Environ. Sci. Technol. 1993, 27,2759-2771
Chemical Contaminants and Hepatic Lesions in Winter Flounder (Pleuronectes americanus) from the Northeast Coast of the United States Lyndal L. Johnson,' Carla M. Stehr, 0. Paul Olson, Mark S. Myers, Susan M. Pierce, Catherlne A. Wlgren, Bruce B. McCaln, and Usha Varanasl
Northwest Fisheries Science Center, Environmental Conservation Division, National Marine Fisheries Service, NOAA, 2725 Montlake Boulevard East, Seattle, Washington 981 12 Relationships between hepatic lesions and chemical contaminant concentrations in sediments, stomach contents, and tissues were examined in winter flounder (Pleuronectes americanus) collectedfrom 22 sites in eight major embayments on the Northeast Coast (i.e., Salem Harbor, Boston Harbor, Plymouth Bay, Buzzards Bay, New Bedford Harbor, Narragansett Bay, Long Island Sound, and Raritan Bay). Prevalences of a number of pathological conditions, including neoplasms, preneoplastic lesions, hydropic vacuolation, and other necrotic and proliferative lesions, were significantly elevated in fish from contaminated urban embayments such as Boston Harbor and Raritan Bay. Results of logistic regression analyses indicated that polycyclic aromatic hydrocarbons, DDTs, or chlordanes in sediments, stomach contents, liver, or bile of winter flounder were significant risk factors for the development of several lesion types, including hydropic vacuolation and proliferative and necrotic lesions. However, concentrations of PCBs in sediments and tissue were not significant risk factors for any of the lesions observed. In addition to chemical contaminants, fish age and sampling season had a significant influence on disease occurrence. The risk of hepatic disease increased with age, and lesion prevalences were higher in animals collected during the spring than in winter when spawning migration was taking place. The relationships observed in this study strongly suggestan associationbetween exposure to certain chemical contaminants and the development of particular liver lesions in winter flounder. Introduction
Histopathology has been increasingly recognized as a valuable tool for field assessment of the impact of environmental pollutants on marine life. It provides a rapid method for the detection of adverse chronic effects of contaminants on various tissues and organs of marine organisms and has been employed successfullyin a variety of national and regional programs designed to assess the environmental quality of coastal and estuarine waters (14). Probably the most commonly used histopathologic indicators of contaminant-associated stress in marine fish are neoplastic and preneoplastic liver lesions, although recent studies indicate that certain associated degenerative and regenerative conditions may be equally or even more useful as bioindicators of contaminant exposure and related effects (5-7). Liver cancer and related lesions have been reported in several species of bottomfish from chemically contaminated coastal areas, including English sole (Pleuronectes vetulus) (1, 8-11), white croaker (Geneonymus lineatus) (1,3,4,12-14); Atlantic tomcod (Gadus tomcod) (15, 161, and mummichog (Fundulus heteroclitis) (17). Associations between concentrations of chemical contaminants in sediments and fish tissues Thls article not subJectto U S . Copyrlght.
and certain of these lesions, particularly liver neoplasms and associated preneoplastic and degenerative conditions, have been observed in field surveys (1,3,9,10,17), and in English sole these associations have been further substantiated by statistical analyses and epizootiological models (5, 13, 14, 18-20). Moreover, in long-term laboratory studies with English sole, cause-and-effect relationships between selected contaminants (e.g., polycyclic aromatic compounds) and lesionssimilar to those observed in field-sampledanimals have been demonstrated (21,22). Previous field studies suggest that liver lesions in winter flounder (Pleuronectes americanus) may be equally reliable bioindicators of environmental degradation on the East Coast. The occurrence of neoplastic and nonneoplastic liver disease in flounder from contaminated areas, especially from sites within Boston Harbor, is welldocumented (23-31). At present, however, there is relatively little information available on how levels of specific contaminants in sediments and tissues are related to disease occurrence or on the effects of biological factors such as age and gender on disease prevalence in this species. The documentation of such relationships using statistical analyses and epizootiological models is particularly important for regenerative or degenerative lesions such as hepatocellular or biliary vacuolation (23-251, which has been described only in fish and whose etiologyis not clearly known. Moreover, although several studies have been conducted examining either levels of chemical contaminants or fish disease in selected estuaries in the Northeastern United States (26, 27, 32-35), few studies have characterized chemical contamination in sediments and in winter flounder throughout the Northeast using standardized procedures for sample collection and analysis or employed a multidisciplinary approach examining the prevalences of different types of lesions in combination with levels of a broad spectrum of contaminants and their metabolites in sediments and fish tissues. In this study, relationships between hepatic lesions and chemical contaminant concentrations in sediments, stomach contents, and tissues were examined in winter flounder collected from 22 Northeast Coast sites in conjunction with the National Benthic Surveillance Project (NBSP), a segment of NOAA's National Status and Trends Program. Logistic regression, a multivariate statistical method frequently applied to epidemiological or epizootiological data (36,37), was used to assess the influence of various biological (e.g., age, gender, and season) and contaminant-associated risk factors (e.g., site of capture and concentrations of selected chemical contaminants in sediment and fish) on lesion occurrence. Results of these analyses and their relationship to other studies of pollutionassociated disease in winter flounder will be discussed.
