U.S. Dept Commerce/NOAA/NMFS/NWFSC/Publications NOAA-NWFSC Tech Memo-4: National Benthic Surveillance Project: Northeast Coast MATERIALS AND METHODS
Field Sampling
Samples were collected from 22 Northeast Coast sites. Site names and abbreviations, the locations of these sites, the types of samples collected, and the frequency of sampling are presented in Table 1. Figure 1 illustrates the locations of the sampling sites. Fifteen of these sites were located in or near urban embayments, and the seven remaining sites were in nonurban embayments, one of which (Long Island Sound, Rocky Point) served as a comparison site. Comparison sites were selected in order to collect from minimally contaminated environments the same fish species obtained at urban sites. Observations of these "reference" fish were used to help interpret the significance of pathological conditions observed in the respective species from urban sites. 
Site selection was based on 1) the availability of bottom-feeding fish, 2) location in a subtidal, sedimentary-depositional zone, 3) location outside of a point source or authorized dumpsite, and 4) a location not subject to dredging, scouring or slumping. 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. In addition to standard NBSP sites, areas sampled during intensive studies of Long Island Sound in 1987 and Boston Harbor in 1988 were also included, to increase the sample size for statistical analyses and thus improve our ability to detect any relationships between contaminant levels and fish lesions. The target species was winter flounder, a bottom-feeding flatfish species. Winter flounder was chosen as a target species because it is widely distributed in both urban and nonurban estuaries along the northern Atlantic coast. Additionally, suspected toxicopathic lesions had been previously reported in flounder from heavily urbanized embayments such as Boston Harbor (e.g., Murchelano and Wolke 1985), indicating that the species was sensitive to environmental toxicants.

The sites along the Northeast Coast were sampled using the NOAA ship Ferrel, and research vessels Gloria Michele and Kyma. To maximize comparability of data over space and time, standardized collection gear and sampling methods were used. All research vessels were equipped with navigational equipment to determine the precise location (latitude/longitude) of the sampling stations.

Fish were collected with otter trawls equipped with netting that was not chemically treated. Fish equal to or above 15 cm in length were randomly selected from each haul and assigned a unique identification number. These fish were kept alive in fresh seawater to prevent autolysis of tissues until necropsies were performed. The necropsy procedure involved 1) weighing (total weight in grams) and measuring (total length in millimeters) each fish; 2) removing otoliths for age determination; and 3) excision of the gall bladder, liver, kidney, gonad and stomach contents. Approximately 30 winter flounder were necropsied at each site in 1987, and the number was increased to a maximum of 60 animals in 1988 and 1989. If the target number of animals could not be captured, as many animals were sampled as possible. Sites where less than 15 animals could be obtained are not included in this report. For flounder sampled as part of NBSP or the 1987 Long Island Sound study, tissue sections of the central portion of the liver and the posterior portion of the kidney, and a sagittal section of the gonad were taken and preserved in Dietrich's fixative (Gray 1954) for routine histological processing and histopathological examination. Only liver and gonad samples were collected during the Boston Harbor reproductive study conducted in winter 1988.

The bile from the gall bladder and a portion of the remaining liver tissue were placed in separate, solvent-rinsed glass vials and frozen at -80�C for later analyses for fluorescent aromatic compounds (FACs) and organic compounds, respectively. Another portion of the liver was placed in a plastic vial and stored frozen until analyzed for selected metals. Approximately 60 liver samples for organic chemistry, 30 liver samples for metals, and a minimum of 10 bile samples were collected per site. Individual liver samples were subsequently composited in the laboratory into 3 composites of 10-20 individuals each per site, and chemical analyses were conducted on the composite samples. The total contents of stomachs from at least 10 fish per target species at each site were removed and composited in a solvent-rinsed glass jar and frozen for chemical analyses. In a similar manner, the stomach contents from another five fish at each site were composited into a jar containing 10% buffered formalin. These latter composites were returned to the laboratory for taxonomic classification of the food organisms.

Surface sediments were collected as described in the previous NBSP reports (Varanasi et al. 1988, 1989a; McCain et al. 1989).
Laboratory AnalysesFish Age Determination 
In selected animals collected over NBSP Cycles 5 and 6, and in the Long Island Sound and Boston Harbor studies, fish age was estimated by counting the number of clearly defined opaque zones of whole otoliths under a binocular dissecting microscope (Kimura et al. 1979, Chilton and Beamish 1982). Ages were determined by examination of otoliths in all animals collected at each site for at least one sampling cycle. For the remaining animals age was estimated from length based on age-length curves constructed from data on fish whose ages had been determined from otoliths. Both gender-specific and gender-combined age-length curves were generated for each major geographic region sampled (e.g., winter flounder females, Boston Harbor) using simple or polynomial regression techniques. Resultant age-length curves and equations (Appendix Table A2) were then chosen on the basis of best fit to the data (i.e., highest adjusted R-squared value). Fish age was then estimated to the nearest year from these gender-specific age-length equations. If no gender data were available, or insufficient numbers of a particular gender existed to perform meaningful age-length regression, ages were estimated by applying age-length equations for both genders combined.

Histopathological Analyses

Fish tissues preserved in the field for histopathological examination were processed for paraffin embedding by routine histological procedures (Preece 1972). After embedding, the tissues were sectioned at 4-5 mm thicknesses. Paraffin sections were stained with Mayer's hematoxylin and eosin-phloxine (Luna 1968). For further characterization of specific lesions, additional sections were stained using special staining methods (Thompson 1966, Luna 1968, Preece 1972). Lesion classification followed previously described diagnostic criteria (Jones and Butler 1975, Stewart et al. 1980, Myers et al. 1987, Robbins et al. 1984, Squire and Levitt 1975).

