APPENDIX A

SAMPLING AND ANALYSIS PLAN

FOR

HYLEBOS WATERWAY FISH INJURY STUDIES
122
123

SAMPLING AND ANALYSIS PLAN

FOR

HYLEBOS WATERWAY FISH INJURY

STUDIES

Prepared By

Environmental Conservation Division

Northwest Fisheries Science Center

National Marine Fisheries Service

National Oceanic and Atmospheric Administration

2725 Montlake Boulevard East

Seattle, WA 98112

For

Damage Assessment Center

National Oceanic and Atmospheric Administration

Washington, D.C.

December 1995
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Project Background and Objectives

Previous investigations in the Hylebos Waterway and other waterways in Puget Sound with

similar suites and levels of contaminants have identified a variety of contaminant-related

injuries in indigenous fish species. In the early 1980s, elevated levels of a variety of toxic

chemicals were found in tissues of English sole and rock sole from the Hylebos Waterway.

These fish species also had fin erosion and a variety of liver lesions, including liver tumors

(Malins et al. 1982, 1984). In studies conducted in 1989 and 1990, juvenile chinook salmon

from other waterways in Commencement Bay were found to have concentrations of

polycyclic aromatic hydrocarbons (PAHs), chlorinated hydrocarbons (CHs) or their

derivatives in tissues, fluids or stomach contents that were similar to those found in juvenile

chinook salmon from the highly contaminated Duwamish Waterway in Seattle (Varanasi et

al. 1993). Juveniles from this latter urban waterway have been found to have significant

concentrations of PAHs and CHs, and exhibited impaired immunocompetence and growth

inhibition (McCain et al. 1990, Arkoosh et al. 1991, Varanasi et al. 1993) Another type of

injury found in flatfish living in contaminated waterways in Puget Sound, such as the

Duwamish Waterway, is reduced reproductive success (Johnson et al. 1988; Casillas et al.

1991). Among the reproductive effects observed are inhibition of oocyte development,

inhibition of spawning, depressed plasma estradiol levels, reduced egg weight, increased

larval abnormalities, and reduced viability of offspring.

More recently a number of biochemical alterations have been found in the liver of a

variety of fish species from several areas in Puget Sound with elevated concentrations of

contaminants in sediment, including Commencement Bay (Stein et al. 1992). Measurement

of these biological markers have been used to improve the assessment of contaminant

exposure and early responses in indigenous fish, and thus strengthen the link between

exposure to toxic chemicals and the injuries described above. One type of alteration, known

as DNA adducts, is a biomarker of exposure to potentially genotoxic compounds and is

positively correlated with indicators of exposure, such as tissue levels of PCBs, and

prevalences of certain liver diseases (Varanasi et al. 1989, Stein et al. 1992, 1994, Myers et

al. 1995). Another alteration involves elevated levels of hepatic cytochrome P4501A

(CYP1A) or associated monooxygenase activities, such as aryl hydrocarbon hydroxylase

(AHH). Many studies have confirmed the induction of hepatic CYP1A activity both as an

indicator of contaminant exposure and as one of the earliest physiological changes detected

after exposure of fish to contaminants (Collier et al. 1991,1995; Payne et al., 1987). A

number of studies have shown that measurement of these biochemical alterations, hereafter

referred to as biomarkers, combined with measurement of the above-mentioned injuries,

give a more consistent assessment of contaminant exposure in indigenous fish than do single

bioeffects measurement.
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The overall objective of the Hylebos Waterway fish injury assessment investigation is to

document the current status of contaminant-associated injuries in fish indigenous to the

Hylebos Waterway, to quantify the extent of these injuries, and evaluate links between

these injuries and chemical contaminants in this waterway. Evidence for evaluating links

will include data on concentrations of selected chemical contaminants in sediments from

where fish are captured and in tissues and fluids of fish. The three primary objectives,

together with more detailed background for each objective, are given below:

Objective 1—Assess Chemical Contamination and Associated Injuries in Juvenile

Salmonids

In studies conducted in 1989 and 1990, juvenile chinook salmon from other waterways

adjoining Commencement Bay were found to have significantly elevated concentrations of

polycyclic aromatic hydrocarbons (PAHs), chlorinated hydrocarbons (CHs) or their

derivatives in tissues, fluids or stomach contents that were similar to those found in juvenile

chinook salmon from the highly contaminated Duwamish Waterway (McCain et al. 1990;

Varanasi et al. 1993; Stein et al. 1995). Juvenile salmon from the Duwamish Waterway

have also been found to have increased induction of hepatic cytochrome P450 1A and higher

levels of DNA damage, and exhibited impaired immunocompetence and growth inhibition,

compared to juveniles from nonurban estuaries (Arkoosh et al. 1991; Varanasi et al. 1993;

Stein et al. 1995). A recent laboratory investigation demonstrated that immunocompetence

of juvenile chinook salmon could be impaired by exposure to an extract of Duwamish

Waterway sediment (Arkoosh et al. 1994). Because the assessment of immunocompetence

and growth is very expensive, we are proposing to initially examine exposure and early

biomarkers (e.g. DNA damage and enzyme induction) in the first phase (year one) of studies

on juvenile salmon from the Hylebos Waterway. If significant exposure is found, then

investigations to assess more serious biological effects will be recommended for the second

year. Data linking specific classes of contaminants to impaired immunocompetence in

salmonids is comparatively scarce, but our recent results suggest that both PAHs and

chlorinated hydrocarbons may be risk factors.

Objective 2—Determine Toxicopathic Conditions in Flatfish

In the early 1980s, elevated levels of a variety of toxic chemicals were found in tissues of

English sole and rock sole from the Hylebos Waterway (Malins et al. 1982, 1984). Many of

these fish species also had a variety of liver lesions, including liver tumors, which are

associated with exposure to carcinogenic chemicals, especially high molecular weight

polynuclear aromatic hydrocarbons,

or PAHs (Myers et al. 1987, 1994a, Myers et al. 1991). These findings contributed to the

eventual designation of Commencement Bay as a Superfund Site. ECD scientists also
126

conducted a long-term laboratory study in which a variety of unique degenerative and

preneoplastic lesions were induced in English sole exposed to benzo[a]pyrene and a PAHrich fraction of contaminants extracted from a contaminated sediment (Schiewe et al. 1991).