Published 1993 by the American Chemical Soclety
Environ. Scl. Technol., Vol. 27, No. 13, 1993 2750
Figure Locations of sampling sites on the Northeast Coast of the United States. Sites in bold type were sampled in spring as part of the lnal Benthic Surveillance Project, and a complete set of samples, including pathology and sediment, tissue, and stomach contents chemistry, was collected and analyzed for at least 1 year; other sites were sampled in winter, and pathology samples only were analyzed. Sites sampled in both winter and spring are indicated by an asterisk (*). The stations were identifiedas Salem Harbor, Folger's Point (42' 32.2' N, 70' 49.6' W) (SALFP); Boston Harbor, Deer Island (42' 19.9' N, 70' 58.1' W) (BOSDI); Boston Harbor, Quincy Bay (42' 18.4' N, 70' 58.4' W)(BOSQB); Boston Harbor, Mystic River (42' 23.2' N, 71' 03.2' W) (BOSMR); Boston Harbor, Hull Bay (42' 17.1' N, 70' 54.4' W) (BOSHB); Boston Harbor, President Roads (42' 20.0' N, 70' 59.0' W) (BOSPR); Massachusetts Bay, outside Boston (42' 20.5'N, 70' 46.0' W) (MASBS); Massachusetts Bay, Plymouth Entrance (41' 59.3' N, 70' 37.6') (MASPE); New Bedford Harbor, Clarks Point (41' 35.0' N, 70' 53.5' W) (NWBCP); Buzzards Bay (41' 35.0' N, 70' 45.0' W) (BUZWI); Narragansett Bay, Prudence Island (41' 40.4' N, 71' 21.2' W) (NARPI); Niantic Bay, Black Point (41' 17.2' N, 72' 11.2' W) (NIABP); Long Island Sound, New Haven (41' 15.3' N, 72' 54.8' W) (LIS"); Long Island Sound, Norwalk (41' 02.3' N, 73' 28.2' W) (LISNO); Long Island Sound Bridgeport (41' 14.9' N, 70' 28.0' W) (LISBR); Long Island Sound, Rocky Point (41' 08.7' N, 72' 24.7' W)(LISRP); Long Island Sound, Lloyd Polnt (40' 58.0' N, 73' 28.9' W) (LISLP); Raritan Bay, East Reach (40' 58.0' N, 73' 28.9' W) (RARER); Raritan Bay, West Reach (40' 30.4' N, 74' 10.2' W) (RARWR); Raritan Bay, Gravesend Bay (40' 35.4' N, 74' 01.6' W) (RARGB); Raritan Bay, Upper Bay (40' 39.4' N, 74' 08.8' W) (RARUB); and Great Bay, Intracoastal Waterway (39' 25.8' N, 74' 25.1 W) (GRTIW).
Materials and Methods Field Sampling. Winter flounder and sediments were collected from 22 sites along the Northeast Coast of the United States (Figure 1). Fourteen of these sites were located in or near urban embayments, and the eight remaining sites were in nonurban embayments, two of which (Rocky Point, Long Island Sound, and Plymouth Entrance, Massachusetts Bay) served as a reference sites for statistical comparisons. These 22 sites were selected for sampling because they were (1) located in subtidal, sedimentary depositional zones; (2) were not subject to dredging, scouring,or slumping; (3)supported populations of winter flounder; (4) exhibited a broad range in types and levels of sediment contamination; and (5) in many cases were located in urban areas where marine pollution is a potential public health concern. Each site consisted of three stations generally located less than 0.4 km apart and positioned in a manner designed to characterize the entire site, but not the entire embayment in which the site was located. Sites for sediment collection were situated along trawl lines for fish collection. Surface sediments were collected using a Smith-MacIntyre grab as described in previous publications (1, 3). Approximately 30-60 fish were collected by otter trawl at each site at each sampling time. Fish less than 15 cm 2760
Environ. Scl. Technol., Voi. 27, No. 13, 1993
in length (generally less than 1 year in age) were not collected because they did not have sufficient tissue and bile for chemical analyses. Animals were kept alive in seawater until necropsies were performed to prevent autolysis of tissues; elapsed time from capture to necropsy did not exceed 3 h. Fish were weighed (total weight in grams), measured (total length in millimeters), and sacrificed by severing the spinal cord. Otoliths were then removed using forceps, cleaned, and stored dry in plastic centrifuge tubes for subsequent age determination. Tissue samples approximately 2-3 mm in thickness were collected from the central axis of the liver and preserved in Dietrich's fixative (38) for histopathological examination. After 2-3 days, tissue samples were transferred from fixative to 70% ethanol for storage. The bile from the gall bladder and a portion of the remaining liver tissue from individual fish were placed in separate,methylene chloriderinsed glass vials and frozen at -80 'C for later analyses for fluorescent aromatic compounds (FACs) and organic compounds, respectively. Liver samples for organic chemistry and histopathology were taken from all animals collected at each site, and a minimum of 10 bile samples were collectedper site. In addition, stomach contents from at least 10 fish per site were removed and composited in a solvent-rinsed glass jar and frozen for chemical analyses. Bile and stomach contents were collected from the same
animals that were sampled for liver chemistry and histopathology. Laboratory Analyses. (A) Fish Age Determination. In selected animals, fish age was estimated by counting the number of clearly defined opaque zones of whole otoliths under a binocular dissecting microscope (39,401. Ages were determined from otoliths in all animals from each site in at least one sampling cycle; a total of 623 animals were aged. For the remaining animals, age was estimated from length based on age-length curves (41) constructed from data on fish whose ages had been determined by examination of otoliths. Separate genderspecificage-length regression curves were constructed for three major geographic areas: Great Bay (Great Bay and Intracoastal Waterway),Raritan Bay, and the Long Island Sound area. In the Massachusetts Bay-Boston Harbor area, the number of males aged was insufficient to calculate a separate regression equation, so a single equation was constructed using data on both males and females. R2 values for the age-length curves ranged from approximately 0.5 to 0.7 (p < 0.01) (41). (B) Histopathological Analyses. Fish tissues preserved in the field for histopathological examination were returned to the laboratory where they were dehydrated, embedded in paraffin, sectioned at 4-5 pm thickness, and stained with Mayer’s hematoxylin and eosin phloxine using routine histological procedures (42, 43). Lesion classification followed previously described diagnostic criteria (11,44-46). For reporting and statistical analyses, hepatic lesions were grouped into the following categories: neoplasms; foci of cellular alteration (FCA), including eosinophilic, basophilic, and clear cell foci; hepatocellular nuclear pleomorphism; spongiosis hepatis; hydropic vacuolation (hepatocellular or biliary hydropic vacuolation); non-neoplastic proliferative lesions (hepatocellular or biliary regeneration, biliary hyperplasia, cholangiofibrosis, and increased hepatocellular mitotic activity) and necrotic lesions (coagulative necrosis, hydropic degeneration, hyalinization, and pyknosis; necrotic lesions associated with parasitic infections were not included in the analyses). A complete description of the types of individual lesions included in these categories is given in previous publications (14, 41). These lesion types were selected because they are well-established as histopathologic biomarkers of contaminant effects in fish and show strong evidence of a contaminant-associated etiology based on previous field or laboratory studies (5-7, 1 1 ) . A variety of other pathological conditions, such as parasitic infections, were noted in flounder examined in this study, but they will not be described here as there is little evidence that they have a chemical etiology (7). (C) Chemical Analyses of Sediments, Stomach Contents, Liver Tissue, and Bile. Prior to analysis, liver samples were pooled into three composites of 10-20 liver samples per site. Stomach contents and sediments were analyzed for a broad spectrum of aromatic hydrocarbons (AHs) and chlorinated hydrocarbons (CHs). Because AHs are rapidly metabolized and do not accumulate in the liver (47),liver tissue was analyzed for CHs only. Chemical analyses were conducted as previously described ( 1 , 2, 48). Exposure to AHs was assessed by measuring aromatic compounds in fish bile fluorescing at BaP wavelengths (FACs-H) and naphthalene wavelengths (FACs-L) using the procedures of Krahn et al. (49, 50). For statistical analyses and reporting, the AHs and CHs
