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The Hylebos Waterway is an industrialized waterway ofCommencement Bay, Tacoma, Washington, that is severelycontaminated with aromatic and chlorinatedhydrocarbons in the sediment. Juvenile chinook (Oncorhynchus keta) and chum salmon (O.tshawytscha) inhabit this waterway for a few days orweeks during their outmigration from freshwaterstreams to saltwater. The purpose of thisinvestigation was to determine to what degree juvenilechum and chinook salmon captured from the HylebosWaterway might bioaccumulate organic contaminants. These levels of exposure will be compared to previousstudies where such exposures have been linked tobiological dysfunction in juvenile salmon. Theresults showed that juvenile chum and chinook salmonfrom the Hylebos Waterway take up a wide range ofchemical contaminants, compared to fish fromhatcheries or reference estuaries. These contaminantsinclude high and low molecular weight polycyclicaromatic hydrocarbons (PAHs), polychlorinatedbiphenyls (PCBs, including the toxic congeners 105 and118), hexachlorobutadiene (HCBD), hexachlorobenzene(HCB), DDTs, heptachlor, and several pesticides. Immunohistochemical examination of the gill and gut injuvenile chum salmon from the Hylebos Waterway showedthe induction of the P450 metabolizing enzyme. Moreover, concentrations of contaminants in juvenilechinook and chum salmon from the Hylebos Waterway arecomparable to levels previously shown to be associatedwith biological injury in juvenile chinook salmon,such as impaired growth, suppression of immunefunction as demonstrated by reduced B cell function,and increased mortality following pathogen exposure.

The objective of this study was to determine if exposure to aromatic hydrocarbons (AHs) or polychlorinated biphenyls (PCBs) was associated with altered ovarian development or reduced spawning success in rock sole Pleuronectes bilineatus. This objective was addressed in two separate phases. In the first study, concentrations of AHs and PCBs were measured in individual fish from four sites in Puget Sound, Washington (Eagle Harbor, Sinclair Inlet, Yukon Harbor, and Pilot Point) with different levels of AH and PCB contamination in sediments and their ovarian development was monitored during the 1989–1991 spawning seasons. In these fish, egg weight was negatively correlated with levels of PCBs in the liver. However, contaminant exposure levels were not significantly associated with the probability of entering vitellogenesis or with gonadosomatic index, plasma estradiol concentrations, or fecundity. In the second study, gravid female soles were taken from Eagle Harbor, Sinclair Inlet, Yukon Harbor, and...

Liver neoplasms are rarely detected in young wild fish. Therefore, other lesions occurring early in the histogenesis of hepatic neoplasia need to be considered as biomarkers of chemical contaminant exposure effects in monitoring studies, especially where adult fish are not available. Moreover, exposure effects may be more reliably assessed in younger fish that have not yet migrated extensively. Accordingly, livers of subadult English sole were histologically examined from nine sites in Puget Sound, WA and the same fish were assessed for contaminant exposure by measurement of fluorescent aromatic compounds (FACs) in bile, hepatic levels of polychlorinated biphenyls (PCBs) and cytochrome P4501A (CYP1A) as catalytic activity of aryl hydrocarbon hydroxylase (AHH), and Hydrophobic DNA adducts in liver by 32P-postlabelling. Although neoplasms were rare, higher prevalences of preneoplastic, regenerative, and unique degenerative/ necrotic lesions were detected in sole from contaminated sites. Prevalences of these early histopathologic biomarkers were significantly higher at the more contaminated sites, and concentration of mean biliary FACs at each capture site was a significant risk factor for most lesions, as determined by stepwise logistic regression. By this statistical method, we also demonstrated that several measures of bioaccumulation or biochemical response to contaminants were significant and near-significant risk factors for prevalences of most hepatic lesion categories. For example, mean hepatic AHH activity was a significant risk factor for prevalence of all lesion types, except neoplasms; hepatic PCB and xenobiotic-DNA adduct concentrations were significant risk factors for the most frequently detected lesion category, hepatocellular nuclear pleomorphism/megalocytic hepatosis, and the inclusive category ‘any early toxicopathic lesion’. These findings further support the utility of certain non-neoplastic liver lesions as early indicators of biological damage in subadult as well as adult fish exposed to xenobiotics in the marine environment.

