U.S. Dept Commerce/NOAA/NMFS/NWFSC/PublicationsNOAA Tech Memo NMFS NWFSC-36: Fish Injury in the Hylebos Waterway (cont): 
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SECTION 2FLATFISH TOXICOPATHIC INJURY STUDYIntroduction
Histopathological examination is widely recognized as a useful, rapid method for assessing injury in marine fishes due to the adverse acute and chronic effects of exposure to anthropogenic contaminants. Certain pathological conditions or lesions in the liver of wild fish, including neoplasms, preneoplastic foci of cellular alteration (FCA), proliferative lesions, and specific or unique degenerative/necrotic lesions (SDN) involved in the histogenesis of hepatic neoplasia morphologically resemble lesions induced by experimental exposure of rodents (Frith and Ward 1980, Stewart et al. 1980, Maronpot et al. 1986, Farber and Cameron 1980, Farber and Sarma 1987) and fish (Meyers and Hendricks 1982, Hinton et al. 1992, Schiewe et al. 1991, Bunton 1990, Hawkins et al. 1990, Hendricks et al. 1984, Nunez et al. 1990) to a variety of anthropogenic chemical toxicants including carcinogens. These lesions also have been shown to be positively associated with exposure to xenobiotic chemical contaminants in field studies. Injuries as represented by these toxicopathic lesions have been reported in bottomfish species from chemically contaminated marine coastal areas, including English sole (Varanasi et al. 1989; McCain et al. 1988; Malins et al. 1984, 1985, 1987a; Myers et al. 1987, 1990, 1991, 1994; Rhodes et al. 1987), rock sole (McCain et al. 1983, Myers and Rhodes 1988, Myers et al. 1992), Atlantic tomcod (Microgadus tomcod) (Smith et al. 1979, Cormier 1986), white croaker (Genyonemus lineatus) (Varanasi et al. 1989; McCain et al. 1988, 1992; Myers et al. 1994; Malins et al. 1987b; Stehr et al. 1997), mummichog (Fundulus heteroclitus) (Vogelbein et al. 1990), winter flounder (Pleuronectes americanus) (Muchelano and Wolke 1991; Moore 1991; Moore et al. 1989; Gronlund et al. 1991; Johnson et al. 1992, 1993a), starry flounder (Platichthys stellatus) (Myers et al. 1992, 1994; Myers and Rhodes 1988; Stehr et al. 1997) and other species (Harshbarger and Clark 1990). Cause-and-effect relationships suggested from field surveys have been supported by statistical analyses and epidemiological models (Malins et al. 1984; Myers et al. 1990, 1991, 1992, 1994; Rhodes et al. 1987; Landahl et al. 1990; Stehr et al. 1997) and by long-term laboratory studies. In these experimental studies, liver lesions identical to those observed in field-captured English sole (i.e., preneoplastic focal lesions, non-neoplastic proliferative lesions, hepatocellular nuclear pleomorphism, and megalocytic hepatosis (the major lesions in the SDN category)) were induced by exposure to an organic-solvent extract of a sediment collected at a Puget Sound site heavily contaminated with PAHs (Schiewe et al. 1991). English sole from this site (Eagle Harbor) are affected by high prevalences of toxicopathic hepatic lesions (Malins et al. 1985, Myers et al. 1987). Moreover, certain hepatic lesions in English sole have been associated with changes in serum chemistry parameters indicative of liver dysfunction (Casillas et al. 1985). 

In the multiple studies done on English sole from Puget Sound and other sites on the Pacific Coast (Varanasi et al. 1989; McCain et al. 1983, 1988; Malins et al. 1984, 1985, 1987a; Myers et al. 1987, 1990, 1991, 1992, 1994; Rhodes et al. 1987; Myers and Rhodes 1988), the chemicals consistently found to strongly influence the probability of toxicopathic hepatic lesion occurrence are PAHs, PCBs, and DDT and its derivatives. The single consistently significant biological risk factor for hepatic lesion occurrence in these studies is fish age, with risk of being affected by neoplasms and preneoplastic FCA rising incrementally with each year of age (Myers et al. 1990, 1991, 1994; Rhodes et al. 1987). Moreover, certain early biochemical responses to contaminant exposure (biomarkers) have been statistically associated with increased risk of toxicopathic liver lesion occurrence. These biomarkers include levels of metabolic conversion products of PAHs detectable as fluorescent aromatic compounds in bile (Myers et al. 1990, 1991, 1992, 1994, 1998; Krahn et al. 1986); hepatic activities of the xenobiotic metabolizing enzyme cytochrome P4501A inducible by PAHs and CHs (Myers et al. 1998, Collier and Varanasi 1991), and hepatic levels of hydrophobic DNA adducts representing reactive intermediates of PAHs covalently bound to DNA bases (Myers et al. 1998, Reichert and French 1994, Stein et al. 1993). This DNA damage by carcinogens and subsequent mutations is a necessary early step in the process of initiation of neoplasia (Stein et al. 1993). Therefore, certain hepatic lesions are currently regarded as having utility as biomarkers of contaminant exposure and effects (Hinton et al. 1992; Myers et al. 1990, 1991, 1992, 1994, 1998; Varanasi et al. 1992) and have become useful as indicators of environmental degradation in marine ecosystems.

English sole and rock sole are both relatively territorial benthic species that occupy their feeding grounds for the majority of the year, exhibit some degree of homing ability, and display only limited seasonal migrations related to reproductive activities (Day 1976, Forrester 1969, Holland 1969). Therefore, they are useful target species in studies assessing potential effects of chemical contaminants. Among the many field studies done on these species, that have focused on histopathological examination and documentation of chemical exposure, several studies done in the late 1970's and early 1980's documented relatively high prevalences of toxicopathic hepatic lesions at multiple sites within Commencement Bay, including sites in the Hylebos Waterway (Malins et al. 1984, McCain et al. 1983, Becker et al. 1987). The data from these earlier studies, in part, formed the basis for the designation of Commencement Bay and several of its waterways, including the Hylebos Waterway, as priority Superfund sites in 1981. The existence of this historical data affords a unique opportunity to test the hypothesis that shore-based cleanup activities, and natural remediation processes in the Hylebos Waterway have significantly altered the prevalences of liver lesions known to be strongly associated with chronic exposure to chemical contaminants. 

These previous studies were used as the basis for the work described in this section. The major objectives of this study were to 1) assess the extent of exposure to various chemical contaminants, and the magnitude of various early biochemical responses to this exposure (biliary FACs, hepatic CYP1A and hydrophobic DNA adducts) primarily in English sole and secondarily in rock sole captured from sites in the Hylebos Waterway and a relatively uncontaminated reference site at Colvos Passage, 2) determine the prevalence of toxicopathic liver lesions in English sole and rock sole captured from these same sites, 3) determine the relationships, if any, among various indices of chemical contaminant exposure, early responses to this exposure, and hepatic lesion occurrence in these species by various statistical methods, and 4) compare lesion prevalences, and the magnitudes of early biochemical responses to contaminant exposure detected in the present study to available historical data from the Hylebos Waterway.
METHODSSample collection and analysis
Adult English sole and rock sole were collected by otter trawl from sites in the Hylebos Waterway, and a reference site in Colvos Passage (Figure 2.1), following methods described in the SAP (Appendix A). Sampling for the Flatfish Toxicopathic Injury Study was conducted in the Hylebos Waterway during July of 1994, and in Colvos Passage in September and October of 1994. Approximately 60 English sole at each site were collected from the Upper Turning Basin, Lower Turning Basin, and 11th St. Bridge sites in the Hylebos Waterway, and from the Colvos Passage site. Approximately 60 rock sole were collected from the 11th St. Bridge site in the Hylebos Waterway, and also from the Colvos Passage reference site. Otoliths were collected for age determination, and liver, kidney, and ovary were collected for histopathological examination; (data on ovarian lesions and other indicators of reproductive dysfunction are discussed in Section 3, the Flatfish Reproductive Injury Study). Liver, bile, and stomach contents were collected for measurement of chemical contaminant levels, and separate portions of liver from individual fish were also collected for measurement of CYP1A activity and DNA damage.

