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## SECTION 1JUVENILE SALMON INJURY STUDY

## Introduction

Estuaries are critical habitats for juveniles of several Pacific salmon species during their transition from life in freshwater to life in the ocean (Healy 1982). Estuarine habitats provide refuge from predators, a rich food supply to support rapid growth, and are where juvenile salmon make the transition from freshwater to marine conditions (Dorcey et al. 1978, Simenstad et al. 1982). Urban estuaries, however, receive inputs of toxic anthropogenic substances from a variety of sources, and many of these chemicals can accumulate in sediments (Dexter et al. 1985) and thus can be retained in the estuary. There is concern that, because juvenile salmon are undergoing numerous physiological adaptations during their residence in estuarine environments, any additional stresses, such as exposure to toxic chemicals, may be injurious.

The Hylebos Waterway of Commencement Bay is a severely contaminated estuary, and juvenile chinook and chum salmon inhabit this waterway in the late spring and early summer. During 1989 and 1990, juvenile chinook salmon were collected from three Commencement Bay waterways other than the Hylebos Waterway (Stein et al. 1995, Varanasi et al. 1993), and found to be substantially exposed to a variety of contaminants, including polycyclic aromatic hydrocarbons (PAHs) and chlorinated hydrocarbons (CHs). Additionally, levels of exposure found in juvenile chinook salmon sampled from the Duwamish Waterway, which runs through an industrialized area of Seattle and empties into Elliott Bay (Stein et al. 1995, Varanasi et al. 1993, McCain et al. 1990), were similar to that found in the 1989 and 1990 studies of Commencement Bay. It is notable that juvenile chinook salmon from the Duwamish Waterway show a variety of biological effects associated with their residence in this contaminated estuary. These effects include reduced immunocompetence, increased mortality after disease challenge, and reduced growth (Varanasi et al. 1993, Arkoosh et al. 1991, Arkoosh et al. 1996). Juvenile salmon from the Duwamish Waterway have also been found to have increased induction of hepatic cytochrome P4501A (CYP1A) and higher levels of DNA damage compared to juveniles from nonurban estuaries (Stein et al. 1995, Varanasi et al. 1993). Additionally, a recent laboratory investigation has demonstrated that immunocompetence of juvenile chinook salmon could be impaired by exposure to CHs and PAHs (Arkoosh et al. 1994). 

Before initiation of the current investigation, however, nothing was known about the potential exposure of chum salmon to chemical contaminants during residence in contaminated estuaries, or the possible effects of any exposure on this species. Most importantly, there was no information on exposure of salmonids in general during residence in the Hylebos Waterway. Accordingly, the objective of the present investigation was to determine to what degree juvenile chum and chinook salmon captured from the Hylebos Waterway might be exposed to organic contaminants, and to compare the levels of exposure observed to previous studies where such exposures have been linked to biological dysfunction.

## Methods

### Sample Collection and Analysis

Juvenile chum and chinook salmon from the Hylebos Waterway were sampled weekly for 6 weeks in May and June 1994. Sampling locations are illustrated in Figure 1.1. Reference samples were obtained from hatcheries, and from estuaries considered to be relatively unimpacted by contaminants (Varanasi et al. 1993). Methods of seining for the salmon, fish holding, necropsy, tissue collection, preservation and processing are described in detail in the Sampling and Analysis Plan (SAP) (Appendix A). Each sample was a composite of tissue from 100-150 fish for chum salmon and 30-60 fish for chinook salmon. The number of fish included in a composite was dependent on the amount of tissue needed for analysis, size of the fish, and number of fish available. Sites and collection dates for each species are shown in Table 1.1. 

Details of compositing methods and analytical techniques are described in the SAP (Appendix A). Sampling in the Hylebos Waterway and reference estuaries ended when outmigrating salmon were no longer being captured in sufficient numbers to complete a composite for analysis. The numbers and types of composite samples collected for each species at each site are shown in Table 1.2. Due to the low numbers of composites collected at individual reference sites, data from analyses of fish collected at reference sites were combined prior to statistical analyses.

### Statistical Methods 

Because environmental chemical concentrations and biomarker data are generally log-normally distributed, the data obtained from analyses of fluorescent aromatic compounds (FACs) in bile, CYP1A and DNA adducts in liver, and organic chemicals in liver and stomach contents were log-transformed prior to statistical analyses. This is a standard approach of our laboratory at the Northwest Fisheries Science Center (NWFSC) (Varanasi et al. 1995, Collier et al. 1986). Analysis of variance (ANOVA) was used to determine the statistical significance of differences between the combined data for fish collected during all sampling periods at the Hylebos Waterway, and the combined data for reference fish. 

