00319
v. 2
°OOR88006
A HISTOPATHOLOGICAL AND CHEMICAL
ASSESSMENT OF WINTER FLOUNDER,
LOBSTER AND SOFT-SHELLED CLAM
INDIGENOUS TO QUINCY BAY,
BOSTON HARBOR AND AN IN SITU
EVALUATION OF OYSTERS INCLUDING
SEDIMENT (SURFACE AND CORES)
CHEMISTRY
Prepared by the United States Environmental Protection Agency,
Environmental Research Laboratory,
Narragansett, Rhode Island
PAHs
Long I
Moon ._
Head--'
113//g/g
S4 S-11
Rainsford I.
8-17 H M2 E
Peddocksl.
QUINCY BAY
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QUINCY BAY STUDY
BOSTON HARBOR
U.S. ENVIRONMENTAL PROTECTION AGENCY
NARRAGANSETT, RHODE ISLAND
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QUINCY BAY
A Histopathological and Chemical Assessment of Winter Flounder,
Lobster and Soft-Shelled Clam Indigenous to Ojaincy Bay,
Boston Harbor and an In Situ Evaluation of Oysters
Including Sediment (Surface and Cores) Chemistry
TASK II AND III REPORT
PRINCIPAL INVESTIGATORS
George R. Gardner
Richard J. Pruell
TECHNICAL ASSISTANTS
Sandra Benyi Eileen McFadden
Warren Boothman Richard McKinney
Doranne Borsay Curtis Norwood
Robert Bowen Frank Osterman
Thomas Daniels Paul Selvitelli
Joseph LiVolsi Jay Terra
HISTOPATHOLOGY CONSULTANT
Paul Yevich
PROJECT OFFICER
Katrina Kipp (EPA Region I, Boston, MA)
TECHNICAL COORDINATORS
George R. Gardner
Walter Galloway
June 1988
U.S. Environmental Protection Agency
Environmental Research Laboratory
Narragansett, Rhode Island 02882
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CONTENTS
DATA SUMMARY iii
LIST OF FIGURES viii
LIST OF TABLES ix
PART I. METHODOLOGY 1
Field Studies of Winter Flounder 1
Field Collection 1
Transportation 3
Histopathological Protocol 3
Histopathological Endpoints 4
Field Studies of Lobster 5
Field Collection 5
Transportation 5
Histopathological Protocol 7
Histopathological Endpoints 7
Field Studies of Soft-shelled Clams 7
Field Collection 7
Transportation 8
Histopathological Protocol 8
Histopathological Endpoints 10
Field Studies of In Situ Transplanted Oysters 10
Oyster Source 10
Oyster Deployment 10
Histopathological Protocol 12
Histopathological Endpoints 14
Chemistry 14
Organic Analysis Methods 14
Tissue Sample Preparation 14
Sediment Sample Preparation 17
Chemical Separations 17
Instrumental Analysis 18
Quality Assurance-Organic Analysis 23
Inorganic Analysis Methods 25
Tissue Sample Preparation 25
Sediment Sample Preparation 27
Instrumental Analysis 28
Quality Assurance-Inorganic Analysis 30
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PART II. RESULTS
Winter Flounder 33
Characteristics of Neoplastic Lesions 46
Liver Neoplasms 46
Intestinal Neoplasms 47
Schwannomas 49
Characteristics of Non-neoplastic Lesions 49
Digestive System Organs (Liver) 49
Digestive (Except Liver), Excretory, Circulatory
(Spleen) and Reproductive Systems 52
Respiratory, Muscular, Integumentary,
Circulatory and Nervous Systems 55
Flounder Peripheral Blood 59
Lobster 62
Characteristics of Cellular Alteration 64
Pathologic Evaluation by Station 66
Soft-shelled Clam 68
Characteristics of Cellular Alteration 70
Oyster 73
ACKNOWLEDGEMENTS 78
REFERENCES 79
APPENDIX A. Winter Flounder Liver Lesion Type and
Frequency 81
APPENDIX B. Chemistry Data 82
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DATA SUMMARY
The purpose of our histopathological and chemical field studies in Quincy
Bay was to assess the prevalence of pathological lesions in four
commercially valuable marine organisms, measure the concentrations of
selected chemicals in edible flesh, and assess the areal extent of chemical
contamination in the sediments. Species collected from Quincy Bay included
the winter flounder (Pseudopleuronectes americanus), lobster (Homarus
americanus) and soft-shelled clam (Mya arenaria) while oysters (Crassostrea
virginica) were evaluated following in situ exposures in Quincy Bay.
Our study provides strong histopathological evidence that winter flounder
and soft-shelled clams in Quincy Bay are in poor health from a histological
standpoint. Lobsters, recently migrated to Quincy Bay (based on commercial
fishing observations) at the time of collection, were in relatively good
health. Oysters deployed for 40 days in the bay developed tumors and
"ovacystis" disease.
Significant pathological changes including neoplastic alterations were
present in Quincy Bay winter flounder. Morphological alterations clearly
neoplastic in character were observed in the liver, the stomach, and the
nerves associated with fin rays of some winter flounder. Eighty three (83t)
of the one hundred flounder collected in Quincy Bay had liver pathology; of
those, twenty three (23%) had liver neoplasms characterized as hepatocytic
lesions. The liver lesions observed in Quincy Bay winter flounder
represented a continuum from early emerging neoplasms to ones with
independently recognizable cellular characteristics that allowed for a
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decisive diagnosis. Seventeen percent (17%) of the liver neoplasms (4) were
considered as emerging or incipient neoplasms. Three of the liver neoplasms
had cholangiocytic characteristics. Stomach lesions in 4 animals were
interpreted as carcinomas in situ. Lesions with adenomatous characteristics
in the gastrointestinal tract occurred in 20, hyperplasia of endocrine
pancreas (nesidioblastosis) 19, gill lesions with the appearance of scuamous
cell carcinoma 7 and a papilloma in 1, and nerve tissue tumors interpreted
as schwannomas in 2 of the fish. The adenomatous appearing lesions in the
gastrointestinal tract, for the present, were interpreted as basophilic
nests or "nidi" that may represent areas of active regeneration. Those
basophilic nests did not meet all the criteria needed to rate them as
neoplasms, based on observations in mammals. The observed alterations may
have been the result of other cellular injury, however, the source of toxins
responsible for inducing the changes could not be identified. Similarly,
lesions in the gills with sguamous cell-like carcinomas also lacked some of
the cardinal features usually associated with malignant tumors. Additional
features might be more cell atypia and amassed cells.
Pathological alterations other than neoplastic or potentially neoplastic
lesions in some flounder involved the circulatory; excretory; muscular;
nervous, including sensory organs (i.e., olfactory, lateral line and stato-
acoustic organs) and integumentary systems. Anatomical sites of those
alterations included kidney, pancreas as pancreatic ductal dysplasia,
spleen, urinary bladder, gonadal organs, arteries (endothelial plaques),
and dermal fibromatosis. An anomalous lesion, a myoadenoma occurred in the
gall bladder of one flounder. Pathological responses in the gills, other
than neoplasms, includes filament bifurcation and parasitic infestation by
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digenetic trematodes (trematodiasis). Trematodiasis of the gills was
observed in ninety percent (90%), while cephalic musculature trematodiasis
was observed in sixty three percent (63%) of the flounder.
Proliferative cellular change in soft-shelled clams from two locations in
Quincy Bay (Moon Head and Moon Islands) was limited to atypical cell
hyperplasia (ACH) in respiratory epithelium and in kidney. Prevalence of ACH
in gills was 56% at Moon Head (n-30) and 62% at the Moon Islands (n-78)
animals; in the kidney, prevalence was 73% at Moon Head and 71% at Moon
Islands. Inflammation was observed in the gills of 77% of the animals at
Moon Head and 83% at Moon Islands. The condition was considered to be
advanced and potentially devastating to the soft-shelled clam community
because 1) the condition alters the respiratory area available to gaseous
exchange (between hemolymph and dissolved oxygen) and 2) partial to advanced
loss of ciliary function alters gill water currents essential to respiration
and feeding. Rickettsia-like prokaryocytes were present in digestive tubules
in approximately 51% of these clams (up to 6 organisms per single tubule).
That presence was extraordinarily high based on ERL/N's twenty-one years of
archived histological data. Reproductive organs of the soft clams appeared
normal morphologically, with the exception of gamete maturation. Ova
development was entirely in the formative stages while male spermatid
development was advanced. Thus, that evidence suggests the reproductive
cycle of Quincy Bay soft-shelled clams was asynchronous. Parasitism was
prominent in Quincy Bay soft-shelled clams in all major organs. Hemato-
poietic neoplasms were absent.
The lobsters examined were generally in good health as related to
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histological parameters. One lobster had Black Spot Gill Disease. Carapace
ulceration was present in lobsters (3 of 5) collected at two stations near
Nut Island. Inflammation and parasitism, present to some degree in collected
animals, was considered minimal.
Tumors occurred in oyster renal excretory (4 of 200 - 2%) and gastro-
intestinal epithelium (8 of 200 - 4%) during the 40 day deployment in
Quincy Bay. Three of the gastrointestinal neoplasms were interpreted as
non-invasive adenocarcinomas in situ that occurred in the rectal segment of
the gut; five neoplasms in the stomach occurred as adenomatous (3) and
papillary (2) formations. Neoplasms were absent in oysters following a 40
day deployment at a reference location near "the Graves" in Massachusetts
Bay. "Ovacystis" disease was identified in oysters deployed at all locations
including the reference location near "the Graves". The causative agent
identified in "ovacystis" disease of oysters is a papovavirus. Ovacystis
disease was absent in pre-exposure control oysters. Oysters for our studies
were obtained from the Cotuit Oyster Company, Cotuit, MA.
Surface sediment chemistry results indicate that stations near Moon Head,
Long Island and Peddocks Island contained the highest concentrations of
polychlorinated biphenyls (PCBs), chlorinated pesticides and the trace
metals except mercury. Different distributions were observed for
coprostanol, polycyclic aromatic hydrocarbons (PAHs) and mercury. The
highest coprostanol levels were seen in samples collected near Long Island.
Elevated coprostanol concentrations were also measured in samples collected
near Nut Island. The PAH concentrations were very high at two sites. One of
these was a sample collected near Moon Head and the highest level was
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measured in a sample collected from near Nut Island. The mercury
concentrations in surface sediment samples varied considerably throughout
the bay.
Sediment cores were collected from four locations in Quincy Bay for detailed
chemical analyses. Trends seen in the contaminant concentrations in sections
of the cores were similar. Differences in the cores were consistent with
differences in net sedimentation rates between stations. In general, the
concentrations of PCBs and chlorinated pesticides showed subsurface
concentration maxima usually in the 2-4 inch section. The concentrations of
PAHs, cadmium, chromium, copper and lead showed more variable trends in the
sediment cores; however, the deepest sections of each core tended to show
the lowest levels. Mercury concentrations were generally higher in the
deeper sections of the cores.
Contaminant concentrations were measured in samples of oysters, soft-shelled
clams, lobster muscle and hepatopancreas, and winter flounder. The highest
concentrations of chlorinated compounds were found in the lobster and
flounder samples; lobster hepatopancreas levels were found to be as high as
113 ug/g dry weight. The highest PAH concentrations were also seen in the
lobster hepatopancreas. PAH concentrations in oysters and clams were
intermediate while concentrations were below the analytical detection limits
in flounder muscle. The trace metal levels (except those of copper in the
lobster samples) were relatively low in all of the tissue samples analyzed.
Mercury concentrations were highest in the lobster muscle samples.
VII
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LIST OF FIGURES
No. Page
—— ,•*
1 Quincy Bay Winter Flounder Trawl Transects 2
2 Quincy Bay Lobster Stations 6
3 Quincy Bay Soft-shelled Clam Collection Sites 9
4 Quincy Bay Caged Oyster Deployment Sites 11
5 Quincy Bay Oyster Cage Design 13
6 Quincy Bay Sediment Core Locations 15
7 Quincy Bay Surface Sediment Sample Locations 16
8 Quincy Bay Composite Station (Biol/Chem) Map 34
8A Quincy Bay Composite Station Map Legends 35
9 Quincy Bay Winter Flounder Transects 36
10 Quincy Bay Winter Flounder Length Vs. Weight 37
11 Quincy Bay Lobster Length Vs. Weight 63
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LIST OF TABLES
NO.
