Mary R. Arkoosh

Effect of Pollution on Fish Diseases: Potential Impacts on Salmonid Populations

Abstract Anthropogenic factors have contributed to the precipitous decline of wild Pacific salmon stocks, although the mechanisms and processes at work are largely unknown. Pollution may be one of these factors. Sediments in estuaries are known to act as repositories for contaminants, and estuaries are important habitats for ocean- and river-migrating salmon. We have shown that juvenile salmon Oncorhynchus spp. and their prey bioaccumulate chlorinated hydrocarbons and aromatic hydrocarbons—important classes of toxic xenobiotics. Furthermore, we have shown that exposure to these pollutants can lead to immunosuppression and increased disease susceptibility in juvenile salmon. Whether pollution influences natural disease outbreaks in host populations, including salmon, is currently unknown. It is postulated that the occurrence of disease depends on the interaction of the host, the environment, and the pathogen. Absence of pathogens would reduce the potential for adverse environments to influence disease outb...

Suppression of B-cell mediated immunity in juvenile chinook salmon (Oncorhynchus tshawytscha) after exposure to either a polycyclic aromatic hydrocarbon or to polychlorinated biphenyls

Juvenile chinook salmon (Oncorhynchus tshawytscha) were injected intraperitoneally with either the polycyclic aromatic hydrocarbon, 7,12-dimethylbenz[a]anthracene (DMBA)1 or with the commercial polychlorinated biphenyl (PCB) mixture, Aroclor 1254, to assess effects on the B-cell mediated immune response. B-cell mediated immunity was assessed by examination of the primary and secondary plaque-forming cell (PFC) responses of anterior kidney and splenic leukocytes to a T-independent antigen, TNP-keyhole limpet hemocyanin (TNP-KLH). Salmon exposed to DMBA at dosages of 20% or 1% of the 96 hr LD50 (12.7 mg and 0.6 mg/kg of salmon, respectively) or to PCBs at a dosage of 20% of the 96 hr LD50 (54.0 mg/kg of salmon) exhibited a suppressed PFC response. The secondary PFC response of anterior kidney and splenic leukocytes to both antigens and the primary splenic PFC response to TNP-LPS were suppressed in salmon exposed to either DMBA or PCBs. However, only the primary PFC response of anterior kidney leukocytes to TNP-LPS was suppressed in salmon exposed to PCBs and no suppression of this response was observed in salmon exposed to DMBA. Neither anterior kidney or splenic leukocytes from salmon exposed to DMBA or PCBs showed an altered primary PFC response to the T-dependent antigen, TNP-KLH. These results suggest that B-cell mediated immunity in salmon is suppressed by known mammalian immunosuppressants and that suppression of the PFC response observed previously in salmon from an urban estuary may be due to contaminant exposure.

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...

Suppression of immunological memory in juvenile chinook salmon (Oncorhynchus tshawytscha) from an urban estuary

In this present study, juvenile chinook salmon were collected from a contaminated urban estuary, the Duwamish Waterway, as well as from a non-urban estuary, the Nisqually River estuary, to determine if exposure to toxic chemicals affects immunocompetency. Juvenile chinook salmon were also sampled from the two hatcheries that release salmon into these estuaries. The ability of anterior kidney (AK) and splenic (SP) leucocytes from primed and unprimed juvenile salmon from the Duwamish Waterway, Nisqually River estuary, and their respective hatcheries to produce a primary and secondary in vitro plaque-forming cell (PFC) response to the hapten, trinitrophenyl (TNP) was examined. Trinitrophenyl-keyhole limpet haemocyanin (TNP-KLH) was added in vitro to AK leucocytes or as TNP-lipopolysaccharide (TNP-LPS) to both AK and SP leucocytes. The primary AK and SP plaque-forming cell response to TNP in salmon from the estuaries or hatcheries was not significantly different. Primed AK leucocytes from salmon collected from the hatcheries and the non-urban estuary were able to produce a heightened secondary response to TNP-KLH; in contrast, primed AK leucocytes from salmon collected from the urban estuary were unable to produce a secondary PFC response to TNP-KLH. Anterior kidney leucocytes from salmon collected from all four areas were able to produce a heightened secondary PFC response to TNP-LPS. However, the AKs plaque-forming cell response generated in primed chinook salmon collected from the urban estuary to TNP-LPS was significantly lower than that produced in salmon from the hatchery. This suppressed PFC response in primed AK cells to TNP-LPS was not observed in primed AK leucocytes from salmon collected from the non-urban estuary and its hatchery. These results suggest that the cells involved in generating immunological memory to TNP-KLH and TNP-LPS were affected in salmon exposed to contaminants in the urban estuary, with cells which generate memory to TNP-KLH being more affected. The PFC responses of primed SP leucocytes from salmon collected from the Duwamish Waterway and Nisqually River estuaries, stimulated in vitro with TNP-LPS were not significantly different. The consequence of suppressed immunological memory in disease resistance of juvenile salmon is currently unknown.