For reporting and statistical analyses, hepatic lesions were grouped into the following categories: neoplasms, foci of cellular alteration (FCA), nuclear pleomorphism, spongiosis hepatis of liver parenchyma, hydropic vacuolation (synonymous with RAM cell areas or atypical vacuolation (Moore et al. 1989, Bodammer and Murchelano 1990)), proliferative lesions, and nonspecific degenerative or necrotic lesions. Kidney lesions were grouped into the following categories: 1) proliferative lesions, 2) necrotic lesions, 3) and sclerotic lesions. A complete description of the types of individual lesions included in these categories is given in Table 2. Data concerning biological characteristics (e.g., length, weight, and sex) and lesions in individual fish were stored in the Reflex1 database system. Lesions were identified using a numerical coding system adapted from File Type 013 of the National Ocean Data Center (NODC) coding system (NODC 1991).

1Mention of tradenames is for information only and does not constitute endorsement by the Department of Commerce.

Chemical Analyses of Sediments, Stomach Contents, Liver Tissue, and Bile

Liver tissue, stomach contents, and sediments were analyzed for a broad spectrum of aromatic hydrocarbons (AHs) and chlorinated hydrocarbons (CHs) (McCain et al. 1988, Varanasi et al. 1988, 1989a, Krahn et al. 1988). Fluorescent aromatic compounds in fish bile were measured using the procedures of Krahn et al. (1984, 1986, 1987). Bile analyses were performed on samples from individual fish, but chemical analyses of liver tissue were perfomed on composite samples. For each site, three 3 g composites of 10-20 livers were analyzed. For statistical analyses, the AHs and CHs whose concentrations were measured in sediments, stomach contents, and fish tissues were grouped into five broad categories: LAHs, HAHs, DDTs, PCBs, and chlordanes. The specific chemicals included in each of these categories are listed in Table 3. As noted previously, trace metal analyses of sediments, liver tissue, and stomach contents were conducted by the Southeast Fisheries Science Center, and data on metal concentrations will not be presented or correlated with disease conditions in this memorandum.

Stomach Contents Taxonomy

Samples of fish stomach contents to be taxonomically characterized were transferred from 10% neutral buffered formalin to 70% ethanol prior to analysis. Food organisms were examined with a dissecting microscope and grouped into four main categories: arthropods, annelid worms, molluscs, and nematodes. For each sample, the organisms in each of these categories were weighed to nearest milligram, and the percentage of organisms (on a wet weight basis) in each taxonomic category was determined.
Statistical AnalysesHeterogeneity in Lesion Prevalence 
To determine if statistically significant differences in lesions prevalences existed between the reference site (Rocky Point, Long Island Sound) and the individual test sites, the G-statistic (Sokal and Rohlf 1981) was computed using the lesions prevalence at the reference site as the expected value. A lesion prevalence was considered to be different (either higher or lower) from the overall mean prevalence for that lesion type if the difference was significant at p < 0.05. Sites at which less than 15 fish were collected were omitted from this analysis.

Logistic Regression and Risk Factor Analysis

Stepwise logistic regression (Breslow and Day 1980, Schlesselman 1982) was used to identify statistically significant relationships between potential risk factors and lesion occurrence in winter flounder. In the logistic model (Fig. 2), the probability of disease (p) is given by:
p = 1/{1 + exp[-(ßo + ß1x1 + . . . + ßpxp)]}where x1, x2, . . . , xp represent potential risk factors, and the ßs are coeffecients that represent the effects of the x's on the risk (probability) of disease. 
Two types of logistic regression 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 lesion prevalences at the sampling sites and mean concentrations of contaminants in sediments, fish stomach contents, and fish tissues. 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 analysis. Consequently, relationships between various classes of chemicals and lesion prevalences were 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 analysis module of the EGRET statistical package (Version III, Statistics and Epidemiology Research Corporation, Seattle, WA) using a procedure similar to stepwise 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 (Fleiss 1981), was calculated from variable coefficients of the logistic regressions (Anderson et al. 1980, Schlesselman 1982). The relative odds or risk of disease in individuals with different values for the risk factors (e.g., in animals of differing ages, or from contaminated vs. reference sites) is given by:
where the ßs are the coeffecients for risk factors x1, x2, . . . , xi, and the x*is and xis represent the values of the risk factors for the two individuals which are being compared (Schlesselman 1982). 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 at 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) proliferative lesions, and 7) non-specific degenerative or necrotic lesions. Additionally, the same analyses were applied to the following categories of renal lesions: 1) proliferative lesions, 2) necrotic lesions, 3) and sclerotic lesions; and to fin erosion. Temporal trends in lesion occurrence were not evaluated because only 2 years of data were available for most sampling sites. The Rocky Point site in Long Island Sound was designated as the reference area, and calculated odds ratios or relative risks associated with site of capture were interpreted relative to this site. 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 winter of 1988-89 during the intensive reproductive success study, and in spring during the routine NBSP sampling. Because no male fish were sampled during the reproductive studies, the analysis of the impact of season of capture on lesion prevalence is limited to female fish only.

Relationships between tissue contaminant concentrations in individual fish and disease risk were not evaluated for this report because data on contaminant exposure levels in individual fish were available only for biliary FACs. In future NBSP samplings, levels of various chlorinated hydrocarbons, including DDTs, PCBs, and selected non-DDT pesticides will be determined in tissues of selected individual fish, and a more comprehensive analysis will be presented when these data are available.
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