1 Selected Organic Compounds 31
2 Selected Polycyclic Aromatic Hydrocarbons 32
3 Quincy Bay Winter Flounder Master Table 38
4 Summary of Neoplasm Prevalence in Quincy Bay
Winter Flounder 44
5 Prevalence of Liver Lesions 48
6 Prevalence of Lesions in Winter Flounder
Visceral Organs Other Than Liver 53
7 Prevalence of Lesions in Gill, Heart,
Sensory Organs and Integument 56
8 Quincy Bay Winter Flounder Differential Mean
Blood Cell Counts 61
9 Histopathology of Quincy Bay Lobster 65
10 Prevalence of Pathologic Lesions in Quincy Bay
Soft-Shelled Clams 69
11 Histopathology of Oysters Exposed _In Situ 77
Appendix A. Winter Flounder Liver Pathology
Prevalence of Liver Pathology 81
Appendix B. Chemistry Data
Key for the abbreviations used 82
PCBs and coprostanol in sediment 83
PCB congeners in sediment 84
Pesticides in sediment 86
PAHs in sediment 87
Metals in surface sediment 90
Metals in sediment cores 91
PCBs in organisms 92
PCB congeners in organisms 93
Pesticides in organisms 95
PAHs in organi sms 96
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Metals in organisms 99
Metals in lobsters 100
PCBs in flounder 101
PCB congeners in flounder 102
Pesticides in flounder 104
PAHs in flounder 105
Metals in flounder 108
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QUINCY BAY STUDY
METHODOLOGY
Personnel at the Environmental Research Laboratory in Narragansett, Rhode
Island (ERL/N), conducted biological and chemical evaluations of selected
biota and sediments in Quincy Bay using the following approach and
methodology.
Biological Study
Indigenous Organisms
Winter Flounder (Pseudopleuronectes americanus)
Field Collection
Winter flounder collections for the Quincy Bay Study started on 8 May 1987
with subsequent collections on 12 May, 13 May, 18 May, 19 May and on 20 May
1987. One hundred winter flounder of random age and size were collected by
otter trawls along transects in the study area (Figure 1). Two transects
were positioned north to south and two in an east to west direction.
North/south transects extended between those sediment sampling stations
identified as S3 to S14 in the Quincy Bay Statement of Work (Tl) and from
West Head to Pig Island (T2). East/west transects were from West Head to
the eastern most part of Peddocks Island (T3) and from Moon Island to the
eastern end of Long Island (T4). Trawling along those transects followed
depth contours or isopleths to the extent possible. Two fifteen minute
trawls were conducted along each transect. Those trawls follow guidelines
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set forth by Mearns and Allen (1978) where practical. The otter trawl type
used was a small low rise having a 24' head rope, 31' foot rope, a 3/16"
chain sweep and a cod end with a 1" mesh. The doors are 3' poly vee doors
with 15' of 1/4" chain on the lower legs and 14' of 3/8" polypropylene
dacron line on the upper leg. At water depths of less than 6 fathoms, a
scope ratio of 8:1 is used.
Immediately after collection the flounder were examined for gross
pathological anomalies and then placed in 30 liter rectangular containers
filled with bay water. Water was replaced in the holding units as required
(i.e., more frequently as flounder density increased during the collection
period).
Transportation
Quincy Bay flounder were periodically transferred from the work boat to
fiberglass tanks mounted on a truck bed. Bay water contained in those
chambers was aerated during on-site holding and transportation to ERL/N.
Compressed air and air stones were used to maintain sufficient dissolved
oxygen content in the water during transport. At the laboratory, flounder
were held overnight in a flow-through sea water facility. All flounder were
prepared for histology and chemical analyses within 24 hours after delivery
to the laboratory.
Histopathological Protocol
Fixation and tissue processing followed procedures developed at ERL/N
(Yevich and Barszcz, 1981). Briefly, fish were weighed to the nearest grar
and total length measured. Trunk musculature was trimmed away with an
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excision that parallels the vertebral column dorsally from the left eye
distally to a point approximately 1-2 cm posterior to the visceral cavity
and mesonephric kidney. The excision was continued with a transverse cut to
the ventral margin, while insuring that reproductive tissue elements
remained intact. Trunk musculature was removed, then wrapped in aluminum
foil, labeled appropriately, and frozen for eventual chemical analyses.
Dorsal musculature enclosing the visceral cavity was then removed and
viscera excised by severing the gastrointestinal tract at the esophagus and
anal opening. Gill opercula were excised, followed by a transverse cut at a
point in the vertebral column that separated head and trunk kidney from the
cephalic region. Those components were then immersed in Dietrich's fixative.
Visceral organs, kidney and the cephalic region were then sagittally and
parasagittally sectioned and reimmersed in fixative. Soft tissues were fixed
for 5 to 10 minutes prior to trimming into 3 to 6 mm thick sections.
Sectioning of the cephalic structure and the vertebral column associated
with the kidney was initiated on a sagittal plane after 30-60 minutes.
Trimmed tissue was decalcified as necessary and then washed overnight in a
water bath, embedded in paraffin, cut at 6 // and stained with Harris
Hematoxylin and Eosin.
Histological Endpoints
Systems evaluated by light microscopy included integumentary, digestive,
circulatory, excretory, endocrine, nervous, respiratory and muscular.
The same protocols were followed using winter flounder collected from
Narragansett Bay and Long Island Sound. Approximately 500 animals frcr
those two locations served as our reference for histopathologice.1
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evaluation of Quincy Bay flounder.
Lobster (Homarus americanus)
Field Collection
Lobster collections occurred at nine locations in the Quincy Bay study area
identified in the US EPA Region I Plan of Study (POS) as A-1,2,3, B-4, C-5,
D-6,7,8, E-9, F-10, G-11,12,13, and 1-15. Station H was relocated to a
position nearer the eastern most point of Peddocks Island (Figure 2).
Lobsters sampled by otter trawl incidental to flounder collections occurred
at two stations (A and B) on May 8, 13 and 20. Lobster collections were
primarily the result of collaboration with a local commercial lobster
fisherman. Commercial lobster traps are set in strings of several units in
the commercial fishing operation. The arrangement with a commercial
fisherman allowed positioning of the lobster traps, on either end of a
lobster pot "string", at a designated POS sampling station. Five animals
were trapped at each of the seven remaining stations (C through I).
Transportation
Lobster claws were banded with different colored rubber bands during
collection to maintain identity with specific stations. Lobsters were held
in rectangular containers on board the commercial lobster boat where
seawater was changed frequently; the insulated containers also served to
maintain ambient temperature during transportation to the laboratory.
Lobsters were delivered to the laboratory on the same day, held in flow-
through seawater systems overnight, then processed for histology and
chemistry the following day.
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Histopathological Protocol
Initially, lobsters were weighed and carapace length measured. The abdominal
region, commonly known as the tail, was removed, wrapped in aluminum foil,
and frozen for chemical analysis. Each lobster was then further processed
by removing other appendages and the thorax carapace covering respiratory
organs. Following a midline cut along the ventral surface the intestine,
reproductive tract, heart and hepatopancreas were removed and placed in
fixative. The hepatopancreas was bisected with one half being fixed for
histology and the other half packaged in aluminum foil, frozen and stored
for chemical analysis. Following a transverse cut just behind the eyestalks,
a cut was made along the dorsal aspect of the thorax. All tissue was
preserved in Kelly's fixative overnight. After fixation for 16-20 hours the
soft tissues were trimmed to 2-3 mm wide sections. All calcified tissue,
including the stomach, was decalcified and trimmed as necessary. Processed
tissues were washed overnight in a water bath, embedded in paraffin, cut at
6 fj and stained with Harris Hematoxylin and Eosin.
Histological Endpoints
Systems observed by light microscopy included integumentary, respiratory,
circulatory, nervous, reproductive and excretory.
Soft-shelled Clams (Mya arenaria)
Field Collection
Field sampling for soft-shelled clams in the study area was within the
intertidal flats extending from Moon Islands ("The Moons", MA Division of
Marine Fisheries Shellfish Definition Chart, Area BH-7) to Nut Island, frorr.
Moon Head east along Long Island and other Quincy Bay island sites including
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Rainsford and Peddocks (Figure 3). Stippled areas within intertidal zones
(i.e., between solid and dotted lines) in Figure 3 represent potential soft-
shelled clam collection sites. Site selection was based on advice from the
local shellfish warden, shellfishermen and soft-shelled clam abundance. A
minimum of twenty animals from viable collection sites were to be prepared
for histopathological evaluation; ten additional animals were to be
collected for chemical analysis. Soft-shelled clams were present in only two
of seven sites examined (noted by "X"; Figure 3). At Moon Head thirty (n-30)
and Moon Islands seventy eight (n-78) clams were collected. Collections for
soft-shelled clams at or near Quincy Great Hill, West Head of Peddocks
Island, Rainsford Island, West Head and Bass Point on Long Island were
unsuccessful. Animals were brought to the laboratory and held in a flow-
through seawater system to purge themselves overnight of sand and
particulate matter to facilitate processing, and to purge themselves of
organic substances contained in the gut that might influence chemical
results.
Transportation
Soft-shelled clams were placed in rectangular containers during on-site
storage and transportation from field to laboratory. Temperature was
controlled using ice-packs; wet paper towels served to keep the animals
moist during transport.
Histopathological Protocol
Soft-shelled clams were opened between the mantle and the valve with a
shellfish knife. Whole animals were then placed in Belly's fixative for 15-
30 minutes, sectioned on a sagittal plane along 95% of the midline, and
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reimmersed in fixative for overnight duration. During final trimming the
midline cuts were finalized and the animals were transectioned. Tissue
quadrants were trimmed to 2-3 mm, washed overnight, embedded in paraffin,
cut at 6 // and stained with Harris Hematoxylin and Eosin.
Histopathological Endpoints
The histopathological protocol was designed to allow evaluation of all major
organ systems with light microscopy.
In-situ Transplanted Oysters
(Crassostrea virginica)
Oyster Source
Oysters for experimental purposes were obtained from the Cotuit Oyster
Company, Inc., Cotuit, MA. Oysters aged from 2h to 3 years represented a
collection obtained from bed six located in Cotuit Bay on May 28,1987.
Oyster Deployment
Oysters were set at a reference area near the Graves (Station 1) in
Massachusetts Bay and four locations (Stations 2, 3, 4 and 5) in the Quincy
Bay study area (Figure 4). Station 2 represents a previously undesignated
site in Quincy Bay. Oysters were deployed from June 5 through July 16, 1987
in the vicinity of previously designated Quincy Bay sediment sampling
locations Cl, C3 and S13 within one half meter of the bottom. Those
locations were selected based on physical characteristics such as depth,
tidal movement and potential contact with water borne organic particulate
emanating from the Nut Island discharge. One hundred oysters were set in
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each location using plastic-coated wire cages (constructed by Ketcham Traps,
New Bedford, MA). The cages are then attached with plastic cable ties to
vinyl coated metal frames set into two rectangular concrete end plates
(Figure 5). The cement end plates served a dual function of keeping the
cages off the bottom and maintaining position even in areas of high current.
In addition, each cage had an acoustic beacon with a pre-set low frequency
radio signal (khz) to facilitate relocation. Cages were relocated by homing
on acoustic beacons attached to the cages and recovered by divers. Water
quality measurements were taken at all oyster deployment stations.
Parameters measured included dissolved oxygen, salinity, conductivity and
temperature. In addition, Christopher Scholl of the Massachusetts Division
of Environmental Quality Engineering (DEQE) collected dissolved oxygen
measurements during the study period.
Histopathological Protocol
The oysters were opened with an oyster knife inserted into the ligament and
twisted to separate the valves. The knife was then used to loosen and
separate the mantle and adductor muscle, removing the dorsal valve. Oysters
were removed from the ventral valve by separating the adductor muscle at
point of attachment. Whole oysters were placed into Helly's fixative for
15-30 minutes, removed and sagittally sectioned along 95% of the midline
and returned to the fixative overnight for 16 to 24 hours. Oysters were
sectioned transversely through the body mass during final trimming. Sections
were cut adjacent to the pericardial cavity oriented towards the anterior
region of the animal. Each half was then cut parasagittally into sections
2-3 mm in thickness, washed overnight in a water bath, embedded in paraffin,
cut at 6 u and stained with Harris Hematoxylin and Eosin.
