
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to the FVP disposal site for BRH-dredged material. They had been selected
by the U.S. Army Corps of Engineers in the FVP Study (Figure 6). The
stations used were (1) the center of the mound, (2) 400 m east of the
center, (3) 1000 m east, and (4) the South Reference Site, approximately
3 km south of the disposal site. In the test, five replicates of n = 10
caged oysters (50 oysters) were deployed at each station for a period of
36 days. Divers deployed the oysters within one meter of the sediment-
water interface on 7 July 1985 and recovered them on 13 August 1985.
Field Studies of Flounder
One hundred winter flounder were collected by otter trawls in BRH
proper [using the R/V Shang Wheeler (National Marine Fisheries Service,
Milford, CT) and a smaller workboat (Boston Whaler)] and at the CLIS
disposal mound (using the R/V Shang Wheeler aided by Loran C navigation).
Winter flounder were also collected at a reference site at Fox Island
in Narragansett Bay, RI. All the flounder had random ages and meristic
parameters.
Chemical Analyses
Experimental Design
Four general areas of chemical support were identified: (1) routine
chemical analyses, (2) additional chemical characterization of BRH sedi-
ment, (3) special analyses or studies, and (4) extraction and fractiona-
tion of BRH sediment for short-term, biological testing. The required
chemical analyses were done at ERL/N where feasible. Over five hundred
laboratory-exposed oysters have bee" archived for chemical analysis;
23
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BLACK ROCK
HARBORS*
1
o
FVP T
DISPOSAL'
SITE
1
Urn
SOUTH REFERENCE
• SITE
72
410
410
92.S 72
1.9
0 9OOm
i.O
92.0 72
FVP OI8PO8AI
«OON
•40OW CNTR 20
•
5003
919 72
SITE
)E 4OOC
910
•
IOOOE
Figure 6. Central Long Island Sound Disposal Site and Field Station Locations
-------
laboratory-exposed winter flounder are yet to be obtained for chemical
analysis.
Two experimental approaches will be used to assess correlations
between BRH sediment and prevalence of neoplastic lesions in marine
organisms. Major classes and of carcinogens known to occur in BRH
sediment, such as polycyclic aromatic hydrocarbons, their metabolites
and certain heavy metals, will be analyzed in exposed tissues and their
correlation with tumor incidence will be determined. For the experimental
controls, sediments from other sources, either contaminated with or free
of these carcinogens, will be used in exposure systems to study the
residue-effect relationship.
Chemical analyses followed procedures adopted by the National
Institutes of Health where those methods could be adapted to animal
experimentation.
Organic Analysis
Extraction. Each sediment sample was mixed using a stainless steel
spatula, and 1-3 g was removed to determine a wet-to-dry-weight ratio.
That portion was placed in a preweighed aluminum pan, which was weighed
again and then dried to a constant weight at 95°C. About 10-20 g of the
original wet sediment was placed into a stainless steel centrifuge bottle
for extraction.
Twenty-five ml of acetone was added to each sediment and the samples
were homogenized at 25,000 rpm for 40 sec using a Polytron (Brinkman
Corp.) with a stainless steel tip and brass bearings. After the first
homogenization, the samples were centrifuged at 10,000 rpm for 5 minutes
in a refrigerated centrifuge at 4°C. The liquid phase (acetone-water)
25
-------
was decanted into a 1-liter separatory funnel containing 150 ml of deion-
ized water that had previously been extracted with freon (1,1,2-trichloro-
trif luoroethane). A second 25-ml volume of acetone was added to the
sample, and the homogenization and centrifugation were repeated. The
supernatant was again added to the separatory funnel. This procedure
was repeated twice more using freon instead of acetone, and the freon
extracts were also added to the separatory funnel. The funnel was shaken,
and the freon layer was drawn off into a 500-ml Erlenmeyer flask. This
partitioning was repeated 2 more times using 50 ml of freon each time.
The freon extracts were combined in the Erlenmeyer flask.
Separation. The freon extracts were passed through a cleanup column.
The column, which was 2 cm x 25 cm with a 500-ml reservoir, contained 50 g
of Biosil A silicic acid (100-200 mesh, BioRad) that had been fully acti-
vated by heating at 120°C for 6 hours. The column was rinsed with 100 ml
of methylene chloride followed by 100 ml of freon. Sample extract was
added to the column at full volume (no volume reduction), and helium
pressure was used to increase the flow rate. After the sample level had
reached the top of the column, 150 ml of methylene chloride was added to
and eluted from the column. All of the column eluent was collected in a
round bottom flask. This extract was volume reduced and solvent exchanged
to hexane and brought to 1 ml in a tube heater. The sample was then
ready for fractionation on the separation column.
Chemical class separations were achieved on a 0.9 cm x 45 cm column
containing 11.5 g of BioSil A silicic acid (100-200 mesh) that had been
fully activated and then 7.5% deactivated with water. This deactivation
step was accomplished by adding an appropriate amount of water to the
26
-------
silica in a glass bottle and rolling it overnight. Consistent column
activity was assured by testing each batch. A mix of PCB and PAH isomers
were separated on a column made up of the newly deactivated silica and
the relative distributions of compounds in 3 column fractions were checked.
The column was cleaned with 50 ml of methylene chloride and 50 ml
of pentane before the samples were added. All sample extracts were
charged to the separation column in 1 ml of hexane and then an additional
container rinse of 1 ml of hexane was also added. The first fraction
(fl) was eluted from the column with 50 ml of pentane. The second frac-
tion (f2) was eluted using 35 ml of 20% methylene chloride in pentane.
A third fraction (f3) was then eluted with 35 ml of methylene chloride.
The fl fraction was treated with copper powder to remove sulfur.
Each fraction was then volume reduced in a round-bottom flask using a
heating mantle and a Kuderna-Danish evaporator with a 3-ball Snyder
column. The sample was solvent exchanged into about 5 ml of hexane and
transferred to a 10 ml concentrator tube fitted with a micro-Snyder
column. An ebulator and 0.8 ml of heptane was added to each tube, and
the fractions were volume reduced to 0.8 ml in a tube heater with a foil
cape. The ebulator was removed and rinsed into the sample with heptane,
which was brought up to 1 ml.
The fl fractions were analyzed for PCBs using electron capture gas
chromatography. The f2 fractions were spiked just before injection with
internal injection standards (2-fluorobiphenyl and 3-fluorofluoranthene)
and then analyzed for polycyclic aromatic hydrocarbons (PAHs) by gas
chromatography-mass spectrometry (GC-MS).
The f3 fractions contained aroe-'tic ketones, quinones, and carbazoles.
27
-------
These extracts were transferred to 25-ml centrifuge tubes and spiked
with internal standards (9-xanthrone and 7,12-dihydro-l-
methylbenz(a)anthracene-7,12-dione). One ml of 90:10, N,N-dimethyl
formamide : methanol was added and the tube was sealed and shaken. The
layers were allowed to separate and then the heptane layer was withdrawn
and discarded. One ml of pentane was added and the process was repeated.
The pentane was removed and discarded and then 15 ml of deionized water
and 5 ml of pentane were added. The sample was shaken and allowed to
settle. The pentane layer was withdrawn and saved in a 25-ml concentrator
tube. An additional 5 ml of pentane was added and the process repeated.
This pentane was combined with the first extract and then concentrated
to 0.5 ml using nitrogen blowdown. The sample and a methylene chloride
rinse of the concentrator tube were combined and then analyzed by GC-MS.
Analysis. The fl fractions were analyzed for PCBs. For these
analyses, 1 ul of each extract (usually at a volume of 1 ml) was injected
into a Hewlett Packard 5840 gas chromatograph equipped with an electron
capture detector and a 30 m DB-5 fused silica capillary column (J + W
Scientific). Helium was used as the carrier gas at a flow rate of about
1.5 ml/min; the flow of a 95:5 mixture of argon:methane to the detector
was 35 ml/min. The oven temperature was held at 80°C for 4 min and then
programmed from 80 to 290°C at 10°C/min. The injector temperature was
270°C, and the detector was maintained at 300°C.
Concentrations were calculated as Aroclor 1254. The amount of
Aroclor 1254 was measured by comparing the sums of the heights of seven
peaks in the sample chromatograms to those of the same peaks in standards
of Aroclor 1254 that were analyzed a*- the beginning and end of each day.
28
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Gas chroroatographic analyses were quality assured in several ways.
Responses of the standards analyzed at the beginning and end of each day
were compared. If the relative percent deviation of these results was
greater than 10%, all of the samples analyzed that day were reanalyzed.
Standard curves were also periodically generated on each instrument to
assure that the response of the detector was linear over the range of
concentrations measured. Also, a known amount of octachloronaphthalene
(OCN) was spiked into each sample at the beginning of the sample analysis
procedure. The recovery of this compound averaged 85% _+ 18% for this
study. The triplicate analysis of a sediment sample indicated that the
precision of our PCB analysis procedure was 4; 8.6%.
The f2 and f3 fractions were analyzed using a Finnigan 4531 quadrapole
gas chromatograph-mass spectrometer (GC-MS) which included a Nova 3
computer with Incos software and a CDC 96-megabyte drive. The Finnigan
GC was operated with a capillary column in the splitless injection mode.
The split flow was approximately 50 ml/min and the septum sweep flow was
approximately 2 ml/min. Both flows were suspended for 1 min just before
the time of injection. The DB-5 (J + W Scientific) fused silica column
was 30 meters in length with a bore of 0.25 mm and a film thickness of
0.25 y. The GC oven was held at an initial temperature of 50°C for 2
minutes, ramped to 330°C at 10°C/min and held for an additional 9 minutes.
The last 40 cm of the column passed through the transfer line oven
area, which was heated to 300°C, and then to within 1 cm of the source
volume in the source area of the MS, which was maintained at 150°C. The
source was operated in the electron impact mode at 70 eV. The filament
emission current was 200 yA and the Carious source potentials were adjusted
29
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to produce a spectrum of decafluorotriphenylphosphine (DFTPP) which
would meet the specifications detailed by Eichelberger et al. (1975).
The MS was scanned from 15 to 650 amu in 1 sec while collecting 10 cen-
troid samples per peak. The continuous dynode electron multiplier was
operated near 1550 V, and the preamplifier sensitivity was set to 10~8 A/V.
Raw data for each sample was stored on disk.
On each day that samples were to be analyzed, the mass scale of the
MS was calibrated by emitting perfluorotributylamine into the source,
acquiring data, and running the software calibration routine. Following
the calibration, a solution of standards was analyzed. This allowed the
determination of the response of the standards, their retention time and
the spectrum of the DFTPP. After all of the samples for the day had
been run, another standards run was made. On days when f2 fractions were
analyzed, the standards were a mixture of PAHs of various molecular
weights. On days when f3 fractions were analyzed, the standards were a
mixture of ketones, quinones, and carbazoles.
Quantitation with the GC-MS involved using the data system to measure
the areas of selected peaks using extracted ion current profiles (EICPs).
In all cases, the peaks were manually integrated; i.e., the baselines
were manually determined on the EICP displayed on the terminal. PAH
quantitation was accomplished using the standards that had been added
just before injection. The f3 compounds were quantified against standards
that were added just prior to the DMF partitioning step. Relative response
factors for those compounds were calculated against standards representing
each chemical group and a range of molecular weights within groups.
That information, the amount of standard added to the sample, and the
30
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dry weight of the original sample were used to calculate concentrations
of the compounds of interest. The calculations were performed by a
Fortran program running on a PDF 11/70 computer, after the quantitation
lists had been transmitted from the Nova 3 to the 11/70 via an RS232
data line.
Inorganic Analysis
Sample Preparation. The initial samples (scraped from the surface
of the sediment) were homogenized and subsampled for analysis. The grab
samples were sectioned, homogenized, and then subsampled for analysis.
The sections analyzed at this time have been 0-2 cm (surface sample) and
5-10 cm (subsurface sample). Aliquots of the wet sediment (approximately
5 g) were transferred to tared 60-ml acid-cleaned polyethylene bottles.
The wet weights of all samples were then determined. The samples were
frozen and freeze-dried in a Virtus lyophilizer (Model 10-145MR-BA) for
2 days. The dry weight of each sample was then determined. The dried
sediment samples were then acidified with 50 ml of 5% HN03 (reagent
grade), sealed with a polyethylene screw cap, and then stored at room
temperature for one week. During the storage period, the samples were
vented daily to prevent rupturing of the plastic containers. After
venting, the samples were mildly shaken to resuspend the sediment in the
closed containers. The samples were then gravity filtered through acid-
washed (5% HN03) Whatman 42 filter paper into 60-ml acid-cleaned poly-
ethylene bottles so that the insoluble residue would not interfere with
the subsequent atomic absorption analysis. A measured portion of each
sample (1 to 5 ml) was treated with 30% 1^2 to oxidize soluble organic
surface active components. Generally, one ml of peroxide was added to
31
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each ml of subsample taken for analysis. This oxidation was necessary
if the sample in question was analyzed by HGA-AA for any of the elements
determined in this study.
Analysis. All flame atomic absorption (FAA) determinations were con-
ducted with a Perkin-Elmer (Model 5000) atomic absorption spectrophotometer.
All heated graphite atomization (HGA) atomic absorption determinations
were conducted with either a Perkin-Elmer Model 500 HGA unit coupled to
a Perkin-Elmer Model 5000 AA spectrophotometer or Model 2100 HGA units
coupled to Perkin-Elmer Model 603 AA spectrophotometer. The model 5000
was retrofitted fitted with a Zeeman HGA background correction unit and
the model 603 was equipped with a D2 arc background correction system.
The model 500 HGA unit was equipped with an auto injector (Model AS-40)
and the 2100 HGA unit was equipped with an auto injector (Model AS-1).
The transient HGA-AA signals were recorded with Perkin-Elmer strip chart
recorders (Model 56) and were also sent automatically to Perkin-Elmer
data station microcomputers (Model 3600). Software supplied with the
data stations reduced the transient signals to a peak height and peak
area for each element determined. The instrument setup procedures for
the FAA and HGA-AA determinations were in accordance with procedures
described in "Methods for Chemical Analysis of Water and Wastes" (EPA,
1979) and are also found in the manufacturer's reference manuals.
The AA instruments were calibrated each time samples were analyzed
for a given element. Instrument calibrations were generally checked
after every five samples had been atomi-zed into the flame unit, or injected
into the HGA units. All samples were analyzed at least twice to determine
32
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signal reproducibility. Most were analyzed three times. Generally, for
each 15 samples processed, one sample was determined by the method of
standard addition and one procedural blank sample was analyzed. All
elements in the BRH sediments were determined by FA-AA. Cadmium concen-
trations in the reference sediment samples (REF from the south reference
site) were too low to be determined by FA-AA and had to be analyzed by
HGA-AA.
Short-Term Biological Testing
As part of the NCI/EPA collaborative project, short-term biological
testing was to be conducted on solvent extracts of BRH and REF sediments.
The purpose of this testing was to further characterize the sediments
relative to potential carcinogenic and tumor-promoting chemicals. Unfrac-
tionated solvent extracts of the sediments were evaluated in two tests:
the Salmonella/mammalian-microsome assay (Ames test) for mutagenic
(initiating) agents, and the Chinese hamster V79 lung fibroblast assay,
a potential assay for tumor promoters. Results obtained with these tests
were consistent with the chemical profile of BRH sediment and suggest
potential cause-effect relationships. Such potential relationships can
be tested in the laboratory ion multistage carcinogenesis studies.
These studies should include assessments of target tissue residues in
relation to neoplastic disease. These types of studies should result
in a better understanding of the origins of neoplastic disease in the
flounder and oyster, further increasing their usefulness as model aquatic
systems for environmental monitoring.
33
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Sample Preparation
Several procedures were explored for extracting the sediments and
preparing such extracts for short-term testing. The final protocol
adopted involved extracting 25 g (dry weight) of freeze-dried sediment
with acetone, which was then: (1) brought to dryness and redisolved in
5 ml of dimethylsulfoxide (DMSO), or (2) solvent-exchanged into 5 ml of
hexane and then partitioned into 5 ml of DMSO. This second procedure
avoided the potential loss of low molecular weight compounds, but resulted
in greater sample toxicity. The above procedures were used for the Ames
test. Methanol extracts of freeze-dried sediments were used in tests
with the metabolic cooperation assay.
Salmonella/microsome assay
The standard Ames assay with preincubation was employed (Ames et
al., 1975; Williams and Preston, 1983). That assay utilizes various
strains of Salmonella typhimurium containing specific types of mutations
in the histidine operon which makes them auxotrophic for histidine.
Mutagens are detected in the assay by reverse mutation at the site of
the original lesion, reverting the strains from histidine dependence to
histidine independence. Reverse mutation is usually lesion-specific,
detecting only those types of mutagens acting by the same mechanism of
action as the agent which induced the original lesion (e.g. , mutagens
inducing base-pair substitutions as opposed to frameshift mutagens).
