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This document is available to the public through:

National Technical Information Service

U.S. Department of Commerce

5285 Port Royal Road

Springfield, VA 22161
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ABSTRACT

More than 500 fish and shellfish samples collected from native

Alaskan fishing grounds were analyzed for aromatic contaminants (ACs)

from petroleum (alkylated and unsubstituted aromatic hydrocarbons with

2-7 benzenoid rings and dibenzothiophenes). Intertidal molluscs (mussels,

clams, chitons, and snails) from Windy Bay, Kodiak (City), Chenega Bay,

and Old Harbor consistently had more than 100 parts-per-billion (ppb) ACs,

with levels in mussels from Windy Bay and Kodiak as high as 12,000 to

18,000 ppb. Levels of ACs in molluscs, crabs, and sea urchins from other

villages were less than 10 ppb, a level comparable to that measured in

shellfish from the designated reference area in Southeast Alaska near

Angoon. Nonparametric statistics on 147 samples of molluscs showed that

levels of ACs in molluscs from Windy Bay, Kodiak, and Chenega Bay were

significantly higher (p ≤ 0.05) than those in the Angoon molluscs sampled.

Of the 210 samples of edible flesh of fish analyzed in 1989, only two

samples of pink salmon (Onchorhynchus gorbuscha) from Kodiak had AC

levels that neared or exceeded 100 ppb. Another 11 samples of pink and

coho salmon (O. kisutch) from Kodiak, Chenega Bay, Tatitlek, and Larsen

Bay exceeded 10 ppb of total, ACs. The levels in the edible flesh of salmon

from other subsistence fishing areas and in bottomfish from all areas were

generally comparable (less than 10 ppb) to the levels detected in the same or

related species from the reference site, near Angoon. Two samples of

smoked salmon, one from Old Harbor and one from Tatitlek, contained

8,200 and 22,000 ppb of ACs, respectively.

In an unofficial advisory opinion, the Food and Drug Administration

has indicated that little risk is involved in the consumption of the nonsmoked subsistence foods studied. The results to date provide important

information on the level of contamination of subsistence fish and shellfish

from fishing areas of native Alaskan villages in and near Prince William

Sound and a reference database against which future temporal changes of

petroleum derived ACs in the edible flesh of fish and shellfish can be

evaluated.
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PREFACE

The Environmental Conservation (EC) Division of the Northwest

Fisheries Center conducts investigations on the fate and effects of organic

contaminants in the marine environment. Because we usually are dealing

with trace levels of toxic chemicals, and because the chemical makeup of

marine environments can be extremely complex, we need sensitive, reliable

analytical methods to produce data with an appropriate level of confidence.

Our analytical chemistry facility for trace organics was originally

established in the mid-1970s to provide NOAA with advanced analytical

capabilities that were not otherwise readily available. This resulted in the

development of state-of-the-art analytical techniques to measure trace

organics and their metabolites. Much of the analytical methodology needed

for NOAA’s environmental projects was developed over the past decade by

the Division’s researchers, with funding support coming from not only the

National Marine Fisheries Service but also other NOAA elements such as

the Outer Continental Shelf Environmental Assessment Program and the

Marine Ecosystems Analysis Program.

During the last decade, the EC Division conducted thousands of

sophisticated analyses of marine samples for trace levels of petroleum

hydrocarbons and other organic chemical contaminants. Early success in

studies conducted in Puget Sound and in the New York Bight laid the

foundations for the Division’s present prominent role in NOAA’s longterm

National Status and Trends Program in analyzing sediments and benthic

fish from U.S. coastal waters for organic contaminants. At the same time,

we have conducted interlaboratory comparisons for analyses of marine

samples. Until 1980, comparisons of analytical results among experienced

laboratories differed by as much as tenfold. We were asked to investigate

ways to improve the intercomparability among such laboratories. Over the

years, precision among experienced laboratories improved substantially to a

range of 14-81% relative standard deviation. Moreover, in response to the

need to analyze large numbers of environmental samples with greater speed,
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we replaced two lengthy manual cleanup procedures with a single high

performance liquid chromatography, cutting cleanup time by 75% and

solvent consumption by 50%. Division scientists also developed a method

to test for petroleum exposure by rapidly screening fish bile for metabolites

of aromatic compounds. These coordinated efforts have placed the EC

Division in an excellent position to provide timely and quality analyses on

subsistence samples related to the oil spilled from the Exxon Valdez in

Prince William Sound.
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INTRODUCTION

The spreading of oil spilled from the Exxon Valdez on 24 March 1989

raised concerns among native Alaskans that their subsistence seafood could have

been contaminated by petroleum hydrocarbons. In response, NOAA entered into

memorandum of understanding (MOU) with Exxon to analyze subsistence fish

and shellfish from native Alaskan villages for aromatic contaminants (ACs)

found spilled in oil. The ACs consist of the aromatic hydrocarbons and dibenzoiophenes listed in Table 1. This report discusses the levels of these chemicals

found in fish and shellfish collected in July, August, and September 1989

(Cycles II, and 1111, respectively). Statistical interpretations are included. The

subsistence fishing grounds sampled in this study appear in Figure 1.

Previous laboratory studies have shown that fish efficiently biotransform

aromatic hydrocarbons to derivatives (metabolites) that are concentrated in bile

for excretion (Statham et al. 1976, Varanasi and Gmur 1981, Stein et al. 1984,

Varanasi et al. 1989a). This means that aromatic hydrocarbons may not readily

cumulate in the edible flesh of fish. Thus, to monitor the exposure of fish to ACs,

we developed a rapid, sensitive method to screen fish bile for presence of

metabolites characteristic of petroleum ACs. This procedure utilizes high

performance liquid chromatography (HPLQ with fluorescence detection (Krahn

et al. 1984, 1986a). It has been employed previously in an oil spill on the

Columbia River (Krahn et al. 1986b) to determine exposure of fish to ACs from

petroleum.

The more specific analyses for individual ACs in tissues, which involve

combined gas chromatography/mass spectrometry (GC/MS), are more costly

than the screening of bile for ACs and their metabolites. Nevertheless, recent

important improvements and automation of the extract cleanup procedure enable

us now to provide high quality analytical data for AC levels in the edible flesh of

fish and shellfish more quickly than before and with less labor (Krahn et al.

1988). These methods can also be used to detect sulfur-containing ACs, such as

the dibenzothiophenes.

Statistical differences of AC levels in edible flesh or bile among sites were

assessed using both parametric and nonparametric tests.
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EXPERIMENTAL METHODS

Details of protocols for the field sampling, chemical analyses, and

statistical evaluation are outlined below. These protocols have also been briefly

described in the periodic reports (Varanasi et al. 1989b-e) issued throughout the

study.

Field Sample Collection

Under contract with Exxon, Dames and Moore biologists collected

samples of fish (Table 2) and shellfish (Table 3) from subsistence fishing areas

near native Alaskan villages (Fig. 1). Generally, at least two sites were sampled

per village with assistance of NOAA or State of Alaska field biologists. Bile

samples from these fish were usually screened for metabolites of ACs (see

below) to indicate the priority by which samples of edible fish flesh should be

subjected to a more detailed analysis; however, Cycle I sampling was completed

before this- protocol could be fully implemented. Otherwise, samples of bile and

flesh were chilled in the field, frozen before shipment, and stored frozen in the

laboratory until analyzed. Shellfish samples were composites of whole individual

animals, while fish were sampled as individuals for shipment.