Methods for chemical and biological analyses (i.e. fish age determination, chemical analyses of liver tissue and stomach contents, measurement of biomarkers in bile and liver, and histopathological examination of fish tissues) are described in detail in Appendix A. Biliary FAC concentrations reported here and used in various correlative statistical analyses were adjusted for protein content of the bile sample. This correction adjusts for variations in bile metabolite levels related to the feeding status of the sampled fish (Collier and Varanasi 1991). 
Statistical methods
Data from these studies were incorporated into an integrated, relational data management system. Statistical analyses (Zar 1984) were performed to: 1) evaluate relationships among indices of contaminant exposure, early biochemical responses to exposure, and liver lesion occurrence, and 2) compare data from English sole and rock sole captured among three unique sites in the Hylebos Waterway, and the Hylebos Waterway as a whole, with data from the same species collected from the Colvos Passage reference site. ANOVA was used to identify statistically significant intersite differences in chemical concentrations in liver and stomach contents, biliary FACs, and hepatic CYP1A and DNA adducts. These data were first plotted and compared to a normal distribution. Because these data were not normally distributed, it was first necessary to normalize them through log-transformation to ensure that data conformed to the assumptions of parametric statistical tests, such as ANOVA. 

Stepwise logistic regression (Breslow and Day 1980) was used to identify statistically significant relationships between potential risk factors and lesion occurrence. This method is appropriate for analysis of data that is binomially distributed or expressed in terms of proportions or percentages (e.g., lesion prevalences), and allows for simultaneous adjustment for several risk factors included in the regression by iterative model fitting. Results are expressed in terms of estimated relative risk of disease associated with a particular risk factor, and the level of statistical significance of this association. This type of analysis of lesion occurrence has been used in retrospective epidemiological studies (Breslow and Day 1980), and more recently as specifically applied to the epizootiology of hepatic and renal lesions in English sole (Myers et al. 1990, 1991, 1994; Rhodes et al. 1987), rock sole, starry flounder (Myers et al. 1992, 1994; Stehr et al. 1997), and winter flounder (Johnson et al. 1993a). This method represents a statistical, epidemiological approach to the examination of the influence of multiple risk factors on the probability of disease occurrence, as well as the investigation of dose-response relationships. 

The logistic regression analyses conducted were of two types: 1) analyses to examine the relative risk of disease, as estimated by the calculated odds ratio in individual fish, in relation to the variables of site of capture, age, biliary FACs levels, and hepatic DNA damage as adduct concentrations, and 2) analyses to assess the significance of the relationships between prevalences of lesions at the sampling sites and discrete risk factors, including levels of contaminants in liver and stomach contents, mean biliary FACs concentrations, mean CYP1A activities, and mean hepatic DNA adducts, while adjusting for mean fish age. In the latter analysis each sampling point for a species at a site was treated as an independent occurrence. These multivariate analyses permitted evaluation of contaminant exposure-lesion occurrence relationships while simultaneously adjusting for fish age, a variable well known for its influence on risk of hepatic lesion occurrence (Myers et al. 1990, 1991, 1994; Rhodes et al. 1987; Moore and Myers 1994). 

The following contaminants, contaminant classes, and biomarkers of early response to contaminant exposure were evaluated as risk factors for hepatic lesions: HACs and LACs in stomach contents (see Appendix B for list of analytes); PCBs (see Appendix B), congener PCB 105, congener PCB 118, DDTs (see Appendix B), HCB, HCBD, chlordanes ( + ), aldrin, dieldrin, heptachlor, and lindane in stomach contents and liver tissue; biliary FACsBaP, FACsNPH, FACsPHN, and hepatic CYP1A and DNA adducts. These contaminants were measured and selected for analyses relating contaminant exposure to disease risk because they are found at high levels in sediments or fish from sites in embayments and estuaries located near major metropolitan centers (Varanasi et al. 1989, Myers et al. 1994), and represent broad classes of chemicals with documented toxic and/or carcinogenic potential in vertebrates (Klaassen et al. 1986) including fish (Meyers and Hendricks 1982). The biomarkers of early response to contaminants were chosen because of their well known sensitivity to PAH exposure (biliary FACs, hepatic CYP1A, hepatic DNA adducts) and CH and pesticide exposure (CYP1A) in fish (Collier and Varanasi 1991; Reichert and French 1994; Stein et al. 1992, 1993, 1994; Payne et al. 1987).

Logistic models were fitted using the PECAN analysis module of the EGRET statistical package, version III (Statistics and Epidemiology Research Corporation, Seattle WA). For all analyses, risk factors were considered significant at p < 0.05. Independent analyses were performed for all major categories of liver lesions. In the first analysis, the odds ratio, or estimated relative risk for the occurrence of a lesion type, represents the degree of association between a risk factor and lesion occurrence (Fleiss 1981), as determined from variable coefficients of the logistic regression equations (Anderson et al. 1980, Schlessleman 1982). Odds ratios for particular lesion categories at a site were calculated and interpreted relative to the lesion occurrence at the Colvos Passage reference site. Increased probabilities of lesion occurrence were indicated by odds ratios greater than 1.000. Calculated odds ratios for age (in years) were interpreted for each additional year of age. For the second set of analyses, separate analyses were performed for each contaminant, contaminant class, biomarker of early response or risk factor discussed above, with results expressed as the significance of the association (p value). Because many of the contaminant exposure-related risk factors assessed in this analysis are known to be highly intercorrelated (Myers et al. 1994), it was not mathematically possible to include all risk factors into a single multivariate analysis. Consequently, relationships between chemicals and chemical response risk factors and lesion prevalences were evaluated separately, while adjusting for potential effects of mean fish age at the sampling sites. 

Pertinent data on liver lesion occurrence, DNA adducts, biliary FACs, stomach contents chemistry, and concentrations of selected CHs in liver from the reproductive injury portion of this study were incorporated into various statistical analyses within this toxicopathic injury report; refer to Section 3 for information on sampling dates, number of English sole collected, and other relevant information. 
Inclusion of additional samples
Rock sole collected within the reproductive injury portion (Section 3) of this study (n = 59) were collected from the Hylebos Waterway in the first week of December 1994, and January, February, and April 1995; rock sole from the Colvos Passage site (n = 46) were collected in the first week of January, February, and March 1995. Rock sole was not the primary target species for the Flatfish Reproductive Injury Study (Section 3). Therefore, a full data set on contaminant exposure and biomarkers is not available for this species, and rock sole are not dealt with in Section 3. However, histopathological examination of liver and age determinations in these individuals were performed, thus these data were used for this section (Section 2) of the fish injury report. Moreover, male English sole captured and necropsied during the reproductive injury sample collections were not dealt with in Section 3, but were included in the larger data set upon which analyses in the present report were based. The inclusion of male English sole collected during the Flatfish Reproductive Injury Study into the overall data analyzed for the present study accounts for the apparent differences in sample sizes reported here and in Section 3. 
Results Chemical Contaminant Exposure
Concentrations of contaminants in stomach contents, liver tissue, and FACs in bile of English sole and rock sole collected from the sampling sites in the Hylebos Waterway and Colvos Passage in July-October 1994 are shown in Figures 2.2 - 2.11.
Stomach contents: aromatic compounds and chlorinated hydrocarbons
English sole and rock sole from sites in the Hylebos Waterway had significantly (p<0.05) higher concentrations (up to two orders of magnitude) of both HACs and LACs in stomach contents than fish from Colvos Passage (Figure 2.2), where mean concentrations ranged from 3-5 ppb (wet wt) for LACs and 9-19 ppb (wet wt) for HACs. In English sole, there was a trend showing progressively increasing concentrations of HACs and LACs from the mouth to the head of the Hylebos Waterway, with HAC concentrations at the Upper Turning Basin site significantly higher than both the Lower Turning Basin and 11th St. Bridge sites; in turn, concentrations at the Lower Turning Basin site were significantly higher than those at the 11th St. Bridge. Although progressively higher levels of LACs in English sole stomach contents were found as one moved up the waterway, these intersite differences were not significant at the p<0.05 level (Figure 2.2). In rock sole (captured only at the 11th St. Bridge site within the Hylebos Waterway), concentrations of HACs and LACs were comparable to the respective values for English sole from that site. It is important to note that the statistically significant differences reported here for LACs and HACs and in the subsequent sections on stomach contents chemistry occurred despite the relatively low sample sizes for each species at each site (n = 2 or 3).