Due to the low numbers of composites collected at individual reference sites (Table 1.2), data from analyses of fish collected at reference sites were combined prior to statistical analyses. Scheffe's multiple comparison test showed no significant differences between the reference estuary and hatchery fish. However, as described below and shown in the Case Narratives (Appendix C), the stomach contents data do suggest some contamination of feed used at the Puyallup State Hatchery and the Puyallup Tribal Hatchery. Low but detectable levels of DDTs, dieldrin, chlordane, and polychlorinated biphenyls (PCBs) were found in the stomach contents of fish sampled at these hatcheries (only one analysis of stomach contents was done for each of these hatcheries, and no stomach contents were collected in fish from the Nisqually Hatchery; see Table 1.2).

A zero value for below detection limit (BDL) samples cannot be log-transformed. Accordingly, we used values that were 50% of the detection limit for the statistical analyses in this report. Analyses of chemical concentrations are limited by the ability of the instrumentation to detect low amounts of the analytes. The limit of detection largely depends on the amount of sample available for analysis and, therefore, can be different for each sample analyzed, depending on the sample weight. A variety of methods have been used to conduct statistical analyses where chemical concentration data lies between the level of detection and zero (Newman et al. 1989). Reporting of 50% of the detection limit has been used in other studies by our laboratory at NWFSC, and by other investigators (Bauer et al. 1992) to examine chemical concentrations in fish tissues and sediment. Analytical results for specific samples, including limits of detection, are given in the Case Narratives (Appendix C).

## Results

### Chemical Contaminant Exposure

### 

### Liver: chlorinated hydrocarbons

Chum salmon 

Juvenile chum salmon captured in the Hylebos Waterway had significantly higher (p<0.0001) concentrations of hexachlorobenzene (HCB), hexachlorobutadiene (HCBD), PCBs, DDTs, chlordanes, dieldrin, and lindane in liver, compared to juvenile chum salmon from reference estuaries and hatcheries (Figures 1.2-1.5). Concentrations were approximately 20 and 9 times higher for HCBD and HCB, respectively (Figure 1.2), and 7 times higher for PCBs, (including toxic congeners 105 and 118; Figure 1.3), compared to reference fish. Concentrations of DDTs, chlordanes, dieldrin (Figure 1.4) and lindane (Figure 1.5) were about twice as high in livers of Hylebos fish compared to reference fish. 

Chinook salmon 

Mean concentrations of HCB and HCBD were about four times higher (Figure 1.2), PCBs were about three times higher (Figure 1.3), and chlordanes were about twice as high (Figure 1.4) in liver of juvenile chinook from the Hylebos Waterway, compared to concentrations in liver from reference fish. These differences were significant at p<0.0001. Hepatic concentrations of dieldrin, chlordanes, and DDTs (Figure 1.4) were 20% to more than 100% higher in chinook salmon from the Hylebos Waterway, compared to values for reference fish, and the differences were statistically significant at p<0.05. Although heptachlor, lindane, and aldrin (Figure 1.5) were often below detection limits, mean concentrations of these chemicals when detected generally were about twice as high in liver tissue of Hylebos Waterway fish compared to reference fish. These differences were statistically significant at p<0.05, using values set at 50% of the detection limit to include samples where these analytes were below limits of detection. Because detection limits can vary depending on sample size, and each sample has a different detection limit, values for individual samples are listed in the Case Narratives (Appendix C).

### Bile: metabolites of aromatic hydrocarbons

Chum salmon 

Concentrations of biliary FACs (Figure 1.6) measured at benzo(a)pyrene (BaP) wavelengths (FACsBaP) and phenanthrene (PHN) and naphthalene (NPH) wavelengths (FACsPHN and FACsNPH respectively), were all significantly increased (p<0.0001) in Hylebos Waterway fish compared to reference fish. Concentrations were 4 to 7 times higher in Hylebos fish compared to reference fish. FACsBaP is a semi-quantitative estimate of metabolites of high molecular weight aromatic compounds (HACs), and FACsPHN and FACsNPH are semi-quantitative estimates of metabolites of low molecular weight aromatic compounds (LACs). 

Chinook salmon 

Concentrations of biliary FACs (Figure 1.6) measured at BaP, PHN, and NPH wavelengths were generally 3 to 6 times higher in the bile of chinook salmon from the Hylebos Waterway compared to values for chinook salmon from the reference areas. These differences were statistically significant at p<0.0001. 