The effect of exposure to xenobiotic compounds on ovarian development was investigated in prespawning female English sole (Pleuronectes vetulus) from the Hylebos Waterway, an industrial site in Commencement Bay, WA, contaminated with aromatic hydrocarbons (AHs), polychlorinated biphenyls (PCBs)and other chlorinated compounds, including hexachlorobutadiene (HCBD) and hexachlorobenzene (HCB). Reference sole were collected from Colvos Passage, a nearby site with minimal sediment contaminant concentrations. English sole from theHylebos Waterway had significantly higher concentrations of fluorescent aromatic compounds (FACs) in bile, polycyclic aromatic compound-DNA adducts in liver, and dioxin-like and other selected PCB congeners in liver than sole from Colvos Passage. The Hylebos Waterway animals also showed significant alterations in their pattern of reproductive development when compared to Colvos Passage sole. Hylebos Waterway sole entered vitellogenesis at a nearlier age than Colvos Passage sole, with about 50%of fish below 5 years of age maturing in the Hylebos Waterway as compared to 20% of Colvos Passage sole in this age range, with corresponding increases in plasma estradiol concentrations and GSI in Hylebosfish. However, while the proportion of maturing Colvos Passage females increased with age to over70% for fish 5 years of age or greater, the proportion of maturing females in the Hylebos Waterway remained at about 50%. Moreover, plasma estradiol concentrations and gonadosomatic indices in these sole were depressed. Inhibited reproductive development and increased oocyte atresia in adult fish were correlated with elevated concentrations of FACs in bile. Enhanced growth, as well as exposure to both aromatic and chlorinated hydrocarbons, were associated with precocious maturation in sub adult Hylebos Waterway sole.

Levels of hepatic DNA adducts and concentrations of fluorescent aromatic compounds (FACs) in bile were measured in English sole (Pleuronectes vetulus) exposed for up to 5 weeks to a reference sediment amended with a sediment containing high concentrations of polycyclic aromatic compounds (PACs) from Eagle Harbor, Puget Sound, WA. Levels of hepatic DNA adducts increased linearly with both concentration of sediment polycyclic aromatic hydrocarbons (PAHs) and length of exposure, whereas concentrations of biliary FACs were dose-responsive to levels of sediment PAHs but attained steady-state concentrations after 2 weeks of exposure. The levels of DNA adducts and concentrations of biliary FACs in fish exposed to the reference sediment remained at baseline levels throughout the exposure. Formation of PAC-DNA adducts was observed in liver of English sole injected with an extract of Eagle Harbor sediment (EHSE) and in English sole hepatocytes incubated with EHSE or the neutral fraction of EHSE containing predominantly PACs. In addition, DNA adduct and biliary FAC levels measured in feral English sole captured from Eagle Harbor, when compared with levels measured in fish from the laboratory sediment exposure study, also suggested accumulation of DNA adducts in the fish captured from Eagle Harbor. These findings, in conjunction with previous studies showing PAC-DNA adducts are persistent in fish, suggest that a substantial proportion of PAC-induced DNA damage was not readily repaired in English sole, and thus, measurement of hepatic DNA adducts can be used as an indicator of cumulative exposure to genotoxic PACs.

Juvenile chinook salmon (Oncorhynchus tshawytscha) were sampled in Puget Sound, Washington, for 2 consecutive years from contaminated urban estuaries, a nonurban estuary, and from the respective hatcheries to assess exposure to anthropogenic chemicals and to determine if biochemical changes were occurring as a consequence of exposure. Exposure to polycyclic aromatic hydrocarbons (PAHs), chlorinated hydrocarbons, and butyltins was determined. The mean concentrations of PAHs and PCBs in stomach contents and PCBs in liver were significantly higher in salmon from the urban estuaries compared to fish from the nonurban estuary in both sampling years. Higher hepatic concentrations of PCBs than DDTs were found in fish from the urban estuaries, but butyltins were rarely detected. Further, mean concentrations of fluorescent aromatic compounds in bile, an estimate of exposure to PAHs, and hepatic cytochrome P4501A and levels of hepatic DNA adducts were also significantly higher in salmon from the urban estuaries compared to either the nonurban estuary or the hatcheries. Results demonstrated increased exposure to chemical contaminants in outmigrant juvenile salmon during their relatively brief residence in urban estuaries of Puget Sound. Moreover, the exposure was sufficient to elicit biochemical responses, which suggest a potential for other biological effects to ensue.