Summed PCBs in stomach contents of both species were also significantly (up to 20 times) higher in fish from Hylebos sites compared to Colvos Passage (Figure 2.3). In English sole, a pattern of progressively increasing PCB levels was evident in sites further up the waterway, with the two upper sites showing significant differences as compared to the 11th St. Bridge site. This same pattern of increasing concentrations at the stations further up the waterway was evident for the toxic coplanar congeners (PCB 105 and 118) that were major components of the fraction of toxic coplanar PCB congeners detected. In rock sole from the 11th St. Bridge site, concentrations of PCBs, PCB 105, and PCB 118 were comparable to the respective values for English sole collected from that site.

Concentrations of HCB and HCBD in stomach contents of fish from Hylebos sites differed significantly from those in both species from the reference site (where near-zero levels were detected), and showed a prominent peak in concentration at the 11th St. Bridge site (Figure 2.4). Both compounds serve as markers for a complex mixture of chlorinated compounds found in the Hylebos Waterway. Concentrations of HCB and HCBD at the 11th St. Bridge site were up to 20 times higher (p <0.001) than levels found in English sole from the other Hylebos sites. In rock sole from the 11th St. Bridge site, concentrations of HCB and HCBD were similar to the respective values in English sole from that site.

Concentrations of DDT and its breakdown products (listed in Appendix B) were also significantly (up to 20 times) higher in English and rock sole from Hylebos sites in comparison to Colvos Passage (Figure 2.5), but were detected at far lower levels than PCBs. In English sole, highest levels were found at the Upper and Lower Turning Basin sites (< 17 ppb), which differed significantly from levels at the 11th St. Bridge site. Concentrations of DDTs were similar in rock and English sole from the 11th St. Bridge site.

While concentrations of chlordanes, dieldrin, heptachlor, lindane, and aldrin in stomach contents of fish from Hylebos sites in some cases differed significantly from levels found in reference fish (Figures 2.5 and 2.6), concentrations of these pesticides and herbicides were relatively low (< 6 ppb, and usually 0.1-1 ppb) and probably not toxicologically meaningful. 
Bile: metabolites of aromatic hydrocarbons
In general, concentrations of biliary FACsBaP wavelengths reflecting metabolites of HACs, and FACsPHN and FACsNPH wavelengths reflecting metabolites of LACs, paralleled concentrations of the aromatic compounds found in stomach contents (Figure 2.7). For all three FACs measures in all cases in both species, levels were significantly higher at the Hylebos sites as compared to Colvos Passage. All three bile FACs measures were intercorrelated (0.77 < r < 0.97). Concentrations of FACsBaP and FACsPHN were highest in bile of English sole from the Upper Turning Basin, and they differed significantly from values at the 11th St. Bridge site, while concentrations of FACsNPH were highest at the Lower Turning Basin site. However, no significant intersite differences existed among the Hylebos sites for this latter FACs measure. As was seen for aromatic compounds (ACs) in stomach contents, levels of FACs in bile of rock sole from the 11th St. Bridge site were similar to those detected in English sole from that site. 
Liver: chlorinated hydrocarbons
Liver concentrations of PCBs, PCB 105, and PCB 118 in both species (Figure 2.8) also paralleled the results seen for this chemical class and these predominant toxic congeners in stomach contents, with significantly higher (up to 25 times) levels in fish from Hylebos sites as compared to reference fish. Progressively increasing levels were detected in English sole captured at sites further up the waterway. Liver PCBs concentrations in English sole and rock sole were ~10-15 times higher than stomach contents concentrations. Concentrations of PCBs were similar in rock and English sole from the 11th St. Bridge site.

Hepatic HCB and HCBD concentrations in liver tissue of both species from Hylebos sites were significantly and approximately 15 to 280 times higher than those in reference fish, with highest concentrations again found in both species from the 11th St. Bridge site (Figure 2.9). Liver tissue/stomach content ratios for HCB and HCBD were approximately 2.5 and 2 respectively for English sole, and approximately 10 and 13 for rock sole, reflecting the higher bioaccumulation of HCBD and HCB in liver of rock sole from the 11th St. Bridge sites as compared to English sole. As noted above, concentrations of these compounds in stomach contents were similar in both species (Figure 2.4). 

Hepatic concentrations of DDTs were also significantly (up to 17 times) higher in fish of both species from Hylebos sites as compared to Colvos fish, with significantly higher levels found in English sole from the two upper Hylebos sites as compared to sole from the 11th St. Bridge site (Figure 2.10). The liver tissue/stomach contents ratios for DDTs in English and rock soles were 10.3 and 13.8, respectively. Hepatic concentrations of DDTs were similar in rock and English soles from the 11th St. Bridge site. 

As in stomach contents, hepatic concentrations of chlordanes, dieldrin, heptachlor, lindane, and aldrin in fish from Hylebos sites in some cases differed significantly from levels found in reference fish (Figures 2.10 and 2.11). However, levels of these pesticides and herbicides were relatively low (< 9 ppb wet wt, and usually between 0.5-4 ppb) and probably not meaningful as potential etiologic agents for the liver lesions detected. 
Biological Indicators of Contaminant ExposureCytochrome P4501A in liver
Activities of hepatic CYP1A were significantly higher in English and rock sole from Hylebos sites than those in the respective species collected from Colvos Passage (Figure 2.12). In English sole, no significant differences among the Hylebos sites were detected. Mean levels of induction at Hylebos sites compared to Colvos Passage were 4.5 times higher in English sole, and 7.3 times higher in rock sole. Mean CYP1A activities in both species from the 11th St. Bridge site were roughly equivalent. 
DNA adducts in liver
Levels of DNA adducts in liver were also markedly and significantly higher (up to 40 times higher in English sole and 10 times higher in rock sole) in fish from Hylebos sites as compared to those in Colvos Passage fish (Figure 2.12). In English sole, highest levels of this DNA damage occurred at the Upper Turning Basin, followed by the Lower Turning Basin, both of which were significantly higher than concentrations in sole from the 11th St. Bridge. Adduct concentrations in rock sole from the 11th St. Bridge site, were comparable to those found in English sole collected at the same location.
Toxicopathic Lesions
Kidney lesions 

Toxicopathic kidney lesions were detected at very low and statistically indistinguishable prevalences (0-3.4%) in both English and rock sole from Hylebos Waterway sites and Colvos Passage. Therefore, kidney lesions are not discussed further. 