### Stomach contents: aromatic compounds and chlorinated hydrocarbons

Many fish collected either had empty stomachs, or the amount of material in the stomach was limited. Therefore, the amount of sample available for analyses allowed only one analysis per site for each of the two reference sites, and two analyses for salmon from the Hylebos Waterway (this was the case for both chum and chinook salmon; see Table 1.2). Accordingly, only limited statistical analyses could be done with these few data points, and thus for this study the chemical analysis of stomach contents should be regarded as a qualitative indicator of contaminant exposure. 

Chum salmon 

Concentrations of HACs and LACs in stomach contents (Figure 1.7) were more than 1000 times and 30 times higher, respectively, in stomach contents of chum salmon from the Hylebos Waterway compared to the reference fish. HCBD and HCB concentrations in stomach contents (Figure 1.8) were approximately 13 times and 5 times higher, respectively, in samples from Hylebos Waterway fish compared to reference fish. The sum of PCBs, as well as toxic PCB congeners 105 and 118 (Figure 1.9), dieldrin (Figure 1.10) and heptachlor (Figure 1.11) were all 3 to 5 times higher in the stomach contents of Hylebos Waterway fish compared to reference fish. Mean concentrations of HACs, HCBD, sum of PCBs, PCB congeners 105 and 118, and heptachlor were significantly different at p<0.05. 

The concentrations of DDTs and chlordanes (Figure 1.10) were not different between juvenile salmon captured in the Hylebos Waterway and juvenile salmon from the reference areas. However, this was largely due to higher levels of these compounds in the stomach contents of salmon from the Puyallup Tribal Hatchery. Those data, submitted to the Damage Assessment Center (DAC) as part of the detailed data submission for this project, suggest that there may have been some contamination of feed in the hatcheries. While the levels of DDTs and chlordanes in stomach contents of Hylebos Waterway fish were approximately 3 and 10 times higher, respectively, compared to values for fish from the Skokomish River estuary alone, the low sample size (n=1) makes this comparison on a statistical basis inappropriate. 

Chinook salmon 

Mean concentrations of HACs in stomach contents (Figure 1.7) of chinook salmon were more than 1500 times higher in fish captured from the Hylebos Waterway compared to fish from the reference areas (Nisqually Estuary and the Puyallup State Hatchery), whereas mean concentrations of LACs (Figure 1.7) were approximately 30 times higher in stomach contents of Hylebos fish compared to reference fish values. Similar comparisons between Hylebos and reference chinook salmon for other compounds are as follows: HCBD (Figure 1.8), 8 times higher; HCB ( Figure 1.8), twice as high; lindane (Figure 1.11), 5 times higher; aldrin (Figure 1.11), 12 times higher; heptachlor (Figure 1.11), twice as high. Differences in concentrations of HACs, HCBD, and lindane were significant at the p<0.05 level.

There tended to be increased concentrations of PCBs (including toxic PCB congeners 105 and 118), DDTs, dieldrin and chlordane in stomach contents of fish from the Puyallup State Hatchery, compared to chinook collected from the Nisqually Estuary, as delineated in the detailed data submitted to DAC. Accordingly, concentrations of these chemicals in stomach contents were not different between the reference samples and the Hylebos Waterway samples (Figures 1.9 and 1.10). However, the Hylebos Waterway chinook salmon generally had increased concentrations of these compounds in their stomach contents as compared to chinook salmon from the Nisqually Estuary. 

### Biological Indicators of Contaminant Exposure

### Cytochrome P4501A in liver

Chum salmon 

CYP1A, a xenobiotic metabolizing enzyme inducible by a broad range of PAHs, CHs, and pesticides, was measured as activity of the CYP1A-dependent enzyme, aryl hydrocarbon hydroxylase (AHH). These activities were approximately 3 times higher in liver tissue of juvenile chum salmon from the Hylebos Waterway, compared to values for reference fish (Figure 1.12). This difference was statistically significant at p<0.0001.

Chinook salmon 

Hepatic AHH activities were approximately 50% higher in juvenile chinook salmon from the Hylebos Waterway, compared to values for reference fish (Figure 1.12). This difference was statistically significant at p<0.2. 

### DNA adducts in liver

Chum salmon 

Concentrations of DNA adducts in liver, measured by the 32P-postlabeling method, serve as a biomarker of DNA damage due to exposure to, metabolism of, and covalent binding to DNA bases of PAHs. These concentrations were about twice as high in Hylebos fish compared to reference fish, and were statistically significant at p<0.02 (Figure 1.12). 