Bile Analyses

Fish bile samples were analyzed for fluorescent ACs (FACs) by the metho

of Krahn et al. (1986a) outlined below. Bile collected from the fish was stored at,

the laboratory at -80°C until analyzed.

Fluorescent Aromatic Contaminant Determinations

The FACs in bile were analyzed on a Waters1

 high performance liquid

chromatograph equipped with a Perkin-Elmer HC-ODS/ PAH column (0.26 X 25

cm.), an automatic injector, and Perkin-Elmer model 40 fluorescence detecto

connected in series.

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 Reference to trade name does not imply endorsement by the National Marine Fisheries

Service NOAA.
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Thawed bile was injected directly into the HPLC and eluted through the

column using a linear gradient from 100% solvent A (water containing 5 ppm

acetic acid) to 100% solvent B (pure methanol) during 15 minutes. The flow rate

was 1.0 mL/min and the column temperature was 50°C. All solvents were

degassed with helium.

The fluorescence responses were recorded at the wavelength pairs for NPH

and PHN, prominent aromatic constituents of Prudhoe Bay crude oil (see Table

1). Fluorescence of NPH metabolites was monitored using excitation and

emission wavelength pairs of 290 and 335 mn, respectively. Fluorescence of

PHN metabolites was monitored using excitation and emission wavelength pairs

of 260 and 380 nm, respectively.

The total integrated area from each detector was then converted to

corresponding units of either NPH or PHN that would give the same integrated

response. Results for FACs in bile are reported on the basis of bile volume and

biliary protein (Fig. 2). The levels of protein in bile (Table 4) were determined

by the method of Lowry et al. (1951) that measures the complex formed with

phenol at 660 nm.

Quality Assurance

Quality assurance procedures included use of NPH and PHN calibration

standards, a “bile pool” reference material, blank analyses, and replicate analyses

to evaluate HPLC/UV fluorescence performance.

Edible Flesh Analyses

Our laboratory procedures for the analysis of toxic organic contaminants

(Krahn et al. 1988a, MacLeod et al. 1985) follow protocols established by the EC

Division. A total of 365 analyses of edible flesh of fish and shellfish were

performed for the ACs listed in Table 1. Summaries of the analytical protocols

are giv en below (for further details, please consult the original publications).

These protocols consist of four ma or steps: a) extraction; b) cleanup (by HPLC);

c) analyte determination (by GC/MS); and d) quality assurance.
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The results of the bile analyses were used to prioritize and composite fish

samples whose edible flesh was to be analyzed by the more quantitative and

costly GC/MS technique. Edible flesh samples from the same fish species were

analyzed as individual samples or as composites according to the levels of FACs

in bile. Thus, for a given species at a given site, flesh from fish showing

relatively high levels of bile FACs was analyzed either from individuals or

composites of individuals with similarly high FACs levels. Samples of flesh

from fish with relatively low levels of bile FACs were generally analyzed as

composites.

Extraction of Aromatic Contaminants

Samples of edible flesh of fish or shellfish were extracted for ACs

according to the procedures of Krahn et al. (1988a). A 3-g sample of flesh is

added to a centrifuge tube containing sodium sulfate and methylene chloride.

The method internal standards (surrogates) for the ACs are added, and the

mixture is macerated with a Tekmar Tissurnizer. The extract is filtered through a

column of silica and alumina, and the extract concentrated to 1 mL for cleanup

by HPLC.

Cleanup of Aromatic Contaminants

The ACs were isolated on a high performance liquid chromatograph. A

Spectra-Physics (Mountain View, CA) model 8800 HPLC was employed,

equipped with an ultraviolet detector (254 nm) and an automatic injector. Two

22.5 x 250-mm stainless-steel (preparatory size) columns containing Phenogel

100-A size-exclusion packing (Phenomenex, Rancho Palos Verdes, CA) were

used in series with a 2-μM Rheodyne model 7302 filter and a 7.8 x 50-mm.

guard column containing the same Phenogel packing. The HPLC precolumn and

column were connected to a six-port valve that allows the guard column to be

backflushed to remove extraneous materials after cleanup of a set of samples

(n ~ 10).

Methylene chloride was used as the solvent and was pumped at a flow rate

of 7 mL/min for 20 minutes at ambient temperature. The HPLC solvent was

degassed by bubbling helium through the solvent. The helium was delivered via

a regulator equipped with a stainless-steel diaphragm and passed through an

in-line charcoal filter (200-cc hydrocarbon trap, Alltech Assoc., Deerfield, IL) to
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eliminate inadvertent contaminants which could be transferred to the HPLC

solvent by the helium.

A 250-μL portion of a 1-mL extract was injected onto the HPLC column

and the fraction containing the ACs was collected according to Krahn et al.

(1988a). The solvent in the HPLC fraction was exchanged into hexane as the

volume was reduced by evaporation to approximately 1 mL. Standards were then

added for analysis by capillary column gas chromatography with mass

spectrometric quantitation.

Aromatic Contaminant Determinations by GC/MS

The ACs were determined according to MacLeod et al. (1985) by GC/MS

quantitation as outlined by Burrows et al. (1990). A 30-m x 0.25-mm DB-5

capilliary column (J & W Scientific) was used in a Hewlett-Packard model 5880

or 5890 gas chromatograph. The GC sample (3 μL) was injected splitless, and

the split valve was opened after 18 seconds (split ratio of 20:1). The oven

temperature of 50°C was held for 1 minute and then programmed to increase at

4°C/min to 170°C, then at 1°C/min to 210°C, and finally at 4°C/min. to 300°C,

where the temperature was held for 10 minutes.

Quality Assurance

Quality assurance measures included analyses of method blanks, spiked

blanks, and matrix spikes. Analyte recoveries were normalized through the use

of internal standards. The recoveries of the following surrogates are reported as

QA information: Naphthalene-d8, acenaphthene-d10, and benzo[a]pyrene-dl2.

Analyte concentrations are reported on the basis of the internal standards

(“surrogates”) added at the beginning of the sample extraction. The HPLC

internal standard (used to determine the fraction of total sample extract that was

used in the analysis for aromatic hydrocarbons) was phenanthrene-dl0.

Hexamethylbenzene was used as the GC internal standard to calculate the

recoveries of the surrogates. The recovery for each surrogate standard was

greater than 50%, but less than 130%, and the relative standard deviations

(RSDs) for surrogate recoveries in a set of samples was less than 25%. When the

recovery of any surrogate was outside these guidelines, corrective action was
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taken, including instrument repair, inlet cleaning, column replacement, and/or

reanalysis.

The GC calibration standards generally included all surrogates and

analytes of interest, except for some classes of alkylated aromatic hydrocarbons

and dibenzothiophenes the corresponding unsubstituted compound was used to

calculate the response factor. Graduated concentrations of GC-calibration-check

standards were used for multilevel response-factor determinations. A GC

calibration standard was analyzed after every six samples to demonstrate the

stability of the calibration. The GC/MS was considered “under control” when the

response for each analyte or surrogate in a GC calibration standard was

reproducible within ± 10% from analysis to analysis.

The detection limits generally were less than 1 ng/g (wet weight basis). A

hyphen (-) is used in the data tables to indicate that the analyte was not detected.