Tester strains have been engineered to maximize their sensitivity to
mutagens. This has been accomplished by introducing into the strains
two additional mutations: one eliminating the polysaccharide side chain
of the cell surface lipopolysaccharide, thus increasing cell permeability,
34
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and a second which results in defective DNA excision repair. Most strains
also contain the R-factor plasmid, pKMlOl, which increases mutagenesis
by triggering the error-prone (SOS) repair system present in bacteria.
In testing sediment extracts, four strains of Salmonella were used,
with and without an exogeneous metabolizing (S-9 microsomal fraction of
aroclor-induced rat liver) system. TA98 and TA100 were used as standard
tester strains for the detection of frameshift and base-pair substituting
mutagens, respectively. Two newer strains were also used. TA102, which
is competent for excision repair, detects a variety of oxidative mutagens,
including X-rays, and many mutagenic ketones and aldehydes: TA104 was
used because of its high sensitivity to some ketones and aldehydes. These
latter strains have been described by Maron and Ames (1983) and Marnett
et al. (1985).
Chinese Hamster V79 Metabolic Cooperation Assay
The Chinese hamster V79 metabolic cooperation (V79/MC) assay is
currently being explored as a short-term test to identify tumor promoters.
This assay is based on the discovery that the phorbol ester tumor promot-
ers inhibit the gap junctional-mediated transfer of low molecular weight
molecules (metabolic cooperation) between cultured cells (Yotti et al.,
1979; Murray and Fitzgerald, 1979). Since this discovery, many structur-
ally diverse promoters have been reported to inhibit metabolic coopera-
tion (MC) between a variety of cell types (Trosko et al. , 1982, Malcolm
and Mills, 1985; Elmore et al., 1985).
The V79/MC assay utilizes co-cultivated mutant (HGPRT-) and wild-type
(HGPRT+) Chinese hamster V79 lung fibroblasts. The mutants are deficient
in the enzyme hypoxanthine-guanine p^osphoribosyl transferase (HGPRTase).
35
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In addition to normal purine bases, HGPRTase catalyzes the biotransforma-
tion of certain abnormal bases, such as 6-thioguanine (6TG), that are
lethal upon incorporation into DNA. Thus, 6TG is toxic to wild-type
cells but not to HGPRTase-deficient cells, unless mutant and wild-type
cells establish physical contact. If contact occurs, gap junctions will
form between cells, allowing the exchange of cytoplasmic components.
Under such conditions, the toxic metabolite of 6TG (6-thioguanine mono-
phosphate) is passed from wild-type to mutant cell, resulting in the
death of both cell types. In the V79/MC assay, this phenomenon of mutant
cell killing via MC in the presence of 6TG is used to identify and quan-
titate the effects of test chemicals on MC. Tests are conducted with an
excess of wild-type cells to ensure physical contact between the two
cell types. Under such conditions, mutant cells are killed upon exposure
to 6TG unless 'rescued' by test chemicals that inhibit MC. Thus, the
capacity of test chemicals to suppress MC is measured as an increase in
mutant cell survival over background. It is also possible for chemicals
to enhance MC. This situation is detected as a decrease in mutant cell
survival below background.
The V79 lung fibroblast has a limited capacity to metabolize xeno-
biotic compounds (Bradley et al., 1981) and no exogenous metabolizing
system is currently used in MC assays. Thus, observed responses are
assumed to result from effects induced by unmetabolized substances.
Although criteria for determining the validity of individual assays and
for assessing the significance of assay responses have been suggested
(Trosko et al., 1981, 1985; Elmore et al., 1985), there is presently no
generally accepted, single set of criteria. In this laboratory, the
36
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effects of chemicals on MC are assessed only at concentrations permitting
at least 70% relative cell survival. Thus, the effects of test chemicals
are assessed essentially at non-cytotoxic concentrations. Responses are
presently evaluated by application of the statistically conservative,
two-fold-increase rule (Ames et al. , 1975). Inhibition of MC is signifi-
cant if experimental mutant survival exceeds solvent control mutant
survival by a factor of two or more. Enhancement of MC is significant
if the reverse is true. Otherwise, test agents are considered to have
no effect on MC. A detailed experimental protocol for the V79/MC assay
as performed in this laboratory has been published (Malcolm et al.,
1985).
37
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PART III. RESULTS
Sediment Chemistry
In chemical analyses conducted on Black Rock Harbor (BRH) and Refer-
ence (REF) sediments, organic compounds from several major chemical classes
were quantified. Those classes included polychlorinated biphenyls (PCBs),
polycyclic aromatic hydrocarbons (PAHs), polycyclic aromatic ketones
(PAKs), polycyclic aromatic quinones (PAQs), and carbazoles. The concen-
trations of several inorganic elements were also quantified, including
iron, chromium, copper, zinc, cadmium, lead, nickel, manganese, and
mercury. Three samples of each of the two sediments were analyzed. The
results are reported for each sediment as mean concentration plus or
minus one standard deviation (Appendix 3). All of the compounds quanti-
fied were found in both sediments except that the ethylan compounds were
not detected in the REF sediment and the REF sediment was not analyzed
for mercury.
All of the compounds quantified except manganese were found in
higher concentrations in the BRH sediment than in the REF sediment.
Manganese is a major crustal element and is not considered a tracer of
anthropogenic activity. All the other compounds or elements except iron
are considered to be anthropogenically produced or mobilized. In all
cases the concentrations of those compounds were elevated in the BRH
sediment.
There was considerable variation between classes of compounds and
between individual compounds within classes as to the extent of enrichment
in the BRH sediment. In general, the low molecular weight PAHs (fluorenes
and dibenzothiophenes) showed the largest differences between sediments,
38
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showing concentrations up to 500 times higher in the BRH sediment. Some
of the higher molecular weight PAHs (benzanthracenes and benzopyrenes)
are elevated only by factors of 10 to 20. PCS concentrations measured
as Aroclor 1254 are about 180 times higher in the BRH sediment. In
general the PAR and PAQ concentrations were about 10 to 20 times higher
in the BRH sediment. The carbazole concentrations were enriched in BRH
sediment to a somewhat greater extent.
Cadmium was enriched in the BRH sediment to a greater extent (by
about a factor of 100) than the other inorganic elements. Copper concen-
trations are about 50 times higher and chromium concentrations are enriched
by about a factor of 30. The concentrations of the remainder of the
inorganic compounds were elevated to lesser degrees.
Laboratory Studies of Oysters
Test I
Histopathological examination of BRH-exposed oysters demonstrated
occurrences of neoplastic lesions involving renal tubular epithelial sur-
faces, gastrointestinal (GI) epithelium, respiratory and water tube epi-
thelium, ventricular cardiac muscle, gonadal duct germinal epithelium and
elements of the oyster nervous system. Polypoid and ulcerative lesions
were also present in stomach and mid-gut of some BRH-exposed oysters.
Overall there were 49 neoplasms in 40 animals of 295, or 13.6%, of
Test I BRH sediment exposed oysters. Multiple forms occurred in nine
oysters as combinations that included Kidney, GI and Gonadal; Kidney, GI
and Neural; Kidney and Neural; Kidney and Gill; and GI and Gonad. The
highest prevalence of lesions by far occurred in renal tubule epithelium
(Table 2).
39
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Table 2
Oyster Tumor Induction Summary Test I *
Tissue
Number of Tumors
(Number and Percent)
BRH Treatment
(Days Exposure and Exp/Latency)
30
60
60/30
30/60
KIDNEY
GI
GILL
HEART
GONAD
NEURAL
n
Total
N with tumors
8
2
5
0
2
0
100
17
12
8%
2%
5%
0%
2%
0%
4 4.7% 10 15.4% 6 13.3%
00% 00% 1 2.2%
2 2.4% 0 0% 1 2.2%
2 2.4% 0 0% 1 2.2%
1 1.2% 0 0% 1 2.2%
1 1.2% 1 1.3% 1 2.2%
85 65 45
10 11 11
10 10 8
* n = number exposed in all replicates per treatment; Total • total
number of "tumors"; N - total number of different animals with tumors.
40
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Twenty-eight of 295, or 9.5% of BRH-exposed Test I oysters examined had
renal lesions characterized by proliferative, indeterminant patterns of
cellular growth. Eight occurred within interlamellar space and filaments
of the respiratory organ, 3 in gastrointestinal mucosa, three in heart
cardiac musclature, 4 in gonadal ducts and 3 in neural tissue elements.
Results of oyster tumor induction following BRH exposures are summarized
by treatment in Appendix 1.
Neoplastic/degenerative lesions were not microscopically detectable
in the kidney, GI, gill, heart, gonad and nerve cells of 338 Test I
oysters examined from 30, 60, 60/30 and 30/60 day REF sediment exposure
treatments. A neoplasm of digestive gland origin occurred in digestive
ducts of a single oyster exposed to the 60/30 reference sediment, however.
That neoplasm was different with respect to the BRH-induced lesions we
observed. The digestive gland ductal neoplasm observed in the oyster
exposed to the reference sediment was not observed in more than 1000
other oysters examined in the present study.
Adult oysters surviving exposures in three replicates for each of
four treatments (i.e., 30, 60, 60/30 and 30/60 days) to two different
sediments totaled 874 of 1200. Of those, 643 were examined histologically
and 231 were archived for chemical analyses. Oysters surviving exposures
to reference sediment totaled 455 of 600; 338 were examined for histo-
pathology and 117 were archived for chemistry. Oysters surviving expo-
sures to Black Rock Harbor sediment totaled 409 of 600; 295 were examined
for histopathology and 114 were archived for chemistry. Mortality in oys-
ters from both sediment treatments increased in parallel demonstrating an
apparent dependence on length of exposure rather than toxicity of sub-
stances in treatments. Histological examinations revealed the presence
41
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of a Haplosporidian parasite designated as "MSX". Observed mortalities
were believed to be MSX-related.
Test II
Oyster Test II, a duplicate of Test I with three replicates of 30,
60, 60/30 and 30/60 versus treatments of REF and BRH sediment, remains
to be assessed for histopathology. At present, the 30- and 60-day REF
and BRH exposure treatments are processed and await interpretation.
Tissue processing is currently being conducted on post-exposure 60/30
treated animals.
Although histological interpretation of Test II has yet to be con-
ducted, macroscopic evidence of tumor development occurred in one 30-day
exposed animal. During post mortem examination, the rectal region of
the gut was noticeably enlarged. Gross observations of the rectal region
followed by selective sectioning was routine since Test I revealed poten-
tial vulnerability of the area to tumor development. Microscopic sections
of the affected oyster confirmed the presence of an adenomatous lesion
in the swollen rectal gut. Furthermore, the same oyster had an advanced
renal cell tumor. Macroscopic evidence of a proliferative lesion was also
found in one other Test II specimen. That lesion was associated with
heart ventricle and pericardium.
Histopathology of Oysters Exposed to BRH Sediment
Noninfectious Lesions
For purposes of the current report, Willis' definition of neoplasia
is used: "A tumor is an abnormal mass of tissue the growth of which
exceeds and is uncoordinated with that of the normal tissues and persists
42
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in the same excessive manner after the cessation of the stimuli which
evoked the change" (Willis, 1960).
Kidney
Histomorphology. The excretory organ of the oyster consists of a
central "pericardial part" and interrenal passage that connects two
nephridial branches, or limbs, according to Galtsoff (1964). The peri-
cardial part functions in concert with the walls of the heart auricles
to remove wastes by ultrafiltration from hemolymph. Filtered wastes are
discharged into renal passages leading to nephridia. The central peri-
cardial part located between pericardium and adductor muscle is the
region most frequently observed in our histological preparations.
Nephridial tubule epithelium is composed of cells that vary in size
from low cuboidal to columnar, dependent on location in relationship to
anterior and posterior portions of the kidney. Epithelial cells that
comprise the functional unit of the kidney have a clear cytoplasm without
granules and a small, central to apically located nucleus (Plate 1).
Nuclei arranged in a regular pattern are uniformly hypochromatic with
dusty appearing chromatin and a small nucleolus.
Renal Cell Tumor. In renal cell neoplasms the prominent feature is
hyperchromatic nuclei of transformed tubular epithelium (Plate 2).
Sporadic cluster or nidi formations of neoplastic epithelial cells is a
trait highly visible in tubules with minimal deviation. Neoplastic tran-
sition occurs variably in nephridial epithelium from sporadic nidal forma-
tions to more advanced, complete tubular involvement. Cytoplasmic to
nuclear ratio is decreased and nuclear chromatin density is increased,
being very hyperchromatic in H and E stain. In an advanced state, coales-
43
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cence of these focal areas occurs, leading to cellular piling, disorganiza-
tion of renal excretory epithelium and tubular swelling. Progression of
the disease is accompanied by invasive tendencies. Tubule limiting or
basal membranes may be breached based on Heidenhain's stain for connective
tissue. Diffusion of neoplastic cells into renal sinusoids is apparent
in the presence or absence of compromised integrity of connective tissue
membranes. In two cases, neoplastic cells invaded the outer sheath
and large neurons of the visceral ganglion (Plates 3 and 4).
Gill Water Transport and Respiration
Histomorphology. The oyster respiratory organ consists of four
folds of tissue or demibranchs suspended from the visceral mass. An
interlamellar space between two lamellae of each demibranch traverses
entire length of the gill. The four interlamellar chambers, divided in
places by septae to form water tubes (Plate 6), merge at the level of
the heart. The functional unit of the gill is a tubular plicate filament
of ciliated epithelium supported by chitinous rods. Interlamellar septae
in the oyster are composed of vascular connective tissue and a compact,
non-ciliated, two-cell epithelial layer interspersed at irregular inter-
vals with eosinophilic and mucous secretory cells. Secretory cells con-
taining red cytoplasmic granules are more regularly arranged as clusters
in comparison to single and multiple occurring mucous cells.
Water Tube and Gill Filament Tumors. Neoplastic development occurred
in interlamellar septae and gill filamental epithelium of oysters exposed
to BRH sediment in the laboratory and field caged oysters from BRH and
CLIS. Gill lesions involving those epithelial surfaces are focal in
nature. Multiple focal areas involving respiratory tissues were observed.
44
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Six occurred in one histological section from a specimen caged 36 days
400 meters east of CLIS, for example. Neoplastic development in respira-
tory organs of the most advanced cases was found to involve connective
tissue elements of interlamellar septae that divide the gill lamellae
and form part of water tubes. Extension to functional respiratory fila-
mental epithelial surfaces was generally evident by serial section in
cases involving the interlamellar septae (Plate 5).
Water Tube Papillomatous/Adenomatous Tumor. An outstanding charac-
teristic of those tumors was the solid replacement of epithelial cells by
variably differentiated cells, with basophilic tinctorial properties.
Clear mucoid-appearing cells usually occurred peripherally in the epithe-
lial neoplasm. Those cells were usually located in a peripheral transi-
tion zone between normal and basophilic neoplastic elements (Plate 5).
Hyperchromatic interlamellar water tube and gill filamental epithelia
transformed by BRH exposures contrast in morphology from small, round to
elongate, tightly packed cells. Nuclei also varied in size from small,
round to elongate, respectively; chromatin was dense and basophilic in
small round cells and dense to scattered in elongate cells. Indistinct
cellular membranes, scanty eosinophilic cytoplasm and a high nuclear to
cytoplasmic ratio characterized small cells while a distinct cellular
outline, basophilic staining cytoplasm and a variable nuclear to cyto-
plasmic ratio characterized the more differentiated, elongated neoplastic
cells.
Morphological characteristics of the neoplastic cells appeared somewhat
related to the amount of supportive stromal elements present. Elongate
neoplastic cells were generally present where there was support by abundant
stromal connective tissue elements. In contrast, smaller blast-like,
45
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less differentiated elements arose in association with thin fibroid-
appearing connective tissue septae. Variable papillary and adenomatous
patterns formed as a result (Plates 5,7, and 8). Stromal elements followed
irregular patterns, in relation to the water tube wall, and supported
papillary formations projecting towards the water tube lumen, while
numerous adenomatous formations occurred within the proliferating tissue
mass. Interlamellar septae in some cases were swollen by adenomatous
formations occurring within septal vasculature and connective tissue
(Plates 5 and 7). Mitotic figures were present throughout the lesion.
Gastrointestinal Tract
Histomorphology. Organs of digestion of food and elimination of
waste consist of the mouth, eosophagus, stomach, crystalline-style sac,
digestive diverticula, midgut, rectum and anus. The ciliated epithelium
of the midgut, including the rectal segment, is invested on a well-defined
basement membrane that is supported by an outer fibrous membrane, muscu-
lature, vesicular connective tissue and a extensive vasculature. The
ciliated, simple columnar epithelium of rectum and anus was one region of
the oyster alimentary tract we found vulnerable to BRH sediment.