More recently a suite of biochemical alterations have been demonstrated in a variety of fish

species from several areas in Puget Sound with elevated concentrations of contaminants in

sediment, including Commencement Bay (Stein et al. 1992). Measurements of these

biological markers have been used to improve the assessment of contaminant exposure and

early responses in indigenous fish (Krahn et al. 1986; Stein et al. 1992; Collier et al. 1993a,b;

Collier et al. 1995), and thus strengthen the link between exposure to toxic chemicals and

the injuries described above. For example, an investigation was conducted in the Hylebos

Waterway, as well as eight other sites in Puget Sound, in 1987 and 1988 to evaluate

relationships among liver lesions and these biomarkers in subadult English sole and rock

sole (Myers et al. 1994b). Substantive correlations were found between the prevalences of

a number of liver lesions and the levels of most of these biomarkers. We have also conducted

several laboratory studies to demonstrate the time- and dose-responsiveness of a number of

these biomarkers (Collier and Varanasi, 1991; Stein et al. 1993).

Objective 3—Determine Contaminant-Induced Reproductive Toxicology in Flatfish

In an effort to assess other biological impacts of the above-mentioned chemical

contaminants on flatfish, the ECD developed a research program to evaluate the effects of

these contaminants on the reproductive status of gonadally maturing adult female fish. Field

studies showed that a significant proportion of female flatfish living in contaminated

waterways of Puget Sound, such as the Duwamish Waterway in Seattle, were found to

exhibit reduced reproductive success (Johnson et al. 1988; Casillas et al. 1991) compared to

females from minimally contaminated sites. Another study suggested that female English

sole from contaminated areas may not be making their spawning migrations at all (Collier et

al. 1992). Among the reproductive effects observed were inhibition of oocyte development,

inhibition of spawning, depressed plasma estradiol levels, reduced egg weight, increased

larval abnormalities, and reduced viability of offspring. These field data have been

incorporated into population projection models, and the results suggest that the decreased

reproductive potential associated with exposure of certain flatfish species (e.g., English

sole) to contaminants may be sufficient to significantly reduce the intrinsic rate of growth in

populations from contaminated areas such as the Duwamish Waterway and Eagle Harbor

(Landahl and Johnson, 1993). Again, similar to results for fish pathology, our data suggest

that exposure to PAHs is a substantial risk factor in determining reduced reproductive

success. Laboratory investigations have shown effects on circulating levels of sex hormones

due to contaminant exposure, consistent with field results (Stein et al. 1991; Johnson et al.

1994).
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Descriptions and scheduling of tasks

General

Juvenile Salmonids. Juveniles of two species of salmonids, chinook and chum salmon,

were collected for injury assessment studies in the spring of 1994. These two species have

the longest residency times in estuaries among Pacific salmon, but they have different

migratory routes into the Hylebos Waterway; a large number of juvenile chum salmon are

released by the Puyallup Tribe into Hylebos Creek at the head of the waterway, whereas

chinook salmon are released from a state hatchery into the Puyallup River and move to the

Hylebos Waterway via Commencement Bay. Fish were collected using a seine deployed

from beaches or docks located around the 11th Street Bridge. Tissues collected for these

studies included liver, stomach contents and bile for measurements of organic contaminants

(CHs, PAHs, and their derivatives); and liver for measurements of biomarkers, e.g. DNA

adducts and CYP1A. Juvenile salmon were sampled from the Hylebos Waterway

approximately every two weeks from May 11 to June 29. A total of 1372 chum and 241

chinook were collected during this time. Reference fish were also sampled from hatcheries

and estuaries considered to be relatively unimpacted. Reference sites for chum include the

Puyallup Tribal Hatchery (n=600) and Skokomish Estuary (n=300). Reference sites for

chinook include the Puyallup State Hatchery (n=200), Kalama Creek Hatchery (n=150),

and Nisqually Estuary (n=170). Location of all sites are shown in Figs. 1 and 2.

Toxicopathic Conditions in Flatfish. English sole and rock sole were collected for assessment

of histopathological conditions of the liver, kidney, and gonad, and measurements of organic

contaminants in liver, bile and stomach contents, and of DNA adducts and CYP1A in liver.

Otoliths were also collected for age determination. Flatfish were collected in the Hylebos

Waterway in July and August of 1994 by otter trawl at three sites — one site near the Upper

Turning Basin, one near the Lower Turning Basin, and one near the 11th Street Bridge (Fig

1). English sole were collected at all three Hylebos sites, whereas rock sole were only

captured at the 11th Street Bridge site. We attempted to collect reference fish in Case Inlet

in August, but were unable to locate fish of a comparable size. Therefore an alternate

possible reference site at Colvos Passage (see Fig. 2 for location) was identified in October,

and both English sole and rock sole were collected for reference samples. Sixty fish of each

species were collected at each site.

Reproductive Toxicology in Flatfish. Adult female English sole were collected by otter

trawl from the Hylebos Waterway and a reference site in Colvos Passage. Sampling was

conducted approximately every 4-6 weeks from October 1994 through April 1995, during

the season when vitellogenesis normally occurs in this species. Approximately 30 fish

were collected at each site for every sampling period. Otoliths were collected for age

determination. Blood samples were collected for estradiol analyses. Liver, kidney and ovary

were collected for histopathological examination and staging of ovarian development. In

January and February when yolk becomes visible in developing eggs, samples of ovary
128

were collected for fecundity measurements. Liver, bile and stomach contents were collected

for measurement of contaminant exposure, and liver was also collected for measurement of

DNA adducts and CYP1A.

Data Analysis, Products and Injury Qualification

The data from these injury assessment investigations will be placed in an integrated

data management system. Statistical tests will be performed 1) to evaluate relationships

between contaminant exposure and bioeffects, 2) to compare values from fish captured in

the Hylebos Waterway with values for fish captured in reference areas, and 3) to compare

concentrations of contaminants in liver tissue, prevalences of liver lesions and biomarker

responses with similar parameters measured in previous studies.

Data acquired and analyzed through this SAP will be used to address the following

questions for each species:

1. Are fish exposed to contaminants in Hylebos Waterway?

2. Are the exposures sufficient to cause injury?

3. What proportion of the population is estimated to be injured by these contaminants?

4. What might be the overall effect of this injury on the population of each species?
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Table 1.

Scheduling of specific tasks

Salmon investigations

(contaminant exposure)

D y uration A s ctivit Numbers of fish and/or analyse

M n ay/June, 1994 Seining for juvenile salmo

(approximately 20 days)

Approximately 1600 fish

M n ay/June, 1994 S h almon necropsy and sample collectio Approximately 1600 fis

J f anuary-October, 1995 Chemical analyses of composites o

livers, stomach contents and bile

35 comps/liver, 8 comps/stomach, 37

comps/bile

J r anuary-October, 1995 Analyses of composites of liver fo

biomarkers of contaminant exposure

37 comps/CYP1A 37 comps/DNA adducts

F g ebruary 1996 D A ata reportin N/

J n une 1996 D A ata interpretatio N/

Flatfish investigations

(Contaminant exposure and

toxicopathic conditions)

D y uration A r ctivit Numbers of fish and/o

analyses

J V uly-August, 1994 Flatfish collection aboard the R

Streeter (approximately 10 days)

Approximately 360 fish

J n uly-August, 1994 F h latfish necropsy and sample collectio Approximately 360 fis

A s pril-July, 1995 S 0 ectioning and staining of fixed tissue Approximately 36

J s uly-November, 1995 H s istological examination of tissue Approximately 360 section

M f arch-October, 1995 Chemical analyses of composites o

livers, stomach contents and bile

36 comps/livers

12 comps/stomach cont.

