NOAA Technical Memorandum NMFS-NWFSC-8 Contaminant Exposure and Associated Biological Effects in Juvenile Chinook Salmon (Oncorhynchus tshawytscha) from Urban and Nonurban Estuaries of Puget Sound Usha Varanasi, Edmundo Casillas, Mary R. Arkoosh, Tom Hom, David A. Misitano, Donald W. Brown, Sin-Lam Chan, Tracy K. Collier, Bruce B. McCain, and John E. Stein National Marine Fisheries Service Northwest Fisheries Science Center Environmental Conservation Division 2725 Montlake Blvd. E., Seattle, WA 98112 April 1993 U.S. DEPARTMENT OF COMMERCE Ronald H. Brown, Secretary National Oceanic and Atmospheric Administration John A. Knauss, Administrator National Marine Fisheries Service William W. Fox, Jr., Assistant Administrator for Fisheries

EXECUTIVE SUMMARY

This report presents and interprets the results of chemical, biochemical, and biological studies on juvenile chinook salmon (Oncorhynchus tshawytscha) outmigrating from urban and nonurban estuaries of Puget Sound, Washington. These studies were conducted between 1989 and 1991 by the National Marine Fisheries Service (NMFS) of the National Oceanic and Atmospheric Administration (NOAA) with sponsorship from the Environmental Protection Agency, Region 10 and NMFS/NOAA. The objective of these studies was to determine the degree of chemical exposure to juvenile chinook salmon as they migrate through urban-associated compared to nonurban estuaries and to evaluate the effects of chemical contaminant exposure on these animals.Urban estuaries studied included the Duwamish Waterway entering Elliott Bay near Seattle, Washington, the Puyallup River entering Commencement Bay near Tacoma, Washington, and the Snohomish River entering Port Gardner Bay near Everett, Washington. Sediments in these aquatic urban environments are known to be highly contaminated, although the sediments from the Duwamish Waterway and the Puyallup estuary, particularly in the waterways, are significantly more contaminated than sediments in the Snohomish estuary. Additionally, juvenile chinook salmon from the Nisqually River estuary, a minimally contaminated nonurban estuary, and from the respective hatcheries of each of the waterways and rivers mentioned above were sampled as reference fish.

The chemical indicators of contaminant exposure include levels of hepatic polychlorinated biphenyls (PCBs) and biliary levels of fluorescent aromatic compounds (FACs), which are semiquantitative measures of exposure to aromatic hydrocarbons (AHs). Stomach contents of juvenile salmon were also analyzed for selected AHs and chlorinated hydrocarbons (CHs) to assess the importance of diet as a possible route of uptake of xenobiotics from polluted estuaries. The study also included measurement of early physiological and biochemical (bioindicator) responses to chemical contaminant exposure. These bioindicators have been shown to reflect the degree of exposure to particular contaminants as well as to indicate some of the biological consequences of chemical exposure. In addition to the chemical indicators, biochemical measures of contaminant-induced responses of the hepatic enzyme system and genotoxic damage were assessed. Measures of the hepatic cytochrome P-450 system included hepatic aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin-O-deethylase (EROD) activities. Hepatic levels of DNA-xenobiotic adducts, detected by ³²P-postlabeling, were measured as a bioindicator of genetic damage. Biological parameters that demonstrate the effects of chemical contaminant exposure were also measured. Initially, considerable effort was expended in understanding and enhancing our fish maintenance and husbandry practices. This allowed us to maintain healthy fish in saltwater for periods of months, which was critical for studies on depuration and measurement of biological effects. Biological effects that were monitored included effects on immune function and effects on growth and long-term survival of juvenile salmon. Alterations in immune function have been shown to be a sensitive index on the effects of contaminants. Suppression of immune function can seriously affect the ability of salmon to face the stresses posed in the saltwater environment. Similarly, effects on growth and survival are more classical measurements on the effects of chemical contaminants; negative effects are more easily interpreted as detrimental to the individual and thus eventually to the population.

Significant Findings

• Concentrations of chemical contaminants (AHs and PCBs) in stomach contents of juvenile chinook salmon from the Duwamish Waterway and the Puyallup estuary were significantly higher than those in stomach contents of fish from the Nisqually River estuary or the hatcheries. However, concentration of chemical contaminants in stomach contents of salmon from the Snohomish estuary were not significantly higher than those in stomach contents of fish from the Nisqually estuary or the hatcheries. These results indicate that diet serves as a potential route of uptake of chemical contaminants in juvenile salmon from polluted estuaries.

• Levels of PCBs and FACs in liver and bile, respectively, were consistently higher in salmon from the Duwamish Waterway and the Puyallup estuary than those in fish from the Nisqually estuary or the hatcheries, thus demonstrating exposure of juvenile salmon from polluted urban sites to potentially toxic chemicals. However, levels of PCBs and FACs were not higher in salmon from the Snohomish estuary, another urban-associated estuary, than in fish from the hatcheries. This is consistent with the lower concentration of contaminants in sediments from this estuary.

