John E. Stein

Bioaccumulation of Polycyclic Aromatic Hydrocarbons by Marine Organisms

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the marine environment, occurring at their highest environmental concentrations around urban centers. While they can occur naturally, the highest concentrations are mainly from human activities, and the primary sources are combustion products and petroleum. Two factors, lipid and organic carbon, control to a large extent the partitioning behavior of PAHs in sediment, water, and tissue; the more hydrophobic a compound, the greater the partitioning to these phases. These two factors, along with the octanol-water partition coefficient, are the best predictors of this partitioning and can be used to determine PAH behavior and its bioavailability in the environment. It is well known that the lipid of organisms contains the highest levels of hydrophobic compounds such as PAHs, and that organic carbon associated with sediment or dissolved in water can have the greatest influence on PAH bioavailability. Partitioning of combustion-derived PAHs between water and sediment may be much less than predicted, possibly because associations with particles are much stronger than expected. This reduced partitioning may produce erroneous results in predicting bioaccumulation where uptake from water is important. Accumulation of PAHs occurs in all marine organisms; however, there is a wide range in tissue concentrations from variable environmental concentrations, level and time of exposure, and species ability to metabolize these compounds. PAHs generally partition into lipid-rich tissues, and their metabolites can be found in most tissues. In fish, liver and bile accumulate the highest levels of parent PAH and metabolites; hence, these are the best tissues to analyze when determining PAH exposure. In invertebrates, the highest concentrations can be found in the internal organs, such as the hepatopancreas, and tissue concentrations appear to follow seasonal cycles, which may be related to variations in lipid content or spawning cycles. The major route of uptake for PAHs has been debated for years. For the more water-soluble PAHs, it is believed that the main route of uptake is through ventilated water and that the more hydrophobic compounds are taken in mainly through ingestion of food or sediment. There are many variables, such as chemical hydrophobicity, uptake efficiency, feeding rate, and ventilatory volume, which may affect the outcome. The route of uptake may be an important issue for short-term events; however, under long-term exposure and equilibrium conditions between water, prey, and sediment, the route of uptake may be immaterial because the same tissue burdens will be achieved regardless of uptake routes.(ABSTRACT TRUNCATED AT 400 WORDS)

Toxicopathic hepatic lesions in subadult English sole (pleuronectes vetuls) from Puget Sound, Washington, USA: Relationships with other biomarkers of contaminant exposure

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.

Influence of life-history parameters on organochlorine concentrations in free-ranging killer whales (Orcinus orca) from Prince William Sound, AK.

Certain populations of killer whales (Orcinus orca) have been extensively studied over the past 30 years, including populations that use Puget Sound, WA, the inside waters of British Columbia, Southeastern Alaska and Kenai Fjords/Prince William Sound, Alaska. Two eco-types of killer whales, transient and resident, occur in all of these regions. These eco-types are genetically distinct and differ in various aspects of morphology, vocalization patterns, diet and habitat use. Various genetic and photo-identification studies of eastern North Pacific killer whales have provided information on the male-female composition of most of these resident pods and transient groups, as well as the approximate ages, reproductive status and putative recruitment order (birth order) of the individual whales. Biopsy blubber samples of free-ranging resident and transient killer whales from the Kenai Fjords/Prince William Sound, AK region were acquired during the 1994-1999 field seasons and analyzed for selected organochlorines (OCs), including dioxin-like CB congeners and DDTs. Concentrations of OCs in transient killer whales (marine mammal-eating) were much higher than those found in resident animals (fish-eating) apparently due to differences in diets of these two killer whale eco-types. Certain life-history parameters such as sex, age and reproductive status also influenced the concentrations of OCs in the Alaskan killer whales. Reproductive female whales contained much lower levels of OCs than sexually immature whales or mature male animals in the same age class likely due to transfer of OCs from the female to her offspring during gestation and lactation. Recruitment order also influenced the concentrations of OCs in the Alaskan killer whales. In adult male residents, first-recruited whales contained much higher OC concentrations than those measured in non-first-recruited (e.g. second recruited, third recruited) resident animals in the same age group. This study provides baseline OC data for free ranging Alaskan killer whales for which there is little contaminant information.