Liver lesions 

Rock sole - 

Contaminant exposure-associated liver lesions detected in rock sole included megalocytic hepatosis and hepatocellular nuclear pleomorphism (SDN); non-neoplastic proliferative lesions such as hepatocellular regeneration, cholangiofibrosis, increased mitotic activity, and oval cell proliferation (proliferative lesions); potentially preneoplastic foci of cellular alteration; and hepatic neoplasms of hepatocellular and biliary epithelial cell origin. These lesions were grouped into an inclusive category called "any toxicopathic hepatic lesion". We also detected hydropic vacuolation of biliary epithelial cells (HydVac) in this species. This lesion has been shown to be associated with contaminant exposure in other species (Myers et al. 1994, Johnson et al. 1993a, Moore and Myers 1994, Stehr et al. 1997). However, previous prevalence data on this lesion in rock sole from multiple sites in Puget Sound have not suggested a clear association with contaminant exposure (Stehr et al. In press, Myers unpubl.2); in this report, it was therefore excluded from the broad category of "any toxicopathic liver lesion". 

Prevalences of these lesion categories in rock sole captured strictly within the toxicopathic injury in flatfish portion of this damage assessment investigation are illustrated in Figure 2.13, with prevalences from the reproductive injury portion shown in Figure 2.14, and prevalences for the combined data shown in Figure 2.15. As in previous studies with this species (Malins et al. 1984, Rhodes et al. 1987, Myers and Rhodes 1988, Johnson unpubl.3, Myers unpubl.2 ) low prevalences of FCA, SDN, and proliferative lesions were detected in rock sole from reference sites, in this case Colvos Passage. No neoplasms were detected in any rock sole within this portion of the current study. The only lesion types showing notably higher prevalences in the Hylebos (11th St. Bridge) as compared to Colvos Passage were SDN and "any toxicopathic liver lesion" (data not adjusted for age). Prevalences of hydropic vacuolation were nearly identical at the Hylebos and Colvos Passage sampling sites.

Rock sole collected during the reproductive injury samplings generally showed higher prevalences of liver lesions (Figure 2.14) than in the toxicopathic injury samplings, because reproductively mature fish of larger size and older age were targeted in the Flatfish Reproductive Injury Study. Higher prevalences of most lesion categories were detected in fish from the Hylebos Waterway. However, neoplasms were detected only at Colvos Passage, where 2 out of 46 rock sole had this lesion. Lesions showing the greatest differential between the reference site and the Hylebos were SDN and "any toxicopathic liver lesion", with prevalences in Hylebos rock sole being ~20% and ~34%, respectively, as compared to ~2% and 13% in rock sole from Colvos Passage.

A more complete and accurate picture of liver lesion prevalence in rock sole from the Hylebos Waterway is provided by combining the lesion occurrence data from the Flatfish Toxicopathic Injury Study and the Flatfish Reproductive Injury Study (Figure 2.15). These combined data were subjected to logistic regression analyses to test the influence of fish age and Hylebos residence on the probability of lesion occurrence, using lesion occurrence probabilities at Colvos Passage as the baseline (Table 2.1), where lesion probability is defined as 1.000. In this analysis, as has been shown for other species in previous studies (Myers et al. 1994, Moore and Myers 1994, Stehr et al. 1997), the occurrence of neoplasms, FCA, and hydropic vacuolation were strongly influenced by fish age, with increased risks of lesion occurrence shown for each additional year of age, ranging from ~1.3 (hydropic vacuolation) to 2.4 (neoplasms). While simultaneously accounting for the influence of fish age on lesion occurrence, residence in the Hylebos Waterway was a highly significant risk factor for increased probability of the lesions SDN and "any toxicopathic hepatic lesion", with an increased estimated relative risk (RRe) of 8.2 for SDN and 2.8 for "any toxicopathic hepatic lesion" (Table 2.1, Figure 2.15). In other words, rock sole from the Hylebos Waterway are 8.2 times more likely to be affected by SDN and 2.8 times more likely to have "any toxicopathic hepatic lesion", than rock sole of comparable age from the Colvos Passage reference site.

English sole - 

Contaminant-associated liver lesions detected in English sole were similar to those in rock sole, with the exception that hydropic vacuolation was not present in English sole, consistent with previous studies. Therefore, except for hydropic vacuolation, the same lesion categories were used. Prevalences for these categories in English sole from the toxicopathic injury portion of the study are shown in Figure 2.13. As in previous studies, low prevalences of neoplasms and FCA were detected in English sole from reference sites, with far higher prevalences detected in sole from the Hylebos sites, especially at the Upper Turning Basin site. Using data unadjusted for potential age differences, prevalences of several lesion categories at Hylebos sites were higher than those at Colvos Passage. For example, prevalences of SDN were higher at all three Hylebos sites, showing an increasing trend at sites further up the waterway; FCA prevalence was higher only at the 11th St. Bridge and Upper Turning Basin; proliferative lesions were higher at all three Hylebos sites, especially at the Upper Turning Basin; and prevalence of "any toxicopathic liver lesion" was also higher at all three Hylebos sites, with the highest prevalence again at the Upper Turning Basin site. 

Lesion prevalences for female English sole collected within the reproductive injury samplings are reported in detail in Section 3. In brief, lesion prevalences unadjusted for fish age differences were always significantly higher in female English sole from the Hylebos Waterway, with prevalence of "any toxicopathic liver lesion" in Hylebos sole being ~31%, as compared to ~8% in fish from Colvos Passage. 

A more accurate representation of intersite differences in lesion prevalences can be derived by simultaneously accounting for possible differences in fish age among the sites, since the risks for several of the lesion types are highly influenced by age. Therefore, the lesion occurrence data were analyzed by logistic regression to test the possible influence of fish age and residence at unique Hylebos sites on the probability of lesion occurrence, using lesion occurrence probabilities at Colvos Passage (combined data from the Flatfish Toxicopathic and Reproductive Injury Studies) as the baseline, where lesion probability is defined as 1.000 (Table 2.2, Figure 2.13). In this analysis, as has been shown for English sole in previous studies (Myers et al. 1990, 1991, 1994; Rhodes et al. 1987; Moore and Myers 1994), the occurrence of neoplasms, FCA, proliferative lesions, and "any toxicopathic liver lesion" were strongly influenced by fish age; relative risks increased for each additional year of age, ranging from ~1.1 (proliferative lesions) to 1.4 (neoplasms, FCA). While simultaneously accounting for the influence of fish age on lesion occurrence, residence at sites in the Hylebos Waterway was a highly significant risk factor for increased probability of several lesion types, with increased estimated relative risks for certain lesions shown at all three sites, and highest relative risks shown at the Upper Turning Basin (Table 2.2, Figure 2.13). Significantly increased relative risks of SDN ranged from 17.6 (11th St. Bridge) to 73.5 (Upper Turning Basin); relative risks for proliferative lesions ranged from 3.7 (Upper Turning Basin) to 3.8 (11th St. Bridge); and relative risks for "any toxicopathic liver lesion" ranged from 2.8 (11th St. Bridge) to 6.9 (Upper Turning Basin). 