whose concentrations were measured in sediments and stomach contents and CHs measured in fish liver were grouped into five broad categories: low molecular weight AHs (LAHs), high molecular weight AHs (HAHs), DDT and its derivatives (DDTs), polychlorinated biphenyls (PCBs),and chlordanes. The specificcompounds included in each of these categories are listed in Johnson et al. (41). It should be noted that in addition to chlordanes, other chlorinated non-DDT pesticides (Le., lindane, mirex, heptachlor, heptachlor expoxide, aldrin, dieldrin and hexachlorobenzene) were present in sediments and tissues from some Northeast Coast sampling sites (51). However, chlordanes were the predominant type of non-DDT pesticide present; others occurred at low concentrations comparable to those found in reference areas (51). Consequently, only chlordanes will be reported and correlated with pathological conditions in this study. Analytical chemistry data generated in conjunction with this study will be reported in greater detail in a subsequent publication (51). Statistical Analyses. (A) Chemical Contaminant Levels in Sediment, Stomach Contents, and Fish. Mean concentrations of LAHs, HAHs, DDTs, PCBs, and chlordanes in sediment and stomach contents; DDTs, PCBs, and chlordanes in liver; and levels of FACs-H and FACs-L in bile were calculated for each sampling site. Contaminant levels at the sampling sites were compared using comparison intervals (52,53)calculated according to the GT2 method (53). The comparison interval is calculated for each mean based on the number of samples for that mean, the variability of the mean, and the number of means being compared. This method allows graphical comparison of means; when the comparison intervals overlap this indicates that the means are not significantly different (p < 0.05). Rocky Point in Long Island Sound was used as a reference site for statistical comparisons of AH concentrations, as it was the site with the lowest levels of these contaminants in sediment. For CHs, Plymouth Entrance in Massachusetts Bay was used as the reference site because levels of PCBs, DDTs, and chlordanes were lower in sediments and tissues from this site than at Rocky Point. (B) Heterogeneity i n Lesion Prevalence. To determine if statistically significant differences in lesion prevalences existed between the reference site (Rocky Point, Long Island Sound) and the individual test sites, the G-statistic (53) was computed using the lesion prevalences at the reference site as the expected values. The significance level for this test was set at p C 0.05. Rocky Point was chosen as the reference site for statistical comparison of lesion prevalences because it was centrally located and had among the lowest levels of organic contaminants in sediment of all the sampling sites. (C) Logistic Regression and Risk Factor Analysis, Stepwise logistic regression (36,37)was used to identify statistically significantrelationships between potential risk factors and hepatic lesion occurrence in winter flounder. This method of analysis is specificallysuited for binomially distributed data and allows simultaneous adjustment for a number of risk factors by iterative model fitting. Two types of analyses were conducted: (1) an analysis to examine the relative risk of disease in individual fish in relation to site of capture, gender, season of capture, and age or estimated age and (2) an analysis to detect significant relationships between mean concentrations of contamiEnvlron. Scl. Technol., Vol. 27, No. 13, 1993
2781
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nantsinsediments,fishstomachcontents,andfishtissues and lesion prevalences at the sampling sites. A number of chemical contaminants were measured in this study, and for the second analysis, it would have been desirable to evaluate their relative importance as risk factors for hepatic disease using a multivariate logistic model. In practice, however, it was not feasible to construct such a model because of the large number of highly intercorrelated risk factors and the relatively low number of samples available for statistical analysis. Consequently, relationships between various classes of chemicals and lesion prevalences wefe examined separately while adjusting for potential effects of the mean fish age and gender ratio at the sampling sites. Logistic regression models were fitted using the PECAN analysismodule of theEGRET statisticalpackage (Version 2182
Envton. Sol. Techml.. Vol. 27. NO. 13. 1993
111, Statistics and Epidemiology Research Corporation, Seattle, WA) usingaprocedure similar tostepwise multiple regression. The estimated odds ratio or relative risk for the occurrence of a lesion in an individual fish, which is a measure of the degree of association between a risk factor and lesion occurrence (54),was calculated from variable coefficientsofthe logistic regressions (37,55).Odds ratios or relative risks greater than 1 indicate an increased probability of disease, and odds ratios less than 1 indicate a decreased probability of disease. For all logistic regression analyses, risk factors were considered significant a t p < 0.05. The statistical analyses described above were applied to the following categories of hepatic lesions: (1) neoplasms, (2) FCA, (3) nuclear pleomorphism and megalocytic hepatosis, (4) spongiosis hepatis, (5)hydropic vacuolation, (6) non-neoplastic proliferative lesions, and
(7)nonspecific degenerativeor necroticlesions. TheRoeky Point site in Long Island Sound was designated as the reference area for calculation of odds ratios and relative disease risks associated with site of capture. Calculated odds ratios for age (in years) were interpreted for each additional year of age. In addition to the risk factors outlined above, the effect of sampling season on lesion prevalence was also examined for stations sampled in both winter and spring of 1 9 8 9 1989. The winter sampling was not part of the NBSP but was part of a separate study investigating the effecta of contaminants on reproductive function in female winter flounder (56), so male fish were not collected. Consequently, the analysis of the impact of season of capture on the risk of lesion occurrence is limited to female fish.