The range of detection limits is included in the explanatory notes for each

appendix.

One method blank was analyzed with each sample set of approximately 10

samples. The aromatic hydrocarbons (except naphthalene) and

dibenzothiophenes in the blanks should not be present above the limit of

detection. A matrix spike (containing 50-80 ng/g of each analyte) or a spiked

blank (containing 40-70 ng/g of each analyte) was analyzed with each set of

approximately 10 samples. The recoveries of analytes should be no less than

50%.

Statistical Methods

The Kruskal-Wallis test nonparametric analysis of variance (Sokal and

Rohlf 1981, Zar 1984) was used to test for differences among villages (sites). If

the null hypothesis of no difference among villages (sites) was rejected at a =

0.05, other nonparametric methods were used, specifically, to a) compare control

results to other groups and b) ascertain differences among groups by multiple

comparisons (Dunn 1964, Hollander and Wolfe 1973, and Zar 1984). The

significance level was set at a = 0.05.
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Because in many cases the sample sizes for individual species were too

small (especially at Angoon) to permit strong statistical conclusions to be drawn,

samples were grouped by taxonomic affiliation and habitat (viz., beach,

midwater, or benthic) for purposes of statistical analyses. For example, it was

necessary to combine data for all intertidal molluscs at each village to obtain a

sample size suitable for statistical analyses. Combining species or taxa with

similar biology and habitat also permitted inclusion of villages that would

otherwise have been omitted from the comparisons because only one sample was

available for a particular species or group.

RESULTS AND DISCUSSION

Fluorescent Aromatic Contaminants in Bile of Fish

Our laboratory studies (Varanasi and Gmur 1981, Stein et al. 1984,

Varanasi et al. 1989a) with benthic fish exposed to naphthalene and

benzo[a]pyrene (BaP) have shown that levels of metabolites of these ACs in bile

can be substantially greater than those in edible flesh. Thus, even though the

values for FACs in bile of field sampled fish may indicate exposure of fish to

petroleum, the levels of aromatic hydrocarbons from petroleum in edible flesh of

fish, if detectable, could remain low. The utility of the bile method is in quickly

identifying those fish that are relatively unexposed to ACs and therefore of less

immediate interest.

The results of the HPLC fluorescence analyses for FACs in bile, at the

PHN wavelengths, are summarized in Figure 2 and Table 4. Presentation of

results at NPH wavelengths is not necessary because of the strong statistical

correlation between FACsPHN and FACsNPH (r = 0.93, P: ≤ 0.0001). In Figure 2

the concentrations of FACsPHN are reported on the basis of bile protein. Previous

laboratory studies (Collier and Varanasi 1987) have shown marked increases in

concentrations of protein in bile of nonfeeding fish compared to feeding fish. It

was shown that the variation between levels of FACs in feeding and nonfeeding

fish were greatly reduced when the differences in bile protein were taken into

account. Hence, the reporting of FACsPHN levels on the basis of bile protein is

appropriate in this study, because the salmon sampled were sexually mature and

may not have been feeding.
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The results from the present study showed that reporting the concentration

of FACs on the basis of bile protein appeared to account for large species

differences in FACSPHNIevels for fish from a village. For example, calculation

of the FACspHN levels for pink salmon (Oncorhynchus gorbuscha) and

bottomfish from Port Graham and English Bay on the basis of bile protein rather

than on the basis of bile volume reduced the difference between the levels from

about tenfold to about 80 % (Table 4). This does not imply that the exposure to

oil is similar for the two species, just that the level of FACs in bile appear

similar. Numerous factors can affect tissue and fluid levels of contaminants;

hence, dose-response studies with each species are required to validate

comparisons between species.

As summarized in Figure 2, the levels of FACPHN in bottomfish from

Chenega Bay, Ouzinkie, and Port Lions were significantly different (higher)

from the levels in bile of bottomfish from Angoon, the reference site. The levels

of FACsPHNin coho salmon (O. kisutch) from Ouzinkie, Akhiok, and Tatitlek

were significantly higher than those in coho salmon from Angoon. For pink

salmon, however, no significant differences were observed when compared to

Angoon. The lack of statistically significant differences with respect to Angoon

was due to a single pink salmon having a bile level of FACsPHN that was four- to

twentyfold greater than the bile levels in the other three pink salmon from

Angoon. What the finding of a high level of FACsPHN in the one pink salmon

from Angoon Figure 2 means is not known. A larger sample size from the

designated reference site or samples from another reference site win be needed

for proper statistical evaluation of salmon exposure to ACs.

The results with bile suggest that a number of fish species from some sites

were exposed to ACs. However, in subsequent analyses of the edible flesh, levels

of total ACs in bottomfish never exceeded 1 ppb and were comparable to those

from Angoon (Fig. 3), while in bile, mean levels of FACsPHN were up to 25 times

greater than those from Angoon (Fig. 2). The results for pink and coho salmon

(Fig. 3) also show low levels of total ACs in muscle (generally less than 7 ppb),

while the level of FACsPHN (Fig. 2) ranged widely, from 80 to 17,000 ng PHN

equivalents/mg bile protein. These results are consistent with the interpretation,

based on laboratory studies, that efficient metabolizing of petroleum-derived

ACs by the liver of fish greatly limits the accumulation of ACs in other tissues

such as muscle (Varanasi et al., 1989a).
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It should also be noted that when the levels of FACsPHN in bile of

individual salmon were less than 1,000 ng PHN equivalents/ing bile protein, no

pink or ccohoo salmon showed levels of total ACs exceeding 5 ppb. A few pink

and coho salmon had AC levels greater than 7 ppb; however, in these fish,the

levels of ACs in muscle were not directly related to levels of FACsPHN in bile,

suggesting that a factor other than exposure was also affecting the tissue

distribution of ACs in these few fish. Most of the salmon sampled in this study

were near spawning. Previous studies (Varanasi et al. 1982; Reichert and

Varanasi 1982) have shown that in marine bottomfish near spawning, aromatic

hydrocarbons are not as effectively metabolized by the liver and that this can

lead to some retention of unaltered aromatic hydrocarbons by tissues such as

muscle.

Aromatic Contaminants in Edible Flesh of Fish and Shellfish

All 548 samples of edible flesh from fish and shellfish received in 1989

are included in the 365 analyses for the ACs reported herein. Summary results

are presented for fish in Table 5 and for shellfish in Table 6. Included are results

from comparison samples collected at Angoon, a designated reference site in

Southeast Alaska. Results are also presented for a sample of smoked salmon each

from Tatitlek and Old Harbor.

The data in Tables 5 and 6 have been summarized according to the lowmolecular-weight ACs (LACs) and the high-molecular-weight ACs (HACs)

listed in Table 1, following a practice we have established (Varanasi et al. 1988,

1989f) in the Benthic Surveillance Project of NOAA’s National Status and

Trends Program. This has the convenience of dividing the ACs approximately

into (a) the more water-soluble and acutely toxic compounds, LACs, and (b) the

less water-soluble and more chronically toxic compounds, HACs. The LACs are

more prone to dissolution, evaporation, and bacterial degradation and, hence,

their levels in the environment generally would decline as the spilled oil

weathers. Moreover, LACs are more rapidly excreted from the body than are

HACs, and LACs are known for their acute toxicity in experimental animals. The

HACs, on the other hand, are more resistant to dissolution, evaporation and

bacterial degradation and, hence, tend to persist in the environment. Carcinogens

generally appear atnong the HACs.
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The tables in the Appendices contain more detailed information. Appendix

A tabulates the summary results (LACs/HACs) according to village, sites, and

species, including our laboratory sample numbers and composite sample

information. Appendix B presents the complete set of analytical data for ACs

from the edible flesh of all samples of fish (including the data for smoked salmon

and the field collector’s sample numbers). Appendix C contains analogous

information for shellfish.