A suite of chemical and biochemical parameters was measured in three species of benthic flatfish (English sole, Parophrys vetulus; rock sole, Lepidopsetta bilineata; and starry flounder, Platichthys stellatus) sampled from up to five sites in Puget Sound, WA, USA, to assess the sensitivity of the parameters to differences in levels of contaminant exposure and the relative merit of the use of a suite of indices for assessing exposure and sublethal effects. The indices examined were hepatic levels of polychlorinated biphenyls (PCBs) and biliary fluorescent aromatic-compound concentrations, hepatic activities of aryl hydrocarbon hydroxylase and ethoxyresorufin-O-deethylase, and levels of total hepatic GSH and hydrophobic DNA-xenobiotic adducts; the last-mentioned were determined by using the 32P-postlabeling assay. The results showed that the indices examined could discriminate among sites exhibiting different degrees of chemical contamination; however, species differences in the range of response of some indices were observed. Additionally, the use of multiple indices appeared to enhance the assessment of contaminant exposure and sublethal effects.

Our studies have shown that early stages of the reproductive process in female English sole (Parophrys vetulus) are disrupted by contaminant exposure. To determine the potential for contaminants to affect later stages of the reproductive cycle, we examined the effects of maternal contaminant exposure on egg and larval viability in this species, in a field study conducted on running ripe fish collected for two consecutive years from spawning grounds. Maternal contaminant exposure was assessed by several methods. Stepwise multiple regression of the data showed that contaminant exposure was only a minor factor in determining egg and larval viability. However, because the range of contaminant exposure in the spawning fish did not encompass the high levels seen in non-spawning fish captured from contaminated areas, it can be hypothesized that female English sole exposed to substantial levels of contaminants might be at least partially excluded from the spawning population.

A suite of chemical and biochemical variables responsive to contaminant exposure was measured in three species of benthic flatfish (English sole, Parophrys vetulus, rock sole, Lepidopsetta bilineata, and starry flounder, Platichthys stellatus) sampled from up to five sites in Puget Sound, Washington, to assess the sensitivity of the parameters to differences in levels of contaminant exposure The examined indexes were levels of (a) polychlorinated biphenyls (PCBs) in liver, (b) fluorescent aromatic compounds in bile, (c) hepatic aryl hydrocarbon hydroxylase and ethoxy-resorufin O deethylase, (d) total hepatic GSH and (e) hydrophobic DNA xenobiotic adducts in liver as measured by 32P postlabeling The sediment concentrations of PCBs and the sum of selected polycyclic aromatic hydrocarbons at the sampling sites ranged from 21 to 570 and 21 to 5,300 ng/g wet weight sediment, respectively The results showed (a) that all the examined indexes could discriminate among sites exhibiting different degrees of chemical contamination, (b) that species differed in the range of response of most of the measured indexes, and (c) that the use of the indexes in concert appeared to enhance the assessment of contaminant exposure and sublethal effects

Our studies have shown that early stages of the reproductive process in female English sole (Parophrys vetulus) are disrupted by contaminant exposure. To determine the potential for contaminants to affect later stages of the reproductive cycle, we examined the effects of maternal contaminant exposure on egg and larval viability in this species, in a field study conducted on running ripe fish collected for two consecutive years from spawning grounds. Maternal contaminant exposure was assessed by measuring levels of polychlorinated biphenyls (PCBs) in ovarian and hepatic tissue, biliary levels of fluorescent aromatic compounds (FACs), and hepatic aryl hydrocarbon hydroxylase (AHH) activities. Additionally, the presence of significant liver disease or ovarian atresia was documented, and levels of plasma estradiol were measured. Stepwise multiple regression of the data showed that contaminant exposure was only a minor factor in determining both egg and larval viability. Hepatic AHH activity showed a slight negative correlation with fertilization success, explaining only 4% of the variability. Increased levels of PCBs in ovaries of spawning female English sole were not associated with reduced reproductive success. However, because the range of contaminant exposure in the spawning fish did not encompass the high levels seen in non-spawning fish captured from contaminated areas, it can be hypothesized that female English sole exposed to substantial levels of contaminants might be at least partially excluded from the spawning population.