sediment PCEE
Results Contaminant Levels in Fish and Sediments. Mean concentrations of organic contaminants in sediments and winter flounder stomach contents, liver and bile are shown in Figures 2-4. It should be noted that in many cases, different classes of chemical contaminantstended to ocm together at polluted urban sites. Concentrations of aromatic hydrocarbons (HAHs and LAHs) measured in sediment were significantly (p < 0.05) and highly intercorrelated (0.8 > Spearman's p > 0.9). Various classes of chlorinated hydrocarbons (i.e., chlordanes, PCBs, and DDTs) were also covariant (0.8 < p < 0.9). Moreover, sedimentAHs were significantly correlated (0.7 > p > 0.9) with concentrations of chlordanes, DDTs, and PCBs in sediment. At all sites, except for Plymouth Entrance in Massachusetts Bay and Buzzards Bay, concentrations of both HAHs and LAHs in sediment (Figure 2) were significantly higher than at the Rocky Point reference site in Long Island Sound. Mean concentrations of AHs in sediments from the Quincy Bay and Mystic River sites in Boston Harbor were among the highest observed in this study (2-4 times higher than concentrations at any of the other sampling sites). BiliiFACs-H and FACs-Llevels (Figure 2) were generally reflective of AH levels in sediment and in stomach contents of winter flounder. Biliary FACs-H levels were significantlyelevated at five sites in comparison with the Rocky Point, Long Island Sound, reference site (i.e., Mystic River and Quincy Bay in Boston Harbor and East Reach, West Reach, and Upper Bay in Raritan Bay); biliary FAC-L levels also tended to be highest in flounder from sites in Boston Harbor and Gravesend Bay. Concentrations of AHs in sediment were positively correlated (Spearman'sp = 0 . 8 ; < ~ 0.05) withconcentrationsofAHs in winter flounder stomach contents. Significant correlations (p < 0.05) were also found between concentrations of HAHs in stomach contents and biliary FACs-H and between LAH concentrations in stomach contents and biliary FACs-L ( p = 0.8). As noted previously, for CHs (i.e., PCBs, chlordanes, and DDTs) Plymouth Entrance in Massachusetts Bay rather than Rocky Point was used as a reference site for statistical comparisonsbecause flounder from this sitehad somewhat lower levels of these contaminantsin tissue than fish from the Rocky Point site. With the exception of Rocky Point in Long Island Sound, Black Point in Niantic Bay, and Buzzards Bay, concentrations of PCBs in sediment were significantly higher at all sampling sites
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than at the Plymouth Entrance reference site (Figure 3a). Levels of PCBs were among the highest in sediments from the Clarks Point site in New Bedford Harbor and the Mystic River site in Boston Harbor. Concentrations of PCBs in liver (Figure 3c) were among the highest in flounder from New Bedford Harbor and Buzzards Bay and were significantly higher in flounder from several additional sites within Boston Harbor, Raritan Bay, and Long Island Sound than in flounder from the Plymouth Entrance reference site. Concentrations ofPCBs in winter flounder stomach contents could not be compared statistically because the number of samples was too small, but they were significantly and positively correlated with PCB levels in sediment ( p = 0.6) and liver ( p = 0.8). Levels of DDTs in sediments at 11of the 19 sampling sites (Figure 4a) were significantly greater than levels at the Plymouth Entrance reference site. Concentrations of DDTs in sedimentswere among the highest a t East Reach in Raritan Bay and a t Quincy Bay and Mystic River in Boston Harbor. Levels of DDTs in winter flounder stomach contents were among the highest in fish from Raritan Bay but were also relatively high a t a number of siteswithinBostonHarborandLongIslandSound(Figure 4b). Concentrations of DDTs in liver were significantly higher in flounder fromseveralsiteswithin Boston Harbor, Raritan Bay, and Long Island Sound than in fish from Plymouth Entrance. Levels of DDTs in liver were among the highest in fish from Upper Bay and Great Bay in Raritan Bay,SalemHarhor. andNew HaveninLong Island Sound. Concentrations ofchlordanes in sediment (Figure 4d) were among the highest in Quincy Bay and Mystic Envkon. Sd. Tednd., VOl. 27, NO. 13. 1993 276.3
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River in Boston and were significantly elevated in comparison to levels at Plymouth Entrance at five additional sites. Concentrations of chlordanes in winter flounder stomach contents, on the other hand, were among the highest in flounder from New Bedford Harbor and Buzzards Bay (Figure 4e). Like liver concentrations of DDTs, concentrations of chlordanes in liver (Figure 40 were lowest in fish from Plymouth Entrance in Massachusetts Bay and Black Point in Niantic Bay but were moderate in flounder from the Rocky Point site. Concentrations of chlordanes in liver were significantly elevated in comparison with Plymouth Entrance fish in flounder from several sites within Boston Harbor, Long Island Sound, and Raritan Bay; levels were among the highest in fish from the Upper Bay site in Raritan Bay, Deer Island in Boston Harbor, Salem Harbor, and New 27M Envkon. Scl. Tsmnol.. Vci. 27. No. 13. 1993
Haven in Long Island Sound. Concentrations of DDTs and chlordanes in stomach contents were significantly correlated with levels of the same contaminants in sediments (0.4 < p < 0.5), as well as with DDT and chlordane levels in liver ( p = 0.8). Chlordanes, DDTs, and PCBs all showed substantial bioconcentration in winter flounder and their prey organisms; levels of these compounds in stomach contents were approximately 10 times as great as in sediments, and levels in liver were 2-3 times as great as in stomach contents (Figures 2-4). Hepatic Lesion Prevalences and Intersite Comparisons. The prevalenceof neoplasmsin winter flounder was significantly elevated in comparison with the Rocky Point reference site at 10 of the 22 sampling sites (Figure 5a,b). These sites included Hull Bay, Deer Island, Long Island, and Mystic River in Boston Harbor; Gravesend
Flgurm 5. Prevalences 01 hepatic neoplasms. foci of cellular aneration (FCAI and proliferativelesions in uinter flounder froom (a)me normern East Coast and (b) the southern East Coast; Prevalences of nuclear pleomorphism. spongiosis hepatis. hydropic vacuolation. and nonspecIRc hepatocellular necrosis in winter fbunder from (cl the northern East Coast and (d) me southern East Coast. Asterisks ('j indicate prevalences m a t are significantiy hipher (G~txti~tIc. p < 0.05)than those at me Rocky Point referencesne in Long Island Sound (in italics). sne abbreviations are definsd in Figure 1