36 comps/bile

M r arch-December, 1995 Analyses of composites of liver fo

biomarkers of contaminant exposure

36 comps/CYP1A

36 comps/ DNA adducts

F g ebruary 1996 D A ata reportin N/

J n une 1996 D A ata interpretatio N/
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Flatfish investigations

(reproductive toxicology)

D y uration A r ctivit Numbers of fish and/o

analyses

O V ctober, 1994-March, 1995 Flatfish collection aboard the R

Streeter (approximately 20 days)

Approximately 360 adult female

fish

O e ctober, 1994-March, 1995 Flatfish necropsy and sampl

collection

Approximately 360 adult female

fish

M d ay-August, 1995 Sectioning and staining of fixe

tissues

Approximately 360 samples

A f ugust-November, 1995 Histological examination o

tissues

Approximately 360 sections

M , ay-December,1995 Chemical analyses of livers

stomach contents and bile.

120 indiv/liver

12 comps/stomach cont.

120 indiv/bile

M s ay 1995 to January 1996 Analyses of liver for biomarker

of contaminant exposure

120 indiv/DNA adducts

J r anuary-August, 1995 Analyses of blood plasma fo

estradiol

Approximately 360 samples

J s anuary-August, 1995 D s etermination of somatic indice Approximately 360 sample

F g ebruary 1996 D A ata reportin N/

J n une 1996 D A ata interpretatio N/
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Standard Operating Procedures

Sample Collection and Analyses for Hylebos Waterway Fish Injury Studies

1994:

Survey of Contaminant Exposure in Juvenile Salmon,

Contaminant Exposure and Toxicopathic Conditions in Flatfish, and

Reproductive Toxicology of Flatfish

Field collections

Juvenile Salmon

Salmon were collected with beach seines generally following the procedures as described

in the Puget Sound Protocols (PTI, 1990), and by Varanasi et al. (1993). Since fish were

often observed to be located near the surface of deeper water, seines were deployed from

docks as well as beaches. Fish captured from the Hylebos Waterway and reference estuaries were held alive in aerated seawater, and fish from hatcheries were maintained in aerated

freshwater until necropsies could be conducted. Fish processing for Hylebos collections

was done either on the NOAA Ship McArthur or the RV Harold W. Streeter. Fish processing for samples from the hatcheries and reference estuaries were done at the NWFSC. Juvenile salmon were collected from the Hylebos Waterway primarily within sight of the 11th

Street Bridge. Latitudes and longitudes were determined with a hand-held GPS and are

listed below:

Latitudes and Longitudes of juvenile salmon collection areas in the Hylebos

47o 16.749 N 122o 23.791 W 47o 16.726 N 122o 23.661 W

47o 16.709 N 122o 23.798 W 47o 16.717 N 122o 23.662 W

47o 16.785 N 122o 23.896 W 47o 16.782 N 122o 23.719 W

47o 16.757 N 122o 23.888 W 47o 16.745 N 122o 23.799 W

47o 16.743 N 122o 23.790 W 47o 16.680 N 122o 23.961 W

47o 16.753 N 122o 23.891 W 47o 16.801 N 122o 23.836 W

47o 16.716 N 122o 23.794 W 47o 16.656 N 122o 23.651 W

47o 16.731 N 122o 23.774 W 47o 16.713 N 122o 23.808 W

47o 16.708 N 122o 23.716 W 47o 16.363 N 122o 24.005 W

47o 16.691 N 122o 23.626 W

Flatfish for Toxicopathic Conditions and Reproductive Toxicology Studies

Flatfish were collected by 25 foot SCCWRP otter trawl similar to that described in

Puget Sound Protocols (PTI, 1990) with the following differences. The net is constructed

of 1.5 inch stretch mesh in the body, and 1.25 inch mesh in the cod end. Five-foot side

panels have been added to increase the net opening. A cable foot rope has been added, and

aluminum doors are now used instead of wood doors. The net was deployed from the 45
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foot NWFSC research vessel Harold W. Streeter and towed for a duration of 5-15 minutes

at a speed of 1.5-2.5 knots.

Fish were identified by trained fisheries biologists, and target species were placed in

holding tanks on board the research vessel. Fish were maintained alive for no more than a

few hours in tanks with flowing seawater until necropsies could be performed in the shipboard laboratory. By-catch was released.

Toxicopathic Conditions Study: Flatfish were collected from three sites within the

Hylebos Waterway including locations near the Upper Turning Basin, the Lower Turning

Basin and the 11th Street Bridge. Fish were also collected from the reference site at Colvos

Passage. Target species were English sole and rock sole. English sole were captured at all

three Hylebos sites and the reference site, while rock sole were available only at the 11th

Street Bridge site in the Hylebos, and the Colvos Passage reference site.

Reproductive Toxicology Study: Beginning in October 1994, female English sole

were collected near the 11th Street Bridge in the Hylebos Waterway and the reference site at

Colvos Passage approximately once every six weeks until April 1995 to monitor reproductive maturation processes over time.

Necropsy and sample collections

Juvenile Salmon

At each site, or for every collection period, approximately 100-150 juvenile chum

and 30-60 chinook were sampled for each composite. The number of fish needed for a

composite was dependent on the size of the fish. We attempted to collect 4 composites/

species at each site for every sampling period, however the number of composites collected

was dependent on the number of fish available. Thirty-seven composites of bile and liver

were collected during the course of the study. This included four composites of chum from

the Puyallup Tribal Hatchery, two composites of chum from the reference site at the

Skokomish Estuary, twelve composites of chum collected over four sampling periods from

the Hylebos, five composites of chinook from the Puyallup State Hatchery, three composites of chinook from the Kalama Creek Hatchery, three composites of chinook from Nisqually