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Histopathological Endpoints
^n situ studies were conducted to assess potential tumor induction in
kidney, gills and gastrointestinal tract in adult oysters.
Chemistry
Sediment chemistry was conducted on edible biological tissue of winter
flounder, lobster, soft shelled clams and oysters, and on sediment core
(Figure 6) and surface sediment (Figure 7) samples collected from selected
locations in Quincy Bay. The stations denoted with smaller lettering were
only analyzed for trace metals as part of the initial sample screening.
ORGANIC ANALYSIS METHODS
Tissue Sample Preparation
Tissue samples were homogenized using a Polytron homogenizer and an aliquot
was taken for a wet/dry weight determination. An appropriate amount of the
remaining tissue homogenate was added to a centrifuge tube along with
internal standards and 50 ml of acetonitrile. The solvent and sample were
mixed with a Polytron homogenizer for twenty seconds. The sample was
centrifuged and the supernatant decanted off into a separatory funnel
containing 300 ml of deionized water. This procedure was repeated two more
times using 50 ml of acetonitrile each time and the extracts were combined
in the separatory funnel.
The acetonitrile/water mixture was extracted three times with 50 ml cf
pentane each time and the extracts combined. Sodium sulfate was added tr
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remove traces of water and then the sample was volume reduced to 10 ml in a
round bottom flask fitted with a Kuderna-Danish evaporator and a 3-ball
Snyder column. The sample was transferred to a 10 ml graduated concentrator
tube containing an ebulator and fitted with a micro-Snyder column and
volume reduced to 1 ml using a tube heater. The extract was then ready for
chemical class separations using silica gel column chromatography as
described below.
Sediment Sample Preparation
Sediment samples were thoroughly mixed and a portion of the sample removed
for a wet/dry weight determination. About 5-10 g of the sample was added to
a centrifuge tube along with internal standards and 50 ml of acetonitrile.
An ultrasonic probe was inserted into the sample and turned on for 60
seconds. The sample was then centrifuged and the supernatant decanted off
into a separatory funnel containing 300 ml of deionized water. This
procedure was repeated two more times using 50 ml of acetonitrile each time
and the extracts were combined in the separatory funnel.
The acetonitrile/water mixture was extracted three times with 50 ml of
pentane each time and the extracts combined. Sodium sulfate was added to
remove traces of water and then the sample was volume reduced and prepared
for silica gel chromatography as described for tissue samples.
Chemical Separations
Chemical class separations were achieved on a 0.9 x 45 cm column which
contained 11.5 g of BioSil A silicic acid (100-200 mesh) that was fully
activated, and then 7.5% deactivated with water. This deactivation step was
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accomplished by adding an appropriate amount of water to the silica in a
glass bottle and placing it on a ball-mill tumbler overnight. Before
samples were added, the column was cleaned with 50 ml of methylene chloride
and 50 ml of pentane. All sample extracts were added 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 45 ml of pentane. A second fraction (f2) was eluted using 35 ml of 30%
methylene chloride in pentane. The third fraction was then eluted with 35 ml
of 30% methanol in methylene chloride. Activated copper powder was added to
the fl fraction to remove any free sulfur.
The volume of each fraction was then 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 volume of the fractions
was reduced to 0.8 ml using a tube heater. The ebulator was removed and
rinsed into the sample and the sample volume was brought up to 1 ml using
heptane. Tables 1 and 2 list the compounds quantified in each of the three
fractions.
Instrumental analysis
Gas Chromatography
The fl fractions were analyzed for PCBs, hexachlorobenzene (HCB) and DDE by
capillary gas chromatography (GC). For these analyses, a 1 ul splitless
injection was made into a Hewlett Packard 5890 gas chromatograph equipped
with a 30 m DB-5 fused silica capillary column (J & W Scientific) and an
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electron capture detector. Helium was used as the carrier gas at a flow
rate of about 1.5 ml/min and the flow of a 95:5 mixture of argon:methane to
the detector was 35 ml/min. The oven temperature was held at 60 °C for 1
min and then programmed from 60 to 315 °C at 10 °C/min. The injector
temperature was 270 °C, and the detector was maintained at 325 °C.
The concentrations of Aroclors 1242 and 1254 were quantified. Two peaks from
each of these Aroclor mixtures were used for quantification. The peaks
chosen included two early eluting peaks from Aroclor 1242 and two late
eluting peaks from Aroclor 1254. The peaks used for each of the Aroclors
were not found (or were very minor) in the other Aroclor mixture. Therefore,
the concentrations of the two Aroclors can be added as a measure of total
PCBs without overestimating the concentrations due to overlaping peaks found
in the two Aroclor mixtures. The Aroclor 1242 measurement could have
included contributions from Aroclor 1016 and the Aroclor 1254 numbers
probably included a contribution from a higher molecular weight Aroclor such
as 1260 or 1262. In addition, thirteen individual PCB congeners were
quantified. These included at least one compound from each chlorination
level ranging from tetrachlorobiphenyls to decachlorobiphenyl.
Chlorinated pesticides including hexachlorocyclohexanes, chlordanes, DDDs
and DDTs were quantified in the f2 fractions. These fractions were analyzed
by capillary gas chromatography with electron capture detection. The
conditions used were the same as those used for the analysis of the fl
fractions.
The f3 fractions were analyzed for coprostanol by capillary gas
19
-------
chromatography with flame ionization detection. The instrument operating
conditions were the same as for the fl and f2 fractions except the oven was
temperature programmed from 150 to 315 °C at 10 °C/min with no initial hold.
Two coprostanol compounds co-eluted using these conditions, 5B-cholestan-3B-
ol (coprostanol) and 5B-cholestan-3A-ol (epicoprostanol). Coprostanol was
the predominant component but the reported concentrations may also include a
contribution of epicoprostanol.
For all of the analyses by gas chromatography, analog data from the
instruments were digitized using a Perkin Elmer LCI-100 integrator and sent
to a Perkin Elmer 3200 LIMS Computer. Perkin Elmer CLAS Chromatography
Software was used for selecting peaks and calculating concentrations.
Results were stored on the Perkin Elmer LIMS systems and after QA checks the
data were shipped to the laboratory VAX computer system.
Gas Chromatography-Mass Spectrometry
The f2 fractions were also analyzed for selected PAHs. The PAHs were
quantified using a Finnigan 4531 quadrupole gas chroma tograph-mass
spectrometer (GC-MS) which included a Nova 3 computer, a CDC 96 megabyte
drive, Tektronix 4010 and 4114B terminals, a Tektronix 4631 hard copy unit
and a Data General 6021 9-track magnetic tape unit.
The Finnigan GC was operated with a capillary column in the splitless
injection mode. Each injection consisted of approximately 1 ul of sample
extract and about 2 ul of solvent backflush. The split flow was
approximately 50 ml/min and the septum sweep flow was approximately 2
20
-------
ml/min. Both flows were suspended for 1 minute just before the time of
injection. The DB-5 fused silica column was 30 m in length with a bore of
0.25 mm and a film thickness of 0.25 microns. The GC oven temperature was
held at an initial temperature of 50 °C for 2 minutes, programmed 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 is heated to 300 °C, and then to within 1 on 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 electron volts. The filament
emission current was 200 microamps and the various source potentials were
adjusted to produce a spectrum of decafluorotriphenylphosphine (DFTPP) which
meet the specifications detailed by Eichelberger et al. (1975). The MS was
scanned from 15 to 650 amu in 1 second while collecting 10 centroid samples
per peak. The continuous dynode electron multiplier was operated near 1550
—8
volts, and the preamplifier sensitivity was set to 10 amps/volt.
The mass scale of the MS was calibrated by emitting perfluorotributylamine
into the source, acquiring data, and running the software calibration
routine. On each day that samples were analyzed, a solution of standards was
also analyzed. This allowed the determination of the response of the
standards, their retention time and the spectrum of the DFTPP. If any of
these determinations were outside of predetermined limits, remedial action
was taken. After the completion of each run, the major peaks were checked
to ensure that the sensitivity was adequate and that no saturation had
occurred. After all of the samples for the day had been run, another
standard run was made.
-------
Areas were manually integrated using peaks displayed as extracted ion
current profiles (EICPs). This better enabled the operator to check that
the compound eluted at the correct time and was not interfered with. For
each sample and standard run, a quantitation list (QL) was compiled. The QL
contained the areas of the various peaks of interest as determined from
their respective EICPs.
Quantitation was accomplished by the method of internal standards (ISs). The
two ISs, DlO-phenanthrene and D12-benz[a]anthracene, were added to the
samples just prior to preparation. The standards solution also contained
the two ISs. The standards runs were used to determine Relative Response
Factors (RRFs); i.e., RF of the standard divided by RF of the IS, where RF
is the Response Factor and is defined as the area counts of the peak divided
by the nanograms injected. Actual concentrations of specific compounds in
the sample were then calculated by using the RF of the IS in the sample
itself, the area of the compound in the sample, the known amount of IS added
to the sample, and the dry weight of the original sample. The calculations
were performed in a fortran program running on the laboratory's Perkin Elmer
3210 LIKS computer, after the QLs had been transmitted from the Nova 3 tc
the LIMS via an RS232 data line.
After the calculations were made, a plot of the concentrations of the
selected compounds versus molecular weight was examined. The distributicr.
of compounds was carefully checked to ensure that it was consistent with the
previous data. Only after this QA step were the data added to the permanent
database resident on the LIMS. The raw GC-KS data were archived on industry
standard magnetic tapes.
-------
Quality Assurance-Organic Analysis
Numerous quality control steps and quality assurance checks are performed
during all phases of the analysis procedures. Many of these are described
in the analytical procedures presented above. Additional information is
provided here.
All glassware that was used for sample analysis was washed with Alconox and
then rinsed with tap water and deionized water. The glassware was capped
with aluminum foil and muffled at 450 °C for 6 hours. It was then stored
capped with foil until used when it was uncapped and rinsed with an
appropriate solvent. Also, blank analyses were conducted with each set of
samples. This averaged to be about one blank analysis for every 6 samples.
None of the blanks analyzed in this study contained significant amounts of
the compounds of interest.
Consistent silicic acid column activity was quality 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
the column fractions were monitored.
Extraction efficiency was quality assured by conducting spike and recovery
studies. The first spike and recovery study conducted with this set of
samples was done by spiking a homogenate made of clams collected from a
'clean' area with representative compounds from each of the chemical classes
studied. The spiking levels chosen were similar to those anticipated in
samples from moderately contaminant sites.
23
-------
Thirteen PCB congeners, 8 chlorinated pesticides and 12 PAH isomers were
spiked into three clam homogenate samples. The background levels of these
compounds were measured in two unspiked homogenate samples and the
background levels were subtracted. A reagent blank was also analyzed along
with this sample set. The results of this experiment showed the overall
recoveries to be 74 ±8 for the PCB congeners, 74 ± 10 for the chlorinated
pesticides and 60 ± 1 for the PAH isomers. These results represent absolute
recoveries for these compounds. However, all of the quantitations of
organic compounds were done by the internal standard method. Therefore, the
results are automatically corrected for losses in the analytical procedure.
Octachloronaphthalene was used as the internal standard or surrogate
compound for PCBs, gama-chlordene was used for chlorinated pesticides, and
DlO-phenanthrene and Dl2-benz[a]anthracene were used as PAH surrogates.
In addition, a spike and recovery study was also done as a quality assurance
procedure for the coprostanol analysis method. For this, two aliquots of a
well characterized sediment from a relatively uncontaminated station were
spiked with coprostanol and its recovery through the analytical procedure
was measured. The recoveries measured were 83 and 86%. The surrogate
compound used for quantitation of coprostanol was 7,(5a)-cholesten-3B-cl.
In the data tables of this report a concentration of 0.00 is indicated for
concentrations below the analytical detection limits. Actual analytical
detection limits are different for each compound and each sample. Tr.e
detection limits are different for each compound because of differences ir,
the instrumental response factors of the compounds. Detection limits fcr a
given compound are different between samples because of differences in the
-------
amount of sample analyzed and the levels of interfering compounds in the
extracts.
Working backward from the instrumental detection limit for each compound,
knowing the final volume of the extracts, allows one to calculate
approximate detection limits in the extract for each compound or class of
compounds. These levels calculated for this study are listed below.