Bay and West Reach in Raritan Bay; Lloyd Point and New Haven in Long Island Sound; and Buzzards Bay. Significantly elevated prevalences of FCA (Figure 5a,b) were found at three sampling sites: Buzzards Bay, Deer Island in Boston Harbor, and Gravesend Bay in Raritan Bay. Of the non-neoplastic toxicopathic lesions, hydropic vacuolation was the most prevalent, occurring in at least 20% of flounder from 11of the 22 sampling sites (Figure 5c,d). Prevalenceswere highest in fish from Mystic River and Deer Island in Boston Harbor, from West Reach and Gravesend Bay in Raritan Bay, and from Norwalk in Long Island Sound. Elevated prevalences of nonspecific necrotic lesions (Figure 5 c,d) were also found in flounder from a number of sites within Boston Harbor and Raritan Bay, from New Haven and Lloyd Point in Long Island Sound, and from Buzzards Bay. Significantlyelevated prevalences of non-neoplastic proliferative liver lesions (Figure 5a,b) were found at only two sites, Deer Island and Mystic River in Boston Harbor. Prevalences of spongiosis hepatis (Figure 5c,d) were significantly elevated at six sampling sites: theHullBay,DeerIsland,QuincyBay,and President Roads sites in Boston Harbor, the Folgers Point site in Salem Harbor, and the New Haven site in Long Island Sound. Megalocytic hepatosis was not observed in any winter flounder examined in this study, although one case of this lesion has been detected in a winter flounder collected from Hull Bay in Boston Harbor in subsequent field sampling (C. Stehr, personal communication). Prev-
alences of hepatocellular nuclear pleomorphism (Figure 5c,d) ranged from 0 to 7 76, but no significant intersite differences were found. Risk Factors Associated with Hepatic Lesion Occurrence in Individual Fish. For all categories of hepatic lesions except nuclear pleomorphism, the risk of lesion occurrence increased significantly @ < 0.05) with increasing fish age (Table I). Also, in comparison to males, females collected at the same time period (i.e., during the spring sampling) had a significantly decreasedrisk (Table I) of developing neoplasms (1.13%of females affected vs 3.2%of males) and spongiosis hepatis (0.55% of females affected vs 1.9% of males). Gender did not have a significant influence on the occurrence of other lesion types. Residence at a number of the sampling sites, particularly those in the Hudson-Raritan Estuary or Boston Harbor, was associated with an increased risk of disease in comparison with the risk in fish from the reference site at Rocky Point, Long Island Sound (Table I). Fish from Deer Island and Hull Bay in Boston Harbor and from West Reach and GravesendBay in Raritan Bay were 4-21 times as likely to develop neoplasms as animals sampled from other areas. An approximately 3-7-fold increase in the risk of FCA was associated with residence at Deer Island and Mystic River in Boston Harbor, Buzzards Bay, and Gravesend Bay in Raritan Bay. Fish from Mystic River and Deer Island in Boston Harbor were approximately 2-4 times as likely to develop proliferative liver Envirm. Scl. Technd.. VoI. 27. No. 13. 1993
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Table I. Calculated Odds Ratio for Significant ( p < 0.05) Risk Factors for Six Categories of Hepatic Lesions in Winter Flounder=
lesion neoplasms GM = 5.773 X 10-4
foci of cellular alteration GM = 2.572 X
risk factor age female Raritan Bay, Gravesend Bay Boston Harbor, Deer Island Raritan Bay, West Reach Boston Harbor, Hull Bay age
odds ratio 1.988 0.1928 21.46 3.887 8.973 5.081 1.373
Raritan Bay, Gravesend Bay 1.778 Boston Harbor, Deer Island 3.500 Boston Harbor, Mystic River 4.368 Buzzards Bay 3.828 proliferative lesions age 1.673 Boston Harbor, Deer Island 3.939 GM = 1.275 X Boston Harbor, Mystic River 1.673 spongiosis hepatis age 2.244 GM = 3.837 X 10-2 Boston Harbor, President Roads 13.06 female 0.1582 nuclear pleomorphism Long Island Sound, New Haven 4.883 GM = 1.463 X 10-2 hydropic vacuolation age 1.345 GM = 2.940 X 10-l Boston Harbor, Mystic River 10.56 Raritan Bay, East Reach 6.594 Boston Harbor, Deer Island 5.403 Long Island Sound, Norwalk 6.752 Long Island Sound, New Haven 5.620 Raritan Bay, Gravesend Bay 8.962 Salem Harbor, Folgers Point 2.481 Boston Harbor, President Roads 4.336 Boston Harbor, Hull Bay 3.875 Boston Harbor, Quincy Bay 4.387 necrosis age 1.184 GM = 4.681 X 10-2 Boston Harbor, Mystic River 2.176 Boston Harbor, Hull Bay 2.010 Boston Harbor, Quincy Bay 3.258 Boston Harbor, President Roads 2.607 Raritan Bay, West Reach 2.826 Odds ratios for the site of capture are interpreted relative to the refernce site as Rocky Point, Long Island Sound. Odds ratios for age (in years) represent the effect of each additional year of age on the odds of disease occurrence. GM = grand mean.
lesions as animals from other sites, while fish from New Haven in Long Island Sound were 4 times as likely to develop nuclear pleomorphism, and fish from President Roads in Boston Harbor were 13times as likely as reference animals to be affected with spongiosis hepatis. The risk of hydropic vacuolation was significantly increased at 10 of the 22 sampling sites. The risk increase ranged from approximately 2.5-fold at Salem Harbor to over 10-foldat Mystic River. Fish from Mystic River, Hull Bay, Quincy Bay, and President Roads in Boston Harbor and from West Reach in Raritan Bay were 2-3 times as likely to be affected with nonspecific necrotic lesions as comparable fish from the reference site. In female fish sampled in both winter and spring (males were sampled in spring only), the season of sampling had a significant influence on prevalences of two categories of lesions: hydropic vacuolation and non-specific necrotic lesions (Table 11). Prevalences of both of these lesions were significantly higher in spring (31% for hydropic vacuolation and 13% for necrotic lesions) than in winter (9 % for hydropic vacuolation and 6 % for necrotic lesions). These differences were particularly notable at contaminated sites such as Deer Island and Mystic River in Boston Harbor. 2766
Environ. Sci. Technol., Vol. 27, No. 13, 1993
Table 11. Liver Lesion Prevalences in Female Winter Flounder Sampled in Winter and Spring.