Disease susceptibility of salmon exposed to polybrominated diphenyl ethers (PBDEs)

The health effects of the flame retardant polybrominated diphenyl ethers (PBDEs) in fish are not well understood. To determine the potential effects of this ubiquitous contaminant class on fish health, juvenile subyearling Chinook salmon (Oncorhynchus tshawytscha) were fed a diet that reflected the PBDE congeners found in the stomach contents of subyearling Chinook salmon collected from the highly urbanized and industrialized lower Willamette River in the Columbia River Basin of North America. The diet, consisting of five PBDE congeners (BDE-47, BDE-99, BDE-100, BDE-153 and BDE-154), was fed to the salmon at 2% of their body weight in food per day for 40 days. Two concentrations of the diet (1x and 10x PBDE) were fed to the salmon. The 1x PBDE diet reflected the concentration of PBDEs (190 ng PBDEs/g food) found in the stomach contents of juvenile subyearling Chinook salmon; the 10x diet was prepared at 10 times that concentration. The fish were then exposed to the marine bacterial pathogen Listonella anguillarum to assess susceptibility to infectious disease. Juvenile Chinook salmon fed the 1x PBDE diet were more susceptible to L. anguillarum than salmon fed the control diet. This suggests that juvenile salmonids in the lower Willamette River exposed to PBDEs may be at greater risk for disease than nonexposed juvenile salmonids. In contrast, salmon that consumed the 10x PBDE diet were not more susceptible to the pathogen than salmon fed the control diet. The mechanisms for the dichotomous results observed in disease susceptibility between salmon fed the 1x and 10x PBDE diets are currently not known but have also been observed in other species exposed to PBDEs with respect to immune function.

Cumulative Effects of Natural and Anthropogenic Stress on Immune Function and Disease Resistance in Juvenile Chinook Salmon

Abstract Previous studies have shown that juvenile chinook salmon Oncorhynchus tshawytscha exposed in the field or the laboratory to polychlorinated biphenyls (PCBs), an anthropogenic stressor, are immunosuppressed. It is not known whether simultaneous exposure to natural stressors can increase this immunosuppression. To examine the effects of natural and anthropogenic stressors on immune function, we infected juvenile chinook salmon with metacercariae of the trematode Nanophyetus salmincola by exposing the fish to infected freshwater snails Juga plicifera. Infected (>300 metacercariae per fish) and noninfected salmon were then injected with either the commercial PCB mixture Aroclor 1254 or an acetone–emulphor carrier. B cell function was examined by in vitro hemolytic plaque-forming cell (PFC) assay. Nanophyetus salmincola infection resulted in significantly lower anterior kidney primary PFCs and lower splenic secondary PFCs. The combination of N. salmincola infection and Aroclor 1254 exposure caused a l...

Survey of Pathogens in Juvenile Salmon Oncorhynchus Spp. Migrating through Pacific Northwest Estuaries

Abstract Although the adverse impact of pathogens on salmon populations in the Pacific Northwest is often discussed and recognized, little is currently known regarding the incidence and corresponding significance of delayed disease-induced mortalities. In the study reported herein, we surveyed the presence and prevalence of selected micro- and macroparasites in out-migrant juvenile coho salmon Oncorhynchus kisutch and Chinook salmon O. tshawytscha from 12 coastal estuaries in the Pacific Northwest over a 6-year period (1996–2001). The major finding of this study was the widespread occurrence of pathogens in wild salmon from Pacific Northwest estuaries. The six most prevalent pathogens infecting both juvenile Chinook and coho salmon were Renibacterium salmoninarum, Nanophyetus salmincola, an erythrocytic cytoplasmic virus (erythrocytic inclusion body syndrome or erythrocytic necrosis virus), and three gram-negative bacteria (Listonella anguillarum, Yersinia ruckeri, and Aeromonas salmonicida). The most pre...

Biomarker responses and disease susceptibility in juvenile rainbow trout Oncorhynchus mykiss fed a high molecular weight PAH mixture