Estuary and seven composites of chinook from the Hylebos collected over four sampling

periods. Fish were weighed and measured for fork length. Bile, liver and stomach contents

were collected as described in Varanasi et al. (1993), except that whole livers were collected and later subdivided for each type of analysis. Liver and stomach contents were

composited into glass 20 ml vials previously rinsed with methylene chloride. Bile was

composited into 4 ml vials containing glass limited-volume inserts. If necropsies were conducted on the McArthur or at NWFSC, liver, bile and stomach content composites were

immediately transferred to freezers. For necropsies conducted on the RV Streeter, samples

were maintained on dry ice until they could be transferred to freezers for storage. Liver

samples were stored at -80o C, and bile and stomach contents were stored at -20° C until

analyses were conducted.
133

Flatfish

 Fish were weighed (to the nearest gram) and measured (fork length, to the nearest 1

mm), and otoliths were collected for age determination. Prior to necropsy, each fish was

assigned a unique specimen number, and all sample containers were labeled with this number for sample tracking. Details of necropsy, histology sample collection and fixation, and

chemistry sample collection and preservation are described in Stehr et al. (1993). Samples

of liver, bile and stomach contents collected for organic chemical analysis were maintained

on ice, then transferred to locked freezers for temporary storage at -80° C at the end of each

day. Samples of liver for CYP1A and DNA adducts were immediately placed in liquid

nitrogen and transferred to a locked -80° C freezer at the end of each day. They were temporarily stored in this freezer until they were distributed to the appropriate laboratories. Once

distributed, samples for organic chemical analyses were stored at -20° C, and samples for

CYP1A and DNA adduct analyses were stored at -80° C until analyses were completed.

Individual samples were collected in the field, however liver, stomach contents and bile

were composited in the laboratory for chemical analyses including measurements of organics, CYP1A and DNA adducts, and biliary fluorescent aromatic compounds (FACs).

Toxicopathic Conditions Study: Sixty English sole were collected at each of the

three Hylebos sites (Fig. 2) as well as the reference site in Colvos Passage. Sixty rock sole

were also collected at the 11th St. Bridge site in the Hylebos, and at the reference site. To

determine the applicability of sample size, power analyses were conducted using lesion

data from a previous study of the Duwamish Waterway (Myers et al. 1993), which is a

highly industrialized waterway located about 30 miles north in Elliott Bay. The lesion type

used in the analysis was foci of cellular alteration (FCA) which is considered to be a precursor to neoplastic conditions. This lesion type is less prevalent than most other contaminantrelated lesion types, therefore it serves as a conservative index of power for analyses of

sample size. For a sample size of 60 fish, the statistical power to detect differences in

prevalence of FCA between the Duwamish and the reference site at Nisqually fell between

70% and 80% at a=0.20. For a=0.10, the power to detect fell between 55% and 60%.

Fish were necropsied and tissues preserved according to standard procedures as

described in Stehr et al. (1993). Samples collected included otoliths for age determination,

liver, kidney and gonad for histology; liver for organic contaminant analyses, CYP1A and

DNA adducts; stomach contents for organic contaminant analyses and bile for FACs.

Reproductive Toxicology Study: Approximately thirty female fish were collected at

each site during each sampling period. The test site was located near the 11th St. Bridge in

the Hylebos and the reference site in Colvos passage. Samples collected included otoliths

for age determination; liver, kidney and gonad for histology and to determine ovarian matu-
134

ration; liver for organic contaminant analyses; DNA adducts; stomach contents for organic

contaminant analyses and bile for FACs. Blood was also collected for plasma 17-b estradiol concentrations, and weights of ovaries, liver, and gutted bodies were collected to determine gonadosomatic, hepatosomatic and condition indices. In January and February when

yolk production becomes visible in the oocytes, one ovary was collected for fecundity determination, and portions of the remaining ovary were collected for chlorinated hydrocarbon analyses. Sample collection procedures are described in Johnson et al. (1988, 1994)

and Stehr et al. (1993). Samples collected for fecundity determination are being archived,

and analyses of these samples are not being performed under this SAP.

Sample analyses

Juvenile Salmon Injury (Table 2A)

Organic chemical analyses. Fish liver is analyzed for chlorinated hydrocarbons (CHs) and

pesticides using the methods described by Sloan et al. (1993). Stomach contents are analyzed for aromatic hydrocarbons (AHs) as well as CHs and pesticides, also using the methods of Sloan et al. (1993). Analytes measured are listed in Table 3. Liver or stomach contents are extracted by grinding tissue, sodium sulfate, dichloromethane, and surrogate standards with a Tekmar Tissumizer. Tissue extracts are filtered through silica-alumina and

concentrated to 1 mL for further cleanup using size exclusion chromatography. The extract

is concentrated and exchanged into hexane for analysis using GC/MS for aromatic hydrocarbons and GC with electron capture detection for chlorinated pesticides and hydrocarbons.

Biliary FACs. FACs including benzo[a]pyrene (BaP), phenanthrene (PHN) and naphthalene (NPH) equivalents in bile are analyzed by HPLC according to the methods described

by Krahn et al. (1986) with the following modifications.

FACs in bile are analyzed on a Waters (Milford, MA) HPLC equipped with a Waters

WISPTM model 715 automatic injector, and three Perkin Elmer (Norwalk, CT) model 40

fluorescence detectors connected in series and interfaced to a Waters MilleniumTM data

acquisition workstation. A 0.20- x 2-cm guard column containing PerisorbTM 30- to 44-μm

reverse-phase C18 packing (Upchurch Scientific, Oak Harbor, WA) is used in series with a

Perkin Elmer HC-ODS/PAH 10-μm (0.26- x 25-cm) reverse-phase analytical column

 (Figure 3).
135

Figure 3. High performance liquid chromatography system for analyses of bile for fluorescent

aromatic contaminants (solid lines indicate flow of mobile phase; dashed lines indicate

electronic connections for data flow)

For each sample, 3-5 μL of thawed, untreated bile is injected onto the analytical

column and eluted with an HPLC linear gradient (flow rate of 0.7 mL/min) beginning with

100% solvent A (water containing 5 ppm acetic acid) to a final composition of 100% solvent

B (methanol) during a period of 15 min. After holding the mobile phase at 100% solvent B

for 10 min, solvent conditions are returned to 100% solvent A during a period of 3 min. The

system is then allowed to re-equilibrate for 10 min at 100% solvent A before the next sample

is injected. The total run time, including running of the linear gradient and re-equilibration

of the system, is 38 min. All solvents are degassed with helium and the column temperature

is held at 50°C.

Bile sample identification numbers, HPLC calibration standards (HPLC CS) and

data from the bile reference pools for each analysis set are entered into a database in the

Waters MilleniumTM2010 Chromatograpy Manager software (see below). For each set of

samples, the HPLC CS, containing known concentrations of NPH, PHN and BaP, are
136

analyzed at the beginning of the set and after every 6 or 7 samples. A quality assurance bile

reference pool is analyzed near the beginning and end of each set analyzed by HPLC. Biliary

FACs are monitored by fluorescence at excitation/emission (ex/em) wavelength pairs for

NPH (ex/em 290/335 nm), PHN (ex/em 260/380 nm) and BaP (ex/em 380/430 nm). The

total area for all peaks in the region of the chromatogram where FACs are known to elute (>

9 min) are integrated for each wavelength pair. Quantification of analytes is performed

according to Krahn et al. (1986). The concentrations (ng PAH equivalents per ml of bile or

per mg biliary protein, wet weight) of FACs in the samples are calculated according to the

response of each PAH in the HPLC CS. Biliary protein is measured according to Lowry et

al. (1951). If the fluorescence response in a sample is sufficiently high that a detector

response reaches its maximum (saturated), the sample is re-analyzed using a smaller injection

volume.