Combining the lesion occurrence data in male and female English sole from this study and the Flatfish Reproductive Injury Study, the influence of fish age and Hylebos Waterway residence on the probability of lesion occurrence was examined by logistic regression (Table 2.3, Figure 2.16). Residence in the Hylebos Waterway was a significant risk factor for three lesion categories: SDN (RRe = 26.0), proliferative lesions (RRe = 4.1) and "any toxicopathic liver lesion" (RRe = 4.4). Although prevalences of neoplasms and FCA were higher in the Hylebos Waterway, no significantly increased relative risk was evident for these lesions when the analysis accounted for influence of fish age. 
Chemical Risk Factors for Liver Lesion OccurrenceRock sole
Synoptic data from rock sole on lesion prevalence, mean liver CYP1A and DNA adducts, mean bile FACs, and mean values for chemicals measured in liver and stomach contents exist for only two sampling sites (Hylebos Waterway, 11th St. Bridge, n = 1; Colvos Passage, n = 1). Consequently, a meaningful statistical analysis of the association between lesion occurrence and contaminant exposure is not possible. However, it is worth noting that whenever statistically higher estimated relative risks for lesion occurrence existed in the Hylebos Waterway (SDN, "any toxicopathic hepatic lesion), these increased relative risks were paralleled by significantly higher measures for a number of chemical risk factors and for biochemical responses to contaminant exposure. These risk factors included mean values for HACs and LACs (Figure 2.2), PCBs and PCB 118 (Figure 2.3), HCB and HCBD (Figure 2.4) in stomach contents, FACs in bile (Figure 2.7), and PCBs, PCB 105, PCB 118 (Figure 2.8), HCB and HCBD (Figure 2.9), and DDTs (Figure 2.10) in liver, as well as hepatic CYP1A activity and DNA adduct concentrations (Figure 2.12). 
English sole
The data on lesion occurrence, contaminant exposure, and biochemical response to chemical exposure in English sole were sufficient to perform a statistical analysis of the influence of these chemical risk factors on lesion occurrence, while accounting for the influence of fish age. In individual fish sampled in both studies, the influence of PAH exposure (as estimated by FACsBaP, FACsNPH, and FACsPHN), and formation of DNA adducts on the estimated relative risk of lesion occurrence was assessed by logistic regression analyses. For the subset of female English sole sampled for the reproductive injury study, it was also possible to statistically relate the probability of lesion occurrence to liver concentrations of several chlorinated hydrocarbons (e.g., HCB, PCBs, PCB 105, PCB 118, DDTs, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxic equivalents [TEQ] of PCBs) in individual fish. For male and female English sole sampled from the Flatfish Toxicopathic Injury Study and female sole from the Flatfish Reproductive Injury Study, it was also possible to relate lesion prevalences (while accounting for mean fish age) at a site to mean levels of the following chemical risk factors: stomach contents HACs and LACs, bile FACs, liver DNA adducts, CHs in liver, CHs in stomach contents, and liver CYP1A. In these analyses of site-based data, logistic regression analysis was used because of its ability to account for the influences of individual and mean fish age on the odds of lesion occurrence. 

Influence of PAH Exposure 

Several lesion types consistently showed PAH exposure or biochemical response to PAH exposure as significant risk factors. As shown in Table 2.4 for the 206 English sole for which individual FACs data were available, concentrations of FACs in bile were risk factors for neoplasms, FCA, and "any toxicopathic liver lesion", and nearly significant (0.05 < p < 0.10) for SDN and proliferative lesions. Mean values of these same estimators of PAH exposure were also significant risk factors for prevalences of FCA, proliferative lesions, SDN, and "any toxicopathic lesions" in analyses based on data derived from composited samples at each site for each collecting period (site-based analyses, Table 2.5). The finding that PAHs are risk factors for these lesions is further strengthened by the fact that both HACs and LACs in stomach contents also were strong, highly significant risk factors for the same lesion categories in the latter site-based analyses (Table 2.5). 

Likewise, as has been demonstrated in previous studies on English sole (Myers et al. 1998, Myers unpubl.2), DNA adduct levels (a molecular dosimeter of genotoxic PAH exposure) were significant risk factors for the lesions FCA and SDN (Table 2.6 and 2.7) which are lesions that precede the development of hepatic neoplasms in fish (Hinton et al. 1992, Hawkins et al. 1990, Hendricks et al. 1984, Myers et al. 1987, Moore and Myers 1994). This was especially true for the most prevalent lesion type, SDN, which appears to be the earliest lesion to occur in fish following PAH exposure, manifesting the cytotoxicity of those compounds (Myers et al. 1987). Recent studies (Myers et al. 1998, Stein et al. 1996, Myers et al. 1995a) have also shown DNA adduct levels to be most strongly associated with SDN. Hepatic DNA adducts were also a risk factor for the "any toxicopathic liver lesion" category probably because this lesion category primarily represented fish affected with the most common independent lesion category, SDN. Additionally, mean hepatic CYP1A activity, which also can reflect biochemical response to PAH exposure, was a significant risk factor only for the most prevalent independent lesion category, SDN (Table 2.5). 

Influence of PCB Exposure 

Dietary exposure to and bioaccumulation of PCBs were also highly significant risk factors for hepatic lesion occurrence/prevalence in English sole. These findings are not surprising, considering their existence as risk factors in previous similar studies of English sole (Myers et al. 1991, 1994; Rhodes et al. 1987; Myers et al. 1998), their well known hepatotoxic and neoplasia-promoting properties (reviewed in Myers et al. 1994)), and because as contaminants they commonly co-occur with other chemical risk factors associated with liver lesions, especially the PAHs (Myers et al. 1991, 1994; Rhodes et al. 1987). In the present study, PCBs in stomach contents were strongly correlated (p < 0.05) with both HACs and LACs, and liver PCBs were strongly correlated with all measures of bile FACs. Therefore, it is difficult to separate out the potential influences of PAHs vs. PCBs as risk factors for liver lesions. Nevertheless, the site-based logistic regression analyses in this study clearly showed that dietary exposure to PCBs was a risk factor for FCA, proliferative lesions, SDN, and "any toxicopathic lesion" (Table 2.5). In the case of liver PCBs, where a lower number of data points were available (n = 4, vs. n = 8 for stomach contents), only SDN was significantly linked to PCBs, while "any toxicopathic liver lesion" was significant at p = 0.055.

Concentrations of the toxic, coplanar congeners PCB 105 and PCB 118 in stomach contents and liver were also significant risk factors for the lesions, closely paralleling the results for PCBs (Table 2.5). These congeners were risk factors for the same lesions, and at approximately the same levels of significance as PCBs, with the single exception that PCB 118 was also a near-significant risk factor for proliferative lesions.

The PCBs in liver were also risk factors for hepatic lesions in the smaller subset of female English sole from the Flatfish Reproductive Injury Study, where livers were individually analyzed for CHs (Table 2.8). In that data set, PCBs, PCB 105, PCB 118, and TCDD (dioxin) TEQs of PCBs were all highly significant risk factors for the occurrence of FCA , and "any toxicopathic hepatic lesion". Various measures of PCB exposure were also risk factors for hepatic lesion prevalences in a logistic regression analysis of the mean values computed from the above data set (Table 2.9), showing significantly increased odds ratios for FCA (PCB 105), SDN (PCBs, PCB 105, non-dioxin-like PCBs) and "any toxicopathic hepatic lesion" (PCBs, PCB 105, PCB 118, non-dioxin-like PCBs, dioxin-like PCBs, and TCDD (dioxin) TEQs of PCBs). 

Hepatic concentrations of CHs are usually considered a more reliable indicator of chronic exposure than concentrations in stomach contents. However, it is important to stress that measures in stomach contents are probably a better index to use in statistical analyses of the site-based data in the current study because more data points for stomach contents CHs (n = 8) are available for analysis than liver (n = 4). Because the same analytical methods were used to measure chemicals in stomach contents in the Flatfish Toxicopathic and Reproductive Injury Studies, we were able to combine these data. Consequently, the number of fish upon which the prevalence data are based is far greater in the data set including stomach contents measurement (n = 342) than for liver chemistry as assessed by detailed methods (n = 230, Flatfish Toxicopathic Injury Study only), or by the high-performance liquid chromotography with photodiode array detection (HPLC/PDA) method in individuals (n = 103, Flatfish Reproductive Injury Study only). Because of the different analytical methods used in analyzing CHs in liver reflecting the different goals of the two flatfish injury studies, the resulting data were not strictly comparable and it was not permissible to combine the liver chemistry data from the two studies. Therefore, the finding that certain chemicals measured in stomach contents were significant risk factors for lesions, whereas the same chemicals measured in liver were not risk factors, is mainly a function of differing sample sizes resulting from the inability to combine the liver chemistry data from both studies, and should not be viewed as being particularly significant.

