U.S. Dept Commerce/NOAA/NMFS/NWFSC/Publications
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.
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.
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).
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).
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.
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.
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.
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).
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.