Liver neoplasms are rare in young wild fish; therefore, it is essential to consider toxicopathic liver lesions that occur earlier than neoplasms as biomarkers of contaminant exposure in studies where targeted or available fish are not adults. Also some exposure effects may be more reliably assessed in younger fish that have not yet migrated extensively. We addressed these issues by histologically examining subadult English sole, rock sole, and starry flounder captured from eight sites in Puget Sound, and by measuring fluorescent aromatic compounds (FACs) in bile and polychlorinated hiphenyls (PCBs) in liver. Neoplasms were detected rarely; higher prevalences of preneoplastic, regenerative, and degenerative lesions were detected in most species, primarily from contaminated sites, including hydropic vacuolation of hepatocytes and biliary cells in rock sole and starry flounder. These nomeoplastic lesion types are inducible in fish by exposure to various toxicants in the laboratory, and/or (in the case of hydropic vacuolation) are associated with contaminant exposure and liver neoplasia in adult wild fish. Prevalences of these ‘early’ lesions were significantly higher at the more contaminated sites, as determined by several statistical approaches, including logistic regression. Moreover, prevalences of most lesions in all three species correlated with mean bile FAC levels at the sites, and prevalences of some lesions in English sole correlated with liver PCB levels. The present findings further support the utility of certain liver lesions other than neoplasms as early indicators of biological damage in subadult as well as adult fish exposed to xenobiotics in the marine environment.

1. Effects of a contaminated sediment extract on the disposition and metabolism of 3H-17β-estradiol in juvenile English sole and on plasma levels of endogenous estradiol in gravid females were examined. 2. Plasma estradiol levels were reduced in both extract-treated juvenile and gravid sole compared to controls, and in juvenile sole this decrease preceeded an increase in bile levels of 3H-17β-estradiol. 3. Extract treatment had no significant effect on types or proportions of 17β-estradiol metabolites or levels of related hepatic enzymes. 4. Thus, lower plasma estradiol levels in extract-treated sole were apparently due to alterations in physiological factors (e.g. increased bile flow) and not to enhanced metabolism of 17β-estradiol.

Deep inspiration preceding Valsalva maneuver and rapid expiration immediately following it (DIVE) enhance venous blood flow on color Doppler flow imaging (CDI). The effect of DIVE was assessed in 115 consecutive lower extremity examinations. Of these, 95 or 115 (83%) had negative CDI sonograms, and 20 of 115 (17%) had partially (six of 115) or completely (14 of 115) occluding deep vein thrombosis. DIVE enhanced venous blood flow in 68% of the negative cases, resulting in transient venous distention, and/or more complete color filling, and/or greater spectral flow velocities. The 14 cases with completely occluding thrombi showed no response to DIVE. Six cases with partially occluding thrombi showed moderate to mild response to DIVE, with improved color delineation of the residual patent lumen around the thrombus. The authors conclude that DIVE facilitates deep venous CDI, especially when compression cannot be used to augment venous flow.

Eagle Harbor is a designated Superfund site due to high sediment concentrations of creosote-derived polycyclic aromatic hydrocarbons (PAHs) released chronically from a nearby creosoting facility. Studies with English sole Pleuronectes vetulus from this site (1984–1986) demonstrated high prevalences of toxicopathic liver lesions including neoplasms in resident sole. Inducibility of neoplasia-related lesions by injections of a PAH-rich fraction extracted from Eagle Harbor sediment has also been shown. Further studies (1986–1988) also sampled starry flounder Platichthys stellatus and rock sole Lepidopsetta bilineata, and incorporated biomarkers of PAH exposure and effect, including hepatic CYP1A expression, biliary fluorescent aromatic compounds (FACs), and hydrophobic DNA adducts in liver. Hepatic lesion prevalences and biomarker values in these species from Eagle Harbor were among the highest found at Puget Sound sites. A cap of uncontaminated sediment was placed (September 1993–March 1994) over the most contaminated portions of Eagle Harbor in an attempt to sequester PAH-contaminated sediments. Lesion prevalences and biomarker values just before capping began were generally reduced compared to historical data, consistent with creosoting facility closure and site-based source controls. Similar data from fish collected immediately after capping, and, at 3, 6, 12, 16, 19, 21, 31, 44, and 49 months after cap completion, are presented to determine the efficacy of the capping in ameliorating PAH exposure and associated effects in resident flatfish species.