Influence of DDT Exposure 

Dietary exposure to and hepatic bioaccumulation of DDT and its breakdown products were also risk factors for several toxicopathic liver lesions, including FCA, proliferative lesions, SDN, and "any toxicopathic liver lesion" in the site-based analyses (Table 2.5). Hepatic bioaccumulation of DDT and its breakdown products was also a significant risk factor for FCA and "any toxicopathic liver lesion" in the data subset in which individual livers were analyzed by the HPLC/PDA methodology (Table 2.8), and for SDN and "any toxicopathic liver lesion" when mean site-based exposure and lesion prevalence data were used (Table 2.9). The lesions for which DDTs in stomach contents and liver tissue were risk factors were nearly the same as those associated with PCB exposure. These findings are consistent with the existence of DDTs as risk factors for hepatic disease in English sole in previous studies (Myers et al. 1994). These findings also reflect the positive correlation between DDTs and PCBs in liver tissue (r = 0.94, n = 24) and stomach contents (r = 0.97, n = 23), and with biliary FACs (0.78 < r < 0.87, n = 23) and aromatic compounds in stomach contents (0.82 < r < 0.85, n = 9). Because of these strong intercorrelations, it is not possible to separate the influence of DDTs as etiologic agents for these liver lesions from the influences of PAHs and PCBs. 
Comparisons with Historical Data: Liver Lesion Prevalence and Biochemical Responses to Contaminant ExposureLesion Prevalences
Rock sole 

Historical data on lesion prevalences in adult rock sole from sites within the Hylebos Waterway are limited to those derived from NWFSC studies done between 1979-1985 (Malins et al. 1984, McCain et al. 1983, Myers unpubl.2). Data from these sources are summarized in Figure 2.17, as compared to prevalence data for the current study. In the multiple studies done between 1979 and 1985, low prevalences of neoplasms were not detected and low prevalences of FCA (3%) were reported, with higher prevalences of SDN (23%), and hydropic vacuolation (up to 43%) (Stehr et al. In press, Myers unpubl.2). Because of the various ways in which the inclusive lesion category "any toxicopathic liver lesion" has been formed over the years (i.e., different unique lesion types have been placed into this category over the years), it is difficult to make direct comparisons between data in the present study and past reported values. However, by examining historical data, we are able to accurately determine a prevalence of 28% in rock sole from those combined studies by NWFSC, using the manner in which the prevalence of this inclusive lesion category is currently computed. Comparisons of the prevalence data from historical studies to the current data is further complicated by the issue of differing age distributions of the fish composing the various studies, and the demonstrated age-dependency of the probability of occurrence of neoplasms and FCAs. However, the limited available age data for Hylebos rock sole composing the 1979-85 studies show a mean age of 5.4 years (n = 21), which is comparable to the mean ages for rock sole specifically from the 11th St. Bridge (6.2, n = 59) and throughout the Hylebos Waterway (7.0, n = 118) in the present study. Aside from the findings that no neoplasms were detected in Hylebos rock sole in the present study, the liver lesion prevalence data in this species, especially for the lesion most prevalent and most strongly linked to contaminant exposure (SDN), does not suggest any appreciable reduction in prevalence over the period since our original studies were done in the Hylebos Waterway. 

English sole 

The historical database for toxicopathic liver lesions in English sole from sites within and adjacent to the Hylebos Waterway is much larger than that for rock sole, and is derived from a number of studies done in 1979-85 (Malins et al. 1984, McCain et al. 1983, Myers unpubl.2), followed by a study in 1984 using similar protocols (Becker et al. 1987). These historical prevalence data are summarized in Figure 2.17, for comparison with data from the current study. The age distributions of English sole composing these separate studies were roughly similar, with the mean age for the past NWFSC studies being ~4.0 years, as compared to median ages for sole from sites sampled by Becker in 1984 (Becker 1987) at the 11th St. Bridge (4.9, n = 60), Lower Turning Basin (5.1, n = 60), and Upper Turning Basin (5.2, n = 60). Mean ages were similar for English sole from the 11th St. Bridge (5.6, n = 60), Lower Turning Basin (4.9, n = 59), Upper Turning Basin (5.1, n = 58), and throughout the Hylebos Waterway (5.8, n = 395) for the combined data set of male and female English sole from the current toxicopathic and reproductive injury studies. The method of computing liver lesion prevalences among these studies was also similar. The single exception was the "any toxicopathic liver lesion" category, which was computed differently in the Becker 1984 study (Becker et al. 1987), in which non-neoplastic proliferative lesions were not included in this category (resulting in a slight underestimate, as compared to the present study). Notwithstanding these methodological differences in lesion prevalence computation, there is no clear evidence that lesion prevalences have dropped appreciably in English sole from separate sites or combined sites in the Hylebos Waterway, since the late 1970's and early 1980's (Figure 2.17). The single exception to this finding is the apparent reduction in prevalences of neoplasms, FCA, SDN, and "any toxicopathic lesion" at the Lower Turning Basin site since Becker's 1984 study and the early NWFSC studies. However, if current (1994) liver lesion prevalences at the Lower Turning Basin are compared by the Fisher's Exact Test (Zar 1984) to those found in the previous NWFSC studies (1979-85) (Figure 2.17), there is no statistically significant change over this time period. 
Biliary FACs
Rock sole

Measurement of biliary FACs concentrations (protein-corrected) in rock sole from the Hylebos Waterway was first performed in 1987-88 (Myers et al. 1992, Stein et al. 1992, Hom unpubl.4). Mean values of FACs BaP ranged from 790 to 2500 ng BaP equivalents/mg bile protein (Figure 2.18). These mean values are comparable to those determined in Hylebos rock sole in the present study, indicating that this estimator of exposure to high molecular weight PAHs has not declined appreciably from 1987 to 1994.

English sole 

Historical data on FACsBaP in bile of English sole from the Hylebos Waterway in the same study cited above show mean values ranging from 280 to 720 ng BaP equivalents/mg bile protein, which are well within the range of values determined in English sole from the various Hylebos sites in the present study. This suggests no evidence of a appreciable decline in this indicator of exposure to PAHs since 1987. 
DNA adducts in liver
Comparisons of hepatic DNA adduct levels first determined in flatfish captured from the Hylebos Waterway in May 1988 (Stein et al. 1992) to the present data are problematic, because of important changes in methods of quantitation since that time. Briefly, quantitation of adduct concentrations in 1988 was relatively crude as compared to the current method utilizing computerized phosphorimaging (Reichert and French 1994). Background levels in flatfish from relatively uncontaminated reference sampling sites in 1988 ranged between 30-35 nmol DNA adducts/mol DNA bases (Stein et al. 1992), whereas current methods of sample analysis and quantitation yield background levels for flatfish from the same reference sites of 0-5. Consequently, by subtracting 30 nmol DNA adducts/mol DNA bases from the levels determined in the 1988 analyses, one can arrive at a reasonable estimate for comparisons with current data.

Rock sole 

Using the above method of estimation applied to 1988 data for rock sole from the entrance of the Hylebos Waterway results in an estimated mean value of 25-50 nmol DNA adducts/mol DNA bases (Figure 2.19). The lower level of this range is comparable to the mean DNA adduct concentrations determined in rock sole from the 11th St. Bridge site in the current study.