Fish

As there are significant differences in the rates of uptake, metabolism,

excretion, and types of toxic effects exerted by various ACs, the fish data are

also presented in Figure 4 to show the levels of LACs and HACs separately. The

HACs are metabolized by fish to a greater extent than are the LACs and some

HACs exert chronic toxic effects (e.g., carcinogenicity, tetratogenicity, and

reproductive dysfunction) in experimental animals. To our knowledge, there are

presently no established guidelines for acceptable levels of LACs or HACs in

food products.

Of the 208 samples of edible flesh of unsmoked fish analyzed in this first

year, only two samples of pink salmon from the village of Kodiak had levels of

ACs nearing or slightly exceeding 100 ppb. Eleven samples of pink, coho, and

sockeye salmon (0. nerka) from Kodiak, Chenega Bay, Tatitlek, and Larsen Bay

exceeded 10 ppb of ACs. The levels of ACs in the edible flesh of salmon from

other subsistence fishing areas and bottomfish from an fishing areas were

generally comparable (less than 10 ppb) to the levels detected in the same or

related species from the reference site, Angoon.

Two samples of smoked salmon from Old Harbor and Tatitlek were

analyzed in this study; they contained 7,500 and 20,000 ppb of LACs and 670 to

2,400 ppb HACs, respectively. The concentrations of benzo[alpyrene, a

carcinogenic HAC, in these samples were 6 ppb (Old Harbor) and 20 ppb

(Tatitlek). More samples of smoked fish must be analyzed before proper

comparisons can be drawn.

Molluscs

Analyses of molluscs (mussels, clams, chitons, and snails) from Windy

Bay, Kodiak (village), Chenega Bay, and Old Harbor showed consistent
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evidence of exposures to ACs (tissue levels above 100 ppb). Mussels from

Windy Bay and Kodiak had AC levels as high as 12,000 to 18,000 ppb (Table 6).

Otherwise, levels of ACs in molluscs, crabs, and urchins from the other

collection sites were less than 10 ppb, a level comparable to that measured in

shellfish collected near the village of Angoon, a designated reference area in

southeast Alaska.

Benzo[a]pyrene was detected in molluscs from Windy Bay and Chenega

Bay at levels of 0.1 to 7 ppb, but was not detected in fish.

Statistical Results

In addition to these qualitative interpretations of the AC data, statistical

analyses of the AC data were also conducted. The results of these analyses

revealed, however, that the sample sizes for individual species were generally

too small to draw strong statistical conclusions. This was especially evident for

the reference site, near Angoon, where the sample size was only 1 to 3 for any

single species.

To increase our ability to draw statistical inferences from the data, samples

were grouped according to type (e.g.,. salmonids or bottornfish) or habitat (e.g.,

intertidal or benthic) as shown in Table 7. For example, combining data for all

intertidal molluscs increased the sample size (n = 147) to a reasonable value and

allowed inclusion of most villages in the statistical analyses. While grouping of

different species and samples from different sampling stations is not ideal, such

an approach did enable certain general conclusions to be drawn, as is discussed

below.

Of the taxonomic groupings of species, the sample size was largest for

Pacific salmon, n = 164; this includes 6 samples from Angoon and 11 villages

with 2 or more samples. Next in sampling size were the intertidal molluscs, n =

147, which includes 7 samples from Angoon and 13 villages with 2 or more

samples. The statistical results have the greatest validity for these two groups.

Although it is often convenient to consider the LACs and the HACs

separately, in this study the statistical conclusions for each of these categories

were not different from those for all the ACs together. Hence, the conclusions
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are presented for total ACs. Thus, Figure 3 shows that the highest mean level of

ACs in molluscs (4,700 ppb) was substantially greater than the highest mean

level in salmon (30 ppb) and bottomfish (0.7 ppb).

Fish. The levels of total ACs in edible flesh of salmon and bottomfish and

results of statistical analyses are shown in Figure 3. In salmon and bottomfish,

the levels of ACs in this study were not significantly different from the levels in

fish from Angoon. However, the levels of ACs in salmon from the village of

Kodiak were significantly different (higher) from those in salmon from Chenega

Bay, Old Harbor, and Port Graham and English Bay. The very low levels of ACs

in the edible flesh of bottomfish did not warrant further statistical investigation.

To date, only two samples of smoked salmon from Old Harbor and Tatitlek were

analyzed in this study (see above). More samples of smoked fish must be

analyzed before proper statistical comparisons can be drawn.

Molluscs. The levels of total ACs in edible flesh of intertidal molluscs and

results of statistical analyses are also shown in Figure 3. The AC levels in

molluscs from Windy Bay, Kodiak, and Chenega Bay were significantly higher

than in those from Angoon. No other villages had significantly higher levels than

Angoon, not even Old Harbor, despite the three samples which had greater than

200 ppb of total ACs.

Analytical Chemical Results According to Fishing Village (Site)

Summary results for each subsistence fishing area are discussed with the

sums of the LACs preceding those of the HACs.

Windy Bay --

Fish. None were collected at Windy Bay.

Mussels. In Cycles I and 111, three samples from this area at the tip

of the Kenai Peninsula showed the highest levels of ACs of any mussels

evaluated in this study. The 2,400-16,000 ppb of LACS and 440-2,500 ppb of

HACs are three to fogr orders of magnitude higher than the levels found in two

samples from the designated reference site near Angoon in Southeast Alaska.
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The Angoon reference data showed only 0.6-1 ppb of LACs and up to 0. 1 ppb of

HACs.

Chitons. In Cycle III, 4,000 ppb of LACs and 1,800 ppb of HACs

were found in a composite sample, compared to the 1 ppb and none detected,

respectively, found at Angoon (n = 2). In Cycle I, a sample showed 13 ppb of

LACs and 3 ppb of HACs, respectively.

Littleneck Clams (Protothaca staminea). In Cycle I, 830 ppb of

LACs and 130 ppb of HACs were found in a sample of littleneck clams. No

littleneck clams were sampled at Angoon, but a sample of butter clams from

there showed LAC and HAC levels of 0.5 ppb and none detected, respectively.

Snails. In Cycle I, 620 ppb of LACs and 160 ppb of HACs were

found in a sample of snails. No snails were sampled at Angoon, but other

molluscs there (n = 6) showed LAC and HAC levels of 0.5-1 ppb and up to 0.1

ppb, respectively.

Kodiak Village

Pink Salmon. In seven samples, 4-110 ppb of LACs and 0.2-12 ppb

of HACs were found in Cycle II. In Cycle III, the LAC and HAC levels found in

three samples were 0.5-3 ppb and none detected, respectively, comparable to the

LAC and HAC levels of 2 ppb and none detected, respectively, in pink salmon

from Angoon (n = 2).