Compound Extract Detection Limit Compound Extract Detection Limit
(ng) (ng)
Aroclor 1242 9 Chlorinated Pesticides 0.45-2.86
Aroclor 1254 13 PAHs 0.50-2.0
PCB Congeners 0.72-1.60 Coprostanol 1000
This information along with the dry weight of each sample can be used to
calculate approximate analytical detection limits. In general about 1-3 g
dry weight of tissue were analyzed and about 5 g dry weight of sediment
samples were extracted. Therefore, for example, for a 5 g dry weight
sediment sample, the analytical detection limit for Aroclor 1242 would be
9/5 or about 1.8 ng/g.
INORGANIC ANALYSIS METHODS
Tissue sample preparation
Trace metals
Flounder, lobster tail and hepatopancreas samples were thawed prior t;
dissection. Tissue samples were separated from shell or skin and specimens
(approx. 20 g) removed for analysis using stainless steel instruments. As a
precaution against cross-contamination, the instruments were cleaned be:>ee:'
25
-------
samples by rinsing with deionized water. Portions of each specimen taken
for trace metals analysis were placed in tared, acid cleaned, 100 ml
borosilicate glass beakers and covered with borosilicate watch glasses.
Wet weights were obtained and the samples were dried; lobster samples were
freeze-dried for 48 hr. at -40 °C and 24 hr. at 30 °C, while the flounder
samples were oven-dried for 36 hr. at 95 °C. After obtaining dry weights
for the samples, 10 ml of concentrated HNO, (reagent grade) was added to
each sample. The tissue was digested for 24 hr. at room temperature, after
which the solutions were heated to 60 °C to complete digestion and evaporate
the acid. When the samples were near dryness, 5 ml of 30% H,O, was added
(to decompose organic material) and the samples again heated to dryness.
After cooling, the samples were dissolved in 10.0 ml of 2N HNO, and stored
in acid-washed 20 ml polyethylene vials.
Oyster and clam samples were prepared in a similar manner. Five organisms
from each sampling site were shucked, rinsing the knife with deionized water
between samples, and oven-dried at 95 C. The larger sample mass required
use of 250 ml beakers and decomposition with 100 ml of concentrated HN3,
(reagent grade) added in 20 ml aliguots over a period of 2 days. Samples
were slowly heated and maintained at 60 °C for 8 hr. after which the samples
were evaporated to dryness. Residue from each sample was reconstituted
with 20 ml of 2N HNO, and filtered through acid washed (2N HN03) Whatman 42
filter paper into a 50 ml volumetric flask. The sample beaker was rinsed
with several 10 to 15 ml washes of 5% HNO, which were also filtered and
combined with the initial solution. The final sample solution was brougw:
to 50 ml by addition of 2N HNO,. Sample solutions were stored in 60 ml acid
cleaned, polyethylene bottles for analysis.
-------
Mercury
Samples prepared for mercury analysis underwent a similar procedure, but the
samples were not dried prior to acid digestion. Dry weights for these
samples were calculated from the wet weights measured and the dry-to-wet
weight ratios obtained from the corresponding trace metal samples. When the
digested sample solution volume was reduced to about 1 to 2 ml, 5 ml of 30%
HjO, was added and the samples heated. This step was necessary to decompose
organic matter and surface-active compounds which cause foaming and
interferences in the hydride reaction step of the mercury analysis. The
samples were evaporated to near-dryness, dissolved in 10 ml of 2N HNO, and
stored in acid-washed 20 ml polyethylene vials.
Sediment sample preparation
Trace metals
Sediment samples were thawed completely and homogenized by stirring with a
spatula. Aliquots of wet sediment (approximately 5 g) were transferred tc
tared, acid cleaned, 60 ml polyethylene bottles and wet weights determined.
Sample aliquots were refrozen and freeze-dried in a Virtis lyophilize:
(Model No.#10-145MR-BA) for 2 days. Dry weights were determined and the
dried samples acidified with 50 ml of 2N HNO, (reagent grade). The sample
bottles were sealed with a polyethylene screw cap and stored at room
temperature for two days. Samples were mildly shaken and vented daily tc
resuspend the sediment and prevent rupturing of the plastic containers by
H2S generated. Samples were gravity filtered through acid washed Whatman 42
filter paper into 60 rri acid cleaned polyethylene bottles so that insoluble
residue would not interfere with subsequent atomic absorption analysis.
27
-------
Mercury
Sediment samples for mercury analysis were prepared by the same technique,
but were not freeze-dried. Dry weights for these samples were calculated
from the wet weights and dry-to-wet weight ratios obtained from the
corresponding trace metal samples.
Instrumental analysis
ICP analysis
Trace metal (Cu, Cr, Pb and Cd) determinations in sediment and flounder
samples were performed using a Leeman Labs Plasma-Spec I inductively coupled
plasma (ICP) emission spectrometer at the Narragansett Bay Commission
laboratory in Providence, R.I. The instrument was set up using standard
conditions according to the manufacturer's recommendations and calibrated
with standard solutions before each use. Concentration data for samples was
collected on a Zenith portable computer and transferred to the ERU;
chemistry LIMS system.
Atomic absorption analysis
Lobster, oyster and clam samples were analyzed for trace metals (Cu, Cr, Fr.
and Cd) by flame atomic absorption (FAA) and heated graphite atomization
(HGA) atomic absorption (AA) using a Perkin-Elmer (Model 5000) AA spectro-
photometer. Absorbance signals were recorded with a strip chart recorder
(Perkin-Elmer Model 56) and collected directly at a data station (Perkin-
Elmer Model 3600). Transient signal data was reduced to peak height an~
area for each sample/element determined. Regression curves generated frcr
absorbance data for standards were used to determine concentrations :r
unknown samples. Polynomial regression algorithms used to calculate
26
-------
standard curves were described by Rugg and Feldroan (1980).
Instrument setup conditions were similar to those described in "Methods for
Chemical Analysis of Water and Wastes" (U.S. EPA, 1979) and those found in
manufacturer's reference manuals. AA instruments were calibrated each time
samples were analyzed for a given element. Instrument calibrations were
generally checked after every ten samples. All samples were analyzed at
least twice to determine signal reproducibility. Generally, for each 15
samples processed, one sample was determined by the method of standard
addition, and one procedural blank sample was analyzed.
Mercury analysis
Mercury analyses of all samples were conducted by the cold vapor technique
with a Perkin-Elmer mercury/hydride system (Model MHS-20) equipped with a
gold amalgam attachment on a Perkin-Elmer AA (Model 403). A Perkin-Elmer
integrator (Model LCI-100) was used to collect the output from the AA and
integrate the signal peaks generated by the MHS-20. Peak areas determined
for samples and standards were manually entered into the data reduction
programs used for the atomic absorption data to determine the unknown sample
concentrations. Blank determinations and standard additions were performed
with each set of samples to check instrumental performance.
Quality assurance - inorganic analysis
Procedural blanks were run with each set of samples and analyzed for metals
along with the sample sets. Concentrations reported in the tables are
corrected for blanks where blanks were above detection lir.it. Reporter
below are the blank concentrations found for each type of s arr.pl e, expressed
29
-------
as the equivalent sample concentration.
(ug/g dry wt)
Sample type
Sediments
Oysters, clams
Lobster tail
" hepato.
Flounder
Cu
<.150
<.100
.150
.250
.075
Cr
<.400
.150
.065
.150
.050
Pb
<.550
.300
.350
<.750
.045
Cd
<.30
.025
.030
.040
<.008
Hg
<.40
.008
.008
.015
<.010
For some sets of analyses, samples were spiked after analysis with a spike
of comparable concentration. The recovery of the spike was calculated from
the difference in the concentrations of the spiked and the unspiked samples
as a percentage of the added spike. This indicates the reliability of
determination of sample concentrations by comparison of the sample against
aqueous standards. The recoveries calculated for the different sets of
analyses are given below.
Spike Recoveries
Sample type Cu
Sediments 85-89%
Oysters, clams
Lobster
Flounder
Cr Pb Cd
73-83% 69-81% 84-87%
_
133-167
_ _ —
Hg
79-115%
73
73
54-65
30
-------
Table 1
Organic Compounds Selected for Analysis
Fraction 1
Hexachlorobenzene
p,p'- DDE
2,2',5,5'-PCB
2,2',4,4'-PCB
2,2',4,5,5'-PCB
2,2',3,5,5',6-PCB
2,3',4,4',5-PCB
2,2',4,4',5,5'-PCB
2,2',3,4,4',5'-PCB
2,2',3,3',4,4'-PCB
2,2',3,4,4',5,5'-PCB
2,2',3,3',4,4',5,6-PCB
2,2',3,3',4,4',5,5'-PCB
2,2',3,3',4,4',5,5'e-PCB
CL10-PCB
Aroclor 1242
Aroclor 1254
Fraction 2
a-hexachlorocyclohexane
g-hexachlorocyclohexane
a-chlordane
g-chlordane
p,p'-DDD
p,p'-DDT
PAHs - (see Table 2)
Fraction 3
Coprostanol
31
-------
Table 2
Polycyclic Aromatic Hydrocarbons Selected for Analysis
Fluorene
Phenanthrene
Anthracene
Cl homologs of phenanthrene and anthracene
C2 homologs of phenanthrene and anthracene
C3 homologs of phenanthrene and anthracene
C4 homologs of phenanthrene and anthracene
Fluoranthene
Pyrene
Benz[a]anthracene
Chrysene
Sum of benzofluoranthenes
Benzo[e]pyrene
Benzol ajpyrene
Perylene
Indeno[1,2,3-cd]pyrene
Benzo[ghi Jperylene
Sum of molecular weight 276 PAHs
Dibenz[a,h]anthracene
Sum of molecular weight 278 PAHs
Coronene
Sum of molecular weight 302 PAHs
32
-------
RESULTS
Quincy Bay study area core and surface sediment chemistry sample locations
and biological station locations are presented in Figure 8. The composite
figure provides an overview of the study area and the relative spatial
relationship of biological to chemistry sampling sites.
WINTER FLOUNDER
Otter trawl fishing success for winter flounder in the Quincy Bay study area
along pre-study selected transects T1-T4 (Figure 1) during the time period
of collection was highly variable. Due to lack of fishing success transect
T4 (from Moon Island to the eastern end of Long Island) was eliminated from
the study design in favor of a trawling effort conducted along the Quincy
Bay shoreline from Nut Island to Blacks Creek and then to Wollaston Beach
(Figure 9). The shoreline trawl course combined areas within transects Tl
and T3 and was, therefore, designated as transect T1-T3. Mean total length
of winter flounder collected for study was 34.5 cm. Ninety of the flounder
were 30.4 cm or greater, the length established for a legal recreational
fishery. Mean weight of collected flounder was 587 grams (Figure 10).
Values for meristic parameters, sex characteristic, station location and the
identification of neoplastic and non-neoplastic conditions among individuals
are provided in a master table (Table 3).
33
-------
o
CM
-------
FIGURE 8A
Quincy Bay Symbols for the Sample Sites
in Figures (1-8)
Flounder trawl transect
Lobster capture site
Soft shell clam collection site
Oyster cage site
Sediment core site
Surface water collection site
-------
-------
•s
K.