• Body burden of PCBs were found to persist for at least 3 months after removing fish from a contaminated environment and holding them in the laboratory. This suggested that exposure to some anthropogenic contaminants could potentially continue for at least some time after juvenile salmon migrate to an ocean environment.

• The activity of an enzyme (hepatic AHH), which plays a critical role in activation of several chemical contaminants (including AHs) to toxic intermediates, was increased in juvenile salmon from the Duwamish Waterway and the Puyallup estuary. A consequence of the activation of toxic chemicals to reactive forms is their binding to DNA; this event is believed to be an early step in the process of chemical carcinogenesis and other toxic effects. In the present study, the levels of binding of chemical contaminants to DNA in liver were higher in fish from the Duwamish Waterway and the Puyallup estuary than in fish from the Nisqually estuary and the hatcheries. These results demonstrated that increased exposure to chemical contaminants in outmigrant juvenile salmon during their brief residency in urban estuaries was sufficient to elicit certain biological responses. These responses, commonly referred to as biomarkers, indicate the potential for other serious biological effects to ensue.

• Immune competence of juvenile salmon from the Duwamish Waterway was found to be suppressed when compared to juvenile salmon from the nonurban Nisqually estuary or the hatcheries. Immune competence was assessed by measuring the primary and secondary in vitro B-cell response of splenic and anterior kidney lymphocytes, using the plaque assay, or by measuring their in vivo primary antibody response to an antigen. An altered ability of these cells to produce antibodies to an antigen could be indicative of an animal's potential for increased susceptibility to infections.

• To causally link the relationship between contaminants and altered immune function, juvenile chinook salmon were injected with an organic extract of a contaminated sediment from the Duwamish Waterway. As in the field-exposed juvenile salmon, suppression of immune function was observed in this laboratory study. These findings suggest that the immunosuppression observed with juvenile salmon from the Duwamish Waterway was most likely due to chemical contaminants and not due to other environmental variables.

• Survival of juvenile chinook salmon from the Duwamish Waterway, the only urban estuary tested, was also significantly lower than that of fish from the Nisqually estuary or from the hatcheries, when held in the laboratory for up to 80 days. Similarly, growth, determined by measuring changes in length and weight of individually tagged juvenile salmon over an 80-day period, was lower in juveniles from the urban estuary than in juveniles from its respective hatchery.

Overall, these results demonstrate that increased chemical contaminant exposure in juvenile chinook salmon during their brief residency in urban estuaries of Puget Sound, Washington, was sufficient to elicit reponses at the chemical, biochemical, and biological level. Measurements of this type provide evidence of linkage between complex mixtures of chemical contaminants in the environment and effects on health and survival of fish. This information will be critical in increasing our capabilities for assessing the full spectrum of effects on salmon resulting from exposure to the myriad anthropogenic chemicals that can be present in the near coastal environment. The detection of chronic effects requires sensitive and reliable tools that cover a broad range of important biological functions and that are both cost-effective in their application and amenable for use in studies as described here. The availability of such tools would allow generation of sufficient data to identify and statistically quantify potential risk factors in the etiology of effects observed in fish from contaminated coastal environments.

• EXECUTIVE SUMMARY
  
• PREFACE
  
• INTRODUCTION
  
• METHODS
  
  • Field Sampling
    
  • Fish Collections
    
  • Tissue Sampling
    
  • Laboratory Holding
    
  • Chemical Analyses of Tissues and Stomach Contents
    
  • Biochemical Analyses of Fish Liver and Bile
    
  • Immunological Methods
    
    • Antigen Preparation
      
    • Measurement of Total Immunoglobulin
      
    • Primary in vivo Anti-TNP Response
      
    • In Vitro Immune Response: Field-Exposed Salmon
      
    • In Vitro Immune Response: Laboratory-Exposed Salmon
      
  • Measurement of Rates of Growth and Survival
    
  • Statistical Methods
    
• RESULTS
  
  • Tag Recoveries and Size of Juvenile Salmon
    
  • Contaminant Exposure in Juvenile Chinook Salmon
    
  • Stomach Contents
    
    • Aromatic hydrocarbons
      
      • Chlorinated hydrocarbons
        
    • Bile
      
    • Liver
      
  • Biochemical Measures of Contaminant Exposure
    
  • Hepatic Cytochrome P450 Activity
    
    • Hepatic Xenobiotic-DNA Adducts
      
  • Depuration Studies
    
  • Immunological Analysis
    
  • Primary in vivo Anti-TNP Response
    
    • In Vitro Immune Response: Field-Exposed Salmon
      
    • In Vitro Immune Response: Laboratory-Exposed Salmon
      
  • Survival
    
  • Growth
    
• DISCUSSION
  
  • Success of Capturing Hatchery Fish in the Estuary
    
  • Chemical Exposure and Biochemical Responses
    
  • Biological Effects
    
• CONCLUSIONS
  
• ACKNOWLEGMENTS
  
• CITATIONS
  
• TABLES
  
• FIGURES
  
• APPENDIX A - TABLES AND FIGURES
  
• APPENDIX B - GLOSSARY OF ABBREVIATIONS