Sockeye Salmon. A sample showed 11 ppb of LACs, slightly higher

than the 2-5 ppb of LACs (n = 6) in other species of salmon from Angoon. No

sockeye salmon were collected at Angoon.

Dolly Varden (Salvelinus malma). A trace (1 ppb) of LACs was

found in one sample. No Dolly Varden were collected at Angoon.

Halibut (Hippoglossus stenolepis). Trace levels of LACs (up to 0.9

ppb) in three samples, comparable to the 0.4 ppb found in halibut from Angoon.
30

Rockfish. No ACs were found in rockfish. No rockfish were

collected at Angoon.

Mussels. In Cycle I, a sample contained 1,500 ppb of LACs and 160

ppb of HACs as compared to the two samples from Angoon, which had 0.61 ppb

of LACs and up to 0.1 ppb of HACs.

Butter Clams (Saxidomus giganteus). In Cycle II, a sample

contained 410 ppb of LACs and 50 ppb of HACs compared to the sample from

Angoon, which had LAC and HAC levels of 0.5 ppb and none detected,

respectively.

Littleneck Clams. In Cycle III, two samples contained 120-270 ppb

of LACs and 24-87 ppb of HACs as compared to the other bivalves (mussels and

butter clams, n = 3) from Angoon, which had 0.5-1 ppb of LACs and up to 0.1

ppb of HACs. No littleneck clams were collected at Angoon.

Chitons. In Cycle II, a sample contained 230 ppb of LACs and 27

ppb of HACs, whereas in Cycle IIEI, another sample contained LAC and HAC

levels of 90 ppb and 30 ppb, respectively. The two samples from Angoon

contained 1 ppb LACs and no detectable HACs.

Dungeness Crabs (Cancer magister). Traces of LACs (0.2-0.6 ppb)

were found in two samples. No reference samples were available from Angoon.

Chenega Bay --

Pink Salmon. A sample showed 20 ppb of LACs in Cycle II. The 19

other samples (Cycles II and III) showed only up to 3 ppb of LACs, which is

quite comparable to the 2 ppb of LACs found in two samples from Angoon.

Halibut, Pacific Cod (Gadus macrocephalus) and Rockfish. Traces

of LACs were detected, which were comparable to those from the Angoon

reference area (0.4-0.6 ppb).

Mussels. Levels of ACs were fairly similar in five samples collected

throughout all cycles (96-220 ppb of LACs and 15-26 ppb of HACs). Two
31

samples from Angoon contained LAC and HAC levels of 0.6-1 ppb arrd up to

0.1 ppb, respectively.

Butter Clams. In two samples, 15-37 ppb of LACs and 3-5 ppb of

HACs were found, as compared to a sample from Angoon, which had LAC and

HAC levels of 0.5 ppb and none detected, respectively.

Old Harbor --

Pink Salmon. Up to 4 ppb of LACs in 18 samples were comparable

to two samples from Angoon (2 ppb).

Smoked Salmon. A sample of smoked salmon showed 7,100-7,800

ppb of LACs and 650-700 ppb of HACs, approximately three orders of

magnitude greater than the unsmoked salmon.

Halibut and Pacific Cod. The levels of LACs found in halibut (up to

1 ppb, n = 3) and a Pacific cod (0.7 ppb) were similar to levels found in the same

species from Angoon (LAC and HAC levels of 0.4 and 0.6 ppb, respectively).

Butter Clams and Littleneck Clams. Two samples from Cycle I had

2-4 ppb of LACs and 0.6-7 ppb of HACs, as compared to 0.5 ppb and none

detected, respectively, from an Angoon sample. Four samples from Cycle II

showed LAC and HAC levels of 2-6 ppb and 0.3-5 ppb, respectively. In Cycle III

three samples from site 4 showed 210-280 ppb of LACs and 20-25 ppb of HACs,

while one sample from site 3 had levels comparable to those from Angoon.

Chitons. A sample from Cycle I had 2 ppb of LACs and 0.1 ppb of

HACs, whereas in Cycles III and IH these LAC and HAC levels of were 0.50.9

ppb and none detected, respectively, (n = 2), which is comparable to Angoon

(LAC and HAC levels of 1 ppb and none detected, respectively, n = 2).

Tatitlek --

Pink Salmon. In Cycles I and II, levels of ACs in pink salmon from

Tatitlek were below the 2 ppb of LACs found in pink salmon from Angoon.
32

Coho Salmon. In Cycle III, seven samples showed 3-19 ppb of

LACs and up to 2 ppb of HACs. Three of these samples had 14 ppb or more of

ACs, compared to three samples from Angoon that had LAC and HAC levels of

2-4 ppb and none detected, respectively.

Smoked Salmon. A sample of smoked salmon showed 20,000-

21,000 ppb of LACs and 1,800-3,000 ppb of HACs, some four orders of

magnitude greater than the levels in unsmoked salmon.

Mussels. In Cycle II, the 3 ppb of LACs in three samples was

slightly higher than the 0.6-1 ppb in two samples from Angoon. In Cycle III, the

0.8-1 ppb of LACs in three samples were comparable to those of the Angoon

samples.

Chitons. Low levels of ACs in two samples (up to 1 ppb) were

indistinguishable from those in Angoon samples.

Larsen Bay --

Pink Salmon. In Cycle I, 1-12 ppb of LACs and up to 0.4 ppb of

HACs were found in two samples, compared to LAC and HAC levels of 2 ppb

and none detected, respectively, in two samples from Angoon. In a Cycle II

sample, values were comparable to those from Angoon (2 ppb, n = 2), while in

Cycle III, levels of LACs (up to 1 ppb) in 11 samples were below those of

Angoon.

Sockeye Salmon. In Cycle II, 1-6 ppb of LACs and up to 0.2 ppb of

HACs were found in six samples, quite similar to the LAC and HAC levels of

2-5 ppb and up to 0.1, respectively, in other species of salmon from Angoon (n =

2). No sockeye salmon were collected at Angoon.

Halibut and Pacific Cod. The very low levels of ACs found were

similar to those found in the same species from Angoon (up to 1 ppb).

Butter Clams. Levels of LACs (0.5-4 ppb) in seven samples of

butter clams were comparable to those in a sample of butter clams from Angoon

(0.5 ppb).
33

Chitons, Snails, Limpets, Sea Urchins, and Crabs. The low levels of

ACs found were similar to, or slightly higher than, those found in some of these

species at Angoon (up to 1 ppb).

Ouzinkie -

Pink Salmon. The 0.9-5 ppb of LACs in nine samples in Cycle II were

comparable to, or slightly higher than, those in Angoon samples (2 ppb, n = 2).

Coho Salmon. In Cycle III, the 0.6-6 ppb of LACs in eight samples

were comparable to the 2-4 ppb in Angoon samples (n = 3). Traces of HACs

(0.2-0.6 ppb) were also detected in three samples, as compared to none detected

in the Angoon samples (n = 3).

Halibut. Up to 1 ppb of ACs found in five samples were comparable

to those in Angoon halibut (0.4-1 ppb).

Butter Clams. In four samples, the 0.9-7 ppb of LACs and up to 2

ppb of HACs were comparable to, or slightly higher than, those in Angoon

samples (LAC and HAC levels of 0.5 ppb and none detected, respectively, n =

1).