C
m*
C
EZ
c
>•
. o
. c
w -*
• cr
2 2
h. _
£
>
S g § g
§ § §
^ *^ r>
-------
TABLE 3
QUINCY BAY WINTER FLOUNDER MASTER TABLE
SAMPNUM
75100
75101
75102
75103
75104
75105
75106
75107
75108
75109
75110
75111
75112
75113
75114
75115
75116
75117
75116
75119
75120
SEX
M
F
F
F
M
F
F
M
M
U
F
F
F
U
F
M
F
F
M
M
F
LENGTH
(TL on)
27.0
34.0
33.3
32.2
29.1
31.9
33.8
31.5
32.0
31.0
39.5
37.0
33.7
41.8
36.5
33.2
33.7
41.9
32.9
33.0
41.6
WEIGHT
(gm)
262
569
487
497
264
434
542
376
531
390
877
747
578
849
706
489
534
1000
487
454
1277
TRANSECT NEOPLASTIC
QBT1
QBT2 1
QBT2
QBT2
QBT2
QBT2
QBT2
QBT2
QBT2 2
QBT1
QBT1
QBT1
QBT2
QBT2 1
QBT2 1
QBT2
QBT2
QBT2
QBT1
QBT1
QBT1 1
NON-NEOPLASTIC
4B,D,H,I
5,7A
41
4B,E
4G,I
4B,I;5
4B,I;6A
4H,7A
4D,E,I;5
4A,B,D,F,G,I
4B,5,7A
4A,E,H
4B,C,I;7A
4B,I
4B,C,H,I
4D,H
4D,H?6B,9A
36
-------
75121
75122
75123
75124
75125
75126
75127
75128
75129
75130
75131
75132
75133
75134
75135
75136
75137
75138
75139
75140
75141
75142
75143
75144
75145
75146
75147
F 34.2
F 37.4
M 30.7
M 35.4
F 36.4
F 10.5
M 33.7
F 41.3
M 33.6
M 33.2
M 32.2
F 31.9
M 35.6
U 27.9
F 35.5
F 35.2
M 33.5
M 35.1
M 32.8
M 26.1
F 22.4
M 30.6
F 38.8
M 32.4
M 38.5
F 36.4
K 31.5
482
751
369
540
824
12
555
904
534
455
456
508
577
257
681
628
516
568
442
217
114
399
868
444
804
764
464
QBT1 4B
08X1 41, 7A
QBT1 4I
08X1 1 4C,F,H;6B
QBT1 4I
QBT2 5
08X113 4A,B,D,H;7A
QBT1T3 4B/E
QBT1T3 4H
QBT1T3 4B
QBT1T3 4B
QBT1T3 4A,B,C,F,H;5
QBT1T3 4A,7A
QBT1T3 5
QBT1T3 1
QBT1T3 4F
QBT1T3 4B,D,H
QBT1T3 4B
QBT1T3 4E
QBT1T3
QBT1T3
QBT1T3 5
QBT1T3 4B
QBT1T3 4B
QBT1T3 1 4A,B,H;7A
OET1T3 4B,H,I;5
QBT1T3
39
-------
75148
75149
75150
75151
75152
75153
75154
75155
75156
75157
75158
75159
75160
75161
75162
75163
75164
75165
75166
75167
75168
75169
75170
75171
75172
75173
75174
M 39.0
F 32.7
F 34.8
M 39.7
F 30.8
M 35.1
M 30.0
M 25.8
F 37.1
M 36.0
M 31.8
F 31.0
F 36.5
M 30.9
F 34.1
M 35.9
M 36.0
M 33.7
F 34.3
M 39.3
M 34.1
M 38.4
F 39.3
F 35.6
M 42.1
M 39.5
M 33.8
738
485
593
816
462
552
433
257
722
590
388
502
658
420
514
536
646
588
535
720
474
651
823
519
886
691
553
QBT1T3 1
QBT1T3
QBT1T3
QBT1T3
QBT1T3
QBT1T3
QBT1T3
QBT1T3
QBT1T3
QBT1T3
QBT1T3
QBT1T3
QBT3
QBT3
QBT3 2
QBT3 1
QBT3 1
QBT3
QBT3
QBT3 1
QBT1 1
QBT3 1,3
QBT3 1 , 3
QBT3
QBT3 1
QET3 1
QBT3
4A,B,C,G,H;5
7A
4B,G,H
4B,H;7A
4A
4B
4F,H,I
4H,I
4B,I;7A
4H,I
4B,D,H,I
4D
4A,B,F,I;6B,7B,8A
4H
4B,I;5
5
4B,E,I
4B,D,H;6B
6B
4B,G,H;5,7A
4B,7A
4A,E,G;5,6B,7B
4B,5
4B,D,I
-------
75175
75176
75177
75178
75179
75180
75181
75182
75183
75184
75185
75186
75187
75188
75189
75190
75191
75192
75193
75194
75195
75196
75197
75198
75199
F
M
M
F
F
T
H
F
M
F
F
F
F
M
F
M
F
F
M
M
M
M
M
F
F
30.6
33.9
38.6
34.0
35.5
34.8
33.5
35.6
32.0
24.6
46.0
35.5
36.0
39.0
42.7
31.5
41.1
39.9
30.0
37.2
32.5
32.2
35.0
46.3
44.2
394
516
586
521
635
606
478
710
452
183
1337
621
576
766
849
420
891
1021
381
602
403
480
535
1304
1168
QBT3
QBT2 1
QBT3 1
QBT3 2?
QBT2 2
QBT3
QBT3 1
QBT3
QBT3
QBT3
QBT1 1
QBT1
QBT1
QBT1
QBT1 1
QBT1
QBT1
QBT1 1
QBT1 1
QBT1
QBT1
QBT1
QBT1
QBT1 1
QBT1 1
4F,5
4D,I
4B,7A
4B,H
41
4D,E,H
41
4B,I
41
4A,B,D,E,G,H,I
7A,9A
4B,I
4B,D,H,I
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4G,I
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4D,E,H;6B,7A
41
-------
Neoplasms in winter flounder confirmed by microscopic examination:
1 / Liver parenchyma and/or bile duct
2 / Gastric, Carcinoma .In Situ
3 / Schwannomas
Non-neoplastic lesions in winter flounder:
4A/ Liver /Cholangiofibrosis
4B/ Liver /Vacuolation
4C/ Liver /Bile Duct Vacuolation
4D/ Liver /Basophilic foci
4E/ Liver /Clear Cell Foci
4F/ Liver /Connective Tissue Hyperplasia
4G/ Liver /Spongiosis Hepatis
4H/ Liver /Necrosis
4I/ Liver /Inflammation
5 / Gastrointestinal Tract /Basophilic Nidi
6A/ Respiratory Organ /Papilloma
6B/ Respiratory Organ /Squamous Cell Lesion
7A/ Pancreas /Nesidioblastosis
7B/ Pancreas /Adenoma
8A/ Gall bladder /Myoadenoma
9A/ Circulatory /Arterial Plague
Histopathological results for winter flounder were based on observation cf
external anatomy and examination for macroscopic lesions at necropsy;
microscopic examinations were conducted on integumentary, muscular,
skeletal, respiratory, digestive, circulatory, excretory, endocrine,
reproductive and nervous systems. Those examinations therefore include all
major tissues and organs. One thousand eight hundred forty-five (1845
macroscopic slides stained with Hematoxylin and Eosin resulted free, tne
histclogical work-up of one hundred (100) winter flounder. Special steir.s
should be performed on several cases to strengthen diagnosis, but funding
-------
has not been provided for histochemical analysis in this phase of the Quincy
Bay study.
NEOPLASMS:
Neoplastic response in flounder collected from Quincy Bay occurred in the
liver, gastric mucosa and as schwannomas in fin rays. Those neoplastic
changes total 29 in 27 different winter flounder and are identified in Table
4. Liver neoplasms in four of twenty-three cases were considered to be
early emerging lesions.
43
-------
TABLE 4
SUMMARY OF NEOPLASM PREVALENCE IN QUINCY BAY WINTER FLOUNDER
SAMPNUM
75108
75113
75114
75120
75124
75135
75145
75148
75162
75163
75164
75167
75168
75169
75169
75170
75170
75172
75174
75176
75177
TRANSECT
T2
T2
T2
Tl
Tl
T1T3
T1T3
T1T3
T3
T3
T3
T3
Tl
T3
T3
T3
T3
T3
T3
T2
T3
LOCATION
Stomach
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Stomach
Liver
Liver
Liver
Liver
Liver
Integument
Liver
Integument
Liver
Liver
Liver
Liver
44
TYPE
Carcinoma In Situ
***
Hepatocytic
Hepatocytic
Hepatocytic
Hepatocytic
Hepatocytic
* **
Hepatocytic ,
Hepatocytic
Carcinoma In Situ
Hepatocytic
*
Hepatocytic
Hepatocytic
Hepatocytic
it
Hepatocytic
Schwannoma
Hepatocytic
Schwannoma
Hepatocytic
Hepatocytic
**
Hepatocytic
* **
Hepatocytic ,
-------
75178
75179
75185
75188
75192
75193
75198
75199
T3
T2
Tl
Tl
Tl
Tl
Tl
Tl
Stomach
Stomach
Liver
Liver
Liver
Liver
Liver
Liver
Carcinoma In Situ
Carcinoma In Situ
Hepatocytic
Hepatocytic
Hepatocytic
Hepatocytic
Hepatocytic
Hepatocytic
Synonyms and related terms:
Hepatoma (adenoma; neoplastic nodule)
Schwannoma (neurinoma; perineurial fibroblastoma; neurilemoma;
* Hepatoma
**
***
Low grade
Cholangiocytic characteristics present
-------
CHARACTERISTICS OF NEOPLASTIC LESIONS
LIVER NEOPLASMS:
Hepatocellular neoplasms, those tumors derived from the liver parenchymal
cells, were present in twenty-three of the Quincy Bay winter flounder. Those
lesions were often visible to the naked eye during necropsy. The
hepatocellular proliferative condition was macroscopically detectable in
twelve Quincy Bay animals; they were non-detectable in the remaining eleven.
In general, macroscopic hepatocellular lesions appeared as solid
white/yellow colored, non-glistening foci ranging from 1 to 4 mm in
diameter. Exceptions in size included a 1 x 1.5 cm and a large 4 x 4 x 7 or
mass in two different fish. Those tumors were composed of both soft and
solid coalescing multicystic formations. The latter was umbilicated. Color
of the diseased livers varied from creamy to dark brown, and in two animals
liver parenchyma of the anterior region appeared mottled with green pigment.
Liver color was creamy in flounder with non-detectable gross pathology.
In fish with neoplastic conditions, solitary and/or nodular foci were
disseminated throughout the entire organ; diffuse hepatomas spread at randrr
and had somewhat increased stroma. Microscopic criteria used to identify
neoplastic changes in winter flounder hepatic cells included chromatic
alteration, increased mitotic frequency noted by cellular division,
compression of adjacent tissues in alterations growing by expansion, and
generally, an absence of macrophage aggregates. Well-differentiated
hepatomas with cells having microscopic features much like those froir wr.icr.
they are derived frequently have acinar patterns, but were also present a;
solid and trabecular formations. Hepatocellular carcinomas were farther
46
-------
characterized by pleomorphic cells with morphologic changes in nuclear
chromatin distribution and bizarre mitotic figures. A prominent microscopic
feature of invasive hepatocellular carcinomas was the occurrence of a
spindle-cell variant. Spindle cells were found to line dilatated areas and
converging cyst-like spaces and to form sheets, trabecular and individual
acinar patterns with intervening fibrous stroma containing cellular
infiltrates. Spindle-cell variants occasionally occur in fusiform or
storiform patterns.
Three of the winter flounder livers had characteristics of hepatocytic and
cholangiocytic neoplasms. Only one is noted in Table 4; characteristics in
the two other cases were less prominent. The lesions were interpreted as
hepatocytic pending further investigation.
Table 5 summarizes the major histopathological changes observed in livers of
Quincy Bay winter flounder. Pathological changes, in addition to hepatocytic
(23%) neoplasms, consisted of non-neoplastic cholangiofibrosis (11%), marked
hepatocyte swelling and vacuolation (48%), marked swelling and vacuolation
of bile ducts/ductules (5%), hepatocyte basophilic foci (18%), hepatocyte
clear cell foci (11%), connective tissue hyperplasia (7%), spongiosis
hepatis appearing lesions (11%), hepatocyte necrosis (33%) and periductal
and/or perivascular inflammation (38%). Data presented only indicates
presence or absence of the pathological lesion; extent of the morphological
change may vary considerably among flounder.
INTESTINAL NEOPLASMS:
Neoplastic change was observed in the gastric mucosa of four animals. The
-------
I
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8
ro
-------
observed changes were consistent with the characteristics of carcinoma _in
situ.
SCHWANNOMA:
Schwannomas were associated with fin rays of dorsal and ventral fins in two
cases.
CHARACTERISTICS OF NON-NEOPIASTIC LESIONS
DIGESTIVE SYSTEM ORGANS:
LIVER:
Characteristics of non-neoplastic liver alterations are as follows:
INFLAMMATION:
Periductal:
Intrahepatic bile ductal tracts in 36% of winter flounder examined had
periductal infiltrates. Conditions ranged from mild to severe reactions.
Perivascular:
Perivascular infiltrates accompanied by swollen vasculature occurred in 6\
of winter flounder examined.