Chinook salmon 

There were no significant differences in levels of hepatic DNA adducts between chinook salmon captured from the Hylebos Waterway, compared to those captured from the reference sites (Figure 1.12).

## Discussion of Major Findings

Juvenile chum and chinook salmon from the Hylebos Waterway are clearly showing increased exposure to a wide range of chemical contaminants, compared to fish from hatcheries or reference estuaries.

A wide variety of chemical contaminants show elevated concentrations in the liver and bile of both chum and chinook salmon from the Hylebos Waterway, compared to fish from the reference estuaries and hatcheries. These include high and low molecular weight aromatic compounds and their associated metabolites, PCBs, including toxic congeners 105 and 118, HCBD and HCB, DDTs, hexachlor, lindane, dieldrin, aldrin, and chlordane. There is some evidence of low to moderate contamination with chlorinated compounds, in the feed used at the hatcheries, as shown by analyses of stomach contents (only one analysis per hatchery was conducted, Table 1.2). However, it is apparent that any such contamination is not a major factor in the increased body burdens measured in fish from the Hylebos Waterway, as liver levels of PCBs and chlorinated pesticides were clearly elevated in fish captured from the Hylebos Waterway, compared to fish from either the hatcheries or other contaminated estuaries. The presence of high levels of HCBD in liver tissue and stomach contents provides strong evidence that exposure of these fish originates from the Hylebos Waterway, rather than other waterways in Commencement Bay, as this compound is found in high levels in the sediments of the lower Hylebos Waterway, with dramatically lower levels being found elsewhere in the Commencement Bay ecosystem (Krahn et al. Append. D). In fact, liver concentrations of HCBD in juvenile chum and chinook exceed those found in any previous studies of juvenile salmonids (Varanasi et al. 1993). 

Associated with these increased concentrations of chemicals, there are indications of early biological alterations and damage, as shown by the increases in hepatic CYP1A-associated enzyme activity in both species and increased levels of DNA damage in chum salmon. Increases in both of these measures are well-established as being linked to contaminant exposure (Collier and Varanasi 1991, Varanasi et al. 1992, Stein et al. 1992). However, the measurement of DNA damage is less sensitive than induction of CYP1A for determining comparatively short-term exposure to moderate levels of contaminants (Collier et al. 1988), such as is likely the case for juvenile salmon inhabiting the Hylebos Waterway during their acclimation to oceanic conditions.

Concentrations of contaminants in juvenile chinook and chum salmon from the Hylebos Waterway are comparable to levels previously shown to be associated with biological injury in juvenile chinook salmon.

Contaminant concentrations similar to those measured in liver, stomach contents, and bile of juvenile salmon from the Hylebos Waterway are associated with impaired growth, suppression of immune function, and increased mortality following pathogen exposure in chinook salmon collected from another contaminated estuary in Puget Sound, the Duwamish Waterway (Varanasi et al. 1993, Arkoosh et al. 1991, Arkoosh et al. 1996). For most measures in the current study, the concentrations of contaminants in Hylebos Waterway juvenile salmon are similar to, or in the case of HCBD, substantially higher than, concentrations measured in Duwamish Waterway fish. These comparisons are depicted in Figure 1.13, along with the appropriate references. It is notable that chum salmon from the Hylebos Waterway generally showed higher indices of exposure than did chinook salmon. Whether this is due to increased exposure, decreased elimination, or other species-specific factors cannot be determined from the current data. Nonetheless, the apparent higher contaminant concentrations in chum salmon raises the possibility that this species may be more susceptible to contaminant-induced biological injury than chinook salmon. It is unknown whether the substantial exposure to HCBD, either alone or in concert with exposure to other contaminants, may contribute to biological injury in salmon from the Hylebos Waterway. 

The current study has focused solely on characterizing possible exposure of juvenile salmon, and thus does not specifically answer the question of what the impacts of this exposure might be on the populations of exposed animals. However, it is known that as salmon complete smoltification and move from freshwater habitats to estuarine and marine habitats, they must adapt to a range of different pathogens and prey organisms, and are also subject to predation from different predators. Thus, impaired abilities to withstand pathogenic challenges and altered growth patterns should generally be considered to be deleterious with respect to early ocean survival of juvenile salmon utilizing contaminated habitats. Further steps to be taken in the assessment of injury to these fish should include a determination of the potential for the toxicants present in the Hylebos Waterway to elicit biological dysfunction such as impaired disease resistance and reduced growth. Such studies should focus on the overall mixture of contaminants known to be present, as well determining the effects of specific contaminants or classes of contaminants. 

On to Section 2

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