U.S. Dept Commerce/NOAA/NMFS/NWFSC/PublicationsNOAA Technical Memorandum NMFS-NWFSC-8Contaminant 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 ServiceNorthwest Fisheries Science       CenterEnvironmental Conservation Division2725 Montlake Blvd. E.,       Seattle, WA 98112

April 1993

U.S. DEPARTMENT OF       COMMERCERonald H. Brown, Secretary

National Oceanic and Atmospheric       AdministrationJohn A. Knauss, Administrator

National Marine Fisheries ServiceWilliam W. Fox, Jr.,       Assistant Administrator for Fisheries 
Contributing Scientific InvestigatorsDouglas G. BurrowsEthan ClemonsSusan M. PiercePaul D. PleshaWilliam L. ReichertKaren L. TilburyDouglas WeberCatherine A. Wigren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.
CONTENTS- 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 
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