As stated above, fluorescence response data for HPLC chromatograms is acquired

and processed with the Waters MilleniumTM 2010 chromatography software package. Each

tray of sample vials (bile samples, calibration standards and reference bile pools) analyzed

by HPLC is assigned a unique Sample Set ID. After processing, the chromatograms and

results of peak integration are printed for review to assure the quality of the results. After

checking quality assurance criteria, an electronic copy of the data files is stored on disk and

a hard copy of the data and results are printed for archival purposes. Specific details of the

use of the acquisition software and workstation can be found in the operation manuals

provided by Waters/Millipore Corporation.

CYP1A

Hepatic microsomes are prepared from frozen liver samples as described previously (Collier

et al., 1995) except that microsomes are resuspended in 0.25 M sucrose made up in 80/20 v/

v water/glycerol. The suspensions are frozen at -80°C until CYP1A assays are performed.

Aryl hydrocarbon hydroxylase (AHH) activities are assayed in triplicate at 25° using 14CBaP as the primary substrate (Collier et al., 1995) .

DNA adducts Hepatic xenobiotic DNA adducts are measured according to the methods of

Reichert and French (1994). (Appendix).

Toxicopathic Conditions in Flatfish (Table 2B)

Age determination Fish age is determined to the nearest year by counting the number of

clearly defined opaque zones of whole otoliths under a binocular dissecting microscope

(Chilton and Beamish, 1982).

Histology Livers, kidney and gonad are preserved in Deitrichs’ fixative. Tissues are

embedded in paraffin, sectioned, stained with hematoxylin and eosin and examined

microscopically. Hepatic lesions are classified according to the criteria outlined in Myers et

al. (1987), then grouped into the following categories: neoplasms (hepatocellular carcinoma,
137

cholangiocellular carcinoma, adenoma, and cholangioma); foci of cellular alteration

(eosinophilic foci, basophilic foci, clear cell foci), specific degeneration/ necrosis (nuclear

pleomorphism and/or megalocytic hepatosis), hydropic vacuolation, and non-specific necrotic

lesions. Ovarian lesions and developmental stage are examined and classified as described

in the reproductive toxicology section.

Organic Chemical Analyses of liver tissue and stomach contents are conducted as described

for the juvenile salmon study (see page 12).

Biliary FACs are measured as described for the juvenile salmon study (see pages 12-14).

Biochemical Analyses. CYP1A and hepatic xenobiotic-DNA adduct levels are measured

in liver tissues as described for the juvenile salmon study (see page 14).

Reproductive Toxicology Sample Analysis Techniques (Table 2C)

Plasma 17-b estradiol concentration. A 1-3 mL blood sample was taken from each fish with

a heparinized syringe. Blood samples were centrifuged at 800 x g, and the plasma stored at

-80oC until analyses are conducted. Plasma 17-b estradiol levels are determined by

radioimmunoassay as described in Sower and Schreck (1982).

Histological Analyses. Histology of the liver, kidney and gonad are conducted as described

above in the toxicological conditions study. The developmental stage of ovaries are

determined, and classified according to criteria described in Johnson et al. (1991) into the

following categories: regressed, late regressed, pre-vitellogenic, vitellogenic, hydrated,

spawning, and spent. Ovaries are also examined for follicular atresia, hermaphroditism,

ovarian macrophage aggregates, and other inflammatory lesions associated with oocyte

resorption, including lymphoid or macrophage infiltrates, using criteria described in Johnson

et al. (1991).

Determination of Somatic Indices. Fish are weighed (to the nearest gram) and measured

(fork length, to the nearest mm), and liver and gonads are excised and weighed (to the

nearest gram). All other internal organs are then removed, and the animal is weighed (to the

nearest gram) to determine gutted body weight.

Gonadosomatic index (GSI) (Nikolsky 1963, Shul-man 1974) is calculated according

to the formula:

GSI = (ovary weight (g)/gutted body weight (g)) x 100

Hepatosomatic index (HSI) (Nikolsky 1963, Shul-man 1974) is calculated according

to the formula:

HSI = (liver weight (g)/gutted body weight (g)) x 100

Condition factor. Because low body weight may be associated with suppressed ovarian

development in adult female fish (Burton and Idler, 1987), a condition factor is determined

for all sampled animals so the influence of emaciation on ovarian development can be
138

distinguished from any potential effects of contaminant exposure. Condition factor (Ricker,

1975) is calculated using the formula:

Condition factor = gutted body weight (g)/length3 (cm)

Age determination. Otoliths are examined to determine age of the fish as described for the

toxicopathic condition study (see page 14).

Rapid PCB Analysis by HPLC with photodiode array (PDA) detection

Tissue Extraction and Cleanup. Selected individual samples are analyzed using rapid

HPLC analyses for PCB congeners, DDTs and hexachlorobenzene (Specific analytes listed

in Table 3.). Fish livers are analyzed for PCBs using the extraction and cleanup method of

Krahn et al. (1994). Briefly, 0.3 - 2.0 g tissue, 20-mL hexane/pentane (1:1 v/v), 5 g sodium

sulfate and the surrogate standard (1,7,8-TriCDD; 250 ng) is homogenized, centrifuged and

the extract is decanted into a concentrator tube. The extraction process is repeated, and the

extracts are combined and evaporated to ~1 mL. The sample extract is loaded onto a gravityflow cleanup column that contains a glass wool plug, silica gel, basic silica gel and acidic

silica gel to remove lipids and other interfering compounds. The PCBs are eluted from the

cleanup column with 14 mL hexane/methylene chloride (1:1 v/v) and collected into a

concentrator tube. The HPLC internal standard is added to each sample (1,2,3,4-TCDD;

250 ng) and the solvent volume is reduced to ~ 150 μL.

HPLC/PDA Analyses. Eleven dioxin-like PCB congeners (PCBs 77, 81, 105, 118,

126, 156, 157, 169, 170, 180 and 189), other selected PCBs (PCBs 101, 128, 138 and 153)

and chlorinated pesticides (e.g., DDTs, hexachlorobenzene) were resolved by HPLC on

two (1-pyrenyl) ethyldimethylsilylated silica (PYE) analytical columns cooled to 9°C and

are monitored with a PDA detector (Krahn et al., 1994). The chlorinated hydrocarbons are

identified by comparing their UV spectra (200-310 nm) and retention times to those of

reference standards in a library. Analyte purity is confirmed by comparing UV spectra

collected for a peak to the apex spectrum.