Mussels and Chitons. Levels of ACs found in a sample each of

mussels (0.9 ppb) and chitons (0.5 ppb) were comparable to those for Angoon

(0.4-1 ppb).

Chiniak -

Pink Salmon, Sockeye Salmon, Chum Salmon (0. keta), and Halibut.

The levels of LACs (0.4-4 ppb) in six samples of pink salmon and two of halibut

(0.8-1 ppb) from Chiniak were comparable to those from the same species

sampled at Angoon (2 ppb, n = 2 and 0.4 ppb, n = 1, respectively). Additionally,

samples of sockeye salmon and chum salmon contained 0.8-5 ppb of the LACs,

similar to the levels for pink salmon from Angoon (2 ppb, n = 2).
34

Butter Clams, Littleneck Clams, Cockles, and Chitons. The levels of

LACs (0.1-2 ppb) in 14 samples of such molluscs were comparable to those in

similar mollusc samples from Angoon (0.5-1 ppb, n = 5). In addition, samples of

butter clams and littleneck clams had traces of HACs (0.3-0.4 ppb, n = 3),

compared to none detected for Angoon.

Akhiok --

Pink Salmon. The 1-4 ppb of LACs and 0.1-0.4 ppb of HACs were

comparable to those in samples from Angoon (LAC and HAC levels of 2 ppb

and none detected, respectively, n = 2).

Coho Salmon and Sockeye Salmon. No ACs were found in samples

of either of these species at this site.

Mussels, Butter Clams, Littleneck Clams, and Chitons. Up to 1 ppb

of LACs in seven samples were comparable to the levels in such molluscs from

Angoon (0.5-1 ppb, n 5).

Port Lions

Pink Salmon. In 10 samples from Port Lions, up to 3 ppb of LACs

and up to 0.2 ppb of HACs were comparable to those in samples from Angoon

(LAC and HAC levels of 2 ppb and none detected, respectively, n = 2).

Coho Salmon, Halibut, and Sole. The 0.2-3 ppb of LACs in six

samples of coho salmon, four samples of halibut and one sample of sole from

Port Lions were comparable to the levels in these species from Angoon (0.4-4

ppb, n = 4).

Crab, Butter Clams, Littleneck Clams, Chitons, and Snails. The

0.4-3 ppb of ACs in these samples were comparable to those for samples from

Angoon (0.5-1 ppb, n = 5).
35

Port Graham and English Bay

Pink Salmon. Up to 1 ppb of ACs in seven samples were

comparable to those in samples from Angoon (2 ppb, n = 2).

Halibut. The level of ACs in a sample was similar to that from

Angoon (0.4 ppb; n = 1).

Irish Lord. The levels of ACs in two samples were not much

different from those of other fish from Angoon. No Irish Lords were collected

from Angoon.

Mussels. The 1-5 ppb of LACs in 10 samples were slightly elevated

compared to Angoon (0.6-1 ppb, n = 2). The HACs (up to 12 ppb) were

sometimes moderately elevated compared to Angoon mussels (up to 0.1 ppb, n =

2).

Littleneck Clams and Cockles. One sample of each showed

moderately elevated levels of LACs (3-20 ppb) and HACs (9-10 ppb) compared

to similar molluscs from Angoon (LAC and HAC levels of 0.5-1 ppb and up to

0.1 ppb, respectively, n = 6).

Chitons. Three samples showed 0.2-0.7 ppb of LACs, which is

comparable to the LAC levels in samples from Angoon (1 ppb, n = 2).

Snails. A sample showed 3 ppb of LACs and 0.9 ppb of HACs,

slightly elevated compared to other molluscs from Angoon (LAC and HAC

levels of 0.5-1 ppb and up to 0.1 ppb, respectively, n = 6).

Karluk --

Pink Salmon, Coho Salmon, Sockeye Salmon, Mussels, Butter

Clams, and Chitons. Very low levels of LACs (up to 0.7 ppb) were found, which

were comparable to, or lower than the levels in these species from Angoon (up to

4 ppb, n = 10).
36

Kasitsna --

Mussels. Two samples from Kasitsna showed 0.7-1 ppb of 2-3 ring

ACs, comparable to the 0.6-1 ppb found in Angoon mussels (n = 2).

Butter Clams. The level of LACs (2 ppb) in a sample was similar to

that in a sample from Angoon (0.5 ppb, n = 1).

Chitons. Levels of LACs (2 ppb) in a sample were similar to that in a

sample from Angoon (1 ppb, n = 2).

SUMMARY

After the Exxon Valdez oil spill in Prince William Sound, native Alaskans

were concerned that their seafood could be contaminated by the oil. NOAA, with

funding from Exxon, analyzed edible flesh of fish and shellfish collected from 13

native subsistence fishing grounds. Flesh samples were sub ected to extraction

and GC/MS analysis for aromatic contaminants from petroleum, including

alkylated and unsubstituted aromatic hydrocarbons with 2-7 benzenoid rings and

related dibenzothiophenes (Table 1). The hundreds of analyses reported via

memo in August, October, and November, 1989 are summarized herein.

Molluscs, mainly mussels, clams, chitons, and snails from Windy Bay,

Kodiak, Chenega Bay, and Old Harbor showed consistent GC/MS evidence of

exposure to ACs (tissue levels exceeded 100 ppb). Mussels from Windy Bay and

Kodiak had the highest levels (1,700-18,000 ppb). Otherwise, levels of aromatic

contaminants in molluscs, crabs, and urchins from other areas were generally less

than 10 ppb. This is comparable to the levels found in shellfish collected near the

village of Angoon, a designated reference area in southeast Alaska.

Generally, the edible flesh of fish analyzed contained relatively low levels

of ACs. Three samples of pink salmon from the village of Kodiak averaged

60-100 ppb of ACs, while another eight samples of pink and coho, salmon from

Kodiak, Chenega Bay, Tatitlek, and Larsen Bay had 12-59 ppb of ACs.

Otherwise, the AC levels in the flesh of salmon from other subsistence fishing

areas and the bottomfish from all fishing areas were generally less than 7 ppb,
37

comparable to the levels detected in the same or related species from the

reference site.

Statistical analyses of the data revealed that the sample sizes (n) for

individual species were often too small to draw statistical distinctions with

respect to the reference site, where n = 1-3 for any single species. To broaden the

statistical base, samples were grouped by taxonomic affiliation (e.g., salmon or

bottomfish) and habitat (e.g., intertidal or benthic). The combining of data for all

intertidal molluscs increased the sample size to a reasonable value (n = 147) and

had the added advantage of including most of the villages in the statistical

analyses. The AC levels in edible flesh of molluscs from Windy Bay, Kodiak,

and Chenega Bay were then found to be significantly different from those in

molluscs from the reference site near Angoon. However, the AC levels in salmon

and bottomfish were not significantly different from those in fish from Angoon.

With greater sample numbers available, the AC levels in salmon from Kodiak

were found to be significantly different (higher) from those in salmon from

Chenega Bay, Old Harbor, and Port Graham and English Bay. The very low

levels of ACs in the edible flesh of bottomfish did not warrant further statistical

analysis.