Rectal Adenoma. Neoplasms involving rectal epithelia were striking
due to a multiplicty of distinct luminal or adenomatous formations in
the tumourous mass (Plates 9-12). Adenomatous formations consisted of two
morphologically distinguishable cell types. Cells located on the surface
nearest the visceral mass, in relation to the lumen of adenomatous struc-
tures, appeared to retain many characteristics of the original columnar
cells. Neoplastic cells intermediate in the tumor mass located on the
adenomatous ductal surface nearest to the rectal lumen lacked those distin-
46
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guishing characteristics. Neoplastic cells intermediate in relation to
rectal and adenomatous luminal areas were greatly reduced on their longi-
tudinal axis. Those cells also featured a greatly reduced cytoplasm!c to
nuclear ratio, and stain intensely basophilic. The size difference
between those two cell populations often created a "buckling-over" effect
that was responsible for cresent-shaped adenomatous patterns (Plate 11).
Neoplastic epithelial cells forming the rectal luminal surface area
retained columnar definition, but nuclei had enhanced hyperchromatism.
Mitotic figures in the lesion are numerous; cilia are generally lacking.
Ciliated epithelium of the rectum transitioned to normal body wall
components at the anal rosette (Plate 13). The adenomatous lesion in
the fecal midgut in one case was continuous with ciliated epithelial
surface to the anus and extended to involve body wall epithelial surfaces
(Plate 14).
Neural Tissue
A neuroblastoma was identified in a 30-day BRH-exposed/60-day post-
exposed oyster (Plate 15). The focal lesion was variably located in
proximity to the alimentary tract from esophagus to stomach occurring in
vesicular connective tissue, possibly open sinusoidal vasculature and
the outer sheath of circumpallial connective and branchial nerve fibers.
Foci were also associated with the respiratory organ being located in the
gill axial region.
Histomorphology. Developing neoplastic embryonic cells occurred as
discrete focal areas with random to rosette-appearing cellular organiza-
tion. The pattern of development was difficult to ascertain due to
limited circumference of focal nests. Foci of neoplastic cells were
47
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contained by a dense fibrotic capsule and were generally located among the
Leydig cells of the visceral mass. Cellular attachment to the capsular
wall was presumed in some sections of the lesion. Embryonic neural elements
in those lesions appeared as small/medium round to slight/moderate oval
cells with indistinct cytoplasm. Nuclei were vesicular with scattered
clumps of dense staining chromatin, a prominent nucleolus and a dense
hyperchromatic membrane.
Heart
Oyster heart is a three-chambered structure suspended at three
points in the pericardium. A dorsal aorta supports the ventricle while
two efferent veins support two auricles. A constriction between the
auricles and ventricle marks the location of two auriculo-ventricular
(A/V) valves, the region affected by 60 day laboratory BRH exposure.
The A/V valves consist of longitudinally to obliquely arranged muscle
fibers supported by connective tissue. Fibers are loosely arranged in a
connective tissue matrix. Nuclei of normal A/V myofibers are small
round to oval; they are vesicular with light-staining chromatin and a
distinct nucleolus.
Two 60-day exposed oysters had lesions associated with the A/V
valve. Those lesions were interpreted as a myxoma (Plate 16). The
myxoma featured stellate-shaped cellular elements in an abundant hyaline
ground substance. Circulatory system components of other specimens were
also affected by BRH exposures. In one of those specimens, vasculature
supplying the adductor musculature was found to have cellular aggregates
of hyperchromatic cells arising from the luminal or endothelial surface
of a blood vessel (Plate 17).
48
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Gonad
Oyster gonadal follicles near the surface of the reproductive organs
were affected by BRH exposures. Gonadal follicles located near the
surface of the organ are lined with germinal epithelium on the inner
surface and ciliated epithelium on the outer surface facing the body
mass. Abnormal cellular changes were evident in the germinal follicular
surface. Usual orderly arrangement of germinal epithelium was disrupted
by papillary or polyp formations attached to basement membrane and/or
connective tissue by slender stalks (Plate 18).
Degenerative Lesions
Non-infectious Diseases
Degenerative lesions were observed in laboratory-exposed and BRH
indigenous oysters reminiscent of metal effects previously documented in
an ERL/N laboratory study. Those lesions were associated with the kidney
tubular mucosa.
Laboratory Studies of Flounder
Two tests using young-of-the-year and 1-2 year age class winter
flounder are referred to as Winter Flounder Laboratory Test I and Winter
Flounder Laboratory Test II. Test I began October 17, and November 25,
1985, for 0-1 and 1-2 year winter flounder, respectively, and both tests
concluded on February 24, 1986. Test II began in October, and November,
1986, and is currently in progress.
Test I
A positive tumorigenic effect was histologically detectable in labora-
49
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tory tested winter flounder following approximately three to four months
of continuous exposures to BRH sediment (Table 3). Neoplastic lesions
resulting from exposures to bedded BRH sediment or dietary contamination
or a combination thereof occurred in 0-1 year class winter flounder after
131 days. Following 92 days of continuous exposures, neoplasms occurred
in 1-2 year class winter flounder exposed to a 50/50 mixture of REF and
BRH sediment and dietary-routed BRH sediment exposure. Neoplastic response
was absent in winter flounder exposed to REF sediment control treatments.
These findings represent results of a limited sample size, however
(Appendix 2).
A premature conclusion of Test I occurred as a result of unanticipated
problems with test chamber design and seawater distribution to replicate
test chambers. Two major problems were (1) an exchange of seawater between
contiguous test chambers and (2) the blockage of individual seawater deliv-
ery to test replicate chambers. Loss of seawater delivery to test chambers
generally resulted in rapid reduction of dissolved oxygen levels with
consequent mortality of the test organisms. Appendix 2 contains the
survival results.
Test II
Test II began in June 1986. In July 1986 a laboratory emergency
generator malfunctioned with a resultant loss of seawater to the experi-
mental system delayed Test II start-up. Test II delay caused protraction
of field collection efforts beyond the time for optimum availability of
1-2 year winter flounder. The experimental design for Test II was somewhat
altered from Test I, because the desired age classes of winter flounder
were unavailable in the quantity required for the proposed experimental
50
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Table 3
Winter Flounder Tumor Induction Summary Test I *
Flounder Tumor
Class
0-1
1-2
Type
HEMANGIOMA
NEPHROBLASTOMA*
ADENOMA/BB**
ADENOMA/DUCT***
PAPILLOMA
HEMANGIOMA
NEPHROBLASTOMA
ADENOMA/BB
ADENOMA/DUCT
PAPILLOMA
REF/C
0
0
0
0
0
0
0
0
0
0
Treatment
REF/CT 50/C 50/CT
+
+ + 0
000
000
00 +
0 + 0
0 + +
00 +
0 + 0
0 + +
100/C 100/CT
0 +
+ 0
0 0
+ 0
0 +
-
-
-
-
- -
* 0 « Absence of Neoplasms; + » Presence of Neoplasms; - « Data
Unavailable; * - Blastema occur normally in 0-1 year aged fish.
** Adenoma/BB - Brockman Body.
*** Adenoma/Duct » Pancreatic Exocrine Duct.
51
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design. One contributing factor was that winter flounder stocks were at
an all-time historical low (Jeffries, 1986). Technical problems in the
exposure system encountered in Test I, that contributed to high mortality,
were corrected for Test II.
Age Class 0-1. Test II is ongoing with a full complement of 260
animals in six treatments (REF/C, REF/CT, 50/C, 50/CT, 100/C and 100/CT)
for 0-1 year class animals. Test II is composed of n = 15 per replicate
with three replicates for a total of n = 45 per treatment and is therefore
not altered from the original experimental design used in Test I.
Age Class 1-2. Test II contains 144 winter flounder of the appro-
priate 1-2 age class, or two complete replicates for each treatment
(REF/C, REF/CT, 50/C, 50/CT, 100/C and 100/CT). The original design
called for six treatments with a total of n - 432 winter flounder aged
1-2 years. Because of difficulties in collecting sufficient numbers of
1-2 year age class, it was necessary to use 0-1 year age class winter
flounder. Two complete replicates have been filled with young-of-the-year
fish that meet minimum length requirements for 1-2 year age differentia-
tion. These two replicates contain 480 flounder.
All tests (including 0-1 age class test II) have now been continuous
without significant losses due to system-imposed mortalities. Test II
will be continuous through June 1987.
Histopathology of Flounder Exposed to BRH Sediment
Non-infectious and infectious pathology in winter flounder both can
be correlated with BRH sediment exposures.
52
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Non-infectious Lesions
All neoplastic lesions observed in fish exposed to BRH sediments
appeared as relatively benign formations* One of the most striking neo-
plasms was an adenomatous formation in the Brockman bodies, or pancreatic
islets (Plates 19-22). Other prominent lesions in winter flounder exposed
to BRH sediments, and perhaps the most consistent, were associated with
renal corpuscles, renal artery and other vascular channels of the mesenephic
kidney as capillary hemangiomas (Plate 28), and focal proliferations of
renal blastic cells as nephroblastomas. In one specimen a neoplasm
occurred as a projection into the luminal space of the carotid artery
(Plate 29). Neoplasms in winter flounder other than pancreatic islets
and kidney appeared as squamous cell papillomas on external dermal surfaces,
on buccal cavity epithelium, and adenomatous in mucosa of the juncture
between the pharynx and the stomach, in olfactory sensory epithelium and
possibly as amelioblastomas in odontogenic elements of pharnegeal teeth.
Kidney neoplasms appeared more universally associated with BRH sediment
exposures as they occur in both age classes of winter flounder. Changes
in the Brockman bodies from these limited samples was observed specifically
in 1-2 year 50%/CT treated fish.
Endocrine Pancreas
His tjomprphology. Islets of Langerhans in winter flounder occur in
mesenteric elements associated with extrahepatic bile ducts and portal
vasculature. Acinar exocrine tissue of winter flounder, as in most
other teleosts, surrounds islets as well as biliary and intra- and extra-
hepatic ducts of the liver portal sytem. H and E stain is insufficient
53
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for differential determination of winter flounder islet endocrine cellular
components (Alpha and Beta). Morphological differences suggest the
presence of two cellular components in islets, one type situated centrally
and the other in a peripheral location. Flounder islets have a distinct
outer fibrous capsule and an indistinct vascular network*
Islet Cystic Adenoma. Adenomas in pancreatic islets of 50/CT treated
1-2 aged winter flounder occurred following three months of continuous
exposure (Plates 19-22). Lesions were characterized by an abundant eosino-
philic stromal tissue and multiple intrainsular duct formations. Intrain-
sular lesions in one specimen encompassed approximately one-third of the
endocrine mass. Intrainsular ductal formation consisted of squamoid to
low cuboidal cells with indistinct cellular outlines. Nuclei of epitheloid
cells lining the luminal area was vesicular with a prominent membrane,
variably sized round, oval or elongate, and containing clumped chromatin.
Diffuse islet formation, nesidioblastosis, occurred in extrahepatic
biliary ducts of 1-2 year flounder (Plate 22) and 0-1 year flounder
(Plate 23 and 24). Based on morphological characteristics, those lesions
appeared to be pancreatic in origin, and are thus presented here in asso-
ciation with islet adenomas. Neoplasms occurring in pancreatic ducts
and having possible pancreatic origin were observed in three animals, a
0-1 year BRH 100% C flounder and two BRH 50% C treated flounder. Those
ductal lesions are morphologically similar to lesions detected in BRH,
CLIS and New Bedford Harbor, Massachusetts, field-exposed winter flounder
(Plates 25 and 26).
54
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Kidney
Nephroblastoma. Embryonic blastema of winter flounder appear to be
preferentially sited in zones or areas located adjacent to the metanephric
duct. Normal blastema are composed of compact layers of basophilic,
elongate cells with scanty cytoplasm. Nuclear features include distinct
nucleoli in a homogenous, medium-staining basophilic chromatin (Plate 27).
Atypical blastema located in proximity to the mesonephric duct of
young-of-the-year winter flounder following exposures to BRH sediment
were composed of tightly packed layers of spindle-shaped cells with
marked hyperchromasia (Plate 28). Those qualitative characteristics make
blastema sites highly visible in histological sections of BRH-exposed
flounder. As a result of increased stimulation, the sites are quantita-
tively altered by several-fold increases in area dimension. The unencap-
sulated, non-invasive appearing lesions are typically cellular, pleomorphic,
and demonstrate mitotic activity. Spindle-shaped cells of poor differen-
tiation similar to those observed in blastemic sites also occurred in the
renal interstitium. Spindle cells in interstium, unlike preferential
blastema, were loosely arranged and mixed with hematopoietic tissue ele-
ments. Proliferation of those cells in advanced cases encompasses nephrons,
renal glomeruli and arterial and portal vasculature. Invasiveness and
mitoses are not characteristics of the lesion for flounder, at least in
these rather short exposure periods.
Capillary Hemangioma. Capillary hemangiomas occurred in renal
corpusles, renal vasculature including the renal artery and a major
cephalic artery of young-of-the-year and 1-2 year old winter flounder
exposed to BRH sediment.
55
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The histology of winter flounder renal capsules is typical of teleosts
in general. The renal corpuscle consists of a glomerulus and Bowman's
capsule. Bowman's space is separated by single layers of visceral and
parietal epithelium.
Glomerular capsules appeared to be particularly sensitive to effects
of BRH sediment exposures, as evidenced by dilation of Bowman's space.
Enlargement of Bowman's space generally appears as a several-fold volu-
metric increase based on luminal dimensions (Plate 28). The parietal and
apparently the visceral epithelia formed a contiguous extended surface.
The altered renal corpusle surface was composed of a single to three-cell
compact layer of endothelial or endothelial-like cells and fibroblasts and
appeared to lack a distinct glomerulus. A pericapsular capillary network
often forms to circumscribe the atypical renal capsule, promoting the
above mentioned layered appearance. Neck segment cells aligned on the
expanded luminal surface retained typical morphological characters, however.
Multiple finger-like vascular tufts arose randomly from the epithelial
surface area projecting inward towards a greatly expanded Bowman's space.
Hemagiomatous formations varied in size. They varied from delicate finger-
like projections that were generally equivalent in size to a single capil-
lary with an attendant single layer of endothelium, to more solid exten-
sions having four or five times the surface area. Increased-size of
those formations generally promoted a change in directional orientation
and a resultant "buckling" pattern.
Hemagiomatous tufts also developed outward in association with atypical
pericapsular vasculature and into renal interstitium of flounder exposed to
BRH sediments. In some specimens, liomangiomatous formations developed from
56
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the intima and project into lumina of renal arteries. Fibrosis of renal
arterial system components occurred in some BRH-treated flounder, especially
100/CT. An angiomatous lesion occurred in the cephalic artery of one
specimen (Plate 29). The formation had morphologic qualities similar to
those observed in renal arteries.
Papillomas
Papillary cauliflower-like formations were grossly visible on two
50% BRH (C and CT) treated 1-2 year aged flounder. Numerous similar forma-
tions, approximately 1 mm x 1 mm, were concentrated on periorbital epidermal
surfaces of those two specimens. In addition, keratochromatosis occurred
on the corneal tunic, appearing as multiple small white, ovoid spots.
In histological section a cauliflower-like lesion, in epidermal folds
associated with an eye of a 1-2-year-old fish, occurred as a focal pro-
liferation composed of inactive-appearing squamous epithelial cells.
Epitheliomatous lesions were also associated with stratified squamous
epithelial surfaces of the oral cavity in winter flounder exposed to BRH
sediment. Odontogenic tissue was stimulated in two winter flounder exposed
to BRH-contaminated feed treatments. Considerable mitotic activity asso-
ciated with undifferentiated basophilic blast-like cells was characteristic
of that potential neoplasm. The most aggressive appearing odontogenic
lesion occurred in a BRH 100% CT 0-1 year animal. A similar lesion also
occurred in a BRH 50% CT 1-2 year animal.
Degenerative and Necrotic Lesions
BRH etiopathic degenerative lesions induced in two age groups of
winter flounder as a response to direct and dietary BRH exposures involved
57
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the liver, spleen, thymus, gastric mucosa, thyroid gland, renal tubular
epithelial cells and cephalic and trunk lateral line processes.
Lesions associated with the digestive system, in addition to epi-
theliomatous conditions in the oral cavity, included mucous cell hyper-
plasia of esophageal surfaces. Morphological alterations of esophageal
epithelial cells, that included luminal formations and a high level of
mitotic activity, were indictative of the potential for neoplasia in 1-2
year flounder exposed for 60 days. Lesions in the esophageal lining were
proliferative formations located randomly from the pharyngeal opening to
the fundus in two BRH 100% C exposure treated flounder. Histological sec-
tions of BRH exposed flounder stomachs demonstrated induction of necrotic
lesions in the gastric mucosa, atypical lumen formations in the mucosa
and inflammation of the submucosa as evidenced by lymphocytic infiltration.