Juvenile rainbow trout were fed a diet containing an environmentally relevant mixture of 10 high molecular weight polycyclic aromatic hydrocarbons (PAHs) at a dose of 0.66 or 7.82 µg PAH · g fish(-1) · d(-1). At 3, 7, 14, and 28 d, biomarkers of aryl hydrocarbon receptor activation (AHR), hepatic microsomal ethoxyresorufin-O-deethylase (EROD) activity, and cytochrome P4501A (CYP1A)-associated staining increased 14- to 26-fold and 6- to 14-fold, respectively, in fish fed 7.82 µg PAH · g fish (-1) · d(-1). Cytochrome P4501A-associated staining increased 2- to 9-fold on days 3, 7, and 28 in fish fed 0.66 µg PAH · g fish(-1) · d(-1). Bile fluorescent aromatic compounds served as a biomarker of exposure and confirmed that PAH exposure was consistent over 50 d. DNA damage in blood cells, protein oxidation, and lipid peroxidation in the kidney were biomarkers of oxidative stress and all increased in fish fed 7.82 µg PAH · g fish(-1) · d(-1). Fish fed 0.66 µg PAH · g fish(-1) · d(-1) had elevated DNA damage in blood cells but increased protein oxidation or lipid peroxidation in the kidney were not observed. Challenge with Aeromonas salmonicida, at lethal concentration (LC) 20, decreased survival in fish previously fed either 0.66 µg PAH · g fish(-1) · d(-1) or 7.82 µg PAH · g fish(-1) · d(-1) relative to fish fed the control diet. In general, biomarkers of both AHR activation and oxidative stress peaked at 3 to 14 d then declined at 28 to 50 d of PAH exposure and an increase in susceptibility to disease was observed at 50 d. These results link PAH exposure to biomarker responses that may be useful as early indicators of population level responses, such as mortality resulting from an increase in disease susceptibility.

4 - Effects on Fish of Polycyclic Aromatic HydrocarbonS (PAHS) and Naphthenic Acid Exposures

Polycyclic aromatic hydrocarbons (PAHs) are derived from both natural and anthropogenic sources and are released from a wide range of industries and everyday activities. Unlike many other organic chemical contaminants that are manufactured and regulated, PAHs continue to be released on a global scale because of the worlds dependence on fossil fuels. This chapter briefly reviews the transformation of PAHs in the aquatic environment, highlighting their efficient metabolism in fish and focuses on evidence that links PAH exposure to a wide range of biological dysfunctions in fish. These dysfunctions include neoplasia, reduced reproductive success and other types of endocrine disruption, immunotoxicity, postlarval growth and somatic condition, transgenerational impacts, and finally, recent findings showing that the embryonic development of fish is severely affected by extremely low concentrations of PAH exposure. A brief review of the effects of naphthenic acids on fish is also included because these compounds are increasingly recognized as major factors in the toxicity of process waters from a variety of petroleum sources, most notably the immense oil sands deposits found in Alberta, Canada. It is recommended that future research for understanding and mitigating the effects of PAHs in fish and associated aquatic ecosytems should include the following. • Using models to link molecular-up-to-organismal level effects to population-relevant metrics. • Building on current case studies demonstrating the effects of PAHs on the health of fish in their natural environments in order to derive regulatory approaches. Current approaches that rely on biota to sediment accumulation factors (BSAF) will not work with contaminants that are efficiently metabolized by species of concern. • Focusing considerable resources on better analytical chemistry for both PAHs and naphthenic acids. Currently, our ability to understand and mitigate the effects of these substances is heavily limited by constraints in analysis.

Reduced cytochrome P4501A activity and recovery from oxidative stress during subchronic benzo[a]pyrene and benzo[e]pyrene treatment of rainbow trout

This study assessed the role of aryl hydrocarbon receptor (AHR) affinity, and cytochrome P4501A (CYP1A) protein and activity in polyaromatic hydrocarbon (PAH)-induced oxidative stress. In the 1-100nM concentration range benzo[a]pyrene (BaP) but not benzo[e]pyrene (BeP) competitively displaced 2nM [(3)H]2, 3, 7, 8-tetrachloro-dibenzo-p-dioxin from rainbow trout AHR2α. Based on appearance of fluorescent aromatic compounds in bile over 3, 7, 14, 28 or 50days of feeding 3μg of BaP or BeP/g fish/day, rainbow trout liver readily excreted these polyaromatic hydrocarbons (PAHs) and their metabolites at near steady state rates. CYP1A proteins catalyzed more than 98% of ethoxyresorufin-O-deethylase (EROD) activity in rainbow trout hepatic microsomes. EROD activity of hepatic microsomes initially increased and then decreased to control activities after 50days of feeding both PAHs. Immunohistochemistry of liver confirmed CYP1A protein increased in fish fed both PAHs after 3days and remained elevated for up to 28days. Neither BaP nor BeP increased hepatic DNA adduct concentrations at any time up to 50days of feeding these PAHs. Comet assays of blood cells demonstrated marked DNA damage after 14days of feeding both PAHs that was not significant after 50days. There was a strong positive correlation between hepatic EROD activity and DNA damage in blood cells over time for both PAHs. Neither CYP1A protein nor 3-nitrotyrosine (a biomarker for oxidative stress) immunostaining in trunk kidney were significantly altered by BaP or BeP after 3, 7, 14, or 28days. There was no clear association between AHR2α affinity and BaP and BeP-induced oxidative stress.