As there are significant differences in the rates of uptake, metabolism,

excretion, and types of toxic effects exerted by the various ACs measured, the

data are also presented to show the levels of 2-3 ring aromatic hydrocarbons and

dibenzothiophenes (LACs) separately from the levels of 4-7 ring aromatic

hydrocarbons (HACs). The LACs are more prone to dissolution, evaporation,

and bacterial degradation and hence, their levels in the environment generally

would decline as the spilled oil weathers. The LACs, known for their acute

toxicity in experimental animals, are also more rapidly excreted from the body

than are HACs. Conversely, HACs are more resistant to dissolution, evaporation,

and bacterial degradation. Hence, they tend to persist in the environment. The

HACs are metabolized by fish to a greater extent than are the LACs and they

have been shown to exert chronic toxicity in experimental animals (e.g.,

carcinogenicity, tetratogenicity, and reproductive dysfunction).

In this study, the levels of LACs in molluscs ranged from not detected to

16,000 ppb and levels of HACs ranged from not detected to 2,500 ppb. For fish
38

muscle, the levels of LACs ranged from not detected to 110 ppb, and levels of

HACs ranged from not detected to 12 ppb. Benzo[a]pyrene, a carcinogenic HAC,

was detected in molluscs from Windy Bay and Chenega Bay at levels of 0.1 to 7

ppb, but was not detected in fish. Only two samples of smoked salmon from Old

Harbor and Tatitlek were analyzed in this study; they contained 7,500 and

20,000 ppb of LACs and 670 to 2,400 ppb HACs, respectively. The

concentrations of benzo[alpyrene in these samples were 6 ppb (Old Harbor) and

20 ppb (Tatitlek). At present there are no national guidelines established for

acceptable levels of aromatic contaminants in food products; however in an

unofficial advisory opinion, the Food and Drug Administration has indicated that

little additional risk is involved in the consumption of the nonsmoked

subsistence foods harvested after the Exxon Valdez oil spill.2

The main use of the analyses of bile from fish “ was to assess exposure to

ACs and prioritize the GC/MS analyses of the corresponding samples of edible

flesh. For each species, edible flesh samples were analyzed as individual samples

or as composite samples according to the levels of fluorescent aromatic

compounds (FACs) in bile. Interesingly, unsubstituted ACs predominated in fish

muscle,3 which could be due to the more rapid metabolism of alkylated ACs than

of unsubstituted ACs by fish liver. Conversely, molluscs which have little ability

to metabolize ACs had both alkylated and unsubstituted ACs, and the pattern

more closely resembled that of Prudhoe Bay crude oil.3

The results to date provide a) important information on the level of

contamination of subsistence fish and shellfish from fishing areas of native

Alaskan villages in and near Prince William Sound and b) a reference database

against which future temporal changes of petroleum derived ACs in the edible

flesh of fish and shellfish can be evaluated.

2

 FDA advisory opinion on the safety of aromatic hydrocarbon residues found in subsistence

foods that were affected by the Exxon Valdez oil spill by the Center for Food Safety and

Applied Nutrition’s (CFSAN) Quantitative Risk Assessment Committee (QRAC). Guidelines

(Report of August 9, 1990) transmitted to Chairman of Alaska Oil Spill Task Force, August 21,

1990, from Fred R. Shank, Director, CFSAN, Department of Health and Human Services, Food

and Drug Administration, Washington, D.C. 20204.

3

 GC/MS observations on sample extracts.
39

ACKNOWLEDGMENTS

We are grateful to our colleagues in the National Marine Fisheries Service

(NMFS) and National Ocean Service (NOS), NOAA, who provided valuable

assistance and support in organizing this joint NOAA/Exxon study. Particularly

noteworthy in this respect were the efforts of Dr. Charles Ehler, Director, Office

of Oceanography and Marine Assessement (OMA/NOS), and John Robinson,

Director, Hazardous Materials Response Branch (HazMat) of OMA/NOS, who

were instrumental in initiating and developing the NOAA/Exxon MOU. Other

HazMat members who provided valuable liaison include Jay Field, biologist;

Lew Consiglieri, Coastal Resources Coordinator; and Ann Hayward-Walker,

Scientific Support Coordinator. We also appreciate the support and

encouragement of Dr. Richard Berry, Director, Northwest Fisheries Center

(NWC/NMFS) and Dr. Tom Nighswander, Chair, Oil Spill Health Task Force.

Dave Schmidt and his staff of field biologists from Dames and Moore performed

the field sampling for this study, under contract to Exxon, and forwarded the

samples to us under chain of custody. Pippa Coiley and Craig Mishler of

Alaska’s Department of Fish and Game assisted with field collections, as did

Tim Reilly of RPI International, Inc. Dr. Paul Boehm of Arthur D. Little, Inc.,

under contract to Exxon, conducted the interlaboratory comparisons in which we

participated. Gary Duker and his staff of the NWC publications unit provided

expert review of this manuscript. Finally, a number of EC Division chemists and

technicians ably assisted this study in the sample analyses and the data

management. In alphabetical order they are Lisa Bogatzki, Jennie Bolton,

Richard Boyer, Tara Felix-Slinn, Jennifer Fisher, Barbara French, Deborah

Holstad, Anna Kagley, Ron Pearce, Eunice Schnell, and Dana Whitney.
40
41

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Spectrometry and Allied Topics.

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exposure in flatfish from Puget Sound, WA. In Oceans ’87/proceedings, Vol. 5,

Coastal and estuarine pollution, p. 1544-1549. Available from IEEE, 445 Hoes

Lane Piscataway, NJ 08854.

Dunn, O. J. 1964. Multiple contrasts using rank sums. Technometrics 6: 241252.

Hollander, M., and D. A. Wolfe. 1973. Nonparametric statistical methods. John

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waterways. Xenobiotica 14:633-646.

Krahn, M. M., L. K. Moore, and W. D. MacLeod, Jr. 1986a. Standard analytical

procedures of the NOAA National Analytical Facility, 1986: Metabolites of

aromatic compounds in fish bile. U.S. Dep. Commer. Tech. Memo. NMFS F/

NWC-102,25 p.

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MacLeod, Jr., S-L. Chan, and D. W. Brown. 1988. Standard analytical

procedures of the NOAA National Analytical Facility, 1988: New HPLC cleanup

and revised extraction procedures for organic contaminants. U.S. Dep. Commer.

Tech. Memo. NMFS F/NWC- 153, 52 p.
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Lowry, O. H., W. J. Rosebrough, A. L. Farr and R. S. Randall. 1951. Protein

measurement with the folin phenol reagent. J. Biol. Chem. 193:265-275.

MacLeod, W. D., Jr., D. W. Brown, A. J. Friedman, D. G. Burrows, O. Maynes,

R. W. Pearce, C. A. Wigren, and R. G. Bogar. 1985. Standard analytical

procedures of the NOAA National Analytical Facility, 1984-1985: Extractable

toxic organic compounds, Second Ed., U.S. Dep. Commer. Tech. Memo. NMFS

F/NWC-92,121 p.

Reichert, W. L., and U. Varanasi. 1982. Metabolism of orally administered

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27:316-324.

Sokal, R., and F. Rohlf. 1981. Biometry (2d ed.). W. H. Freeman and Co, San

Francisco. 854 p.

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xenobiotics in trout bile: A proposed monitoring aid for some waterborne

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Stein, J. E., T. Hom, and U. Varanasi. 1984. Simultaneous exposure of English

sole (Parophrys vetulus) to sediment-associated xenobiotics: 1. Uptake and

disposition of [14C]-polychlorinated biphenyls and [3

H]-benzo[a]pyrene. Mar.