Granulocytic/Lymphocytic:
Inflammation primarily composed of granulocytic infiltrates in liver
parenchyma (i.e., associated with cellular necrosis) occurred in 4% of those
animals; lymphocytic inflammatory response in hepatic parenchyma was present
in 3% of the animals.
49
-------
Macrophage aggregates:
Macrophage centers in the liver were increased numerically and dimensionally
in 8% of the animals examined. Macrophage aggregates in one animal
contained cells having a distinct foamy cytosol. The foamy cells and the
imparted lace-like appearance of aggregates was atypical of flounder
collected in Quincy Bay and other New England estuaries.
RETROGRESSIVE CHANGES:
Vacuolated hepatocytes:
Marked cellular swelling, clear cytoplasm and small apically located nuclei
having dense chromatin was a common characteristic of liver parenchymal
cells in winter flounder from Quincy Bay and other estuarine locations
along the North Atlantic coast. Vacuolated cells having those morphologic
characteristics were often arranged in acinar patterns, although cord-like
and/or a diffuse distribution was common. Occurrence was marked by presence
of single or multiple clusters of cells throughout the organ; size of foci
ranges considerably (i.e., from visible to non-visible to the naked eye).
Those vacuolar lesions occurred in 50% of the Quincy Bay flounder. Vacuolar
lesions that appeared uninterrupted with columnar bile ductal epitheliur
occurred in 5% of the Quincy Bay flounder. Of those, neoplasms were
microscopically confirmed for two animals. Further, vacuolar cells were
associated with exocrine pancreatic ducts occurring in a total of 11% of the
animals. Vacuolation of exocrine duct epithelium was a retrogressive
response observed in seven flounder known to have hepatic neoplasms, and
four without neoplasms.
Fibroplasia, stromal: (Connective Tissue Hyperplasia)
50
-------
Acinar cords of vacuolated cells, or of well differentiated parenchymal
cells, were usually enshrouded by hyperplastic connective tissue. When
present, connective tissue hyperplasia was generally distributed throughout
the organ. Hyperpiasia of connective tissue stroma occurred in 14% of
flounder livers.
Basophilic foci:
Basophilic foci are discrete, variably sized areas of liver parenchymal
cells that exhibit a chromatic change. Cellular constituents in these foci
were characterized by a low mitotic index and usually the absence of
macrophage aggregates. High mitotic index, compression and absence of
macrophage aggregates along with a basophilic tinctorial change are criteria
normally associated with hepatomas. Sixteen flounder livers had either
single or multiple basophilic foci. Six of those animals had liver
neoplasms; neoplasms were absent in the ten remaining fish with basophilic
foci, although three of them had moderate to high mitotic indices.
Clear Cell Foci:
Clear cell foci were also discrete focal areas of hepatocytes which lacked
the same degree chromaticity as neighboring cells. Since cell outlines
usually retain normal shape and size, and nuclei have distinct nuclear
membranes and a distinct nucleolus, those areas may represent alterations in
physiological state including fatty metamorphosis.
Spongiosis Hepatis:
Single to multiple variably sized fluid-filled spaces occurred in hepatic
parenchyma of 11% of the Quincy Bay winter flounder. Fluid retenticr
51
-------
promoted severe compression of adjacent parenchyma! cells. The spaces lack a
fibrous capsule; they were confined by a very fragile network of fibers.
Individual compartments within the lesion contained a proteinaceous
substance interspersed with polymorphonuclear cells. The condition was
frequently associated with the intrahepatic ductules and ducts of the
biliary tree.
DIGESTIVE (EXCEPT LIVER), EXCRETORY, CIRCULATORY (SPLEEN) AND REPRODUCTIVE
SYSTEMS:
Prevalence of cellular response in those systems appears in Table 6 as
follows:
INFLAMMATION:
Chronic intestinal inflammation consisting of lymphocytic infiltrates was
observed in 35% of the flounder; acute inflammatory infiltrates (4\<.
Inflammatory response in the kidney and spleen as indicated in Table 6
represents an increased quantitative shift of macrophage aggregates.
Quantitative change was based on number and size of the aggregates and
graded as mild, moderate or advanced.
RETROGESSIVE CHANGES:
Twenty (20) Quincy Bay flounder have non-neoplastic intestinal 'nidi' cr
basophilic nests. In the Quincy Bay winter flounder the nidi tended to orcrur
in the pyloric cecum near juncture with the gastric fundus extending intc
the duodenal region, and in other regions of the intestine having ar.
absorptive epithelial mucosa. The lesion also occurred in gastric lir.irr
(in one case) and in the rectuin (in one case). Microscopically, the r.:c:
52
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consisted of mitotically active hyperchromatic epithelial cells and,
frequently, mucous secretory cells. Menomatous formations, as such, were
characterized by cyst-like spaces with mucous retention. The basophilic
nest-like formations originated at the basement membrane. Mucosal distention
into intestinal lumina may have promoted a reduction of the space between
villus folds. Discontinuity of the basal lamina and extension into the
submucosa was evident in three cases. Advanced cases were characterized by
altered continuity of basal lamina, increased fibrovascular stalks,
hyperplasia and basophilia of mucosal membrane with reduced mucin
production.
Intestinal changes in mucosa/submucosa other than inflammation included
vasodilation (7%); hemorrhage, vacuolation, edema, and cystic formations
occurred in from 1% (ulceration) to 6% (edema) of the animals. Reactive
lesions (parasitic granulomas) in the digestive gastrointestinal region
occurred in 11% while hyperplasia and repair responses occurred in 3 and 4*
of the flounder respectively.
A myoadenoma occurred in the gall bladder of one animal. The malformaticr.
consisted of acinar tubule structures with cuboidal epithelial lining in a
stroma consisting of connective tissue and musculature. The lesion, isolated
on a broad base in the submucosa, did not extend into the muscular coat c:
the lumen. Other alterations in the gall and urinary bladders consisted
primarily of epithelial cell vacuolation.
Pathology in the renal excretory system was limited to some giomerulcpatr..:
lesions, nephroblastic stimulation and nephron proliferation. Presurr::--?
-------
evidence exists that indicates seme of these may be neoplastic disorders.
Disorders of the pancreas included nineteen (19) with nesidioblastosis and
two (2) with isolated ductal adenomatous formations. The ductal complex of
the winter flounder is comprised of intralobular and extralobular ductules
in association with centroacinar cells, and periinsular ductules with
islets. Those ramifying ducts/ductules respond to change as hyperplasia to
the extent of ductal distention and occlusion by islet cells. Formations of
intra and extraductal islets (nesidioblastosis) were greatly increased.
Isolated pancreatic lesions with ductal patterns marked the extensive nature
of pancreatic involvement in two animals. The nature of the lesion, whether
neoplastic or metaplastic, remains to be resolved.
Vacuolated cells were observed in exocrine pancreas of eleven (11%) of the
flounder.
Parasites:
Parasitic infestations, both intra and extragastric, was dominated by the
genus Glugea (presumably) (24%), microsporidia (9%) and unidentified wcrips
in gut lumina (7%).
RESPIRATORY, MUSCULAR, INTEGUMENTARY, CIRCULATORY AND NERVOUS SYSTEMS:
Prevalence of pathological changes associated with respiratory, muscular,
integumentary, circulatory and nervous systems are summarized in Table ~.
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Characteristics of lesions in flounder integument, respiratory organs,
circulatory and nervous systems were as follows:
INFLAMMATION:
Respiratory Organs:
Lyraphocytic infiltrates were present in respiratory epithelium of 34% of the
winter flounder. Mucous cell hyperplasia occurred in the filamental
structure of the organs (25%); increased mucous cell activity was
specifically associated with the respiratory lamellae (26%).
In addition to respiratory epithelium, lymphocytic infiltrates were present
in epidermis and dermis (6%) of integument, and olfactory lamellae (9%);
lesser prevalences occurred in the central nervous system, internal ear and
lateral line structures, and heart in one case. Swelling of the heart,
specifically the bulbous arteriosus, was apparent in 42% of the Ouincy Bay
flounder.
RETROGRESSIVE CHANGES:
"Ballooning" (telangiectasis, lamellar aneurysm, hemorrhagic gill disease,
hemorrhagic globes) did occur in the distal region of gill lamellae (33%).
The condition was a result of vasodilation followed by separation of
respiratory epithelium from underlying supporting elements allowing
extravasation. Blood lakes circumscribed by detached respiratory epitheliur
thus gives the lamellae a bulbus appearance.
In 3< cf the animals there was degeneration of some neuromasts located IT.
stratified squamous epithelium associated with gill arches and the hurral
-------
cavity. Thyroid glandular tissue was present in some microscopic sections
of the supporting and epithelial tissues of the gill. Thyroid presence,
although atypical in the gill respiratory filaments, was not considered to
ectopic and/or pathologic.
Proliferation of stratified squamous epithelium that lines interlamellar
spaces between lamellae at the level of the supporting gill rod was a
prominent cellular alteration in 53% of the winter flounder. A microscopic
feature of the hyperplastic alteration was cellular layering on the simple
lamellar respiratory boundary; progressive stages indicated a tendency
toward occlusion of the interlamellar spaces followed by formation of
papillomatous lesions. Lesions with some squamous cell-like tumor
characteristics were found in seven (7%) and a papilloma in one (1%) of the
winter flounder, in addition, lamellar polypoid formations in a distal
location consisted of blood vessels surrounded by dense collagenous
connective tissue. Those lesions have hemangioma-like characteristics.
Endothelial plagues were observed in mesenteric arteries in two cases. The
lesion has a mesh-like core consisting of collagen and elastic fibers.
Distant metastases or possibly a synovial knot occurred in the ventricular
epicardium of one (1%) flounder. Vacuolated cells were present in bet.-.
types of lesions.
Degenerative alterations in integument included vacuolated and cystic foci
in epidermis (71), hemorrhage (3%) and ulceration (1%). Neuroepithelial
and/or epithelial sustentacular cell vacuolation was observed in interr.;!
ear, lateral line and olfactory organ. The lesion in olfactory organs w~r
56
-------
observed in 21 of 41 flounder. Degenerative alteration of epidermal
neurosensory papillae was evident in three flounder.
Proliferative lesions potentially included olfactory neurosensory placodes
with adenomatous formations present in three cases.
Necrosis:
Necrosis of stratified squamous epithelium in varying degrees occurred in
the basal lamellar region along the axis of the gill filament and gill arch
epithelial surfaces (25%). A lymphocytic infiltration accompanied the
necrotic condition.
Epidermal necrosis occurred in 4% of the animals, but infrequently in
neurosensory elements.
Parasites:
Winter flounder gill structures were infested with metacercarial cysts of a
digenetic trematode (86%). The infestation was severe based on counts of
metacercarial cysts that exceeded 125 in some microscopic sections. The
parasite was generally enveloped by cartilaginous or dense collagenc'js
connective tissue; inflammation was absent. Metacercarial cysts embedded in
the cartilaginous gill rod promote redirection of growth that results in
deformed filaments including bifurcation (6%).
FLOUNDER PERIPHERAL BLOOD:
Peripheral blood differential cell counts conducted on eighty (n=SO,' Quir.ry
Bay winter flounder were compared to a winter flounder reference pcp'jlet:r-
59
-------
collected at Fox Island in Narragansett Bay, Rhode Island. Relative
frequencies of peripheral blood cell types among winter flounder with and
without neoplastic liver lesions were compared to Fox Island fishes serving
as a reference. Results of peripheral blood cell counts, summarized in
Table 8, provided evidence of two major shifts in cellular distribution.
First, mean blood cell counts indicated a strong shift in the ratio of
lymphocytes to thrombocytes in all Quincy Bay flounder sample groups (i.e.,
54% to 37%) in comparison to Fox Island flounder (29% to 66%). Secondly, a
significant change was apparent in the ratio of immature to mature red blood
cells in Quincy Bay flounder. Immature red blood cells comprise 18% of the
total red blood cell population of Quincy Bay collected fishes in comparison
to the normal 2% observed in Fox Island winter flounder.
Correlations of winter flounder pathology and concentrations of contaminants
in edible flesh were considered. Several studies have indicated that levels
of polycyclic aromatic compounds (PACs) in sediments (PAHs and nitrogen
substituted PACs) correlate with the incidence of tumors in fish (Malins e_t
al., 1984; 1985; 1988). Fish from contaminated locations have been shown to
have elevated levels of enzymes that metabolize PACs (Fabacher and Baurnar--.,
1985) and metabolites of PACs in fish bile have been shown to correlate with
PAC levels in the sediment (Krahn e_t al., 1987). PAC metabolites in bile
were also shown to positively correlate with the occurrence of hepatic
lesions (Krahn et al., 1986). Therefore, there is some evidence that PATs
may play a role in the development of tumors in fish.