Accessory digestive organs of the flounder include the liver and
exocrine pancreas. Positive indications of incipient neoplasia in hepatic
parenchyma and biliary components of the liver were absent, with the excep-
tion of basophilic foci (1 mm or less). Other nonspecific degenerative
changes were observed. In general, those changes included dilation of
hepatic veins and sinusoids, perivascular inflammation, atypical macrophage
accumulations, fibrosis, and occasional basophilic foci as previously men-
tioned. Those conditions were most consistent in 0-1 year winter flounder
having exposures to BRH 100% C and CT treatments. Effects in the exocrine
pancreas of flounder exposed to BRH sediment were histologically undetect-
able by light microscopy with the possible exception of one case. Megacytic
cells were apparent in one 0-1 year REF CT treated flounder.
Circulatory system responses to BRH sediment exposures was varied and
inconsistent. Degenerative effects observed in flounder exposed to BRH
58
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sediment included depletion of hemopoietic tissue in the head kidney and
depletion of RBCs in the spleen.
Excretory system effects induced by BRH sediment exposures in addition
to the above-described neoplastic changes included hylanization and fibrosis
of renal corpuscles. That condition occurred in 0-1 year REF CT, 100% C
and 100% CT and 1-2 year 50% C and CT treated winter flounder. Membranous
elements of renal tubules thickened and became fibrotic in some flounder
exposed to 50% BRH sediment. The condition usually involved a group, or
cluster of adjoining, closely associated nephrons. Varied patterns of
fibrosis occurred in many of those lesions as a result of distorted tubule
configurations. Excretory epithelial cells of those tubules often assumed
an uncharacteristic basophilic stain property. In addition, renal tubular
epithelial surfaces in animals exposed to BRH sediment occassionally were
clear, lacking any apparent staining properties. The condition was most
evident in 1-2 year BRH 50% CT animals. Cystic formations occurring in
some 0-1 year class flounder exposed to BRH sediment contained a mucoid
material (Plate 28).
Changes in endocrine tissue, in addition to pancreatic islets, included
the thyroid and corpuscles of Stannius. Thyroid follicles appeared to be
functional, although cellular components of many fish exposed to BRH sedi-
ment from the standpoint of tinctoral staining properties were atypical.
Stannius corpuscles, in a very limited number of cases, were found to have
degenerative changes such as dilation of blood vasculature. There was
alteration in corpuscle of Stannius endocrine parenchyma occurring as
luminal formations in a limited number of animals.
Nervous system effects were present and included dilation of blood
vasculature and blood congestion in association with the menix, and
59
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possibly necrosis and other changes of neuroglial cells of all major
regions of the brain, spinal cord and optic tract in some winter flounder
exposed to BRH sediment. Degenerative changes were also observed in
neurosensory components including taste buds, cephalic and trunk lateral
line sensory receptors, and olfactory neurosensory epithelium. Lateral
line canal epithelial surfaces underwent alteration including hyperplasia
concomitantly with increased cellular volume. Advent of those changes
caused reduction of canal luminal diameter. Necrosis of the epithelial
mucosa also occurred in those flounder exposed to BRH sediments. Olfactory
neurosensory lesions possibly represented early neoplastic development
based on observed morphological changes.
Infectious Lesions
Infectious lesions in flounder exposed to BRH sediment that appeared
as BRH-related responses included parasitic granulomatous reactions to
microsporidian protozoans and trematodiasis. One unidentified microspo-
ridian had an apparent affinity for Bowman's space in renal corpuscles of
flounder exposed to 50% BRH sediment and of some field-collected animals.
Glugea stephani, a spore-forming microsporidian, was identified in labo-
ratory exposed and BRH field-collected winter flounder.
Field Studies of Oysters
Oysters Indigenous to BRH
Histological examination failed to reveal neoplastic lesions in 84
oysters indigenous to BRH obtained in oyster beds at the entrance to BRH.
During gross examination many of those animals were light green in color.
Green coloration in oysters represents a phenomenon generally accepted
60
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as due to the presence of copper adsorbed from the water column by
amebocytes. Degenerative lesions observed most frequently in the indig-
enous oysters involved renal tubules and the stomach/mid-gut regions of
the gastrointestinal tract. Degenerative changes in the renal tubules
were characterized by a change in epithelium from columnar or cuboidal to
a squamous-like, highly basophilic cell. Changes in the gut were primarily
ulcerative in nature.
Oysters Caged in BRH
30- and 60-Day Exposures. One tumor was confirmed in 90 oysters
surviving deployment in BRH. The tumor was identified as a water tube
papilloma in an oyster exposed for 30 days at the North Dock station.
Survival of caged oysters held at two different BRH locations, Buoy
12 and North Dock, was greatly reduced in comparison to the reference
site at Milford estuary (Appendix 1). Two attempts were made to study
In situ effects of BRH water quality, but results were less than optimal
due to low oyster survival rates. Although high mortality of those
field-deployed animals influenced the number of histological evaluations,
43 oysters were archived for chemical analysis.
Oysters Caged in CLIS
A gill water tube papilloma occurring in multiple locations was
confirmed by serial sections of an oyster recovered from the field study
station located 400 m east of the CLIS disposal mound.
Sixty-six percent of the oysters deployed at four stations in CLIS
survived 36 days of caged exposure. Survival was similar for all the
treatments. An exception was the station located 1000 m east of the
61
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disposal mound. There, one replicate was recovered damaged, without
oysters, and was thus lost to biological interpretations. Ten oysters
from each of the four stations were archived for future chemical residue
analysis.
Field Studies of Flounder
Winter flounder field studies assessed animals collected by otter
trawl at CLIS and BRH for histological variations. Those field sampling
efforts to establish an index for occurrence and distribution of tumors
in winter flounder from CLIS BRH were compared to a reference population
of winter flounder collected near Fox Island in Narragansett Bay, Rhode
Island.
Central Long Island Sound (CLIS)
Ninety-four winter flounder caught by otter trawl near the disposal
mound have been processed for histopathological examination. Although
those fishes await histopathological interpretation, preliminary evalua-
tion indicates the presence of both preneoplastic and neoplastic lesions
in organ systems such as the liver.
Black Rock Harbor (BRH)
Fifty winter flounder caught by otter trawls in BRH have been proc-
essed for pathological evaluation; nineteen others await processing.
The integumentary system of many BRH-collected winter flounder was char-
acterized by fin hemmorhage, parasitic petechia on the ventral body
surface and frank ulceration. Glugea stephani, a spore-forming protozoan
parasite that infects the intestinal connective tissue of winter flounder,
was present in many of the BRH collected fish. Several flounder collected
62
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from BRH had mycosis, evidenced by the presence of a branching fungus in
association with kidney, spleen and cephalic neural and muscular elements.
A high prevalence of bifurcated gill filaments occurred in BRH collected
flounder.
Oyster Tissue Chemistry
Chemical analyses of oyster tissue residues remain incomplete.
Flounder Tissue Chemistry
Chemical analyses of tissue residues in laboratory-exposed winter
flounder have not yet been initiated.
Short-Term Biological Tests
Salmonella/Microsome Mutagenicity Assay
BRH and REF sediment samples in DMSO were tested for mutagenic activ-
ity in the preincubation version of the Ames test with strains TA98, TA100,
TA102 and TA104. Results for unfractionated solvent extracts are presented
in Figures 7-10.
Toxicity data for acetone extracts exchanged into hexane and parti-
tioned into DMSO indicated high toxicity to all tester strains at volumes
of extract above 50 yl per plate, while acetone extracts brought to
dryness and redissolved in DMSO were testable at sample volumes up to
300 ul per plate. In experiments with BRH sediment extracts prepared by
the latter method, a strong mutagenic response was obtained in TA100
with S-9 activation. A weak but positive mutagenic response was also
obtained with TA104 in the presence of S-9 metabolism.
63
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lOOn
UJ
80-
01 60H
0
I I I I
40 80 120 160 200 240 28O
/1L PER PLATE
Figure 7. Results of Ames Testing with Salmonella Strain TA98 and S-9 Metabolic Activation.
Open Circles = Solvent Blank; Closed Circles = Extract of Black Rock Harbor
Sediment; Open Triangles = Extract of Reference Sediment
-------
1000
UJ
40
80
120
160 200 240
280
flL PER PLATE
Figure 8. Results of Ames Testing with Salmonella Strain TA100 and S-9 Metabolic Activation.
Open Circles - Solvent Blank; Closed Circles - Extract of Black Rock Harbor
Sediment; Open Triangles - Extract of Reference Sediment
-------
a-
liJ
a
UJ
a
a:
ui
>
UJ
(T
50On
400-
300-
zoo^
—1—
100
200
300
//L PER PLATE
Figure 9. Results of Ames Testing with Salmonella Strain TA102 and S-9 Metabolic Activation.
Open Circles - Solvent Blank; Closed Circles - Extract of Black Rock Harbor
Sediment; Open Triangles - Extract of Reference Sediment
-------
//L PER PLATE
Figure 10. Results of Ames Testing with Salmonella Strain TA104 and S-9 Metabolic Activation.
Open Circles - Solvent Blank; Closed Circles - Extract of Black Rock Harbor
Sediment; Open Triangles - Extract of Reference Sediment
-------
Spiking experiments (data not shown) were done in which standard
reference mutagens (sodium azide, 2-aminofluorene, 2-nitrofluorene and
3,4-benzopyrene) were added to varying concentrations of BRH and REF
sediment extracts. The purpose of those experiments was to determine if
the mutagenic activity of standard compounds could be detected when
standard mutagens were added to complex mixtures such as solvent extracts
of marine sediments. Mutagenic activity of those compounds requiring
bioactivation (3,4-benzopyrene and 2-aminofluorene) was almost completely
suppressed in extracts of BRH sediment, and significantly suppressed in
REF sediment extracts. The activity of the direct-acting mutagens (sodium
azide and 2-nitrofluorene) was suppressed only at cytotoxic concentrations
of extract.
Our findings support the hypothesis that inhibitors of microsomal
metabolism are present in the extracts and that such inhibitors block
the bioactivation of promutagens to mutagenic forms. Consequently, frac-
tionation procedures are likely to be required for detection of mutagenic
activity in such samples. These results also suggest that the concentra-
tion of direct-acting mutagens in these extracts is relatively low.
V79/Metabolic Cooperation Assay
Freeze-dried, methanol-extracted sediments from BRH and the REF
site were tested in the V79/MC assay for potential tumor promoters.
Results are shown in Figures 11 and 12. Evidence of significant,
concentration-dependent, but weak inhibition of MC was found in the
methanol extracts of Black Rock Harbor sediment. Concentration-dependent,
but insignificant effects on MC were observed in methanol extracts of
reference sediment.
68
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20-,
1180 |:I6O I 140
DILUTION OF DRY-EXTRACTED REFERENCE SEDIMENT
I 120
Figure 11. Effects of Extracts of Reference Sediment on Cell Survival and Mutant Recovery
in Two Independent V79/MC Assays. Circles Represent the Results of One
Experiment; Triangles Represent the Results of a Second Experiment.
Open Symbols Indicate Cytoxicity; Closed Symbols Indicate Mutant Recovery
(i.e., Inhibition of Metabolic Cooperation)
-------
1000
tr
80-
5 7OH
o:
ui
>
o
o
UI
a:
t-
20-i
10-
0 I:4OO C340 1280
DILUTION OF DRY-EXTRACTED BLACK ROCK HARBOR SEDIMENT
1:220
Figure 12. Effects of Extracts of Black Rock Harbor Sediment on Cell Survival and Mutant
Recovery in Two Independent V79/MC Assays. Circles Represent the Results of One
Experiment; Triangles Represent the Results of a Second Experiment. Open Symbols
Indicate Cytoxicity; Closed Symbols Indicate Mutant Recovery (i.e., Inhibition of
Metabolic Cooperation)
-------
Preliminary Ames-V79/MC results with Fractionated Solvent Extracts
(The data are not given here.)
Recently, a protocol for the fractionation of sediment extracts was
developed based on compound polarity. Briefly, sediments extracted with
acetonitrile were placed on a silica gel column, and then sequentially
eluted in four steps: The fl fraction was elated with pentane and contains
highly non-polar compounds such as PCBs; f2, a PAH fraction, was eluted
with an 80:20 pentane:methylene chloride mixture; f3, eluted with methylene
chloride only, contained polycyclic aromatic ketones, carbazoles and
phthalates; f4, eluted with methanol, was largely uncharacterized, but
contained generally polar compounds. Preliminary results (not given in
this report) with the Ames test indicated the presence of mutagenic
agents in all fractions but fl, with most of the activity contained in
f2, for which all strains (TA98, TA100, TA102 and TA104) showed mutagenic
responses. Weak responses were obtained in the V79/MC assay with f3 and
potent responses with f4. Although preliminary, these findings support
the need to test fractionated extracts in characterizing sediments with
short-term assays, and thereby increase the probability that such tests
may be predictive of organism-level responses.
71
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PART IV. DISCUSSION
The etiopathic relationship established between BRH sediment and
neoplastic disorders in oysters and winter flounder permits us to conclude
the industrially polluted Black Rock Harbor in Connecticut is a source
of biologically available oncogenic substances. Experimental neoplasia
in oysters and winter flounder resulting from NCI/EPA sponsored research
represented the first demonstration of a direct causal relationship
between neoplasms in marine fauna and exposures to contaminated marine
sediment (Gardner and Yevich, 1986; Gardner et al., 1986). Furthermore,
these results also represent the first field investigations concurrent
with laboratory study to verify neoplasia in a marine bivalve mollusc
exposed in situ at an environmentally impacted site. Laboratory-field
comparisons of winter flounder demonstrated experimental hyperplasia
(nesidioblastosis) of islets, characterized by endocrine cell prolifera-
tion in pancreatic ducts of the exocrine system in laboratory BRH sedi-
ment treated animals. The condition was verified in BRH and CLIS field-
collected animals. Laboratory and field investigations together supported
our preliminary histological evidence, suggesting that BRH contaminated
sediment is potentially tumorigenic to oysters and winter flounder.
Chemical characterization of BRH sediment, which confirmed the presence
of carcinogens, co-carcinogens, and tumor promoters (Rogerson et al.,
1985) is further supported by short-term tests for genotoxic agents (Ames
assay) and the Chinese hamster V79 cell metabolic cooperation assay for
potential tumor promoters. Increased frequencies of sister chromatid
exchange were also observed in a marine worm, Nephtys incisa, exposed to
whole BRH sediment in laboratory tests and at the field disposal site
72
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(Pesch et al., 1986; Pesch et al. , 1987a, 1987b).
The success of these pathological investigations is due to a unique
approach in aquatic animal laboratory experimentation that departs from
the traditional method of testing water column pollutants to a method
focused on sediment contaminants. Indeed, traditional short-term (28 day)
tests with various species of invertebrate and vertebrate marine fauna
during laboratory FVP investigations failed to demonstrate an overtly
toxic endpoint to BRH exposures, with the exception of amphipods. Because
of the large numbers of oysters with tumors (13.6 Z of Test I), we believe
that these results strongly justify additional studies.
American Oyster
Tumors in oyster renal excretory epithelial constituents provided
the strongest demonstration of a direct causal relationship with BRH sedi-
ment. Six different types of experimental tumors were expressed within
30 and/or 60 days after onset to BRH sediment exposure. A high degree
of variability in the neoplastic response of the oyster to BRH material
was observed. Frequency data also demonstrated that experimental tumors
occur most often in organs performing excretory roles in the oyster - in
tubular nephridial elements of kidneys, gill, heart and the rectal odd-gut.
Persistent development of these lesions occurred after discontinuation of
BRH exposures and 30 and/or 60 day latency post-exposure periods. Reduc-
tion of neoplastic lesions was not statistically evident after discontinued
BRH stimulation. These proliferative conditions thus appear to be auton-
omous and thereby fulfill an important criterion for neoplasia.
Excretion in the oyster begins with hemolymph ultrafiltration through
pericardial glands and heart auricles followed by resorption of carbohydrates
73
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in nephridia and waste discharge to the external environment. The prolif-
erative kidney disorder in oysters exposed to BRH sediments in addition
to nephridial tubules, often involve renopericardial openings, funnels
and canals. These nephridial units serve to transport metabolic filtrates
to renal excretory epithelium. Epithelial surfaces of the renal reservoir,
that retains waste fluids until discharge to the external environment, is
also involved in some cases. Neoplastic disorders also occurred in the
heart. Polypoid formations developed on the ventricles and on the roots
of anterior and posterior aortas, anatomically within the pericardial
cavity, of oysters exposed to BRH sediment. A polypoid formation observed
on the ventricle of a Test II oyster appeared as a gross lesion approxi-
mately one-third the dimension of the ventricle itself and was an example
of the potential for rapid growth following introduction to BRH sediment.