Biliary fluorescent aromatic compounds are measured for selected individuals as

described for the juvenile salmon study (see pages 12-14).

Biochemical Analyses. Hepatic xenobiotic-DNA adduct levels are measured in liver

tissues of selected individuals as described for the juvenile salmon study (see page 14).

Chain of Custody Procedures

all samples collected and stored are accompanied by a chain of custody form. Each box

of samples is sealed with security tape, and transferred to the laboratory in secured

containers. Samples are stored in locked freezers. Chain of custody is maintained

throughout storage, distribution and analyses of samples. Samples will be stored by the

QA officer for one year after the data are accepted.
139

Quality Assurance Procedures

Hylebos Waterway Fish Injury Studies 1994:

Survey of Contaminant Exposure in Juvenile Salmon,

Toxicopathic Conditions in Flatfish, and

Reproductive Toxicology of Flatfish

Quality Assurance procedures will be followed to monitor (1) the performance of the

measurement systems to maintain statistical control, and provide rapid feedback so that

corrective measures can be taken before data quality is compromised and (2) verify that

reported data are sufficiently complete, comparable, representative, unbiased and precise so

as to be suitable for their intended use. Procedures for QA are summarized in Table 4.

Organic Chemical Analyses of Liver and Stomach Contents

Quality Assurance Procedures are described in detail for the detailed Organic Chemical

analyses of liver and stomach contents in the Commencement Bay Damage Assessment

Quality Assurance Plan (December 1995).

Rapid PCB Analysis by HPLC/PDA

The HPLC/PDA is calibrated daily (see Table 4, continuing calibration). Each sample

set consists of 11-14 samples and the following quality assurance samples: a whale blubber

control material, a method blank and a duplicate.

CYP1A

Quality assurance procedures include duplicate zero-time and boiled enzyme blanks

for each set of assays. Each sample is run in triplicate and if samples show > 30% absolute

difference between triplicates and >20 units (pmoles benzo[a]pyrene metabolized/mg

microsomal protein/minute) difference between triplicates the outlying replicate will be

omitted from the calculations. If the coefficient of variation (CV) still exceeds 30%, the

analysis of that sample will be repeated.

DNA Adducts

Salmon testes DNA prepared from coho (Oncorhynchus kisutch), is used for measuring

the efficiency of DNA hydrolysis and as a sample blank throughout the assay to assess

background. The compound 7R,8S,9S,10R-(N2-deoxyguanosyl-3'-phosphate)-7,8,9,10-

tetrahydrobenzo[a]pyrene (BaPDE-dG-3’p) is used as an internal standard to monitor the

efficiency of enzyme-mediated transfer of the 32P-phosphate from [g-32P]ATP to DNA

adducts and as an alternate means of determining the specific activity of the [g-32P]ATP

used in the assay. In addition, the postlabeled BaPDE-dG-3’p is used as a chromatography

standard. To assess reproducibility, every 10th tissue sample is analyzed in duplicate. The

storage phosphor screens used to image the chromatograms in the assay are calibrated with

dilutions of 32P at each use.
140

Histopathology

Tissue sections of liver, kidney and gonad are examined for lesions by fish

histopathologists with 3 or more years of experience. Standard diagnostic criteria are used

to identify lesions (Myers et al. 1987, Johnson et al. 1991). Each individual specimen is

coded so that the location of the sample collection is unknown to the histopathologists

reading the slides. To maintain consistency and quality of interpretation, all pathologists

routinely consult with the chief histopathologist on any questionable lesions. If cases remain

problematic, the chief histopathologist will consult with colleagues at other agencies. In

addition, any new lesion types are sent to the Registry of Tumors for lower vertebrates in

Washington D.C. The chief histopathologist re-examines 10% of the slides read by the

other histopathologists for quality control.

Bile FACs

The quality assurance procedures include analyses of HPLC calibration standards

(CS), method blanks, replicates, and the bile reference pool materials. Before analyzing

samples, the stability of the HPLC performance is verified by replicate analysis of an HPLC

CS (3 or more times, as necessary), then comparing the area of each compound in the

calibration standard from analysis to analysis. Before analyzing sample sets, the HPLC

performance is considered stable when the mean response for each compound in the HPLC

CS has a relative standard deviation (RSD) < 15%. If the RSD for any analyte in the

continuing calibration is > 25% (during the period of analyzing a sample set), corrective

maintenance will be performed and the bile samples re-analyzed.

One method blank is analyzed at the beginning of each set of samples. If the

fluorescence response of the blank exceeds the usual background noise, appropriate column

maintenance is performed before proceeding with analyses of samples.

A bile reference material (BRM) is also analyzed in duplicate with each set of samples.

The BRM is a pooled fish bile that was analyzed as part of an interlaboratory quality assurance

exercise, which included two laboratories from the Environmental Conservation Division

and one laboratory from Texas A & M (Krahn et al. 1991). The concentrations of FACs

from each analysis of the BRM are then compared to the results from previous analyses of

the material. If the BRM concentrations vary by more than ±2 standard deviations from the

historical mean of previously analyzed BRMs, corrective action is taken, including instrument

maintenance or repair and re-analysis of samples.

One replicate bile sample is analyzed for each 10 field samples.

Reproductive Steroid RIAs- Plasma 17-b Estradiol Concentrations

Steroid RIAs are competitive binding assays which require the use of radiolabeled

standards and steroid specific antibodies. In this type of assay, a standard curve is generated

from known concentrations of 17-b estradiol, based on the binding characteristics of the

standards in the presence of antibody and radiolabeled hormone. The steroid concentrations
141

in plasma are then predicted by comparison with the standard curve using logistic regression

analyses. In order to assure that reproductive steroid RIAs are conducted properly, a number

of criteria are examined:

1) Standard curve: Standards typically range from 0 - 2000 pg estradiol 17-b/ml; if

necessary the range of standards may be modified somewhat to accommodate the

concentration range in the test samples. The regression coefficient, slope, Y-intercept, RSD

of the triplicates, and the binding efficiency and binding range of the standard curve are

calculated. Binding efficiency of antibody to labeled antigen (0standard (cpm) - NSB (cpm)/

Total (cpm) -NSB (cpm) x 100] should be approximately 30-35%, and the range of binding

of the standard curve should be from approximately 20-80% of B0; this constitutes the

effective concentration range of the assay. These are typical values used in most radio- and

enzyme-linked immunoassay procedures, and are documented in the literature (cf. see Sower

and Schreck, 1982 and Chard, 1982). If binding efficiency falls outside the 25-40% range,

or if the regression coefficient of the standard curve is < 0.95, the quality of the reagents is

checked and the assay is re-run with appropriate modifications.