However, because fish metabolize many of these compounds, the tiss-.
concentrations of PACs are not good indicators of exposure. In fart, FAhr
60
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were not detected in the Quincy Bay fish flesh samples although high PAH
levels were found in the sediments from some of the stations in Quincy Bay.
Therefore, although PAHs were not detected in the flesh samples, these
organisms may have been significantly exposed to these compounds. Numerous
chlorinated hydrocarbons including PCBs, DDTs and chlordanes were found in
the fish samples; however, these compounds are generally not considered to
be potent tumor initiators. In estuaries where the levels of contaminants
from different chemical classes generally follow the same trends such
compounds could be used as indicators of the overall extent of exposure of
an organis- In Quincy Bay, however, the sediment chemistry results
indicated that the trends seen for the chlorinated hydrocarbons were
different from those of the PAHs. Therefore, it was felt that the
concentrations of the chlorinated pesticides in the fish would not be good
indicators of overall exposure to carcinogenic compounds. Because of this
and because PAHs could not be detected in the fish samples no correlations
of contaminant levels and disease conditions were attempted.
LOBSTER
Lobster collections at nine stations (see methods) in Quincy Bay began on 6
May 1987. Subsequent collections for lobster occurred on 13 and 20 May, and
2 and 16 June 1987. Carapace length to weight (in grams) data was presented
in Figure 11. Histopathological evaluation indicated their general well-
being as good. Our conclusion was based on macroscopic and microscopic
examinations of carapace including appendages (uropod, mouth or mandibuiar
and walking) respiratory organs, stomach and alimentary canal, heart,
62
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reproductive tract and thorax associated muscle. Responses to disease and
cytopathic alterations including proliferative conditions were generally
lacking; two animals had presumptive lesions in digestive gland and kidney.
A comprehensive evaluation of the lobster from Quincy Bay is presented in
Table 9.
CHARACTERISTICS OF CELLULAR ALTERATIONS IN THE LOBSTER
INFLAMMATION:
Limited inflammatory infiltration did occur in lobster gill lamellae. The
inflammatory response was primarily in early formation stages, based or.
number of blood cells in the vascular lumina. Total occlusion of some gill
lamellae capillaries by inflammatory cells did occur, however. The
inflammatory response was insufficiently advanced to pose a debilitating
effect upon the respiratory process of those animals. The inflammatory cell
response in those animals was, as pointed out previously, in very early
developmental stages. Based upon previous examination of lobsters at ERL /K
the gill condition could develop into Black Spot Gill Disease (BSGD). ES3^
is a disease usually associated with the bacteria (Gaffkemia hpmarusi, cr
other parasitic infestations in the gill lamellae. We are of the opinicr.
that a higher prevalence of BSGD, and the associated gill inflammatory
response would be likely during mid-summer,
RETROGRESSIVE CHANGES:
The two animals with lesions in the digestive gland and kidney were
considered to be in reasonably good health because the lesions were net very
extensive and probably involved the organs and normal functions in a rrir-.ir.2l
64
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fashion. it is plausible that lesions in digestive gland and kidney were
associated with parasitism; in one case the digestive gland had a parasitic
granuloma. Ulceration of carapace chitin was noted in only one animal.
Appendage chitin in several other animals was eroded slightly.
Pathological evaluations of lobster on a station by station basis is as
follows:
QB Lobster Station A
N=6 Sex:2 female, 4 male.
Gills-Two animals showed inflammatory response in the gill lamellae and in
one animal the brachial vein. In those animals black particulate debris was
also noted in the basal portions of the gill lamellae. One animal had a
bacterial growth on one of the gill rakers. A protozoal form was noted
between the gill lamellae in all animals. It was a commensal protozoal
form with a stalk attachment to gill lamellar chitin.
QB Lobster Station B
N=5 Sex:3 males, 1 female, 1 unknown.
Gills-Three of the animals showed early stages of inflammatory response in a
few of the gill lamellae. Protozoa were noted among the gill lamellae in
all of the animals. All animals but one were in good health. One animal had
extensive involvement of the gill lamellae with inflammatory cells and
accumulation of fine dark particulate on the basal portion of the gill
lamellae. In the animal there was also noted ulceration of the chitin in
one of the many claws.
66
-------
QB Lobster Station C
N= 8 Sex:4 males, 4 females.
All animals were in good health. The gills of five of the animals showed
some early stages of inflammation in the gill lamellae. A protozoan was
noted between the gill lamellae.
QB Lobster Station D
N*5 Sex: 5 males.
All animals were in good health. Two of the animals showed some early stages
of inflammation in the gill lamellae. Protozoa were noted between the gill
lamellae in all animals.
QB Lobster Station E
N=5 Sex: 3 males, 2 females.
Three animals were in good health. In the other two animals there was an
inflammatory response in organs other than the gills. One animal had
necrosis and an inflammatory cell response in the labrynth epithelium of the
kidney. In the other animal there was intertubular inflammation as well as
necrosis and sloughing of the epithelium of the hepatopancreas. There was
also inflammatory cell infiltration of the connective tissue of the
labyrinth of the kidney.
QB Lobster Station F
N=5 Sex: 3 males, 2 females.
All animals were in good health. Protozoa were noted between gill lamellae
in ail animals. The intestine of one animal showed two cross sections c:
worms in the connective tissue between the muscle bundles.
67
-------
QB Lobster Station G
N*7 Sex: 4 males, 3 females
All animals were in good health. Protozoa were noted between gill lamellae
in all animals. The intestine of one of the animals has a section of a worm
in muscle bundle connective tissue.
QB Lobster Station I
N=5 Sex: 4 males, 1 female
All animals were in good health although inflammation was noted in the
hepatopancreas of one animal and in the gill lamellae of two of the animals.
Protozoa were found between gill lamellae in four of the animals.
QB Lobster Station J
N=7 Sex: 4 females, 2 males, 1 unknown.
All animals were in good health. Protozoa were noted between gill lamellae
in all animals. The gills of one animal showed early stages of inflammation
in the gill lamellae.
SOFT-SHELLED CLAM
Significant histopathological lesions in the soft-shelled clam included gill
inflammation (~ 80%), atypical cell hyperplasia in gill (~ 60%) and kidney
(~ 72%), and hyperparasitism with rickettsia in digestive ducts/tubules
(~ 51%) and general parasitism. Prevalence of those conditions at the tw:
collection sites was summarized in Table 10.
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CHARACTERISTICS OF CELLULAR ALTERATIONS IN THE SOFT-SHELLED CLAM
INFLAMMATION:
Inflammation as a process was stimulated to a high degree in Quincy Bay
soft-shelled clams evidenced primarily by histopathologic changes in
respiratory organs. Seventy seven percent (77%) of clams collected froir,
Moon Head had gill inflammation. Of those, the reaction was considered an
extensive response in ten cases (43%). In comparison, the same histologic
inflammatory reaction was present in eighty three percent (83%) of the clams
collected at the Moons. Of those, the reaction was considered to range from
a baseline level to an intensity considered extensive (27%) to massive
(10%).
Microscopically, the principal cytologic element in gill inflammatory
responses was the amebocyte. Amebocytes, a white blood cell, react to
stimulation (physical, chemical etc.) in the gills by infiltrating the
interlamellar connective supporting tissue of blood vessels, chitin and
epithelium. Macrophages and a general proliferation of raucous secretory
cells (hyperplasia) were also among the cytological observations associated
with the reaction in gill filamental and water tube regions cf the
respiratory organ. The reaction, from a progressive standpoint, ranged frorr
a mild response without microscopically visible cytological alteration
(other than amebocytes) to extreme change having obvious physiological and
morphological ramifications.
Proliferation cf mucous secretcry cells by itself does net necess2i_l^
constitute a pathologic condition, but it is considered a defensive respcr.s-.
-------
to environmental irritants (i.e., the greater the irritant the greater
increase in the number of mucous secretory cells). Thus the result was
presented in the general category of "inflammation". Mucous secretory cells,
interspersed with squamous epithelial cells, were dispersed along the length
of gill filaments and water tubes. Mucous secretory cell activity increased
above normal levels in eighty percent (80%) of the clams collected at the
Moon Head station. In comparison, the increase in activity at "the Moons" is
fifty six percent (56%).
RETROGRESSIVE CHANGES:
Atypical cell hyperplasia (ACH) was present in 73% (22 of 30) of the animals
from Moon Head; ACH in 4 of those animals (23%) was extensive. Prevalence
of ACH in clams collected at "The Moons" was 71% (56 of 78).
Atypical cell hyperplasia in marine bivalve molluscs has cytological
features that include hyperplastic alteration of normal ciliated and non-
ciliated gill filamental columnar epithelial cells accompanied by changes in
cellular chromaticity. Cellular proliferation and piling presenting a
stratified appearance was accompanied by changes in a normal pink staining
homogeneous cytoplasm to one that chromatically was dark purple (basophilic)
in an H and E stain. There was also loss of cilia as those cells undergoing
morphological change. Once proliferation of columnar epithelial cells was
initiated it spread along the gill filament eventually extending into the
water tubes. Adhesion of the gill filaments and replacement of blood
vessels, water tubes, and chitinous rods by fibrous connective tissue was a
hallmark of severe ACH. Further, atrophy (shrinkage) was evident ir.
stimulated tissue creating polyp-like formations.
71
-------
Parasites:
Worms were present in the gill filaments. The worms were embedded in a
calcareous shell and did not cause any inflammatory response; eventually the
worm does calcify and becomes a variety of a "pearl". At Moon Head, fifty
three percent (53%) (16 of 30) of the animals had parasites in their gills.
Sixty two percent (62%) (49 of 78) of the clams from "the Moons" had gill
associated parasites.
Digestive Diverticula:
Inflammatory and otherwise regressive lesions were lacking in the
gastrointestinal system of Quincy Bay soft-shelled clams.
Parasites:
Rickettsia, an endocytoplasmic organism known to occur in marine bivalves,
were present in epithelial mucosal cells lining digestive ducts and tubules.
Rickettsial organisms were present as membrane bound inclusion bodies which
stain a very deep blue basophilic color and are called ABI (amorphous
basophilic inclusions).
Fifty percent (15 of 30 animals) of the soft-shelled clams collected at Moon
Head had rickettsia in epithelium of ducts and tubules of the digestive
diverticula. Rickettsia in 1.3% (2 of 15) Moon Head clams were numerous
(five to six inclusions in a single tubule). In comparison, 561 (44 of 76
animals) of the clams collected at "The Moons" hosted the organism. The
rickettsial infestation in 1.5% (7 of 44) of the clams from "the Moons" were
rated as numerous.
72
-------
Calcium concretions of various sizes were noted between the epithelial folds
of the kidney. Concretions occurred in 15 of 30 animals (50%) collected
near Moon Head and in 23 of 78 animals (29%) collected from "the Moons".
Reproductive Tract:
Sex of Moon Head collected animals (30) consisted of 11 males, 16 females, 1
hermaphrodite, and 2 that were neutral. All female reproductive follicles
were in a state of product development (i.e., germ cell, primary, secondary
and mature ova). Thus, female germinal follicles were entirely formative.
Male reproductive follicles were, in contrast, full or partially full cf
mature spermatozoa and in the process of spawning.
Sex of clams collected at "the Moons" (78 animals) consisted of 40 males and
38 females. At the time of collection all reproductive follicles in females
showed development of some ova in follicles, while male reproductive
follicles contained mature spermatozoa. The male segment of the population
was in the process of spawning. Thus, spawning in the male and female soft-
shelled clams within those two Quincy Bay populations was asynchronous.
OYSTERS:
Tumor induction occurred in renal excretory tubules and gastrointestinal
mucosa of oysters exposed in situ 40 days at four stations in Quincy Bay.
Kidney tumors occurred in three oysters (3 of 50) exposed near Nut Island at
station 2 (QB Southwest) near Veazie Rocks and one (1 of 50) exposed at
station 3 (Nut Island Short Sewage Outfall). The three renal necplasr;
observed in oysters exposed at station 2 were in an early deveioprer.ta.