Formation and persistence of benzo[a]pyrene-diolepoxide-DNA adducts in liver of English sole (Parophrys vetulus)

The formation of DNA adducts from the carcinogenic environmental pollutant benzo[a]pyrene (BaP) was investigated in liver of English sole (Parophrys vetulus), a fish species that exhibits a high prevalence of liver neoplasms in several polycyclic aromatic hydrocarbon (PAH)-contaminated areas of Puget Sound, WA. Analysis by the 32P-postlabeling assay of hepatic DNA digests from English sole exposed parenterally to BaP showed the presence of BaP-diol epoxide (BaPDE)-DNA adducts. When English sole were injected with 2-15 mg BaP/kg body wt., one major adduct was detected and was identified as the anti-BaPDE-DNA adduct. Moreover, in English sole sampled at 1, 28 and 60 days post-exposure to 15 mg BaP/kg body wt., there was no significant change in the level of the anti-BaPDE-DNA adduct. The autoradiographs of 32P-labeled hepatic DNA digests from fish exposed to 100 mg BaP/kg body wt. showed an elongated spot suggesting the presence of more than one adduct. Chromatography on large polyethyleneimine sheets (20 x 20 cm) showed 2 spots with the same chromatographic characteristics as those of syn- and anti-BaPDE-deoxyguanosine adduct standards. Mild acid hydrolysis of hepatic DNA of English sole, exposed to 100 mg BaP/kg body wt., also revealed the presence of tetrols derived from both anti- and syn-BaPDE, thus confirming the presence of syn- and anti-BaPDE. In fish exposed to 2-100 mg BaP/kg body wt., a linear (0.996) dose response for anti-BaPDE-DNA adduct formation was observed. The results from this study offer the first direct evidence for the formation of the suspected ultimate carcinogen, BaPDE, in liver of English sole exposed to BaP in vivo and thus further support the hypothesis that exposure to PAHs is an important factor in the etiology of hepatic neoplasms in English sole from contaminated sites.

DISPOSITION OF XENOBIOTIC CHEMICALS AND METABOLITES IN MARINE ORGANISMS

Studies with several bottom fish species from urban waterways show that of the identified xenobiotic chemicals in bottom sediments, polycylic aromatic hydrocarbons (PAHs) are the most strongly associated with the prevalence of liver lesions, including neoplasms. Accordingly, there is concern about the transfer of contaminants, such as PAHs, from aquatic species to humans. Because PAHs exert their toxicity only after being biotransformed, increasing attention has been focused on the ability of aquatic organisms to metabolize these chemicals. Overall, the results of both laboratory and field studies show that generally low levels (nanograms per gram wet weight) of a few low molecular weight PAHs may be present in edible tissue of fish from contaminated areas and that high molecular weight PAHs, such as the carcinogen benzo(a)pyrene, will rarely be detected because of extensive metabolism. Additionally, the results from a few studies suggest that even though interactions between xenobiotics can affect both biochemical and physiological systems to alter the disposition of PAHs in fish, these interactions do not markedly change the relative proportions of metabolites to parent PAH in tissues. Thus, these studies clearly demonstrate that to obtain some insight into the questions of whether there is any risk to human health from consuming fish and crustaceans from urban areas, techniques must be developed that measure metabolites of carcinogens, such as PAHs, in edible tissue. Initial attempts may focus on semiquantitative methods that permit rapid assessment of the level of metabolites in edible tissues of fish and crustaceans from many urban areas.(ABSTRACT TRUNCATED AT 250 WORDS) ImagesFIGURE 4.FIGURE 4.FIGURE 4.

Accumulation and dose-response of hepatic DNA adducts in English sole (Pleuronectes vetulus) exposed to a gradient of contaminated sediments

Abstract 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.

Overview of studies on liver carcinogenesis in English sole from Puget Sound; evidence for a xenobiotic chemical etiology II: Biochemical studies

The levels of aromatic hydrocarbons in sediments of Puget Sound, Washington, are positively correlated with the prevalence of hepatic neoplasms and related lesions in English sole (Parophrys vetulus). To investigate the biochemical processes involved in chemical carcinogenesis in fish from Puget Sound, we have studied the uptake, activation, and detoxication of polycyclic aromatic hydrocarbons (PAHs) in English sole, and have compared these data to PAH metabolism in a related species, starry flounder (Platichthys stellatus), which shows a lower prevalence of hepatic neoplasms than sole. The results of both laboratory and field studies show that sediment-associated PAHs are biologically available to both flatfish species, and that both species accumulate similar levels of PAHs. Analyses of hepatic DNA from sole using the 32P-postlabeling technique indicate that xenobiotic chemicals were adducted to hepatic DNA of fish from the contaminated sites but not to the DNA of fish from reference sites. Studies of the ability of English sole and starry flounder to metabolize benzo(a)pyrene (BaP) and bind reactive BaP intermediates to hepatic DNA indicate that biochemical differences in the metabolism of carcinogenic PAHs may explain, at least in part, the apparent lower susceptibility of starry flounder than English sole to chemically induced hepatocarcinogenesis.