2) Samples: Samples are also tested in triplicate. Samples that do not fall in 20-80%

binding range of the standard curve are retested by adjusting sample volume; additionally,

if the within-assay RSD of the triplicates is > 20%, the sample is re-run.

3) Quality Control: Triplicates of pooled plasma from male and/or gravid female

English sole collected in Puget Sound are tested with each assay as reference material for

quality control. The pooled reference plasma from the male sole contains very low or

undetectable concentrations of estradiol 17-b, while the pooled reference plasma from the

female sole contains elevated concentrations of estradiol 17-b. The concentrations of estradiol

from each analysis of the plasma reference pool material are then compared to the results

from analysis of the material in the previous sample sets. If interassay (i.e., day to day)

RSD of quality control samples exceeds 30%, the assay is re-run after making appropriate

modifications to reagents or other assay conditions.

Chain of Custody Procedures

These are described in detail in the Commencement Bay Damage Assessment

Quality Assurance Plan. Briefly, chain of custody procedures will be used for all samples

and for all data and data documentation, whether in hard copy or electronic format. Each

container is considered to be an individual sample and is assigned a unique ID number. A

sample is considered in “custody” if: a) it is in the custodian’s actual possession or view,

b) it is retained in a secured place (under lock) with restricted access, or c) it is placed in a

container and secured with an official seal(s) such that the sample cannot be reached without

breaking the seal(s).
142

Samples are kept in the custody of designated sampling and/or field personnel until

transfer to the laboratory. The original signed and dated chain of custody record accompanies

the sample(s). The laboratory sample custodian or designee maintains a laboratory sampletracking record, similar to the chain of custody record, that will follow each sample through

all stages of laboratory processing.

All unanalyzed samples and unutilized sample aliquots or extracts are held by the

laboratory in a manner to preserve sample integrity at a secure location with chain of custody

procedures for one (1) year after the QA Contractor has validated the data package for that

particular set of samples.

All data and data documentation, whether in hard copy or electronic format, is the

responsibility of the QA Coordinator acting on behalf of Counsel to the Case Management

Team. These materials will all be clearly marked with “Attorney Work Product.”
143

Budget Summary Table

Sampling and Analysis Plan

Hylebos Waterway Fish Injury Study

* up to 10 stomach organics samples are analyzed in a set. For every two sets, 5 or more QA

samples are needed (see Table 4). In some cases, there will not be enough samples to complete a

set of 10, however QA is required for partial sets. Since stomach contents are analyzed for both

AHs and CHs, these samples cannot be combined with sample sets of liver organics.

** up to 10 liver organics samples are analyzed in a set. For every two sets, 5 or more QA samples

are needed (see Table 4).

Juvenile Salmonid Assessment

# s Analyses T e ype of Analysi C $ ost/sampl Total

3 A 7 C 3 YP1 1 1 2 455

3 s 6 D 2 NA adduct 2 2 6 943

3 s 7 b 4 ile FAC 1 8 2 458

8 ) * 3 Stomach organic Chem (1 set 1 4 00 802

3 A * 3 Stomach organic Chem Q 1 9 00 300

3 ) 7 * 9 *Liver organic Chem (4 sets 7 3 7 2882

1 A 3 * 9 *Liver organic Chem Q 7 7 7 1012

2 s 0 S 0 ampling day 3 0 56 7120

S 4 almon Study Total = 13975

Toxicopathic Conditions in Flatfish

# s Analyses T e ype of Analysi C $ ost/sampl Total

3 A 6 C 3 YP1 1 8 2 442

3 s 6 D 2 NA adduct 2 2 6 943

3 s 6 B 4 ile FAC 1 4 2 446

1 ) 2 * 3 Stomach organic Chem (2 sets 1 6 00 1203

7 A * 3 Stomach organic Chem Q 1 1 00 702

3 ) 6 * 9 *Liver organic Chem. (4 sets 7 4 7 2804

1 A 4 * 9 *Liver organic Chem. Q 7 6 7 1090

3 y 60 F 3 ish histopatholog 4 0 1548

1 s 0 S 9 ampling day 3 0 36 3369

T 1 oxicopathic Study Total = 12550
144

Budget Summary Table (cont.)

Sampling and Analysis Plan

Hylebos Waterway Fish Injury Study

 1995 Hylebos Proj. Total = 463271

* up to 10 stomach organics samples are analyzed in a set. For every two sets, 5 or more QA

samples are needed (see Table 4). In some cases, there will not be enough samples to

complete a set of 10, however QA is required for partial sets.

*** up to 11 liver PCB samples are analyzed in a set. For every set, 3 QA samples are

needed (see Table 4.)

Reproductive Toxicology in Flatfish

# s samples T e ype of Analysi C $ ost/sampl Total

3 l 60 E 7 stradio 4 0 1692

1 s 20 D 2 NA adduct 2 0 6 3144

1 s 20 B 4 ile FAC 1 0 2 1488

1 ) 2 * 3 Stomach organic Chem. (2 sets 1 6 00 1203

7 A * 3 Stomach organic Chem Q 1 1 00 702

1 ) 20 * 3 **Liver PCB screens (11 sets 2 0 0 2436

3 A 3 * 3 **Liver PCB screens Q 2 9 0 669

3 y 60 F 3 ish histopatholog 4 0 1548

2 s 0 S 9 ampling day 3 0 36 6738

R 6 epro. Study Total = 19801
145

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150

Table 2A. Summary of Analytic Methods for Assessment of Injury to Juvenile Salmon.

Table 2B. Summary of Analytic Methods for Toxicological Conditions in Flatfish.

Table 2C. Summary of Analytic Methods for Reproductive Toxicology in Flatfish.

S s ample type A e nalysi Referenc

l s iver CHs, pesticide

CYP1A

DNA adducts

Sloan et al. 1993

Collier et al. 1995

Reichert et al. 1994

s s tomach contents A 3 Hs, CHs, pesticide Sloan et al. 199

b s ile A 6 H metabolite Krahn et al. 198

S s ample type A e nalysi Referenc

l ) iver CHs, pesticides (detailed

CYP1A DNA adducts

Sloan et al. 1993

Collier et al. 1995

Reichert et al. 1994

s s tomach contents A 3 Hs, CHs, pesticide Sloan et al. 199

b s ile A 6 H metabolite Krahn et al. 198

l y iver, kidney, gonad Histopatholog Liver lesions classified after

Myers et al. (1987). Ovarian

stages and lesions classified after

Johnson et al. (1991)

O n toliths A 2 ge determinatio Chilton and Beamish 198

S s ediment A 3 Hs, CHs, pesticide Sloan et al. 199

S s ample type A e nalysi Referenc

l ) iver CHs, pesticides (screening

DNA adducts

Krahn et al. 1994 Reichert et

al. 1994

s s tomach contents A 3 Hs, CHs, pesticide Sloan et al. 199

b s ile A 6 H Metabolite Krahn et al. 198

L y iver, kidney Histopatholog Liver lesions classified after Myers

et al. (1987)

O e vary determine maturation stag Ovarian stages and lesions

classified after Johnson et al.