Numerous neoplastic disorders evoked in heart and kidney by elements in
BRH sediments suggest that biological metabolic wastes contain complete
and/or transformed substances that include tumor initiators and promoters.
The blue mussel, Mytilus edulis, exposed to BRH particulate (10 mg/ml for
28 days) in the same manner as oysters during FVP studies at ERL/N, also
demonstrated disorders of the heart (Yevich et al., 1986).
Gill and rectal adenomatous lesions involve non-ciliated and ciliated
epithelial surfaces that have constant physical contact with organic/
inorganic particulate matter from the moment when the oyster filters it
as a potential food item until elimination to the surrounding aquatic
environment. In the gill, organic and inorganic particules are retained
in close proximity to respiratory surfaces with mucous and moved by cilia
along tracts leading to the food grc-ve and labial palps. Sorting of
74
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particulate occurs in the labial palp and at the entrance to the stomach,
where material is shunted to digestive diverticula or passed for elimina-
tion. The nature of lesions in these tissues suggests prolonged contact
with BRH chemically laden particulate whether it is diverted to digestive
diverticula or passed is a factor in the development of neoplasms. Pre-
sumably, additional exposure of these surfaces to toxic substances may
occur as a result of elevated chemical waste removal via diapedesis. The
oyster cellular excretory mechanism of diapedesis removes heavy metals
through gill filaments and rectal midgut (Galtsoff, 1964). Toxic action
of BRH sediment transported into the alimentary canal also manifests
papillary and polypoid formations and occasionally massive stomach ulcera-
tions. Logically, the mechanics of food collection, digestion and waste
elimination in combination with physical and chemical properties of the
particulate are important keys in the rapid uptake and possible accumula-
tion of BRH contaminants that have influenced neoplastic development and
other degenerative changes in organs of digestion in the oyster. We
cannot comment on accumulation of BRH sediment contaminant residues at
the present time, although evidence of rapid uptake and accumulation is
demonstrated in continuous BRH particulate exposures to the mussel. In
uptake and accumulation studies with the mussel, steady-state bioaccumula-
tion was attained after seven days (Rogerson et al., 1985). In situ PAH
uptake studies in oysters transplanted from a clean to a contaminated
area in the Elizabeth River demonstrated residue steady state with stabi-
lization and/or decline in 3 days (Pittinger et al., 1986). Our studies
also demonstrated the presence of mutagenic activity using the Ames test.
Renal carcinoma in oysters was *"he pivotal element that prompted
75
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the present NCI/EPA collaborative research effort to study tumorigenic
potential of BRH sediment. Renal carcinoma in two of ten oysters exposed
31 days in a preliminary study (Gardner and Yevich, 1986) provided strong
histological evidence of the carcinogenic potential of those substances
contained in BRH sediment. The hallmark of oyster renal carcinoma was
neoplastic cellular invasion into interrenal sinusoids, the visceral
ganglion and the outer sheath of the visceral ganglion. Extension to
the visceral ganglion also involved branchial and/or cerebro-visceral
connectives, major tracts leading from the visceral ganglion.
Evidence of cellular infiltration to visceral ganglion in Test I
oysters was limited to one case even though invasion of neoplastic cells
into interrenal sinuses occurred frequently. We are uncertain why the
same degree of accelerated growth and invasiveness observed in preliminary
study was not apparent in Test I oysters. The degree of cellular trans-
formation may be linked to subtle differences in exposure conditions,
primarily those influencing rate of aeration and sediment oxidation
prior to and during dosing. Chemical data to support this contention
are lacking, however. Nevertheless, these experimental renal carcinomas
in oysters represent the first cases reported for these marine bivalve
molluscs. Other than the oyster, renal carcinomas have been reported
in a fresh water amphibian and mussel. In the leopard frog (Rana pipens)
spontaneous renal adenocarcinomas were discovered by Luck'e and associates
near Philadelphia, PA, in 1932 (Stewart et al., 1959). A single renal
carcinoma, described as a poorly differentiated carcinoma, was reported
to have occurred in a fresh water mussel following experimental water
column exposures to N-Nitroso compounds (Khudoley and Syrenko, 1978).
Experimental gill tumor induction in the oyster at locations in BRH
76
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and CLIS was morphologically characteristic of those oysters exposed to
BRH sediments in the laboratory. In comparison to counterparts in
laboratory-exposed oyters, the CLIS case appeared to be more active as
evidenced by numerous areas of focal involvement with very distinct
adenomatous formations. As many as six areas of involvement occurred in
one tissue section, for example. Water tube and gill neoplasms occuring
at a 1:90 and 1:24 ratios in BRH and CLIS, respectively, demonstrated
that CLIS in situ exposure results were within the realm of the 1:50
ratio in laboratory exposed oysters. A single occurrence at the CLIS
location was insufficient evidence for an etiological linkage to BRH
dredged/disposed sediment as the source. That occurrence, reinforced by
the historical record, and in concert with our laboratory-field compari-
sons, suggests that BRH sediment disposal in CLIS is a possible cause for
the neoplasms observed in fauna collected from that area. Due to the
potential ramifications with regard to commercial oyster activity, we are
of the opinion that more thorough field investigations are warranted to
further explore the potential for occurrence of neoplasia in situ.
BRH in_ situ studies demonstrated a lower average number of gill
tumors than in the laboratory study. A partial explanation could be
reduced biological functions as a result of the extremely harsh, toxic
environment in BRH. Compromised water quality conditions within the
inner harbor visually reduced health and well-being of oysters caged at
BRH. Oysters held at the Milford, CT. reference and CLIS disposal area,
in comparison, appeared to be in good condition. However, the oysters we
examined that were indigenous to the BRH entrance were free of neoplasms.
The oyster beds at the entrance are geographically contiguous with the
harbor per se, but remote in terms of comparative physical characteristics
77
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of the two areas. Oysters indigenous to entrance to BRH are located in a
more spacious and well-flushed environment in comparison to the constricted
harbor area with its minimal channel flushing and compromised water
quality. The greatest dichotomy between inner and outer harbor is related
to the enriched organic appearance of bottom sustrate (i.e., homogenous,
"black mayonnaise" consistency vs. granular sand) and odiferous and
visual asthetics associated with inner harbor water quality. As such,
physical and chemical factors that influence biological availability of
contaminants contrast greatly between inner and outer Black Rock Harbor.
Although neoplastic change is absent in these oysters, other pathological
conditions existed that we consider to be reminescent of heavy metal
exposures conducted at ERL/N (Yevich, personal communication), and possi-
bly relevant to the tumorigenic process.
Proliferative neoplastic lesions encountered in various molluscan
species during the past two decades has heightened scientific and public
awareness of ever-widening occurrences of such oncological conditions
(Farley, 1975; Pauley, 1969; Sparks, 1985). The etiology of these condi-
tions remains elusive, although conclusions developed during discussion
in scientific forums and reviews of pertinent literature imply an associa-
tion with highly polluted environments (Scarpelli and Rosenfield, 1976;
Couch and Harshbarger, 1985; Mix, 1986a, 1986b; Kraybill et al., 1977;
Yevich and Barszcz, 1977). Couch and Harshbarger (1985), in a recent
review, discussed the dominant form of neoplasia in bivalve molluscs as
a group of disorders variously termed sarcomas, hematopoietic neoplasms
and blood cell proliferations, with numerous hypothetical causes advanced
including chemical and viral etiologies. Despite these apparent associa-
tions of neoplasms encountered in soft-shelled clams, mussels and oysters
78
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with polluted environments, Mix (I986a), in a recent review, stated that
there is little evidence suggesting an association with environmental
pollution. Historic records critically reviewed by Mix (1986) and Couch
and Harshbarger (1985) also demonstrated an inconclusive relationship
between environmental pollution and neoplasia in indigenous aquatic
species. In contrast to these reviews, this study has shown contaminated
sediment from BRH demonstrates carcinogenic potential in oysters and
winter flounder and strongly directs our attention to the benthic environ-
ment. Also, Farrington et al. (1986) recently stated that aquatic research
historically has been preoccupied with the water column, and must make a
transition from "the-top-down to the bottom-up," suggesting that sediment
substrates deserve more investigative attention. We agree with the views
of Farrington (1986) and Anderson (1986) that future research must place
more emphasis on sediment and laboratory studies, respectively. In our
opinion the approach taken at ERL/N can guide us to a better understanding
of the causes of neoplasms and their relationships to environmental
pollutants.
In situ monitoring can be an effective means of monitoring pollutant
discharges and enviromental quality, such as the "Mussel Watch" program.
Bivalve molluscs are considered to be a sensitive indicator of water
quality in near-coastal marine environments (Yevich and Barszcz, 1983).
As such, "mussel watch" became a national program for monitoring pollu-
tants. Mussel sensitivity to pollutants also prompted exposure studies
with BRH particulate sediment, concurrent with the NCI/EPA research
agreement and other programs at ERL/N. A neoplastic disorder, induced
in myocardial tissue elements by the~e exposures, is documented in studies
79
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by Yevich et al. (1986). The heart disorder in missels and our in situ
observations with oysters reinforce the relative sensitivity of these two
species of shellfish to particulate borne chemical contaminants. Compar-
atively speaking, other bivalve species, such as the scallop and soft-
shelled clam, are more sensitive to hydrophobic contaminants than are the
oyster and mussel, based on research conducted at ERL/N. Comparative
toxicity differences among these species may plausibly be related to
anatomical differences in the excretory system. Sensitivity combined
with particular anatomical features, serve as a recommendation for in
situ studies with the oyster and mussel, as these fauna can withstand the
longer periods of exposure necessary for tumor induction. Both the
oyster and mussel appear to be a promising means for in situ pollutant
monitoring of particulate-bound oncogenous substances.
Winter Flounder
BRH sediment-induced pathology in winter flounder is unusual,
based on our historical records of ERL/N toxicological programs. Neo-
plastic disorders of the type recorded in the NCI/EPA program are either
conspicuously absent or few in number in reviews of neoplasia in fishes
(Couch and Barshbarger, 1985; Kraybill et al., 1977; Mawdesley-Thomas,
1975; Mix, 1986). Angiomatous lesions in kidney and islet adenomas in
teleosts are unreported in major documentations of fish neoplasia in
reviewed literature. Nephroblastomas are reported for striped bass
(Helmboldt and Wyand, 1971) and steelhead trout (Mawdesley-Thomas, 1975),
and can be induced in great number in rainbow trout with aflatoxin
(Hendricks, personal communication), however.
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Experimentally induced neoplasms in winter flounder, especially
nephroblastic lesions, are recognized as minimal deviations. It is
important for the reader to recognize that research results with the
flounder are representative of a prematurely concluded test. Sample size
was, as a result, too small to make any definitive statistical inferences.
Further, continuous exposures to six treatments in two age classes is
currently in progress and has equalled or surpassed the exposure time of
the earlier study. In our opinion, neoplastic development reported to
date is in early stages and we feel more progressive formations will be
apparent provided that longer exposure or latent periods are possible.
Of the neoplastic disorders observed, we consider the most signifi-
cant development to be intrainsular adenomas in pancreatic islets and
hyperplasia (nesidroblastosis) in exocrine ducts of the pancreatic system.
At the present time we have not been able to determine the origin of
focal proliferations in exocrine glands, but suspect that it is pancreatic
endocrine tissue. Pancreatic intrainsular adenomatous formations observed
in 1-2 year old flounder occurred following treatment with 50% REF/50% BRH
sediment test media and a diet of mussels contaminated with BRH sediment.
From these observations we conclude that dietary exposures had an obvious
and significant role in the development of islet neoplasia in winter
flounder exposed to BRH sediments in the laboratory. The response observed
was similar to that in Syrian hamsters treated with the pancreatic carcin-
ogen N-nitrosobis(2-oxopropyl)amine by Pour (1978). In hamsters the
neoplastic process was initiated with hyperplasia of intercalated ductal
cells (Nesidioblastosis), metaplasia, and malignant alteration. Because
the response in flounder was most prominent with the contaminated food
81
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treatment, we share John Black's view that dietary exposure is one of the
most promising avenues for fish neoplasia study (Black, personal communi-
cation).
These results are significant because they establish the first demon-
stration of neoplasia to occur in a benthic fish as a result of continuous
direct contact exposure with a "generic", pollutant-laden sediment and
the apparent added effect through trophic transfer of biologically avail-
able carcinogens and co-carcinogens. Indirect evidence that neoplasia
in benthic fish is causally associated with polluted sediment, and pos-
sibly dietary transfer, is presumed from results of various field inves-
tigative studies (Dawe and Couch, 1984; Malins et al., 1985; Murchelano
and Wolke, 1985; Baumman, 1984; 0'Conner and Huggett, 1986; Fabacher et
al., 1986) with the exception of research conducted by Black (1983).
Black demonstrated carcinogenicity of sediment extracted chemicals from
the Black River near Buffalo, New York, using brown bullheads and mice
as test animals. The experimental approach used by Black to study car-
cinogenic properties of contaminated sediment differs significantly from
our investigations, however. Papillomas induced on the skin of bullheads
and mice by Black was accomplished using repeated applications of concen-
trated chemical extracts from a sample of Black River sediment. Although
Black's investigations demonstrate that extracts of Black River sediment
induce papillomatous lesions in bullheads following twelve months of
repeated application, the exposures must be considered atypical with
respect to expected concentrations in the source environment. In our
experimental approach, both direct sediment contact and dietary exposures,
in comparison, are representative of realistic environmental concentrations
82
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expected for flounder indigenous to BRH. Indeed, BRH sediment mixed 1:1
with REF sediment demonstrated the relatively high potency of BRH in a
less concentrated form. In our view, the concentrations used experimentally
with the oyster were also realistic, especially in view of the results
obtained from iin situ CLIS investigations.
Field Studies
Winter flounder collections from BRH and CLIS provide histological
evidence of liver tumor development. Hepatic clear cell lesions thought
by some investigators (Murchelano and WoIke, 1985) to be "preneoplasia"
in winter flounder collected from various other Northeast Atlantic coastal
embayments are present in BRH and CLIS collected specimens. These inves-
tigators consider such clear cell lesions to be highly predictive of
hepatocellular tumors. It is their contention that serial sectioning of
livers will demonstrate the presence of hepatomas in animals with affected
hepatocytes. Histological interpretation of BRH and CLIS field-collected
specimens in our study is incomplete at the present time, and therefore,
we cannot provide a statistical measure of prevalence. Otter trawl
collections in BRH for winter flounder are now complete in consideration
of our goal of 100 animals. Field collection trips were made on five
and eight occassions to BRH in 1985 and 1986, respectively, to obtain
flounder for these comparisons. Water quality at BRH seemingly has
deteriorated further during the course of our studies, because we experi-
enced limited success in 1985 and virtually no success during 1986 field
collection efforts. These qualitative observations that demonstrate an
even less habitable environment for -quatic life now than in 1985 are
83
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also supported by survival rates of field deployed oysters (Test II),
although hard data measurements of water quality in support of our con-
tentions are lacking.
Incidental to the NCI/EPA collaborative research effort, we have
histological evidence supporting the presence of a high prevalence of
tumors in other field exposed winter flounder. Winter flounder we obtained
from New Bedford Harbor (NBH), Massachusetts, had a 26% prevalence of
hepatomas, an 80% prevalence of "preneoplastic" hepatic clear cell lesions,
and other lesions we consider relevant to the NCI/EPA project. Neoplastic
foci occurred in pancreatic ductal epithelium of a NBH specimen with
hepatocellular carcinoma. We are of the opinion that these ductal lesions
compare to and are possibly of similar origins to ductal lesions experi-
mentally induced in flounder fed contaminated food. We consider these
lesions to have possible pancreatic origins based on light microscopic
histomorphology of cellular elements.
Numerous field investigative efforts in recent history point out a
rather widespread occurrence of fish neoplasms on a national and global
scale (Couch and Harshbarger, 1985). Investigators generally infer from
their studies that organismal behavior and bottom sediment represent a
probable mechanism and source for neoplasia due to the presence of chemical
contaminants and disease potential. Malins et al. (1985) demonstrated
positive correlations for hepatomas in benthic-orientated fish species
to toxic chemicals associated with bottom sediments in urban embayments
of Puget Sound. Thorough field investigative efforts, such as those of
Malins et al. (1985) that provide correlations of tumors to toxic chemi-
cals in sediment, must at some point be complemented with laboratory
experimentation to establish the direct causal link to contaminated
84
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sediment. We suggest that Malins1 approach employing extensive chemical
analyses of sediment, tissue and metabolites in bile and our field and
laboratory investigations complement each other in establishing the
causal link. Malins et al. (1985), by virtue of a demonstrated positive
correlation of hepatomas in English sole to specific chemical groups in
Puget Sound sediment, Blacks' interpretations with Black River sediment
extracts, and our evidence of experimental tumor induction are strong
indictments of sediment-bound chemicals being etiological agents repon-
sible for neoplasias in aquatic animals. It is our opinion laboratory-
field comparisons for oysters and winter flounder together provide irref-
utable evidence that chemically contaminated sediment is linked to neo-
plasias in marine vertebrate and invertebrate organisms.