English sole

Estimates of adduct concentrations in English sole liver from the above 1988 study range from 60-90 nmol DNA adducts/mol DNA bases (Figure 2.19), which are also comparable to mean DNA adduct concentrations in English sole from the two upper sites in the Hylebos Waterway. As in rock sole, there is no clear evidence of a reduction in this dosimeter of PAH exposure and metabolism in English sole from the Hylebos Waterway since the original measurements done in 1988.
CYP1A activity in liver
Rock sole 

Induction of this xenobiotic metabolizing enzyme indicates recent exposure and response to a broad range of PAHs, CHs, herbicides, and pesticides (Collier and Varanasi 1991, Payne et al. 1987). Activity levels in Hylebos rock sole from the current study were similar to levels determined in Hylebos rock sole in 1987-88 (Stein et al. 1992, Collier and Anulacion unpubl.5), where mean activity levels ranged from 480-880 pmol/mg/min (Figure 2.20). There is no evidence in rock sole of a decline in this general biomarker of organic chemical contaminant exposure since that time.

English sole 

Similarly, the range of CYP1A activity levels in Hylebos Waterway English sole determined in 1987-88 (410-1200 pmol/mg/min) encompasses the activity levels determined in the same species in the present study (Figure 2.20). As in rock sole, no consistent pattern of decline in hepatic CYP1A activity since 1987 is apparent in English sole. 
Discussion of Major Findings
English sole and rock sole residing in the Hylebos Waterway show clear evidence of exposure to several covariant classes of toxic chemical contaminants, including PAHs, and CHs such as PCBs, HCB, HCBD, and DDT and its derivatives. These exposures are also clearly reflected in elevated levels of biliary FACs and hepatic DNA adducts, and induction of CYP1A activities in liver.

Adult English sole and rock sole living in the Hylebos Waterway are clearly exposed to low and high molecular weight PAHs, PCBs, DDTs, HCB, and HCBD, regardless of whether the exposure is assessed by analyzing stomach contents (all contaminants), liver (CHs) or bile (PAH metabolites). Concentrations of these contaminants were generally 1 to 2 orders of magnitude higher in stomach contents of rock and English sole from Hylebos Waterway sites as compared to Colvos Passage reference fish. Among indices of exposure to PAHs, dietary exposure (stomach content LACs and HACs) was up to 2 orders of magnitude higher in English and rock sole from the Hylebos sites, and biliary FACs were 4 to 10 times higher at the Hylebos Waterway sites, with a clear pattern of increasing PAH exposure by both indices in English sole captured from the sites further up the waterway. Levels of exposure to PAHs in rock sole (captured only from the 11th St. Bridge site, the site closest to the entrance of the Hylebos Waterway) were very similar to those in English sole from the same site. As a molecular dosimeter of exposure to genotoxic PAHs (Stein et al. 1992, 1994, 1996), DNA adduct levels in liver were also significantly higher in English and rock sole from Hylebos Waterway sites, showing progressively increasing levels in English sole from sites further up the waterway, as well as similar levels in livers of both species captured at the 11th St. Bridge site. 

Levels of dietary exposure to and hepatic bioaccumulation of PCBs and DDTs were 15 to 25 times higher in rock and English sole from Hylebos sites compared to Colvos Passage reference fish, and like the PAHs, also showed a clear pattern of increasing exposure in English sole from the upper two sampling sites in the Hylebos Waterway. Levels of PCBs and DDTs exposure in rock sole from the 11th St. Bridge site were again similar to levels determined in English sole from that site. Exposure to HCB and HCBD, chemicals that are markers for the complex mixture of CHs in Hylebos Waterway sediments (Malins et al. 1982), was most pronounced in both English and rock sole from the 11th St. Bridge site, with comparable levels of dietary exposure in the two target species and somewhat higher levels of hepatic bioaccumulation in rock sole. 

Levels of hepatic CYP1A induction, a biochemical response to organic contaminants such as PAHs and CHs, were significantly higher than reference values for both species living in the Hylebos Waterway, regardless of where in the waterway they were captured. 

Overall, there was an exceptional degree of consistency for all exposure indices measured (except those for HCB and HCBD, and CYP1A in liver), demonstrating increased exposure and biochemical response to exposure moving from the near the mouth to the head of the Hylebos Waterway. This consistency was also evident in the close similarity in exposure levels among rock and English soles from the single common sampling site in the Hylebos (11th St. Bridge). The significant and often marked differences in many exposure parameters for fish captured among the three sites sampled in the Hylebos Waterway, the consistency among different measures of exposure to the same chemical class, and site-specific parallels in exposure data between the two target species clearly show that exposure closely reflects the site of capture within the Hylebos Waterway. These data lead us to conclude that there is fidelity in feeding areas and limited movement of fish among the Hylebos sites.

Higher prevalences of injury in the form of toxicopathic liver lesions were detected in rock, and especially English sole from the Hylebos Waterway, in comparison to the reference site. 

Significantly increased risks for occurrence of several toxicopathic liver lesion categories were associated with residence at sites within the Hylebos Waterway in both fish species, using statistical analyses that account for the important variable of fish age in determining risk of liver lesion occurrence. Although low prevalences of certain lesion categories occurred in both species at the reference site, this is not a unique finding based on previous studies on these species in Puget Sound (Malins et al. 1984, 1987a; Myers et al. 1990, 1991, 1992, 1995b, 1998; Rhodes et al. 1987; McCain et al. 1983; Myers and Rhodes 1988; Casillas et al. 1985; Krahn et al. 1986; Becker et al. 1987). Significantly increased estimated relative risks of SDN and "any toxicopathic liver lesion" were shown in Hylebos Waterway rock sole, and far more cases of increased risk of lesion occurrence were shown for English sole from sites in the Hylebos Waterway relative to Colvos Passage. For example, RRes of 2.7 to 74 times that for comparably aged fish from Colvos Passage were determined for SDN, proliferative lesions, and "any toxicopathic liver lesion" at the three separate Hylebos sites. Estimated relative risks for SDN were significantly and consistently higher at all three sites, with progressively higher relative risks as one moved up the waterway. In analyses combining all data from both the Flatfish Toxicopathic and Reproductive Injury Studies, increased estimated relative risks were shown for these same lesion categories, at values up to 26 times the baseline risk at Colvos Passage. 

In prior studies of English sole from Puget Sound using this same statistical method, RRe for hepatic lesion occurrence ranged from 2.7 (SDN, South Seattle Waterfront) to 8.7 (neoplasms, Upper Duwamish Waterway) when fish from Port Madison were used as the reference population (Rhodes et al. 1987). In studies incorporating data from the Puget Sound Ambient Monitoring Program (data collected 1989-1993) and using a much larger database from several reference sites to establish a baseline (Myers et al. 1995b), significantly increased estimated relative risks of lesion occurrence at a sampling site in Commencement Bay were shown for neoplasms (RRe = 7.7), FCA (RRe = 7.7) and SDN (RRe = 11). Similar RRes occurred at the Elliott Bay site (ranging from 2 to 20), and much higher ones (ranging from 31 to 71) at the Duwamish Waterway site. If the lesion occurrence and age data in Hylebos English sole from the present study were to be analyzed and compared to the reference site data from either of these prior studies (where lesion prevalences were much lower than those shown in Colvos Passage English sole), it is highly probable that the RRes of lesion occurrence in English sole from the Hylebos sites in the present study would have approached or exceeded those shown for the Commencement Bay and Elliott Bay sites in that prior study. For example, prevalences in English sole from Commencement Bay in that study were ~5% for SDN, 12% for FCA, and 5% for neoplasms, and in Elliott Bay English sole were 10% for SDN, 11% for FCA, and 5% for neoplasms. Prevalences of the same lesion categories in English sole from the Hylebos sites in the current study were similar, and in some cases higher.

