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
               S-1<{
               
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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
                                    11

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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



                                    iv

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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



                                    vi

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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
                                   Vlll

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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

                                    ix

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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



                                    10

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o
o
o
in
•
(M
     C
     N-

-------
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.



                                    12

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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



                                    14

-------

-------

-------
 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




                                    17

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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



                                    18

-------
 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

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 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

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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

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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

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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

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 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

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 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

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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

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                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

-------

-------
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        K.
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        C
        EZ

        c
        >•
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                      . 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
4B,F,G,H,I
4B,E,G,H,I;5
4B,H,I;7A
4A,B,I

4B,G,H,I;7A
4G,I
4D,I
4H
4B,D;7A
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
  (X

  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.
                                    6E

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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
      Chemistry   2: 395-410.

 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
      Science and Technology 18: 705-713.

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
     11: 43-67.

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.

Yevich,   P.P. and C.A.  Barszcz.  1981.  Preparation of Aquatic Animals for
     Histopathological Examination.   DN  0543A, Aquatic Biol.  Sect., Biol.
     Meth. Branch,   Environmental  Monitoring and  Support Laboratory,  U.S.
     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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