hydropic vacuolation (%) spring winter
site name Massachusetts Bay, Plymouth Entrance Narragansett Bay, Prudence Island Boston Harbor, Deer Island Boston Harbor, Mystic River Niantic Bay, Black Point total
necrotic lesions (%) spring winter
0 (n = 25)
0
16 (n = 25)
5
(n = 22)
4
10
2
3
(n = 22)
(n = 39) (n = 51) (n = 39) 11* 10 O* (n = 100) (n = 27) (n = 100) ( n = 27) 59 20* 27 30 (n = 37) (n = 30) (n = 37) (n = 30) 20 4* 6 O* (n = 81) (n = 69) (n = 81) (n = 69) 30 9* 10 6' ( n = 294) (n = 187) (n = 294) ( n = 187) Asterisks (*) indicate that prevalences for fish collected in winter are significantly different from prevalences for fish collected at the same site or sites in spring. (n = 51)
47
Relationships between Levels of Contaminants in Sediments and Tissues and Hepatic Lesion Prevalences. Of the lesions monitored in this study, hydropic vacuolation showed the strongest and most consistent relationships with contaminants in sediment, stomach contents, and liver (Table 11). Concentrations of LAHs, HAHs, DDTs, and chlordanes in sediments and stomach contents were significant risk factors for this lesion, as well as levels of biliary FACs-H and FACs-L, arid chlordanes and DDTs in liver tissue. However, no relationship was observed between hydropic vacuolation and PCB concentrations in sediment, stomach contents, or liver tissue. Prevalences of necrotic lesions were associated with all classes of contaminants in sediment except for PCBs (Table 111). Other risk factors for this lesion were LAHs, DDTs, and chlordanes in stomach contents and biliary FAC levels. No relationship was found, however, between necrotic lesion prevalences and any class of chlorinated hydrocarbon measured in winter flounder liver. No significant positive associations were found between prevalences of neoplasms or FCA and levels of measured contaminants in sediment, liver tissue, or bile. However, both neoplasms and FCA were positively associated with concentrations of LAHs in stomach contents. Levels of LAHs in stomach contents accounted for 7% of variation in neoplasm prevalence (p = 0.05), and 22% of FCA prevalence (p = 0.009). Chlordanes in stomach contents also emerged as a significant risk factor for neoplasms (22% of variation in prevalence explained, p = 0.031). Prevalences of other categories of degenerative or regenerative hepatic lesions showed relatively weak and inconsistent associations with concentrations of organic contaminants in sediments and fish. Levels of chlordanes in stomach contents accounted for 47 % of variation in the prevalence of spongiosis hepatis (p = 0.018), but no contaminants in liver or bile emerged as risk factors for this lesion. Concentrations of chlordanes in stomach contents and liver accounted for 16% (p = 0.023) and 15% (p = 0.006),respectively,of variation in the prevalence of non-neoplastic proliferative lesions. However, proliferative lesions showed no relationship with chlordane concentrations in sediments or with other contaminants in any compartment, Nuclear pleomorphism showed no
Table 111. Chemical Compounds or Classes of Compounds in Sediment Showing Significant Positive Relationships (Logistic Regression, p I 0.05) with Hydropic Vacuolation, Non-neoplastic Proliferative Lesions, and Necrotic Liver Lesions.
chemical class
hydropic vacuolation n
(%)
24
p = 0.001
proliferative lesions
necrotis lesions
LAHs sediment
ns
p = 0.001
ns
p = 0.002
ns
p = 0.001 36
ns
p = 0.001
ns
20 ns
ns
p = 0.001
22
16
stomach contents
24
p = 0.001
bile (FACs-L)
24
p = 0.001
9
27
16 HAHs sediment
24
p = 0.001
29 stomach contents
24
p = 0.001
bile (FACs-H)
24
p = 0.001
29
32
14 PCBs ns ns ns
ns ns ns
ns
p = 0.001
ns
p = 0.001
ns
19 ns
ns
p = 0.001
p = 0.001
p = 0.006
43
15 p = 0.023
p = 0.014 8
sediment stomach contents liver
24 24 24
ns ns ns
sediment
24
p = 0.001
DDTs
19
18 stomach contents
24
p = 0.001
liver
24
sediment
24
p = 0.001 7 Chlordanes p = 0.001
18
24
30 stomach contents
24
ns 16 9 0 Chemical risk factors for neoplasms and pre-neoplastic lesions are presented in the text. Analyses were performed separately for each major class of Contaminants, while adjusting for the possible influence of gender and age. Percentages indicate percent of total variation in lesion prevalence explained. A total of 19 sites were sampled, and some sites were sampled more than once. Each sampling was considered a separate data point for statistical analyses, resulting in n = 24. FACs-H = aromatic compounds in bile fluorescing at BaP wavelengths; FACs-L = aromatic compounds in bile fluorescing at BaP wavelengths;PCBs = total polychlorinated biphenyls; chlordanes = a-chlordane and tram-nonachlor;DDTs = DDT and its derivatives; ns = not significant. liver
24
p = 0.001
relationship to concentrationsof any class of contaminants in sediment, stomach contents, bile, or liver tissue.
Discussion Chemical Contaminant Levels in Sediments and Fish. Concentrations of organic contaminants observed in this study were consistent with values reported for sediments and fish from sites and embayments in the Northeast that have been sampled in previous investigations (26, 27). Concentrations of AHs, PCBs, and chlordanes were particularly high, with levels at some of the Boston Harbor and Raritan Bay sites among the highest reported in the nation (1,3,4).Generally, different types of organic contaminants tended to co-occur in sediments from urban embayments, a trend which has been observed in other studies (1-4); the Clark’s Point site in New Bedford Harbor, where sediment concentrations of PCBs were extremely high but levels of other
contaminants were moderate or low, was a notable exception. In most cases there were strong positive correlations between levels of contaminants in sediment and levels in stomach contents, liver, or bile, providing confirmation that organic contaminants in sediment are bioavailable to marine fish and their prey organisms and suggesting that diet is an important route of contaminant uptake in winter flounder. The CHs, in particular, showed evidence of bioaccumulation and bioconcentration (41, 57). There were a few cases in which concentrations of contaminants in sediments were at variance with contaminant levels in stomach contents or fish tissues. At the Buzzards Bay site, for example, levels of PCBs in sediments were relatively low, yet concentrations of these compounds in winter flounder stomach contents and liver were among the highest observed in this study. The reason for this lack of consistency between levels of contaminants in sediments and fish is not clear, but could be the result of patchy distribution of contaminants in sediment. Migration of fish from the nearby New Bedford Harbor site, where levels of PCBs are high, could also be a contributing factor. Patterns of Lesion Occurrence. In general, the types and prevalences of lesions observed were consistent with earlier findings at previously sampled sites (23-25,58).At heavily contaminated sites within Boston Harbor, for example, Murchelano and Wolke (23,24)and Moore (25) reported pre-neoplastic and neoplastic lesions in 10-15 % and hydropic vacuolation in 56-74% of winter flounder sampled. As in the present study, at minimally contaminated sites in Massachusetts Bay, neoplasms were not observed, and vacuolated cell lesions were found in less than 10% of flounder examined (24,25). Of the lesions monitored in this study, hydropic vacuolation and nonspecific necrotic lesions appeared to be particularly useful as biomarkers of contaminant effects. These lesions were extremely common in a wide range of age classes of winter flounder collected from heavily contaminated sites in Boston Harbor, Raritan Bay, and Long Island Sound and were strongly associated with contaminant concentrations in fish and sediments. AIthough prevalencesof other hepatic lesions (i.e., neoplasms, FCA, non-neoplastic proliferative lesions, and spongiosis hepatis) were substantially