Environ. Res. 13:97-119.

Varanasi, U. and D. J. Gmur. 1981. Hydrocarbons and metabolites in English

sole (Parophrys vetulus) exposed simultaneously to 3H-B(a)P and 14Cnaphthalene in sediment. Aquat. Toxicol. 1:49-67.

Varanasi, U., M. Nishimoto, W. L. Reichert and J. E. Stein. 1982. Metabolism

and subsequent covalent binding of benzo[alpyrene to macromolecules in gonads

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Varanasi, U., S-L. Chan,, B. B. McCain, M. H. Schiewe, R. C. Clark,-D. W.

Brown, J. T. Landahl, M. M. Krahn, W. D. Gronlund, and W. D. MacLeod. 1988.

National Benthic surveillance Project: Part I summary and overview of the

results for cycles I to 111 (1984-1986). U. S. Dep. of Commerce Tech. Memo.

NMFS F/NWC-156,71 p.

Varanasi, U., J. E. Stein, and M. Nishimoto. 1989a. Biotransformation and

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envirorunent, p. 93-149. CRC Press, Inc., Boca Raton, FL.

Varanasi, U., S-L. Chan, W. D. MacLeod, J. E. Stein, D. W. Brown, D. G.

Burrows, K. L. Tilbury, C. A. Wigren, and T. Hom. 1989b. Survey of subsistence

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Varanasi, U., S-L. Chan, W. D. MacLeod, J. E. Stein, D. W. Brown, D. G.

Burrows, K. L. Tilbury, C. A. Wigren, and T. Hom. 1989c. Survey of subsistence

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44

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45

APPENDIX A

SUMARY OF CONCENTRATIONS: LACS/HACS,

PLUS FIELD AND LABORATORY SAMPLE NUMBERS
46

Explanatory notes for Tables A-1 through- A-26,

Results on sample extracts were determined by gas chromatography/mass

spectrometry (GC/MS) using sequenced multiple ion detection.

Cycles I, II, and III refer to samples collected in July, August, and September

1989, respectively.

A hyphen (-) indicates that the analyte was not detected above the limit of

detection which ranged from 0.02 to 1 ng/g (ppb) wet weight. This applies to

individual contaminants, as well as groupings of contaminants (e.g., C2-

phenanthrenes/anthracenes).

Low levels of naphthalene found were indistinguishable from those of blank

analyses and were not included in the sums.
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
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APPENDIX B

CONCENTRATIONS OF INDIVIDUAL AROMATIC CONTAMINANTS

IN FISH
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Explanatory Notes for Tables B-1 through B-15.

Naphthalene-d8 was the internal standard for naphthalene through C4-

naphthalenes. Acenaphthene-d10 was the internal standard for acenaphthylene

through C1-fluoranthenes/pyrene. Benzo[a]pyrene-d12 was the internal standard

for benz[a]anthracene through benzo[ghi]perylene. Percent recoveries for the

internal standards (surrogates) averaged 91%, RSD = 19%, n = 662. Percent

recoveries of the surrogates include split or duplicate samples.

Results on sample extracts were determined by gas chromatography/mass

spectrometry (GC/MS), using sequenced multiple ion detection.

A hyphen (-) indicates that the analyte was not detected above the limit of

detection which ranged from 0.02 to 1 ng/g (ppb) wet weight. This applies to

individual contaminants, as well as groupings of contaminants (e.g., C2-

phenanthrenes/anthracenes).

Low levels of naphthalene found (a) were indistinguishable from those of blank

analyses and are unlikely to indicate exposure to petroleum in the absence of 1-

and 2-methylnaphthalene.
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131

APPENDIX C

CONCENTRATIONS OF INDIVIDUAL CONTAMINANTS

IN SHELLFISH
132

Explanatory Notes for Tables C-1 through C-12.

Naphthalene-d8 was the internal standard for naphthalene through C4-

naphthalenes. Acenaphthene-d10 was the internal standard for acenaphthylene

through C1-fluoranthenes/pyrene. Benzo[a]pyrene-d12 was the internal

standard for benz[a]anthracene through benzo[ghi]perylene. Percent

recoveries for the internal standards (surrogates) averaged 88%, RSD = 15%,

n = 452. Percent recoveries of the surrogates include split or duplicate

samples.

Results on sample extracts were determined by gas chromatography/mass

spectrometry (GC/MS), using sequenced multiple ion detection.

A hyphen (-) indicates that the analyte was not detected above the limit of

detection which ranged from 0.02 to 1 ng/g (ppb) wet weight. This applies to

individual contaminants, as well as groupings of contaminants (e.g., C2-

phenanthrenes/anthracenes).

Low levels of naphthalene (a) found were indistinguishable from those of

blank analyses and are unlikely to indicate exposure to petroleum in the

absence of 1- and 2-methylnaphthalene.
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141
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143
144
145

APPENDIX D

QUALITY ASSURANCE FOR INDIVIDUAL AROMATIC

CONTAMINANTS IN FISH AND SHELLFISH
146

Quality Assurance for Individual Aromatic Contaminants in Fish and

Shellfish:

Matrix Spikes, Spiked Blanks, and Method Blanks

For each set of 10 samples, a method blank and a matrix spike or a method spiked blank

were analyzed for selected aromatic contaminants (ACs) in Table 1.

Matrix Spikes

Thirty-one matrix spikes were analyzed for this project. The results are summarized in

Table D-1. Mean percent recoveries for the ACs ranged from 79 to 110% and the

relative standard deviations (RSDs) ranged from 7 to 34%.

Spiked Blanks

Spiked method blanks were analyzed with 6 sets of samples (Table D-2). Mean percent

recoveries for the ACs ranged from 95 to 120% and the RSDs ranged from 2 to 19%.

Method Blanks

Thirty-seven method blanks were analyzed (Tables D-3a,b). Naphthalene was present in

each method blank at about 1 ppb (1 ng/g). The source of the naphthalene appeared to

be the dichloromethane solvent, despite the dichloromethane being the highest grade

available. Traces of methy1naphthalenes and phenanthrene were also found in some

method blanks.
147

Explanatory Notes for Tables D-1 through D-3a,b.

Naphthalene-d8 was the internal standard for naphthalene through C4-naphthalenes.

Acenaphthene-d10 was the internal standard for acenaphthylene through

Cl-fluoranthenes/pyrene. Benzo[a]pyrene-d12 was the internal standard for

benz[a]anthracene through benzo[ghi]perylene. Percent recoveries for the internal

standards (surrogates) averaged 87%, RSD = 15%, n = 216.

Results on sample extracts were determined by gas chromatography/mass spectrometry

(GC/MS) using sequenced multiple ion detection.

A hyphen (-) indicates that the analyte was not detected above the limit of detection

which ranged from 0.02 to 1 ng/g (ppb) wet weight. This applies to individual

contaminants, as well as groupings of contaminants (e.g., C2-phenanthrenes/

anthracenes).

The matrix spike values are the percent recoveries of analytes added to a sample

comparable to the type of tissue being analyzed. The matrix spike was then analyzed as

a sample.

The spiked blank values are the percent recoveries of analytes added to a method blank

and analyzed as a sample.

The relative standard deviation (RSD) is the standard deviation divided by the mean and

expressed as a percent.
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