Risk factors of chemical exposure most commonly associated with hepatic lesion occurrence in Hylebos flatfish were those reflecting exposure or biochemical response to PAHs, PCBs, and DDTs. The frequency of injury in the form of certain hepatic lesions closely paralleled measures of DNA damage and CYP1A induction in liver in fish from the same sites. 

The results of logistic regression analyses assessing potential relationships between contaminant exposure and injury in the form of toxicopathic liver lesions were quite similar in the current study to those previously conducted on English sole in Puget Sound and elsewhere (Myers et al. 1990, 1991, 1994, 1995b; Rhodes et al. 1987). Exposure to PAHs, PCBs (including mono-ortho substituted toxic coplanar congeners PCB 105 and PCB 118), and DDTs were the risk factors most commonly associated with hepatic lesion occurrence. However, as in those previous studies, because of the covariance of these contaminants it is not possible to quantify the proportional risk attributable to each of these chemical classes. Nonetheless, PAHs are toxicologically meaningful risk factors in field studies such as this because of the experimentally demonstrated hepatotoxicity and carcinogenicity of these compounds and their prominent role as genotoxicants in the initiation of carcinogenesis. In contrast, the mode of action for PCBs and DDTs appears to be as nongenotoxic promoters of carcinogenesis by virtue of their cytotoxicity and subsequent stimulation of cell proliferation (reviewed in Myers et al. 1987, 1994; Moore and Myers 1994). In addition, experimental exposures of fish to these CHs have resulted in the formation of liver lesions identical to those included in the SDN category, including megalocytic hepatosis and nuclear pleomorphism (reviewed in Myers et al. 1987, 1994; Moore and Myers 1994). Neither HCB nor HCBD were strong risk factors for toxicopathic hepatic lesions in English sole in the current study, primarily because levels of dietary exposure and hepatic bioaccumulation of these compounds were comparatively low (relative to those at the 11th St. Bridge site) at other sites in the Hylebos displaying higher lesion prevalences. 

The importance of chemical contaminant exposure in the Hylebos Waterway to liver lesion development in rock sole was more equivocal, for two reasons. First, chemical exposure data generated for this secondary target species was insufficient to support a robust statistical evaluation of such a relationship. Second, evidence from other studies done in Puget Sound shows that the same lesions strongly linked to contaminant exposure in English sole are less consistently shown to have a toxicopathic etiology in rock sole (Malins et al. 1984, Myers and Rhodes 1988, Myers et al. 1992, Johnson unpubl.3). Nonetheless, higher prevalences of certain lesions were detected in fish from the Hylebos Waterway, where exposure to a number of contaminants was significantly higher than in Colvos Passage rock sole. The lesion categories in this study showing significantly higher RRes in Hylebos rock sole compared to Colvos rock sole (SDN, "any toxicopathic lesion") have also shown higher relative risks associated with residence in other contaminated urban sites in Puget Sound (Myers et al. 1992), and have been statistically associated with PAH exposure reflected by measurements of bile FACs (Myers et al. 1992, Johnson unpubl.3). This is particularly the case for lesions in the SDN lesion category, which are the most frequently detected toxicopathic lesions in this species. Moreover, DNA adduct levels in liver have been shown to be a highly significant risk factor for occurrence of lesions in this category in rock sole from Eagle Harbor, WA (Myers et al. 1995a). The overall evidence from the current study and other Puget Sound studies targeting rock sole is that occurrence of the most prevalent lesion in this species, SDN, is strongly linked to various indices of exposure to PAHs.

Results of logistic regression analyses relating mean hepatic CYP1A activity levels to lesion prevalences for English sole in the Flatfish Toxicopathic Injury Study also showed CYP1A induction as a significant risk factor for the most common hepatic lesion, SDN. These results are consistent with other studies in English sole from multiple sites in Puget Sound that have shown SDN prevalence to be significantly influenced by mean hepatic CYP1A activity. This finding is consistent with the role of CYP1A in the metabolism and bioactivation of PAHs to their genotoxic and cytotoxic intermediates, as well as its inducibility by and metabolism of CHs (Collier and Varanasi 1991).

Finally, lesion prevalences in rock and English sole from Hylebos Waterway sites generally paralleled levels of DNA adducts in liver from the same fish and same sites. Highest mean levels of adducts were detected in English sole at the Upper Turning Basin site where hepatic lesion prevalences were also highest. Moreover, in logistic regression analyses examining the increased risk of lesion occurrence in English sole attributable to hepatic DNA damage, adduct levels were significant risk factors for FCA, SDN, and "any toxicopathic hepatic lesion". These results parallel those of prior studies (Myers et al. 1998, 1995a; Myers unpubl.2; Stein et al 1996) and provide further evidence linking measures of PAC exposure to hepatic lesion occurrence in benthic fish, especially for lesions occurring early in the histogenesis of hepatic neoplasia (SDN, FCA) (Myers et al. 1987, Moore and Myers 1994). Levels of DNA adducts in livers of Hylebos Waterway English sole from the current study were similar to those determined by comparable quantitation methods in sole from Eagle Harbor in Puget Sound prior to capping of PAH-contaminated sediments (Myers et al. 1995a, Collier and Myers 1997), and those estimated from previous 1988 data (Stein et al. 1992) in sole from the Duwamish Waterway. Both of these sites are also highly contaminated (primarily with PACs and/or PCBs) urban sites in Puget Sound exhibiting high prevalences of hepatic lesions in resident flatfish species (Malins et al. 1984, 1985, 1987a; Myers et al. 1987, 1990, 1991, 1992, 1995a, 1995b, 1998; Rhodes et al. 1987; McCain et al. 1983; Krahn et al. 1986; Collier and Myers 1997). 

Lesion prevalences and biochemical measures of early response to contaminant exposure (bile FACs, liver CYP1A/DNA adducts) have not changed appreciably since the late 1970's, and 1980's, respectively. With respect to fish injury, there has been no appreciable improvement in the Hylebos Waterway since those initial studies. 

The only available historical data on lesion prevalences in rock sole from the Hylebos Waterway come from NWFSC studies conducted in 1979-85 (Malins et al. 1984, McCain et al. 1983, Myers and Rhodes 1988, Myers unpubl.2) showing prevalence of the most common toxicopathic hepatic lesion in this species (SDN) to be ~23%, with prevalence of "any toxicopathic lesion" as defined in the present report of ~28%. These prevalences are not substantially different than those detected in the current study (14% and 22%, respectively). For the biochemical measures of response to exposure, historical data is first available in NWFSC studies done in 1987-88 on subadult rock sole. In the current study, mean concentrations of biliary FACsBaP and DNA adducts are somewhat lower than the upper range of values measured in 1987-88. However, the current levels of these indices measuring exposure and response to PAHs are well within the range of those mean values determined in 1987-1988. Historical mean activity levels for hepatic CYP1A in this species in 1987-88 are actually slightly lower than the current measures of this integrative biomarker of PAH and CH exposure.

Drawing from data from the several studies done on English sole between 1979 and 1985, there is neither a substantial decrease in injury, as overall toxicopathic liver lesion prevalence, at the specific Hylebos sites, nor in the Hylebos Waterway as a whole. More importantly, prevalences of most of the toxicopathic hepatic lesions in English sole from the Hylebos Waterway continue to far exceed those found in sole from relatively uncontaminated reference sites in Puget Sound. Current concentrations of biliary FACsBaP in English sole are well within the range of values determined in 1987-88, and in the case of the Upper Turning Basin, are much higher than historical values. Similarly, the mean DNA adduct levels and CYP1A activities in liver found in the current fish injury study are within or exceed the mean values determined in 1987-88. Overall, the data for rock and English sole do not provide evidence that natural or engineered remediation processes over the last 10-15 years have resulted in a substantial improvement in the extent of chemical exposure and associated injury to flatfish in the Hylebos Waterway. 

On to Section 3

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