(1991)

o n toliths A 2 ge determinatio Chilton and Beamish 198

Ovary, liver and gutted

body weight

Gonadosomatic index

Hepatosomatic index

Condition index

Johnson et al. 1991

B s lood plasma p . lasma E2 level Sower and Schreck (1982)
151

Table 3. List of Organic Analytes

Detailed Organic Analyses (liver and stomach content tissues for juvenile salmon and

toxicopathological studies, and for stomach contents for the reproductive toxicology study).

Low molecular weight AHs (stomach contents)

Naphthalene

Acenaphthylene

Fluorene

Anthracene

Acenaphthene

Phenanthrene

2-Methylnaphthalene

LAH [sum of low molecular weight AHs]

High molecular weight AHs (stomach contents)

Fluoranthene

Pyrene

Chrysene

Benzo(a)pyrene

Dibenz(ah)anthracene

Benz(a)anthracene

Benzo(b+k)fluoranthenes

ldeno(1,2,3-cd)pyrene

Benzo(ghi)perylene

HAHs [sum of high molecular weight AHs]

PCBs (stomach contents, liver)

PCB congeners (Nos. 18, 28, 44, 52, 66, 101, 118, 128, 138, 153, 170, 180, 187, 195,

206,209)

DDTs (stomach contents.. liver)

p,p’-DDE

p,p’-DDT

p,p’-DDD

Pesticides (stomach contents, liver)

Aldrin

Chlordane [ sum of a andy]

Heptachlor

Dieldrin

Lindane

Hexachlorobenzene
152

Table 3. cont.

Hexachlorobutadiene (stomach contents, liver); see p 19 of December, 1995 Commencement

Bay Damage Assessment Quality Assurance Plan

Analytes for Rapid CB analysis by HPLC/PDA

(liver tissues for reproductive toxicology study)

Dioxin-like PCB Congeners

77,105,118,126,156,157,169,170,180,189

Other PCB Congeners

101,128,138,153

DDTs

pp’-DDE, op’-DDT, op’-DDT, op’-DDD, pp’ DDD

Hexachlorobenzene (HCB)
153
154
155
156
157
158
159
160
161

Addendum for the

Hylebos Fish Injury Sampling and Analysis Plan (SAP)

1/28/97

This addendum contains changes to the SAP, primarily to, reflect differences between the work

as originally proposed and the work as conducted. It also corrects minor errors noted in the SAP

as originally submitted.

1) Standard length vs Fork length. Correction to the SAP: Standard length of flatfish was

measured for the Toxicopathic Conditions Study, and the Reproductive Toxicology Study. Fork

length of juvenile salmon was measured for the Juvenile Salmon, Injury Study.

2) Number of flatfish collected. The SAP originally stated that approximately 360 fish would

be sampled for each of the flatfish studies (Toxicopathic Conditions Study, and the Reproductive

Toxicology Study). Although 60 fish/site was the goal for the Toxicopathic, Conditions Study,

and 30 fish/site/sampling period was the goal, for the, Reproductive Toxicology Study, the

number of fish available at any given sampling period was sometimes fewer than anticipated.

The most significant -change from the expected number of fish collected occurred in the March,

1995 Reproductive Toxicology sampling. No English sole were present at the Hylebos site

during this sampling period, most likely due to normal migration patterns that occur during the

spawning season. As a result, the reference site was also not sampled for that month.

The actual number of fish collected for the Toxicopathic; Conditions Study was 120 Rock sole

and 233 English. sole for a total of 353. An additional 30 English sole were also collected at

Case Inlet, which was the original choice for a reference site, but these fish were too small to be

comparable with Hylebos fish. Therefore, these fish were not analyzed, and a new reference site

(Colvos Passage) was identified and successfully sampled. The actual number of female English

sole caught during the Reproductive Toxicology Study was 286. Of those, 275 were used in the

Reproductive Toxicology Interpretive Report (11 fish were not used due to missing or

questionable data).

In addition, extra fish not included in the SAP were collected during the Reproductive

Toxicology Study, including male English sole, and female and male rock sole. These fish were

not originally intended to be part of the Fish Injury Study, however the histopathology samples

were processed and analyzed as outlined in the SAP, and these samples were also maintained

under chain of custody. Histopathology data from these extra fish were ultimately included in the

Toxicopathic Conditions Interpretive report to increase the size of the data set used to evaluate

lesion prevalences and risk assessment. The number of extra fish added to the histopathology

data was 7 male rock sole, 100 female rock sole and 110 male English sole. These additional fish

are included in the histopathology case narrative. However, they are not included in the

Summary reports of average site values; the Toxicopathic Conditions Summary Report includes

only data specific to that study, while the Reproductive Toxicology Summary Report includes

only female English sole. The Toxicopathic Conditions Study Interpretive Report (Section 2 of

the Final Report) takes this data one step further by combining histopathology data from both
162

studies, as well as data from the extra fish described above, and includes additional bile FAC

data described below.

3) Additional individual bile analyses: In addition to the bile analyses of individual fish

collected from the December and January samplings for the Reproductive Toxicology Study as

described in the SAP, a miscommunication resulted in the analysis of bile from an extra 106

individual fish. These additional bile samples were from some of the extra male English sole that

were collected during the December and January samplings for the Reproductive Toxicology

Study, and from both male and female English sole collected during months other than

December and January. These additional bile samples were processed as described in the SAP,

handled under chain of custody and met QA criteria. We therefore took advantage of this

opportunity to increase the size of the data set, and included the extra bile FAC data in the lesion

risk assessment portion of the Toxicopathic Conditions Interpretive Report (Section 2 of the

Final Report). These additional bile samples are included in the revised case narrative (Appendix

3 to the Final Report).

4) Correction of salmon bile FAC data for protein content of bile: During collection of the

bile from juvenile salmon, it was noted that it was difficult to ensure that there was no

contamination of the collected bile with other fluids, such as blood or mucus. This was due to the

very small sizes of the gall bladders, especially from the chum salmon. Because of this, protein

correction was not used for the salmon bile data, as contamination by blood or mucus could

substantially affect this value. For purposes of comparisons to data from other sites (Figure 1.13

in Final Report), only non-protein corrected values were used. For other portions of the fish

injury studies [Toxicopathic and Reproductive Injury in Flatfish (Sections 2 and 3,

respectively)], protein-corrected bile data were used for interpretation of results.