lower (e.g., 2-1096) than prevalences of hydropic vacuolation and hepatocellular necrosis, their pattern of distribution was reflective of an association with contaminant exposure. Hepatocellular nuclear pleomorphism, on the other hand, was infrequently observed in winter flounder and showed no relationship with contaminant concentrations in sediments or fish. As this lesion is a characteristic toxicopathic response to AH exposure in English sole, as well as in several other bottomfish species (5, 1 1 , 1 3 , 1 4 , 1 9 ) ,its rarity in winter flounder exposed to organic contaminants is a striking finding. Biological Risk Factors Associated with Hepatic Lesions in Winter Flounder. The most important biological risk factor associated with the occurrence of hepatic lesions in winter flounder was age. An increase in the risk of neoplasia and other hepatic lesions with age has been documented in winter flounder (24,25),as well as in several other fish species (13,14, 18, 19, 59-61) in previous studies. It was also observed that winter flounder females appeared to exhibit a decreased probability of hepatic disease when sampled during the spawning season. Environ. Sci. Technol., Vol. 27, No. 13, 1993 2767
The most likely explanation for this phenomenon is that spawning populations are partially composed of fish migrating from other, less contaminated areas that are not normally resident at the sampling sites at other times of the year (62-66) and may not experience the same level of chronic exposure. It is also possible that certain lesions may have regressed in association with changes in hormone levels, water temperature, or immunological response during the period of prespawning, but this seems unlikely as regression of suspected toxicopathic lesions (i.e,, neoplasms, FCA, biliary proliferation, and hydropic vacuolation) was not observed in winter flounder held for up to 1 year in the laboratory (25). The data from the present study also suggestthat female winter flounder are slightly less likely to develop certain lesions, including neoplasms and spongiosis hepatis, than male winter flounder. This finding was somewhat surprising, as other studies have found little or no effect of gender on lesion prevalence in winter flounder (24,251or other Pleuronectid fish such as English sole (13, 14, 18, 19). Because prevalences of both of these lesions were so low, the apparent decreased risk of lesion occurrence in females may represent a sampling anomaly rather than a true biological difference. Relationships between Chemical Contaminants and Lesion Prevalences. Of the lesions monitored in this study, hydropic vacuolation showed the strongest and most consistent relationships with levels of organic contaminants measured in sediments and fish. Concentrations of DDTs, chlordanes, and AHs (or metabolites) measured in all compartments emerged as significant risk factors for the development of hydropic vacuolation in winter flounder, strongly suggesting that this lesion is chemically induced and helping to establish its validity as a biomarker of the effects of contaminant exposure. However, because concentrations of AHs, DDTs, and chlordanes in sediments were highly intercorrelated and winter flounder were exposed to all three contaminants simultaneously, their relative importance as etiologic agents for hydropic vacuolation is difficult to assess. The problem is further compounded by the fact that in a recent laboratory study (25),this lesion was not induced in winter flounder by exposure to either chlordane or the AH, benzo[alpyrene (BaP). It may be that exposure to the complex mixture of chemicals present in urban sediments is necessary for the generation of hydropic vacuolation. Alternatively, hydropic vacuolation may be caused by an unknown and unmeasured etiologic agent that covaries with the measured contaminants. Whatever the case, it would be premature to dismiss AHs, chlordanes, or DDTs as potential risk factors for this lesion without further investigation, especially as hydropic vacuolation has also been associated with exposure to AH8 and CHs in starry flounder (Platichthys stellatus) and white croaker from contaminated sites sampled on the West Coast in other studies (14). Laboratory exposure studies with multiple species using intact sediments, organic extracts of urban sediments, and model compounds may be needed to determine how various classes of AHs and CHs, alone or in concert, contribute to the development of this lesion. Prevalences of nonspecific necrotic lesions showed positive and consistent associations with AH concentrations in sediment and stomach contents and with levels of FACs in bile. Moreover, laboratory data confirm the role of AHs as necrogenic agents in winter flounder (25) 2788
Environ. Sci. Technol., Voi. 27, No. 13, 1993
and other fish species (67-69). Necrotic lesions were also associated with elevated concentrations of DDTs and chlordanes in sediments and stomach contents, but showed no relationship with concentrations of these contaminants in liver tissue. As concentrations of chlordanes and DDTs in liver are a more reliable measure of chronic exposure than concentrations of these compounds in either sediment or stomach contents, the lack of correlation between concentrations of DDTs and chlordanes in liver and the prevalences of necrotic lesions suggest that these contaminants are not major risk factors for the development of these lesions. However, laboratory exposure to chlordane has induced necrotic lesions in winter flounder (251, and both chlordanes and DDTs have been shown to have necrogenic effects on the liver in mammals (70, 71), so these compounds should not be ruled out as necrogenic agents in winter flounder liver without further investigation. In the present study, prevalences of neoplastic and preneoplastic lesions in winter flounder were not strongly associated with concentrations of organic contaminants in sediments, stomach contents, or biliary FACs. As AHs are well-established genotoxic carcinogens in mammals (72)and there is substantial evidencethat they act similarly in fish (21, 471, it is not entirely clear why they did not emerge as a significant risk factor for FCA and neoplasms in winter flounder. Certainly, the relatively low prevalences of neoplasms and FCA in winter flounder examined in this study made it difficult to establish a relationship between AH exposure and these lesions. Another fact to consider is that neoplasms are induced only after longterm exposure to chemical carcinogens and/or promoting agents; however, we currently have no reliable measure of chronic AH exposure because AHs are largely metabolized and do not accumulate in fish tissue (47, 73). Levels of FACs in bile are reliable indicators of short-term exposure to AHs in flatfish (74, 75), but are likely to be elevated only when animals are in or have recently left contaminated areas (75) and may not necessarily reflect long-term exposure. The difficulty of obtaining an accurate assessment of chronic AH exposure in winter flounder may at least partially account for the relatively weak and inconsistent relationships observed in this study between neoplastic and pre-neoplastic lesions and AH exposure. While the association between chlordane levels in stomach contents and hepatic neoplasms is too weak to be considered as evidence for involvement of chlordanes in the development of neoplasia in winter flounder, an association between chlordanes and neoplasms is not inconsistent with current theories on carcinogenesis. Pesticides such as chlordanes are generally not considered to be primary carcinogens (76,77),but in both mammals (78) and fish (14, 79) they are considered to be possible promoters that facilitate tumor development by cells which have been altered by genotoxic compounds Proliferative lesions such as biliary and hepatocellular regeneration were significantly associated only with levels of chlordanes in stomach contents and liver tissue. However, in laboratory studies, these lesions have been induced in winter flounder (25)and other fish species (22, 80,81)not only by chlordanes but also by other hepatotoxic or hepatocarcinogenic compounds as well, including AHs. As with neoplasms, the relatively low prevalence of proliferative lesions, coupled with the lack of a reliable measurement of chronic AH exposure, may have obscured