Concentrations of selected anthropogenic chemical contaminants and levels of pollution-related biological effects were measured during three consecutive years (1990–1992) in hardhead catfish (Arius felis), Gulf killifish (Fundulus grandis), longnose killifish (F. majalis), and red drum (Scieaenops ocellatus) from 12 subtidal and intertidal sites in Tampa Bay and nearby Sarasota Bay. Each species was collected from at least four sites. Compared to nonindustrialized sites, concentrations of PCBs, DDTs, and alpha-chlordane in liver, and of fluorescent aromatic compounds in bile, were highest in fish from sites in or near Hillsborough Bay, the most industrialized portion of Tampa Bay. The results of analyses for two biochemical markers of contaminant-induced effects in fish, hepatic cytochrome P4501A activities and levels of hepatic DNA adducts, also showed the highest levels to be in all four fish species from sites in the vicinity of Hillsborough Bay. Liver lesions, considered to be pollution-associated in several other bottom-feeding fish species, were found in hardhead catfish and longnose killifish, exclusively from sites in Hillsborough Bay. Overall, concentrations of selected contaminants and their derivatives in the four target fish species generally reflected concentrations of these contaminants found in sediment. The biochemical and histopathological responses demonstrated that chemical contaminant concentrations in the vicinity of Hillsborough Bay are sufficiently high to cause adverse effects in indigenous fish species. The results, collectively, showed that the extent of contaminant exposure and biological effects in fish from sites in Tampa Bay were low to moderate compared to more urbanized coastal sites of the United States. *** DIRECT SUPPORT *** A01BY073 00009

English sole ( Parophrys vetulus ) were exposed to environmentally realistic levels of sediment-associated 3 H-benzo[a]pyrene (3 μg BaP/g sediment, dry weight) and 14 C-Aroclor 1254 (1 μg PCBs/g), separately and together, for up to 10 days. BaP and its metabolites in tissues reached steady-state concentrations by the first day of the exposure, whereas PCBs did not approach steady-state concentrations until the tenth day of exposure. Simultaneous exposure of sole to BaP and PCBs, relative to separate exposure to the xenobiotics, significantly increased the concentrations of BaP-derived radioactivity and decreased the concentrations of PCB-derived radioactivity in some tissues and bile. Accumulation of PCB-derived radioactivity, estimated as the burden in tissues, was significantly greater (4- to 13-fold) than that of BaP-derived radioactivity throughout the experiments and regardless of the type of exposure. The rank of the concentrations of PCB-derived radioactivity in tissues and bile was: bile ∼ liver > brain > kidney ∼ gill > skin ∼ blood ∼ muscle and for BaP-derived radioactivity the order was: bile > liver > gill > kidney > skin ∼ blood > muscle > brain. BaP-derived radioactivity in liver and bile was present primary (85–99%) as metabolites, whereas PCB-derived radioactivity was present equally as parent compounds and metabolites in bile and primarily (98 %) as parent compounds in liver. Hydrolysis of bile from PCB/BaP-exposed sole with β-glucuronidase/arylsulfatase released 35 % of the BaP-derived radioactivity and 32 % of the PCB-derived radioactivity as primary metabolites. A much higher proportion of the BaP-derived radioactivity (64 %) than the PCB-derived radioactivity (13 %) in bile was unaffected by the enzyme treatment, indicating differences in the conjugation of the primary metabolites of PCBs and BaP. The results suggest that in contaminated environments the tissue to sediment concentration ratios for PCBs would exceed that for BaP and its metabolites; however, BaP would be continually absorbed and metabolized by sole to potentially carcinogenic and mutagenic compounds. In addition, the tissue levels of these toxic compounds may be increased by simultaneous exposure to PCBs.

Benzo[a]pyrene became bound to the hepatic DNA in juvenile English sole ( Parophrys vetulus ) force fed tritiated benzo[a]pyrene. No statistically significant change was observed in the level of the binding from 16 h to 2 wk after the single exposure. Specific activities of binding were similar for both DNA and protein. Moreover, a binding index was calculated to represent the number of benzo[a]pyrene molecules bound per 106 nucleotides after administration of a theoretical dose of 1 mmole of hydrocarbon per kg body weight. The value for English sole liver DNA was of the same order of magnitude as the values reported for mouse skin and mammary gland in which benzo[a]pyrene is carcinogenic.