Human Health
Winter flounder exposed to BRH sediment exhibited aberrant visual or
other nervous behavior during feeding stimulation. During investigation
of PCBs relative to human health in Dutch mothers and infants born to
mothers in high PCB fish-consumption categories, Swain (1986) noted
deviations in developmental and behavioral patterns of newborn children.
One behavioral observation in these affected infants at the age of seven
months was of interest to us for comparison with behavioral effects in
winter flounder exposed to BRH sediments. Swain noted that infants born
of mothers with high PCB content had altered lability and increased
startle reflex at birth followed by substantial alterations in fixation
novelty at seven months. Swain further noted that the tendency of an
infant to respond to a new stimulus decreased in direct proportion to the
level of maternal exposure to contaminants in fish consumed, suggesting a
85
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greater than 10% decline in visual recognition memory. Winter flounder
in BRH direct sediment exposures demonstrated somewhat similar behavioral
tendencies associated with visual fixation. Flounder had obvious diffi-
culty with the visual coordination required during feeding behavior
approximately two weeks after onset of direct contact exposure with BRH
sediment. Flounder's ability to feed when stimulated with chopped clam
offered to them between the tips of forceps was greatly reduced as evi-
denced by uncoordinated occular motor activity and an inordinate amount
of time to visually fix on the object. Flounder with these symptoms of
impaired nervous reaction strike erringly by missing the offered food.
Errant strikes deviate from the target by as much as 2 to 4 cm. We feel
that there are significant pathological changes occurring in flounder
nerve tissues; a neuroblastoma did occur in an oyster exposed to BRH
sediment. Swain suggested that his observations demonstrate an effect of
contaminants upon the centers of higher integration in infants secondarily
exposed. We are of the opinion that winter flounder directly exposed to
BRH sediment contaminants, not necessarily PGBs, experience similar
integration problems in these nerve centers as suggested by thier unusual
behavioral patterns.
Future Studies
Based on the results of our testing to date we suggest several
additional areas for future study.
1. A latency study of tumor induction in the oyster and winter
flounder in (15 cm total length, 2-3 year age class) laboratory
sequential (start-stop) testing using BRH sediment. Proposed
exposure treatments will be those used in present NCI/EPA tests.
86
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2. Assessment of Che prevalence of nesidioblastosis and hepatocel-
lular lesions in winter flounder for potential use as models in
human medicine. We propose to investigate prevalence of nesidio-
blastosis in winter flounder from near coastal environs other
than BRH such as New Bedford and Boston Harbors. Investigation
will also include endocrine function tests to determine circu-
lating levels of insulin and other blood chemistry parameters
associated with the condition. Secondly, we propose to investi-
gate the relationships between clear hepatocytes, termed RAM
cells, and cholangiomas in liver, and lesions in exocrine pan-
creatic ductals of flounder. These lesions present an opportu-
nity for the study of a disease that could model human liver
pathology.
3. Continue validation and application of the oyster and winter
flounder as model marine organisms, with emphasis on the investi-
gation of carcinogenesis as a multistage (initiation/promotion)
process in these animals. Contaminated marine environments con-
tain complete carcinogens, co-carcinogens and tumor promoters,
all of which may act synergistically to enhance tumor development
in the oyster, flounder and other susceptible species. In fact,
mammalian studies suggest that many classes of chemicals impor-
tant in experimental carcinogenesis and which persist in aquatic
environments (i.e., PCBs, chlorinated hydrocarbons, dioxins and
phthalates) are more important as tumor promoters than as initi-
ators or complete carcinogens. Future research with the oyster
and flounder will provide uiique opportunities to better understand
87
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the role of these substances in aquatic carcinogenesis. Such
knowledge will be essential to the use of these animals for
environmental monitoring.
4. Complete short-term testing with fractionated sediment extracts.
Use test data to construct a biomarkers/waste characterization
matrix for predicting multistage carcinogenic effects in suscep-
tible aquatic organisms and for identifying potential cause-effect
relationships. These potential relationships would then be tested
in the oyster and flounder to further validate the use of the
tumor endpoint in these animals as an index of environmental
quality.
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PART V, CONCLUSIONS
1. Oysters and winter flounder were sensitive to oncogenic substances in
BRH sediment as evidenced by several specific neoplastic disorders
experimentally induced during laboratory study.
2. Neoplastic responses of oysters to suspended BRH sediment particulates
within 30 and/or 60 days of onset of exposure varied greatly with
regard to anatomical location and site of origin. The highest prev-
alence of tumors occurred in kidney followed by gill, heart, GI tract,
gonadal germinal epithelium and embryonic nerve tissue. Success of
the sediment exposures was directly linked to a unique experimental
approach that used state-of-the-art electronics.
3. Post-exposure effects studies with oysters failed to demonstrate a
statistically significant reduction of experimental neoplasia during
30 and/or 60 days latent exposure indicating autonomous progression.
4. Oyster gill neoplasia occurred within 30 and 36 days during in situ
studies in BRH, the source of contaminated sediment, and at the
selected CLIS site 400 m east of the disposal location. Laboratory
experimental evidence verified by in situ study established the
first positive laboratory-field comparison linking contaminated
sediment to tumor induction in a bivalve mollusc.
5. Neoplasia developed in experimental winter flounder within four months
of onset to direct contact exposures to sediments containing 50% BRH
and 50% REF or 100% BRH. Experimental pancreatic intrainsular neoplasia
appeared related to direct BRH sediment and dietary exposures via
blue mussels contaminated with BRH sediments.
89
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6. Winter flounder from field stations in BRH and CLIS had a high
prevalence of preneoplastic hepatic lesions based on preliminary
histopathological evaluations. Neoplasia in the pancreatic ductule
tree of field-exposed animals establishes a laboratory-field compar-
ison linking the pathological condition to sediment-related pollution
and exposures.
7. Effects studies at ERL/N with marine organisms in addition to the
oyster and winter flounder give added dimensions to the demonstrated
toxic nature of BRH sediment contaminants. Experience with multiple
species studies suggests to us that the oyster is a sensitive biolog-
ical model for study of the neoplastic process in marine invertebrates.
NCI/EPA research results demonstrate that it is now technically
possible to advance invertebrate oncology, especially in relation to
particulate sequestered toxics. Oysters offer a means of systematic-
ally determining carcinogenic sediment-bound pollutants and the
added advantage of jLn situ monitoring where natural populations are
at risk. Winter flounder offers similar unique qualities for study
of cause-and-effect relationship of demersal fishes.
8. Evidence for the presence of genotoxic agents (Ames test; Nephtys
incisa) and tumor-promoting substances (Chinese hamster V79 cell/
metabolic cooperation assay) were consistent with the chemical charac-
teristics of BRH sediment and predictive of the neoplastic disease
induced in the flounder and oyster via laboratory exposures.
90
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ACKNOWLEDGMENTS
We wish to thank Dr. John Harshbarger, Director of the Registry of
Tumors in Lower Animals, Smithsonian Institution, Washington, D.C., for
his assistance with the diagnosis and/or verification of neoplastic
lesions during the course of the NCI program.
We would like to give special thanks to the following staff of the
histopathology unit at ERL/N: Sandra Beyni (URI), David Borrus (URI),
Dorrane Borsay (SAIC), Tom Daniels (SAIC), Mary Lello (URI), Susan Ryan
(URI), Paul Selvitelli (URI) and Joseph Terra (URI). The histology
staff shared responsibility for the conduct of field collections of
organisms, laboratory testing, and histological processing. Photography
for the report was done by Mr. Selvitelli.
Personnel of the ERL/N chemistry department integral to the NCI
research include Curt Norwood (EPA), Richard McKinney (SAIC), and Robert
Bowen (SAIC).
Special thanks are also due to Dr. Anthony Calabrese, Director, NOAA,
Northeast Fisheries Center, Milford, CT, and staff involved in field
collections of winter flounder, oyster, and reference sediment with the
R/V Shang-Wheeler.
91
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PLATE LEGENDS
1. Light micrograph of kidney and visceral ganglion of a reference or
control oyster held in suspended CLIS sediment for 30 days. Normal
kidney tubules consist of a clear, simple columnar epithelium (Arrow
A). Normal visceral ganglion and nerve tract (Arrow B). Normal
Large neurons are indicated by Arrows C and D. (Scale bar - 45 y)
2. Oyster kidney tubules with a neoplastic lesion following continuous
exposure to suspended BRH sediment for 30 days. Normal kidney epi-
thelium is replaced by clusters or nidi of proliferating, hyperchro-
matic kidney epithelial cells. Arrows indicate clusters of hyper-
chromatic cells; Arrow A indicates an area of invasiveness into the
renal sinusoidal spaces. (Scale bar » 22 u)
3. Invasion into visceral ganglion and attendant nerve fiber by neoplastic
cells. Clusters of proliferating hyperchromatic cells in the visceral
ganglion are highlighted by Arrow A. These cells have replaced the
larger neurons of the ganglion. Arrow B points to a representative
area of the nerve outer sheath having invasive neoplastic cells.
Kidney tubules with clusters of neoplastic excretory epithelial
cells are located nearby (Arrow C). Tissue is from the oyster case
presented in Plate 2. (Scale bar - 22 u)
4. Neoplastic cells in the visceral ganglion of an oyster following
60 days exposure and 30 days post-exposure to suspended BRH sediment.
Arrows deliniate clusters of neoplastic cells. (Scale bar - 9 p)
5. Adenomatous lesion on interlamellar wall and septae of a gill water
tube. Adenomatous formation is also present in gill filamental struc-
ture of the 30 day BRH sediment exposed specimen. (Scale bar « 52 y)
100
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6. Interlamellar septae of a control oyster at a higher magnification.
Characteristics of normal columnar secretory epithelium (Arrow A),
Water tube (Arrow B) and vesicular connective tissue (Arrow C) are
demonstrated for comparison to Plate 7. (Scale bar - 22 y)
7. Interlamellar septae with adenomatous formation on outer septal wall
of case demonstrated in Plate 5 at a higher magnification (Arrow).
Note adenoma in connective tissue of septal wall. (Scale bar ™ 35 y)
8. Involvement in another area of gill tissue from the same 30 day BRH
sediment exposed oyster. (Scale bar - 22 y)
9. Adenomatous lesion in epithelium of rectum and anal rosette in a
30 day BRH exposed oyster. Luminal formations that characterize the
adenomatous lesion are indicated by Arrow A. Normal epithelium is
indicated by Arrow B. (Scale bar - 19 y)
10. Adenomatous lesion in rectum of same 30 day BRH exposed oyster.
Arrows indicate areas of hyperchromatic proliferative cells. (Scale
bar - 21 y)
11. Adenomatous lesion in rectum of same BRH case. Arrows indicate
areas of hyperchromatic neoplastic cells. Note the extensiveness of
luminal formation and buckling over of the adenomatous structure.
(Scale bar - 21 y)
12. Adenomatous formation in rectum of same BRH case demonstrating yet
another variation of the lesion. (Scale bar * 21 y)
13. Anal rosette region of a control oyster demonstrating usual anatomical
characteristic of epithelial surfaces. (Scale bar ™ 52 y)
14. Anal rosette region of case demonstrated in Plates 9-12. Adenomatous
formation is continuous from rectum to anus and extends to outer
body wall. (Scale bar - 21 y)
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15. Neuroblastoma in vesicular connective tissue located in a fold near
stomach wall (Arrow). BRH 30/60 exposure/post-exposure. (Scale bar
- 26 y)
16. Heart atrioventricular valve with swollen cardiac muscle. Note
ground substance and stellate-shaped cells (Arrows). The lesion is
interpreted as a myxoma. BRH 60 day exposure. (Scale bar - 13 y)
17. Blood vessel in adductor muscle of BRH 30 day exposed oyster. Arrows
point to foci of neoplastic cells that appear to arise from luminal
surface of the vessel. (Scale bar » 22 y)
18. Atypical germinal cells and polypoid-like formation in gonadal duct.
The lesion is from same BRH 30 day specimen for Plates 10, 11, 12,
14 and 17. (Scale bar - 32 y)
19. Brockman body or pancreatic islet of a BRH 50/CT 92 day exposed 1-2
year class winter flounder. Arrows point to location of adenomatous
lesions in two different islets. (Scale bar - 66 y)
20. Brockman body in figure 19 at a higher magnification. Normal endocrine
insular tissue surrounds the adenomatous lesion. (Scale bar » 34 y)
21. Brockman body of another BRH 50/CT three month exposed 1-2 year
class winter flounder with adenoma (Arrows). (Scale bar « 14 y)
22. Lesion formation in Brockman body in same case (Plate 21) (Arrow A).
Intraductal formation of pancreatic tissue (nesidioblastosis) in
pancreatic duct indicated by Arrow B. (Scale bar » 14 y)
23. Pancreatic duct of BRH 100/C 131 day exposed 0-1 class winter flounder.
Focal areas of insular cells in ductal connective tissue is indicated
by Arrows. (Scale bar - 34 y)
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24. Same lesion as in Plate 24 from a different section. Note lack of
orderly arrangement or organization (Arrow). Also note erosion of
ductal epithelial surface. (Scale bar - 11 y)
25. Pancreatic duct of a field exposed winter flounder (New Bedford
Harbor). Multiple neoplastic foci of pancreatic insular tissue
indicated by Arrows. (Scale bar - 52 u)
26. Winter flounder depicted in Plate 25 with higher magnification demon-
strates erosion of ductal epithelium by tumor mass. Some of these
cells appear organized and are recognizable as duct formations (Arrow
A). Disorganization is also apparent (Arrow B). Neoplastic cells
occur randomly in ductal epithelium (Arrow C). (Scale bar « 16 u)
27. Winter flounder (0-1 class) REF-exposed metanephric kidney with
normal tubular biastendc cells demonstrating an orderly arrangement.
(Scale bar - 14 y)
28. Winter flounder (0-1 class) BRH 50/C four month exposed with sites
of proliferating blastemic cells (Arrows at top of photo). Centrally
located Arrows point out a greatly enlarged Bowman's space with finger-
like glomerular tufts. The latter condition is representative of
capillary hemangiomas resulting from BRH exposures. (Scale bar « 21 u)
29. Hemangioma in cephalic artery of a BRH 100/CT (0-1 class) winter
flounder. Here the hemangioma appears to arise from the arterial
wall. (Scale bar - 13 u)
103
-------
-------
s
-------
-------
J
'
-------
-------
v.
^^&
%*x v.
> > - ^otu
't^^'v*
•-1* - *-^r v
•.
^^^:^-
•• ^
^oSS**s«8aBl
C^g?^|^^^
*1
-------
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v^
-------
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V A
i--.V4t? - ~ *-
***•.&, ;&<
?
• #»,*•« -^ -»
•» ** » t. -^ **. * «*
* %*si** ^.^*
_»
-* 22
-------
v"---^...""-^-^ -; '••'{*-'
24
-------
• •". t-Fl.< -
K-«t--ijsr - * *.
-------
••*
••*-.. *Hs;27
*»». v«^»v
?j» .•
*
-------
-------
APPENDIX 1. Oyster Tumor Data
BRH 30 DAYS TEST I
TISSUE
KIDNEY
GI
GILL
HEART
GONAD
NEURAL
Number of Tumors
REP1 REP2 REP3
4 3 1
1 1 0
1 3 1
o o o
1 1 0
o o o
BRH 60 DAYS TEST I
TISSUE
KIDNEY
GI
GILL
HEART
GONAD
NEURAL
Number of Tumors
REPl REP2 REP3
202
0 0 P a
200
1 1 P
0 0 1
0 0 1
a p - POLYP.
104
-------
BRH 60/30 DAYS TEST I
TISSUE
KIDNEY
GI
GILL
HEART
GONAD
NEURAL
a U - ULCER.
TISSUE
KIDNEY
GI
GILL
HEART
GONAD 100
NEURAL
REP1
7
U a
0
0
0
1
BRH 30/60
REP1
3
1
1
1
l*a
Number of Tumors
REP2
2
0
0
0
0
0
DAYS TEST I
Number of Tumors
REP2
2
0
0
0
0
REP3
1
U
0
0
0
0
REP3
1
0
0
0
0
NEUROBLASTOMA.