Inducibility of spawning and reproductive success of female english sole (parophrys vetulus) from urban and nonurban areas of puget sound, Washington

Abstract Vitellogenic female English sole were sampled from four areas in Puget Sound that varied in the nature and degree of chemical contamination. The fish were then injected with an analogue of Luteinizing Hormone Releasing Hormone (LHRH) in the laboratory to induce spawning. Ability to spawn, time to spawn, larval viability, and initial concentrations of plasma estradiol and vitellogenin [measured as alkaline-labile protein associated phosphate (ALP)] were assessed. In general, low initial plasma estradiol and ALP concentrations and subsequent reproductive impairment were most common in English sole from sites where levels of sediment contaminants [e.g. polycyclic aromatic hydrocarbons and polychlorinated biphenyls (PCB)] and measures of contaminant exposure in fish (hepatic PCB concentrations and aryl hydrocarbon hydroxylase activity) were highest, and where pollution-associated liver lesions in fish (i.e. neoplasms, foci of cellular alterations, specific degeneration/necrosis, and storage disorders) were most prevalent. Additionally, the time to spawn was found to be inversely correlated with initial plasma estradiol concentrations. Spawning success was found to be positively correlated with initial plasma estradiol and ALP concentrations as determined by logistic regression analysis; nearly 62% of the variability in the spawning response could be accounted for by these factors. Fertilization success was also found to be positively correlated with initial ALP concentrations, whereas fish captured from contaminated sites produced lower proportions of normal larvae. Overall, these findings suggest that contaminant exposure may result in poor reproductive success of female English sole. This may be related to a hormone imbalance or to slower ovarian development (non-synchronous timing) of female English sole from contaminated sites.

Assessing the effects of anthropogenic stressors on Puget Sound flatfish populations

Puget Sound is an estuary in the northwestern United States which serves as the habitat for a number of recreationally and commercially important species of flatfish. Over the past 100 years, there has been substantial urban and industrial development within this region, resulting in heavy inputs of chemical contaminants at selected sites, as well as significant loss or alteration of marine habitat. Studies show that feral flatfish in Puget Sound are experiencing a range of biological effects due to chemical contaminant exposure, including reproductive dysfunction, altered immune competence, and development of toxicopathic diseases, and there is some evidence of reduced survival in fish from urban areas of Puget Sound from increased infectious and toxicopathic disease. Puget Sound sole are also subject to other anthropogenic stressors, such as fishing pressure or alteration of nearshore nursery habitats. The cumulative impact of these stressors on flatfish abundance in Puget Sound, however, is poorly understood. In a series of field and laboratory studies, we determined vital rates and other life history parameters in English sole (Pleuronectes vetulus) subpopulations from urban and non-urban sites in Puget Sound, and are using this information to estimate potential population level impacts of anthropogenic stressors, with age and stage-based Leslie-matrix models. Initial results suggest that declines in the fecundity component of the model, as observed in field studies of fish from contaminated sites, could reduce the size of sub-populations in these areas if the loss of recruits is not offset by density-dependent changes in recruitment, immigration, or other compensating mechanisms. Studies on flatfish species from a variety of sites in Europe and North America suggest that contaminant-related disease and reproductive impairment are widespread in this group of fish, although substantial differences in sensitivity have been observed, even among closely related species. Comparative studies with a variety of Pleuronectid species will enable us to better evaluate the risk posed by anthropogenic stressors to flatfish, and contribute to improved assessment and management of this important fisheries resource.

Increased Susceptibility of Juvenile Chinook Salmon to Vibriosis after Exposure to Chlorinated and Aromatic Compounds Found in Contaminated Urban Estuaries

Abstract Saltwater-adapted juvenile chinook salmon Oncorhynchus tshawytscha exposed to aromatic and chlorinated compounds, representative of contaminants found in urban estuaries in Puget Sound, have a higher susceptibility to vibriosis than do fish exposed only to the solvent vehicle. Susceptibility to vibriosis was assessed by examining the percent cumulative mortality of the salmon after exposure to the bacterial pathogen Vibrio anguillarum. The aromatic and chlorinated compounds examined consisted of a sediment extract from the Hylebos Waterway that was enriched in butadienelike compounds (chlorinated-enriched Hylebos Waterway sediment extract (CHWSE)), a model mixture of polycyclic aromatic hydrocarbons (PAHs), a polychlorinated biphenyl mixture (Aroclor 1254), hexachlorobutadiene (HCBD), and 7,12-dimethylbenz(a)anthracene (DMBA). Two trials were conducted. In trial l, the percent cumulative mortality of juvenile chinook salmon exposed to V. anguillarum after receiving either CHWSE, HCBD, or the mode...