-------
stage, while the single renal neoplasm in an animal exposed at station 3 was
advanced. Adenomatous neoplasms occurred in the gastrointestinal mucosa of
oysters exposed at all four Quincy Bay stations. Of these, one (1 of 50)
occurred in the rectal segment of an oyster exposed at station 2 (QB
Southwest), one (1 of 50) in stomach or gastric nucosa at station 3 (Nut
Island Short Sewage Outfall), one (1 of 50) in gastric mucosa at station 4
(Nut Island Long Sewage Outfall) and five (5 of 50) at station S(Rainsford).
Of these five two (2) were rectal adenomas and three (3) were gastric
adenomatous lesions. A papilloma was observed in the stomach of an oyster
exposed at station 4. Tumors were not found in oysters exposed at the
reference location near "The Graves", or pre-exposure controls.
Renal Cell Tumor
In renal cell neoplasms the prominent feature was hyperchromatic nuclei of
transformed tubular epithelium. Sporadic cluster or nidi formations of
neoplastic epithelial cells were a trait highly visual in tubules with
minimal deviation. Neoplastic transition occurs variably in nephridial
epithelium from sporadic nidal formations to more advanced, complete tubular
involvement. Cytoplasmic to nuclear ratio was decreased and nuclear
chromatin density was increased being very hyperchromatic in H and E stair,.
In an advanced state, coalescence of these focal areas occurs, leading tc
cellular piling, disorganization of renal excretory epithelium and tubular
swelling. Invasive tendencies of the disease were cited in studies at tne
Environmental Research Laboratory, Narragansett, Rhode Island (Gardner, ej.
al., 1987).
-------
GASTROINTESTINAL TRACT
Rectal Adenoma
Neoplasms involving rectal epithelia were striking due to a multiplicity of
distinct luminal or adenomatous formations in the timorous mass. Adenomatous
formations that were well advanced consist of two morphologically
distinguishable cell types. Cells located on the surface nearest the
visceral mass, in relation to the lumen of adenomatous structures, appear 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 lack these distinguishing characteristics.
Neoplastic cells intermediate in relation to rectal and adenomatous luminal
areas were greatly reduced on their longitudinal axis. These cells alsc
feature a greatly reduced cytoplasmic to nuclear ratio, and stain intense
basophilic. Size difference in these two cell populations often creates a
"buckling-over" effect with a resultant crescent-shaped adenomatous pattern.
Neoplastic epithelial cells forming the rectal luminal surface area retain
columnar definition, but nuclei have enhanced hyperchromatism. Kitotic
figures in these lesion were numerous; cilia are generally lacking.
DEGENERATIVE:
Focal degenerative changes occurred in the kidney tubules of oysters exposed
at all stations, including pre-exposure controls. These changes occurred ir.
2%, 4%, 6%, 6%, 2% and 2% at stations one, two, three, four, five and pre-
exposure controls, respectively. Normal columnar epithelium was replaced
(metaplasia) by a squamous-to-cuboidal epithelial lining. Necrotic cells
were present in these lesions. Also, oysters at stations three, feu: a-.;
five were found to have very limited necrotic foci in mucosal cells c:
75
-------
digestive tubules. Those changes represent 6% or less of the total number
of oysters exposed (See Table 11).
Parasites:
Pathogenic ciliates and papovaviruses occurred in oysters exposed at all
Quincy Bay stations including the reference location near "The Graves".
Ciliates were present in pre-exposure control oysters; viruses were absent
from pre-exposure control oysters. Rickettsia were also present in oysters
exposed at stations one, three and four.
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ACKNOWLEDGEMENTS
We would like to thank Gary Lipson from the U.S. Environmental Protection
Agency Laboratory in Lexington, MA for collecting the sediment samples and
Bruce Reynolds and David Borrus from ERL/N for assisting in dive operations.
We would also like to thank Dr. John Harshbarger, Director, Registry of
Tumors in Lover Animals, Smithsonian Institution, Washington, D.C. and Toir.
LeFoley and colleagues at the Department of Pathology, Rhode Island
Hospital, Providence, RI for reviewing microscopic slides.
76
-------
REFERENCES
Eichelberger, J.W., L.E. Harris and W.L. Budde. 1975. Chromatography-mass
spectrometry systems. Analytical Chemistry 47: 995-1000.
Fabacher, D.L. and P.C. Baumann. 1985. Enlarged livers and hepatic
microsomal mixed-function oxidase components in tumor-bearing brown
bullheads from a chemically contaminated river. Environmental
Toxicology and Chemistry 4: 703-710.
Galloway, W.B., J.L. Lake, D.K. Phelps, P.F. Rogerson, V.T. Bowen, J.W.
Farrington, E.D. Goldberg, J.L. Lasiter, G.C. Lawler, J.H. Martin and
R.W. Risebrough. 1983. The Mussel Watch: Intercomparison of trace
level constituent determinations. Environmental Toxicology and
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Gardner, G.R., P.P. Yevich, A.R. Malcolm, R.J. Pruell, P. Rogerson, T.C.
Lee, A. Senecal, J. Heltshe and L.J. Mills. 1987. Carcinogenic
Effects of Black Rock Harbor Sediment on American Oysters and Winter
Flounder. Report to National Cancer Institute. U.S. EPA,
Narragansett, RI. May 17. 127 pp.
Krahn, M.K., M.S. Myers, D.G. Burrows and D.C. Malins. 1984. Determination
of metabolites of xenobiotics in the bile of fish from polluted
waterways. Xenobiotica 14: 633-646.
Krahn, K.M., L.D. Rhodes, M.S. Myers, L.K. Moore, W.D. MacLeod, Jr. and D.C.
Malins. 1986. Associations between metabolites of aromatic compounds in
bile and the occurrence of hepatic lesions in English Sole (Parophrys
vetulus) from Puget Sound, Washington. Archives of Environmental
Contamination and Toxicology 15: 61-67.
Krahn, M.M., D.G. Burrows, W.D. MacLeod, Jr. and D.C. Malins. 195".
Determination of individual metabolites of aromatic compounds ir.
hydrolyzed bile of English Sole (Parophrys vetulus) from polluted sites
in Puget Sound, Washington. Archives of Environmental Contamination and
Toxicology 16: 511-522.
Malins, D.C., B.B. McCain, D.W. Brown, S.-L. Chan, M.S. Myers, J.T. Landahl,
P.G. Prohaska, A.J. Friedman, L.D. Rhodes, D.G. Burrows, W.D. Gror.lur.d
and H.O. Hodgins. 1984. Chemical pollutants in sediments and diseases
of bottom-dwelling fish in Puget Sound, Washington. Environmental
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Malins, D.C., M.M. Krahn, M.S. Myers, L.D. Rhodes, D.W. Brown, C.A. Krone,
B.B. McCain and S.-L. Chan. 1985. Toxic chemicals in sediments ar.~
biota fror. a creosote-polluted harbor: relationships witr. hep-tir
neoplasms and other hepatic lesions in English scle (Parcphr-r
vetulus'. Carcinogenesis 6: 1463-1469.
79
-------
Malins, D.C., B.B. McCain, J.T. Landahl, M.S. Myers, M.M. Krahn, D.W. Brown,
S.-L. Chan and W.T. Roubal. 1988. Neoplastic and other diseases in
fish in relation to toxic chemicals: an overview. Aquatic Toxicology
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Mearns, A.J. and M.J. Allen. 1978. The use of small otter trawl in coastal
biological surveys. Rep. No. 600/3-78-083. U.S. EPA, Corvallis, OR 34
pp.
Rugg, T. and P. Feldraan. 1980. Mathematics programs in TRS-80 programs.
Dilithium Press, Forest Grove, Oregon, pp. 185-194.
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EPA, Cincinnati, Ohio.
-------
APPENDIX A
PREVALENCE OF LIVER LESIONS
Samp Gross
75101
75105
75106
75108
75109
75110
75111
75112
75113 U
75114 F
75115
75116
75117
75118
75119
75120 F
75121
75122
75123
75124 M
75125
75127
75128
75129
75130
75131
75133
75135 F
75136
75137
75138
75139
75143
75144
75145
75146
75148 M
75150
75151
75152
75153
75154
75156
7515"
75156
7516:
Neoplasm
Hepato Cholfib Vac BDVac Basof
F F
F
U
F
F
U U U U
F F
M
M M
F
M M
M
F F
F
M M
MM M
F
M
M
MM M
F
M M
M
F
M
F F F
F
M MM M
F
M
F
M
M
r. M
K
CCF CTH SpongH Necros Inflam
F
F
M
F
F
U U
M M
M
M
F
M M
M
F
M
M M
M
M
M
F
F
M M
F F
M
M M
F
K
K
F
F
M
U
F
F
U
M
F
M
F
M
F
F
''
V
> -
i
-------
PREVALENCE OF LIVER LESIONS (CONTINUED)
Samp
75164
75165
75167
75168
75169
75170
75171
75172
75173
75174
75175
75176
75177
75178
75179
75180
75181
75182
75183
75184
75185
75187
75188
75189
75190
75191
75192
75193
75194
75195
75196
75197
75198
75199
TOTAL
Gross
Hepato
Choi fib
Vac
BDVac
Gross Hepato Cholfib
M
M
M M
M
F F
M M M
M
M
M M
M M
F F F
M
F
F F F
M
F F
F F
16 19 11
Vac
M
M
M
F
F
M
M
F
F
F
M
F
F
M
F
M
F
F
M
F
48
•= Gross Pathology
= Hepatocellular
= Cholangiofibrosis
= Vacuolation
= Bile Duct Vacuolation
BDVac Basof CCF CTH SpongH
M
M
F
M M
M
M
M M
F F F
M
F F
M M
M
M
M
F
F F
5 18 11 7 11
Necros
M
F
F
M
F
M
F
M
F
M
M
F
33
Inflan-.
tt
K
M
M
K
F
F
M
F
F
F
K
F
K
F
F
K
K
K
38
Basof - Basophilic Foci
CCF = Clear Cell Foci
CTH = Connective Tissue Hyperplasia
SpongH « Spongiosis Hepatis
Necros m Necrosis
Inflam - Inflammation
M - Male
F = Female
U = Unidentified
-------
APPENDIX B
Table Bl. Key for the abbreviations used.
SAMPNUM - ERLN sample number
0.00 - Value below the analytical detection limit.
CB052 - 2,2',5,5'-PCB
CB047 - 2,2',4,4'-PCB
CB101 - 2,2',4,5,5'-PCB
CB151 - 2,2',3,5,5',6-PCB
CB118 - 2,3',4,4',5-PCB
CB153 - 2,2',4,4',5,5'-PCB
CB138 - 2,2',3,4,4',5'-PCB
CB128 - 2,2',3,3',4,4'-PCB
CB180 - 2,2',3,4,4',5,5'-PCB
CB195 - 2,2',3,3',4,4',5,6-PCB
CB194 - 2,2',3,3',4,4',5,5'-PCB
CB206 - 2,2',3,3',4,4',5,5',6-PCB
CB209 - CL10-PCB
HCB - Hexachlorobenzene
A-HCH - alpha-hexachlorocyclohexane
G-HCH - gamma-hexachlorocyclohexane
A-CHLOR - alpha-chlordane
G-CHLOR - garama-chlordane
FL - Fluorene
PHEN' - Phenanthrene
ANTH - Anthracene
C1PA - Cl homologs of phenanthrene and anthracene
C2PA - C2 homologs of phenanthrene and anthracene
C3PA - C3 homologs of phenanthrene and anthracene
C4PA - C4 homologs of phenanthrene and anthracene
FLUO - Fluoranthene
PYR - Pyrene
B[a]A - Benz[a]anthracene
CHRY - Chrysene
BFL - Sum of benzofluoranthenes
B[e]P - Benzo[e]pyrene
B[a]P - Benzo[aIpyrene
PERY - Perylene
IND - Indeno[l,2,3-cd]pyrene
B[ghi]P - BenzolghiJperylene
S276 - Sum of molecular weight 276 PAHs
D[ah]A - Dibenzla,h]anthracene
S278 - Sum of molecular weight 278 PAHs
COF. - Coronene
S302 - Sum of molecular weight 302 PAHs
TOTAL - Total of measured PAHs
(ItO and B[ghi]F are
S278)
included in S276, D[ah]A is included :r
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