105
-------
TUMOR OCCURRENCE IN BRH OYSTER FIELD TEST I
TISSUE
KIDNEY
GI
GILL
HEART
GONAD
NEURAL
TISSUE
KIDNEY
GI
GILL
HEART
GONAD
NEURAL
BRH
BUOY 12
0
0
0
0
0
0
TUMOR OCCURRENCE
60
BUOY 12
0
0
0
0
0
0
30 DAYS EXPOSURE
Number of Tumors
NO. DOCK
0
0
1
0
0
0
IN BRH OYSTER FIELD TEST
DAYS EXPOSURE
Number of Tumors
NO. DOCK
0
0
0
0
0
0
MILFORD
0
0
0
0
0
0
I
MILFORD
0
0
0
0
0
0
106
-------
TUMOR OCCURRENCE IN BRH OYSTER FIELD TEST II
30 DAYS EXPOSURE
TISSUE
KIDNEY
GI
GILL
HEART
GONAD
NEURAL
BUOY 12
0
0
0
0
0
0
Number of Tumors
NO. DOCK
0
0
0
0
0
0
MILFORD
*
*
*
*
*
A
* Oyster histopatholocigal examinations not performed.
TUMOR OCCURRENCE IN CLIS-DEPLOYED OYSTERS
Number of Tumors
TISSUE
KIDNEY
GI
GILL
HEART
GONAD
NEURAL
MOUND
0
0
0
0
0
0
400m
0
0
1
0
0
0
1000m
0
0
0
0
0
0
SO. REF SITE
0
0
0
0
0
0
107
-------
PERCENT SURVIVAL 30 AND 60 DAY CAGED OYSTERS
TEST PERIOD
I
30 DAYS
60 DAYS
II
30 DAYS
60 days
Percent Survival
BUOY 12 NO. DOCK MILFORD
84 70 92
24 24 64
28 15 *
0 0 *
* Oysters were not retrieved at 30 days.
108
-------
APPENDIX 2. Winter Flounder Tumor Data - Test I
PERCENT TUMORS FOR YOUNG-OF-YEAR FLOUNDER
TUMOR TYPE
HEMANGIOMA
NEPHROGENIC*
ADENOMA/DUCT
PAPILLOMA
R/C
0
26
0
0
R/CT
18
35
0
0
50 /C
2.9
24
0
0
50 /CT
27
18
0
0
BRH/C
6
13
6
0
BRH/CT
27
20
0
0
* Young-of-Year Flounder normally have nephroblastemic cells
during the 0-1 year period of development.
PERCENT TUMORS FOR ONE-YEAR-OLD FLOUNDER
TUMOR TYPE
HEMANGIOMA
NEPHROBLASTOMA
ADENOMA/BB
ADENOMA/DUCT
PAPILLOMA
R/C
0
0
0
0
0
R/CT
16
8
0
0
0
50 /C
16
43
0
25
0
50 /CT BRH/C
14
73
13
0
6
BRH/CT
—
—
—
—
— —
109
-------
WINTER FLOUNDER SURVIVAL TEST I
TREATMENT
(number %)
AGE
R/C
50/C
BRH/C R/CT
50/CT BRH/CT
0-1 YEAR 23 51% 21 47% 15 33% 17 38% 11 24% 15 33%
1-2 YEAR 6 8% 7 10% 0 0% 12 17% 15 21% 0 0%
NOTE: Winter flounder initially tested per treatment 0-1 year is
n = 45; for 1-2 year winter flounder n - 72. Fifty-eight other winter
flounder removed from the test and observed to be moribund were processed
for pathological examination.
110
-------
APPENDIX 3. Sediment Chemistry
Contaminant Concentrations in Black Rock Harbor (BRH) and Reference (REF)
Sediments. Concentrations as ng/g Dry Weight.
Concentration
Contaminant
PCBs as Aroclor 1254
Black Rock Harbor
MEAN
7170
Polycyclic Aromatic
Fluorene
Cl fluorenes
C2 fluorenes
C3 fluorenes
C4 fluorenes
Di benzo thiophene
Cl dibenzothiophenes
C2 dibenzothiophenes
C3 dibenzothiophenes
C4 dibenzothiophenes
Phenanthrene
Anthracene
Cl phenanthrenes + anthracenes
C2 phenanthrenes + anthracenes
C3 phenanthrenes + anthracenes
635
1520
2270
2580
2180
375
1310
2740
2540
1640
4020
1330
6910
8060
6030
Std. Dev
566
Hydrocarbons (PAHs)
113
268
287
282
287
72
183
295
183
50
617
288
791
863
516
Reference
MEAN
39.80
4.93
4.07
5.04
7.00
3.33
3.56
5.46
9.19
8.10
3.21
70.40
10.80
56.00
51.70
31.70
Std. Dev
4.41
0.62
0.82
1.17
1.81
0.91
0.83
1.21
1.64
1.99
1.13
7.83
1.54
6.39
7.94
5.63
(Continued)
111
-------
APPENDIX 3. (Continued)
C4 phenanthrenes + anthracenes 2980
Fluoranthene 5800
Pyrene 7250
Cl f luoranthenes + pyrenes 6800
C2 f luoranthenes + pyrenes 4330
C3 f luoranthenes + pyrenes 3010
C4 f luoranthenes + pyrenes 1220
Benz [ajanthracene 3450
Chrysene 4450
Cl benzanthracenes +• chrysenes 5220
C2 benzanthracenes + chrysenes 3210
C3 benzanthracenes + chrysenes 1900
C4 benzanthracenes + chrysenes 885
Sum of Benzf luoranthenes 5970
Benz[e]pyrene 2880
Benz[a]pyrene 3160
Perylene 504
Cl homologs of raw 252 PAHs 4450
C2 homologs of mw 252 PAHs 2200
C3 homologs of mw 252 PAHs 925
C4 hohologs of mw 252 PAHs 283
Sum of mw 276 parent PAHs 6670
Sum of mw 278 parent PAHs 3430
Sum of mw 300 parent PAHs 678
4
372
449
409
283
156
70
336
377
454
313
224
87
542
270
311
127
453
258
77
18
862
426
93
12.30
208.00
249.00
131.00
67.50
45.00
19.70
122.00
174.00
161.00
96.20
56.90
30.60
470.00
217.00
243.00
66.70
259.00
116.00
46.60
13.20
604.00
246.00
59.30
1.94
24.90
27.90
11.60
8.43
7.02
2.31
15.70
23.00
24.20
14.60
11.20
6.15
106.00
44.80
46.80
12.20
52.00
29.80
10.90
5.99
118.00
35.10
12.90
(Continued)
112
-------
APPENDIX 3. (Continued)
Sum of mw 302 parent PAHs
Isomer of Ethylan
Isomer of Ethylan
Isomer of Ethylan
Sum of parent PAHs
Sum of PAH homo logs
3550
367
373
3880
55600
75300
Polycyclic Aromatic Ketones
Fluorenone
Cl fluorenone
Anthraquinone
Cl anthraquinones
Cyclopent a [de f ] phenanthrenone
7H-benzo [ c ] f luoren-7-one
1 IH-benzo [ b] f luoren-1 1-one
1 IH-benzo [ a ] f luoren-1 1-one
7H-benz[de]anthracen-7-one
Cl homologs of mw 230 PAKs
Benz [ a Janthraquinone
mw 254 ketone
mw 254 ketone
mw 254 ketone
6H-benzo[cd]pyren-6-one
mw 278 ketone
63.0
34.3
421
156
83.5
288
139
500
1120
985
126
100
81.0
206
627
103
703
37
13
35
4590
5290
(PAKs)
10
2
63
19
7
12
6
29
149
77
16
3
0
10
197
8
232.00 45.50
0.00 0.00
0.00 0.00
0.00 0.00
3020.00 444.00
1240.00 176.00
and Quinones
3.47 1.19
3.25 1.90
26.50 9.99
9.44 3.15
8.35 2.72
33.30 7.52
12.60 3.36
41.70 11.50
43.50 20.50
75.30 23.40
15.80 4.03
13.40 2.84
14.00 4.29
28.60 7.82
24.90 10.70
19.50 5.64
(Continued)
113
-------
APPENDIX 3. (Continued)
Sum of mw 280 parent PAKs
Sum of ke tones + quinones
Carbazole
Cl carbazoles
C2 carbazoles
I IH-benzo [a ] carbazo le
5H-benzo[b]carbazole
7H-benzo [ c] carbazo le
CL benzcarbazoles
Dibenzcarbazole isomer
1 3H-dibenz [ai ] carbazole
Dibenzcarbazole isomer
Dibenzcarbazole isomer
Dibenzcarbazole isomer
Dibenzcarbazole isomer
Dibenzcarbazole isomer
Sum of carbazoles
700
5890
Carbazoles
182
72.5
95.4
245
53.6
156.7
205
64.5
54.4
148
63.8
22.4
26.9
30.3
1420
27
518
19
9
12
20
9
20
16
5
4
15
14
3
7
5
133
83.00
466.00
4.25
1.71
1.83
8.43
1.65
2.82
6.19
3.94
3.45
5.88
2.09
0.67
0.74
0.57
44.20
35.40
155.00
2.99
0.97
0.76
5.60
1.55
3.98
3.63
1.81
1.60
5.64
2.74
0.94
1.04
0.67
33.80
Iron
Chromium
Inorganic Compounds
(Concentrations as yg/g dry weight.)
30800 2820
1480 104
(Continued)
114
21400.00 1320.00
50.30 13.80
-------
APPENDIX 3. (Continued)
Copper
Zinc
Cadmium
Lead
Nickel
Manganese
Mercury
2860
1330
23.7
420
170
311
1.72
311
162
1
29
16
40
1
60.90
164.00
0.23
54.60
25.50
462.90
-*
3.87
7.18
0.05
4.18
2.62
23.80
-
* - Not analyzed.
115
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APPENDIX 4. PATHOLOGY DATA BASE
Description of the Automated Data Entry System for the
National Cancer Institute Research Program
by
Jeffrey S. Rosen
and
Diane Sheehan
116
-------
Introduction
A data system was designed and implemented with three objectives in
mind.
I. The data system had to be consistent and compatible with the data
system in use for all work done at the Environmental Research Labo-
ratory in Narragansett. This was necessary to allow the NCI project
to interface results of the histopathological research with related
results from:
A. the original Field Verification Study (Black Rock Harbor disposal)
B. chemical analyses done by the analytical group
C. other related research
II. The system had to have on-line capabilities of tracking individual
organisms and slides made from those organisms, from collection to
summarization of observations. The desired summarizations had to be
done at a number of different levels including:
A. summarization by treatment (station data)
B. summarization of pathology for an individual organism
C. summarization of pathology within an organ in a treatment
III. Allow input of raw data directly into the computer while working at
the microscope. All observations needed to be associated with the
animal} the slide and the treatment from which it was taken.
117
-------
Approach
These objectives were met using the DATMAN relational data management
system. The tracking of samples and slides and the interface with other
projects and services within the laboratory were accomplished using the
data set SAMPLOG which identified all samples and treatments. The path-
ologies observed were entered into a separate data set called HISTRAW.
Within the HISTRAW data set each observation was entered detailing the
origin of the sample, the specific animal being observed and the partic-
ular slide in which the observed pathology occurred. Each organism was
identified by a separate value called the PATHNUM. Each slide was iden-
tified by a unique value for a variable called SLIDENUM. A user friendly
front end was created to facilitate the entry of raw data at the micro-
scope. For each slide read the investigator was prompted to enter the
sample number associated with the slide, the PATHNUM and the SLIDENUM.
All the other values which are detailed below for the data set HISTRAW
were input via screen prompts. The screen prompts eleviated the need
for the investigators to type observations and to remember codes. Input
screens were developed which included all possible inputs at a particular
point in the observations. By pointing to a choice on the screen the
investigator directs the computer to code the observation and display
the next set of appropriate choices. The screen lists the entire descrip-
tion of the structures and the pathologies. When a choice is made on
the screen the computer automatically codes the observation. Definitions
of all codes are available on-line. A comprehensive list of these codes
is included below. These codes were developed as closely as possible to
Farley's codes (Farley, A., NMFS, NOAA, Oxford, Maryland). When all
118
-------
observations on a set of animals are completed summaries are done either
by individual organism or by treatment. Summaries include number of
organisms which included a certain pathology and percent of all organisms
in which the pathology was noted. Additional data sets contain associated
data about the organisms observed. These data sets contain size, age,
weight, salinity, temperature, and other data pertinent to the study.
The data base design is unique in its approach as it is developed to
accomodate both shellfish and fish.
Designs of the Data Sets
List of names for data set HISTLOG
variable name
SAMPNUM
DATE
CODE
DAY
KIND
LORF
STATION
TIME
EXP
TESTNUM
STARTDATE
CONG
MEASCONC
METAL
ORGANIC
HISTCOM1
HISTCOM2
length
4
4
15
4
3
1
10
4
10
4
4
15
8
3
3
80
80
type
i
i
a
i
a
a
a
a
a
i
i
a
r
a
a
a
a
type definition
E.R.L.N. approved sample number
75000 - 79999
the date the sample was taken
a species code for animal samples
the number of elapsed days from
start of experiment
the kind of sample, o-organism,
s^sediment
L » lab sample, F « field sample
the field station designation
a time grouping variable, e.g.