toxicologic relationships between AH exposure and lesion development. Aside from a relatively weak correlation with chlordane levels in stomach contents, prevalences of spongiosis hepatis showed no relationship with levels of measured organic contaminants sediments, stomach contents, or tissues. Although this lesion has been observed in laboratory exposures of medaka (Oryzias latipes) and sheepshead minnow (Cyprinidon uariegatus) to carcinogens (82-85), and in winter flounder exposed to BaP in the laboratory (25), it did not show strong evidence of a chemical etiology in winter flounder in the present study. The lesion does occur primarily in urban sites, but because of its low prevalence ( < 5 % ) , it may be less useful as a biomarker in field studies than more frequently observed lesions such as hydropic vacuolation. PCBs and Liver Disease in Winter Flounder. In this study PCBs did not emerge as a significant risk factor for the development of any category of hepatic lesion in winter flounder, The lack of a relationship between concentrations of PCBs in tissue and sediments and prevalences of idiopathic disease in winter flounder is in contrast to earlier findings. Johnson et al. (561, for example, found that PCB concentrations in the liver and ovary of female winter flounder were positively correlated with prevalences of both hydropic vacuolation and biliary proliferation. Moreover, in starry flounder and white croaker examined as part of the West Coast NBSP, PCBs were found to be significant risk factors for hydropic vacuolation and other degenerative lesions (14),as well as contributoryrisk factors for the development of neoplasms in English sole. However, in these investigations (14,47) fish were captured from sites where PCBs were present in combination with other contaminants such as AHs, DDTs, or pesticides. In the present study, flounder were not only sampled from sites where sediments contained complex mixtures of PCBs and other contaminants but also were collected from New Bedford Harbor where concentrations of PCBs in sediments and flounder liver were extremely high, but levels of other measured organic contaminants in sediments and fish tissue were moderate to low. These fish, which were exposed to PCBs in the absence of high levels of other toxicants, such as AHs or pesticides, did not exhibit significantly elevated prevalences of any category of hepatic lesion in comparison to animals from minimally contaminated reference sites. As noted previously, chlorinated hydrocarbons such as PCBs are generally considered to be promoting agents rather than primary genotoxic carcinogens in fish and mammals (72,86, as?, so it is not surprising that winter flounder from the New Bedford site which were exposed to high levels of PCBs but not AHs did not exhibit high prevalences of neoplastic or pre-neoplastic lesions. On the other hand, the finding that winter flounder from the New Bedford site did not develop degenerative or regenerative lesions, such as biliary hyperplasia and hydropic vacuolation, was unexpected, as previous studies had suggested that PCBs could be possible etiologic agents for these lesions in winter flounder (56). Moreover, laboratory exposure to PCBs has led to the development of biliary proliferative lesions in mammals (88-90). It is important to note, however, than in the present study, total PCB concentrations were used in the statistical analyses rather than in the concentrations of specific PCB congeners, which in recent studies have been shown to be responsible
for much of the toxicity of PCB mixtures in mammals and possibly fish (90-92). Although total PCB concentrations were very high at New Bedford Harbor, it is possible that levels of toxic coplanar congeners at this site were not high enough to cause significant pathological damage. Laboratory studies in winter flounder and other fish species are clearly needed to clarify the role that PCBs, especially the toxic congeners, may play in the development of nonneoplastic degenerative or proliferative lesions such as hydropic vacuolation. In summary, AHs and chlorinated pesticides such as DDTs and chlordanes emerged as the most important risk factors for hepatic disease in winter flounder. However, because these classes of contaminants generally occurred together in sediments and animals were exposed to them simultaneously, it was difficult to evaluate the specific roles or to quantitate the relative contributions of these toxicants in the etiology of pollution-associated disease in winter flounder. Somewhat surprisingly, PCBs did not appear to be significant risk factors for any of the lesions monitored in this study, suggesting that while they may play a role in the development of certain hepatic lesions in winter flounder, they are not the primary etiologic agents. Relationships with chemical contaminants were strongest for those lesions which developed relatively early in the life cycle and were observable at high prevalences, such as hydropic vacuolation and nonspecific necrotic lesions. Proliferative, pre-neoplastic, and neoplastic lesions were not strongly associated with contaminant levels in fish or sediments, in spite of substantial evidence of a chemical etiology for these conditions on the basis of laboratory and field studies in other fish species. This may be attributable to their relatively low prevalences in winter flounder examined in this study and to the fact that they are generally induced only after long-term exposure to chemical carcinogensand/or promoting agents. Two lesions,spongiosishepatis and megalocytic hepato&/ nuclear pleomorphism, which are closely associated with exposure to chemical contaminants in other species, were relatively rare in winter flounder and did not prove to be useful bioindicators of contaminant effects. Our ability to identify relationships between environmental toxicants and disease conditions in benthic fish would be further enhanced by the utilization of bioindicators (e.g., xenobiotic-DNA adducts in liver tissue) that measure chronic exposure to labile toxicants, such as AHs, and by the determination of concentrations of selected contaminants, including CHs, in individual fish. Such data are currently being gathered by investigators in our laboratory and will be used to develop predictive dose-response models linking contaminant levels in individuals with the risk of disease. Acknowledgments
We thank Maryjean Willis and Tom Lee for processing histological samples; Ken Carrasco for assistance in histopathological examination of tissue sections and diagnosis of hepatic lesions; Tom Hom, Anna Kagley, and Margaret Krahn for performing HPLC analyses of bile; Donald Brown, Karen Tilbury, Douglas Burrows, Ronald Pearce, Jennie Bolton, Deborah Holstad, Richard Boyer, Tara Felix-Slinn, Eunice Schnell, and Dana Whitney for organic chemistry analyses; Carol Airut, Bich-Thuy Le Eberhart, William Gronlund, Jennifer Hagen, Tom Hom, Paul Plesha, and Herbert Sanborn for their assistance in field sampling operations; and Joy Evered, Marc NishEnvlron. Scl. Technol., Vol. 27, No. 13, 1993 2768
imoto. and two anonvmous reviewers for their helpful comments on the maniscript. These studies were partially supported by the NOAA’s Coastal Ocean Program and by the Office of Ocean Resources Conservation and Assessment (NOAA’s National Ocean Services) as part of the National Status and Trends Program.
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