't+81
for lab experiment - experiment
name
for lab experiment - the test
number
the date the laboratory test was
started
the concentration plan for this
treatment
the measured concentration in a
laboratory experiment
'*' if sample is to be analyzed
for metals
'*' if sample is to be analyzed
for organics
comment 1 - 80-character field
comment 2 - 80-character field
119
-------
List of names for data base HISTRAW
variable name
SAMPNUM
PATHNUM
INDEX
READAT
SEX
CODE
SYSTEM
ANASTRUCT
ANADIV
ANASUBDIV
HISTO
TISCELL
CATEGORY
GROUP
PATHOLOGY
DEGREE
PATHCOM1
PATHCOM2
SLIDENUM
length type definition
4 i E.R.L.N. approved sample number
representing a treatment
4 i pathology number-reps, a single
animal w/in a group (6 digits
yynnnn i.e. 860001)
4 i an index into the database
4 i the date the slide was read
1 a male or female
10 a a code describing the type of
organism
8 a a code defining the system being
observed
8 a a code defining the anatomical
structure
being observed
8 a a code defining the anatomical
division being observed
8 a a code defining the anatomical
subdivision being observed
8 a a code defining the histological
elements
8 a a code defining the tissue/cell
being observed
15 a the category of pathology
8 a the heading under a category
4 i a pathology integer code in the
range 0 - 999
4 i degree of pathology observed -
range 1-4
80 a first 80 column alpha numeric
field for comments
80 a second 80 column alpha numeric
field for comments
5 a slide number - represents a slide
within a pathology number
Codes Used in N.C.I. Black Rock Harbor Program
EA - EXTERNAL ANATOMY
IT - INTEGUMENTARY SYSTEM
MS - MUSCULAR SYSTEM
RS - RESPIRATORY SYSTEM
GI - GASTROINTESTINAL SYSTEM
EN - ENDOCRINE
SYSTEM
CR
EX
RE
NE
SK
CIRCULATORY SYSTEM
EXCRETORY SYSTEM
REPRODUCTIVE SYSTEM
NERVOUS SYSTEM
SKELETAL
120
-------
ANATOMICAL STRUCTURE
RV =• RIGHT VALVE
LV - LEFT VALVE
HI - HINGE
MS » MUSCLE SCAR
MA - MANTLE
AD - ADDUCTOR
RT - RETRACTOR
FO - FOOT
BY - BYSSUS
PR = PROMYAL CHAMBER
GL = GILLS
PT - PLYCATE
SN - SIPHON
NE = NERVE TRACTS/FIBERS
GA • GANGLIONS
EPAX =• EPAXIAL/HYPAXIAL
MYO - MYOMERES
MSHEAD - MUSCLES OF HEAD
BRCHL - BRANCHIAL
NEURO - NEUROCRANIUM
HEMA » HEMATOPOIETIC TISSUES
BV - BLOOD VESSELS
OP - OPISTHONEPHRIC DUCT
PIT - PITUITARY
HK - HEAD KIDNEY
UG - ULTIMOBRANCHIAL GLAND
SENSE » SENSE ORGANS
CRAN - CRANIAL NERVES
AL = ALIMENTARY TRACT
HA - HEART
AR = ARTERIES
VN - VEINS
SI - SINUSES
PC = PERICARDIUM
EPID - EPIDERMIS
KI = KIDNEY
PG - PERICARDIAL GLAND
RG - RED GLAND
TS - TESTES
OY = OVARY
GD • GONODUCTS
OT » OTHER
LATLN - LATERAL LINE
SEXCHAR - SEXUAL CHARACTERISTICS
MSFIN - MUSCLES OF FINS
ACDI - ACCESORY DIGESTIVE ORGANS
VERT - VERTEBRAL COLUMN
PHNYX - PHARYNX
PER - PERIPHERAL BLOOD ELEMENTS
ALI - ALIMENTARY CANAL
UB - URINARY BLADDER
THY - THYROID
ENPAN - ENDOCRINE PANCREAS
CAUDAL - CAUDAL NEUROSECTORY SYSTEM
CNS - CENTRAL NERVOUS SYSTEM
ANATOMICAL DIVISION
WM - WHITE MUSCLE
DM - DARK MUSCLE
OS - OSTIA
FL - FILAMENTS
CL - CILIARY TRACTS
VE - VENTRICLE
AT - ATRIUM
MC - MACROPHAGES
EPID =• EPIDERMIS
LEPID - LEPIDOTRICHIA
VERT - VERTEBRAE
ARCH - GILL ARCH
LAM - GILL LAMELLAE
ORAL - ORAL CAVITY
PAN - PANCREAS
GB - GALL BLADDER
BA - BULBUS ARTERIOSUS
SPL - SPLEEN
ERY - ERYTHROCYTES
PL - LABIAL PALPS
MO - MOUTH
EP » ESOPHAGUS
ST - STOMACH
RE - MIDGET/RECTUM
DD - DIGESTIVE DIVERTICULA
AM - AMEBOCYTES/HEMOCYTES
GN - GRANULOAMEBOCYTES
LATLN - LATERAL LINE CANAL
AT • ATRIUM
TYPE - FIN TYPE
FILA - GILL FILAMENTS/RAKERS
PSEUD - PSEUDOBRANCH
IN » INTESTINE
LIV » LIVER
SV - SINUS VENOSUS
HK - HEAD KIDNEY
THY - THYMUS
LEU - LEUCOCYTES
121
-------
AR = ARTERIES
CAP - CAPILLARIES
RTUBE = RENAL TUBULE
ADENO - ADENOHYPOPHYSIS
DAHL - DAHLGREN CELLS
URO - UROPHYSIS
SP = SPERMATOZOA
OF - OVARIAN FOLLICLES
TE - TELENCEPHALON
DI - DIENCEPHALON
ME - MESENCEPHALON
OL = OLFACTORY
OC - OCCULOMOTOR
TRIG - TRIGEMINAL
FA = FACIAL
GL - GLOSSOPHARYNGEAL
EY - EYE
TB = TASTE BUDS
EA - EAR
VN - VEINS
RCORP - RENAL CORPUSCLE
NEURO =» NEUROHYPOPHYSIS
THYFOL - THYROID FOLLICLES
INTIS - INTERRENAL TISSUE
CHRMAF - CHROMAFFIN TISSUE
PANIS - PANCREATIC ISLETS
CS - CORPUSCLE OF STANNIUS
ST - SEMINIFEROUS TUBULES
SC - SPERMATOGENIC CELLS
CE - CEREBELLUM
OP - OPTIC
TROC - TROCHLEAR
AB - ABDUCENS
AC - ACOUSTIC
VA - VAGUS
OLL - OLFACTORY LAMELLAE
LL - LATERAL LINE SYSTEM
ANATOMICAL SUBDIVISION
DD - DIGESTIVE DUCTS
DT - DIGESTIVE TUBULES
PY = PYLORIC
DE - DESCENDING
EX - EXOCRINE
HP = HEPATIC PARENCHYMA
HD - HEPATIC DUCTS
LYM - LYMPHOCYTES
NEU - NEUTROPHILES
BAS - BASOPHILES
PROX - PROXIMAL TUBULE
DUCT - COLLECTING DUCTS
PARSDIS - PARS DISTALIS
ON = OLFACTORY NERVES
EP = EPITHALAMUS
HY - HYPOTHALAMUS
TEG » TEGMENTUM
VV - VALVULA
SCL - SCLERA
CHOR - CHOROID GLAND
NEUR - NEUROEPITHELIUM
EPID - EPIDERMAL
LP - LATERALIS PROPER
00 - OTOLITH ORGANS
RMUSC - RETRACTOR MUSCLE/
SUSPENSORY LIGAMENT
HYPO - HYPODERMIS
CAR - CARDIAC/FUNDIC
AS - ASCENDING
RECTM - RECTUM
EN - ENDOCRINE
HV - HEPATIC VEINS
HEM - HEMOBLASTS
THM - THROMBOCYTES
EOS - EOSINPHILES
NECK - NECK SEGMENT
DIST - DISTAL TUBULE
PARSINT - PARS INTERMEDIA
OB » OLFACTORY BULBS
OL - OLFACTORY LOBES
TH - THALAMUS
OPTM - OPTIC TECTUM
CC » CORPUS CEREBELLIS
CORN = CORNEA
IRIS m CHOROID/IRIS
RET - RETINA
OM - ORAL MUCOSA
CL - CEPHALIC LATERALIS
SN - SUPERFICIAL NEUROMASTS
ML - MEMBRANOUS LABYRINTHS
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HISTOLOGICAL ELEMENT
CN =« CONNECTIVE TISSUE OT - OTHER
LC - LEYDIG CELLS CART - CARTILAGE
NOTO - NOTOCHORD GLRAY - GILL-RAY CARTILAGE
CHLOR - CHLORIDE CELLS PIT - PITUICYTES
MAT - MATURATION STAGES VM - VITELLINE MEMBRANE
MO = MEDULLA OBLONGATA SC - SPINAL CORD
TISSUE/CELL
PUR - PURKINJE CELLS PFLOBE - PAIRED FACIAL LOBE
WM - WHITE MATTER ACLOBE - ACUSTICOLATERALIS LOBES
GM - GREY MATTER CC - CENTRAL CANAL
CATEGORY
GP - GROSS PATHOLOGY
IN =» INFLAMMATION
RP - REPAIR AND PROLIFERATIVE PROCESSES
DE • DEGENERATIVE CHANGES, CELL AND TISSUE DEATH
NE = NEOPLASIA
TR - TRAUMA AND RELATED INSULTS
PI - PIGMENTS
GF = IMMUNOLOGIC AND GENETIC FACTORS
EX = EXOGENOUS FACTORS
PA - PARASITES
GROUP
ES = EXTERNAL SHELL LESIONS (101-126)
IS - INTERNAL SHELL LESIONS (127-139)
ET - EXTERNAL TISSUE LESIONS AND PARASITES (140-169)
IT - INTERNAL TISSUE LESIONS AND PARASITES (170-184)
ACUTE - ACUTE INFLAMMATION
CHRON - CHRONIC INFLAMMATION
REPAIR - REPAIR PROCESSES
PROLIF - PROLIFERATIVE PROCESSES
DE - DEGENERATION
CT - CELL AND TISSUE DEATH
BA - BEHAVIOR ASPECTS
BN - BENIGN NEOPLASMS
MN - MALIGNANT NEOPLASMS
TR - TRAUMA AND RELATED INSULTS
PHY - PHYSIOLOGICAL FACTORS
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Pathology:
101 « gaper
103 » malformation
105 - hole
107 - sponge
109 - ascidians
111 • algae
113 - hydroids
115 - Odostomia
117 = Crepidula
127 » loss of mantle fluid
129 - drill hole
131 - Cliona perforations
133 = irregular growth
140 • lesion
142 -
144 •
146
148
150
152
154
156
158
160
162 •
164 i
170 •
172 •
174 >
176 •
178 «
185 •
187 =
190
192 •
194
> green lesion
« ulcer
nodule
muscle detachment
pearls
foreign body
watery cyst
papilloma
nematode
cestode
• Pinotheres
• striped sea snail
• pale digestive
• white dg
• orange dg
• black dg
• ripe gonad
• moribund
» behavior
• tumors
« hemorrhage
• eyes (sunken)
gland
201 » acute inflammation
205 - perivascular infiltration
207 - edema
209 » granulocytic infiltration
102 - new growth
104 ™ fracture
106 « hydrogen sulfide
oxidation
108 =» barnacles
110 • tube worms
112 - drill egg cases
114 - Hydractinia
116 - Diplothyra
118 » anemone
128 a sediment accumulation
130 - mud blister
132 • Polydora tunnels
134 - conchiolin deposit
141 - yellow inflammatory
lesion
143 - pustule
145 » abcess
147 - weak muscle
149 - hyperemia
151 - fibrosis
153 =» odor
155 » tumor
157 » xenoma
159 - trematode
161 - copepod
163 - fish
165 - hake
171 » tan dg
173 « yellow dg
175 - brown dg
177 « ripening gonad
179 - Mytilicola sp.
186 - dead
188 » color
191 » ulceration
193 » parasitism
195 - evagination
204 = exudation
206 • phagocytosis
208 - abcess
211 * degranulation
124
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251
253
255
257
259
261
301
303
305
351
353
355
357
359
361
363
401
403
405
407
409
411
416
418
450
452
454
456
459
501
503
505
507
520
522
524
526
528
530
550
hyalin hemocyte infiltration 252
(focal)
hyalin hemocyte infiltration 254
(systemic)
melanization 256
concretion
pearl formation
macrophages
repair
collagen deposition
sclerosis
hypertrophy
dysplasia
fibroplasia
maturation arrest
anaplasia
polyploidy
enlarged nuclei
atrophy
metaplasia
erosion
vacuolation
cyst
fatty degeneration
nuclear inclusion
embolism
necrosis
karyorrhexis
coagulative necrosis
hyaline necrosis
lysis
benign
locally invasive
metastatic
anaplastic
embryonal
neuroma
rhabdomyoma
lipoma
germanoma
papilloma
sarcoma
258
260
302
304
306
352
354
356
358
360
362
402
404
406
408
410
415
417
419
451
453
455
457
460
502
504
506
521
523
525
527
529
hyalin hemocyte
(perivascular)
pigmented aggregation
collagenous
encapsulation
calcification
lymphocytic
regeneration
granulation tissue
cellular clot
hyperplasia
xenoma
adenohyperplasia
mitosis
anaploidy
chromosome abnormality
ceroidosis
ulceration
sloughage
syncitia
edema
cytoplasmic inclusion
feulgen + inclusion
infarct
pyknosis
karyolsis
caseous necrosis
liquefactive necrosis
necrobiosis
multifocal
diffusely invasive
differientiated
epithelioraa
leomyoma
fibroma
teratoma
hemangioma
570 » carcinoma
125
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600 = trauma 601 - abrasion
602 = encision 603 » laceration
604 = compression 605 = burn
606 • hemorrhage
651 - mucous production 652 - diarrhea
654 • dietary deficiency
700 - pigment 701 » lipofuscin
702 - melanin 703 » feritin
704 = hemosiderin 705 «• hemocyanin
706 = chlorophyll
800 = immunological 850 » genetic
900 = heavy metal 901 = copper
902 - zinc 903 =• lead
904 • cadmium
910 - viruses 911 » fungi
912 = sporozoans 913 » nematodes
914 =« cestodes 915 - Turbellaria
916 - trematodes
degree:
1 - moderate 2 - definite 3 - severe 4 - fatal
126
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APPENDIX 5. IMMUNE REPRESSION TEST SUMMARY
Repression of Scale Graft Rejection as an Index of
Immunosuppression in Fish
Richard E. WoIke
Comparative Aquatic Pathology Laboratory
University of Rhode Island - East Farm
Kingston, Rhode Island 02882
Progress Report of Work Performed on Cooperative Agreement
CR-912807-01-1
127
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INTRODUCTION
The relationship of the mechanisms and cells involved in graft
rejection immunity to those involved in tumor immunity (TSTA) is well
recognized (Lackmann and Mitchison, 1982). In both graft rejection and
tumor immunity, the macrophage plays a pivotal role in recognition of
cell surfact antigens and as an activated effector cell (Fidler, 1985;
Keller, 1985; Normann, 1985; Dy et al., 1979). These cells, and the
lymphocytes they activate, may be repressed by various immunosuppressive
agents, including many environmental agents (Vos, 1981). Pesticides,
industrial compounds and heavy metals have been related to immune repres-
sion in homeotherms (Sharma, 1981). An immune suppression of similar
cause has been reported to occur in fish (Avtalion, 1981; Zeeman and
Brindley, 1981; Bennett, 1984). Allograft rejection as a test to detect
immune function in mice has been suggested (Vos, 1981). Interestingly,
scale grafting in fish is a simple, rapid, and temperature dependent
procedure that is well documented (Hildemann, 1957; Hildemann and Haas,
1960). It is therefore hypothesized that repression of scale graft
rejection in fish may be related to aqueous anthropogenic pollutants, and
in turn, to repression of tumor immunity. To this end, scale graft
rejection and its repression are being studied in the winter flounder
(Pseudopleuronectes americanus).
The objectives of this on-going study are: 1) to determine the ease
and feasibility of scale transplantation in winter flounder, 2) to deter-
mine the effect of temperature on graft survival time, and 3) to measure
the effects of exposure to Black Rock Harbor sediment on graft survival
time as an indicator of immune repression.
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MATERIALS AND METHODS
One to two winter flounder (1 to 2 year class; 11 to 18 cm total
length) were randomly placed in 42 x 42 x 20 cm flow-through, salt water
compartments, fed daily, and held in the dark. The compartments were in
sets of four and each set received 10, 15 and 20°C water. To determine
the ease and feasibility of scale transplantation procedures, experimental
fish were acclimated to, and held at, 20°C water. The effects of temper-
ature were determined in acclimated fish simultaneously at 10, 15 and
20°C. A pilot exposure experiment using Black Rock Harbor sediment and
an anamnestic experiment were both conducted at 15°C. For all experiments,
56 fish with 168 allographs and 56 homographs were examined.
In each experiment, graphs were obtained from a single donor. Donor
scales were placed in empty recipient scale pockets just above the lateral
line of the caudal penduncle on the pigmented side. Three allographs and
one homograph were made per fish with a minimum of two normal scales left
between each graft. Rejection was determined after the method of Hildemann
and Haas (1960) based upon "clearing" of the donor scale and characterized
by loss of donor epidermis and dissolution of epidermal melanophores.
Grafting and graft rejection determinations were made with a 6 to 12
power disecting microscope while holding anesthetized animals in a shallow,
water-filled container. Initially, mean scale rejection times were
determined by the rapid graphic method of Litchfield (1949); later data
were reexamined using a simple one-way ANOVA or a Student T-test.
129
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RESULTS
Scale transplantation feasibility with the aide of a dissecting
microscope and ophthalmic microforceps, scales easily transplanted from
donor to recipient fish. It is estimated that 10 to 20 fish receiving
four scales each can be grafted per hour. Grafts of the correct size fit
tightly within the host scale pocket and are revascularized rapidly. No
homographs were rejected in any instance. Determination of scale rejec-
tion is somewhat subjective, but not so subjective as to affect test
validity. Reproducibility of results was excellent and more dependent
upon water water temperature or prior acclimation time than upon tech-
nique. To test the technique, transplants were made at 20°C in November
and December of 1985 and in January and April of 1986 (fish N • 20).
Mean time in hours to rejection were: November: 184, SD 9.7; December:
187, SD 1A.5; January: 210, SD 12.5; and April: 172, SD 7.7. Rejection
times for November, December, and April are not significantly different,
whereas January rejection times are significantly different from the
other months.
Effect of temperature on graft rejection time
Temperature significantly altered graft rejection time. In the
experiment to measure temperature effect, grafts to fish held at 20°C had
a mean rejection time of 172.4 hours, SD 11.5; in fish held at 15°C,
217.3 hours, SD 23.8; and in fish held at 108C, 424.0 hours, SD 27.3.
Statistically, these means were significantly different.
130
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Effect of exposure to Black Rock Harbor sediment on graft rejection time
In a pilot study, three control fish were placed on a reference
sediment and three test fish were placed on undiluted Black Rock Harbor
sediment at 15°C for a period of 30 days prior to scale transplantation.
Following grafting, fish were kept in clean, sediment-free water. Two of
the control fish died shortly after grafting. Mean rejection time in the
remaining control fish was 296 hours, whereas mean time to rejection in
the exposed fish was 536 hours with an SD of 84.2 hours.
DISCUSSION
To date, results of this study indicate that scale transplantation
and determination of time to graft rejection in winter flounder is a
feasible, reproducible and promising procedure to measure immune repres-
sion. The fish lends itself well to the surgical procedure, is of eco-
nomic importance, is native to polluted and potentially polluted coastal
areas, and has recently been shown to develop hepatocellular carcinoma
(Murchelano and Wolke, 1985).
The pilot study to evaluate the effect of a highly polluted sediment
indicated a doubling time to graft rejection in exposed fish when compared
to animals kept in clean, sediment-free water. Controls comparisons
cannot be safely made as two of the three control fish died and the
remaining animal was cachexic. However, time to graft rejection in the
cachexic animal was 340 hours less than in the exposed animals. Because
this project is on-going, a larger, more comprehensive exposure experiment
is scheduled to begin in January 1987.
131
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