NOAA Technical Memorandum NMFS-NWFSC-12

Volume I: Survey of Alaskan subsistence fish, marine mammal, and invertebrate samples collected 1989-91 for exposure to oil spilled from the Exxon Valdez Usha Varanasi, Donald W. Brown, Tom Hom, Douglas G. Burrows, Catherine A. Sloan, L. Jay Field*, John E. Stein, Karen L. Tilbury, Bruce B. McCain, & Sin-Lam Chan National Marine Fisheries Service Northwest Fisheries Science Center Coastal Zone and Estuarine Studies Division 2725 Montlake Blvd. E. Seattle WA 98112-2097 *Hazardous Materials Reponse and Assessment Division Office of Resources Conservation and Assessment October 1993 U.S. DEPARTMENT OF COMMERCE Ronald H. Brown, Secretary National Oceanic and Atmospheric Administration D. James Baker, Administrator National Marine Fisheries Service Rolland A. Schmitten, Assistant Administrator for Fisheries

CONTENTS

Sample Collection

Fish and Shellfish

Marine Mammals

Bile Analyses

Analyses of Aromatic Compounds in Tissue (Edible Flesh)

Extraction of Aromatic Compounds

Fractionation of Extract to Isolate Aromatic Compounds

Aromatic Compound Determinations by Gas Chromatography/Mass Spectrometry-Sequenced Selected Ion Monitoring

Statistical Methods

Aromatic Compounds in Petroleum and Petroleum Products

Fluorescent Aromatic Compounds in Bile of Fish

Biliary FACs_PHN in Salmon

Biliary FACs_PHN in Bottomfish

Temporal Changes in Biliary FACs in Salmon

Aromatic Compounds in Edible Flesh of Fish

Fluorescent Aromatic Compounds in Bile of Marine Mammals

Aromatic Compounds in Edible Flesh of Marine Mammals

Aromatic Compounds in Invertebrates

Comparison of ACs in Molluscs by Area

Comparison of ACs in Molluscs by Species

Aromatic Compounds in Mussels

Aromatic Compounds in Butter Clams

Aromatic Compounds in Littleneck Clams

Aromatic Compounds in Chitons

Aromatic Compounds in Other Invertebrate Species and Miscellaneous Samples

Temporal Changes in the Concentrations of Aromatic Compounds in Molluscs

Patterns of Aromatic Compounds in Invertebrates

Executive Summary

Based on the concentrations of FACs in bile, it was evident that pink salmon, halibut, and Pacific cod from the Chenega area had been exposed to ACs during 1989, as were pink salmon from Tatitlek, Kodiak, and Old Harbor. The bile method was useful because it quickly identified those fish that were relatively unexposed to ACs and, therefore, of less immediate interest for analysis by the more detailed method for ACs in tissue.

As expected, most fish muscle samples were not contaminated with ACs (<10 ng/g). In fact, the highest concentration of ACs found in muscle samples of fish caught during this study was 100 ng/g in a pink salmon caught near Kodiak (city) in 1989. In contrast, two samples of smoked salmon obtained from Tatitlek and Old Harbor contained 23,000 and 8,100 ng/g ACs, respectively.

Bile and tissue samples were collected from 33 harbor seals and 10 sea lions in 1989 and 1990. As with fish, the concentrations of FACs in bile of harbor seals varied considerably. Nine of the 12 bile samples with the highest concentrations of FACs were from animals collected in 1989 that were visibly oiled. With two minor exceptions, samples of muscle, liver, and kidney from harbor seals and sea lions, as well as blubber from sea lions, were not contaminated (<10 ng/g) with ACs. Samples of blubber from 12 of the harbor seals were minimally contaminated (10 to 99 ng/g), and samples of blubber from 4 harbor seals were moderately contaminated with ACs (100 to 1,000 ng/g).

Invertebrates from most of the sampling areas were not contaminated or were minimally contaminated by ACs (<100 ng/g). Therefore, results are presented for only those few stations where higher concentrations of ACs were found. Molluscs from some stations at the Chenega, Windy Bay, Kodiak, and Old Harbor sampling areas were moderately or highly contaminated (>100 ng/g) with ACs. For example, some of the mollusc samples from 4 of the 12 stations in the Chenega area (CHE1, CHE7, CHE10, CHE24) were moderately or highly contaminated with ACs. Mollusc samples from Windy Bay stations WNB1 and WNB3 contained concentrations of ACs as high as 18,000 ng/g; 34 of 106 invertebrate samples contained concentrations of ACs greater than 100 ng/g. The only two stations on Kodiak Island where mollusc samples had mean concentrations of ACs (by year for individual species) greater than 100 ng/g (moderately or highly contaminated) were KOD3 and OHA4. Most of the mollusc samples (26 of 30) from station KOD3 (located on Near Island about 1/4 mile from Kodiak's boat harbor) were moderately or heavily contaminated with ACs (>100 ng/g). Station OHA4 was adjacent to the village of Old Harbor near the boat harbor, and the concentrations of ACs in molluscs collected at this site in 1989 and 1990 were just within the moderately contaminated category or lower.

Aromatic compounds were present in molluscs at concentrations high enough to evaluate in terms of temporal trends only at some stations. For example, the concentrations of ACs declined significantly with time in mussels at: 1) Chenega stations CHE9 and CHE10 (1990 to 1991), and 2) at the combined Windy Bay stations WNB1/WNB3 (1989 to 1991). The degree of contamination of invertebrates from WNB1 and WNB3 stations varied with sampling year and by species. Specifically, some of the mussel samples from Windy Bay station WNB1 (1989) and from WNB3 (1990) were highly contaminated (>1,000 ng/g), whereas the concentrations of ACs in mussels from these stations in 1991 were minimally to moderately contaminated (10 ng/g to 1,000 ng/g). The decline in concentrations of ACs in these molluscs probably related to decreased exposure which resulted from weathering of the spilled oil at the particular stations. The concentrations of ACs in molluscs at some other stations did not decline significantly with time. For example, the concentrations of ACs in butter clams from Kodiak station KOD3 did not consistently decline over four sampling periods during 1990.

The relative concentrations of the hundreds of different ACs in various petroleums and petroleum products can vary considerably. The patterns of these concentrations can be useful for purposes of comparison. The patterns of some ACs (phenanthrenes and dibenzothiophenes) in selected mollusc samples from Chenega area stations CHE1 and CHE10 and Windy Bay stations WNB1 and WNB3 were similar to that of weathered PBCO. Because the overall patterns of ACs in molluscs did not exactly match that of PBCO, other observations were also important in considering sources. For example, following the spill, oil was observed in the area of station CHE1 and at CHE10, a tar mat about 1 m wide extended the length of the beach at the high tide line. Also, stations WNB1 and WNB3 were observed to be moderately to heavily oiled. Thus, based on the patterns of ACs in molluscs and the known proximity of the spilled oil to these areas, oil from the Exxon Valdez may have been the source of ACs in mollusc samples from CHE1, and most likely was the source of ACs in mollusc samples from CHE10, WNB1, and WNB3. The patterns of ACs in selected samples from Kodiak Island stations KOD3 and OHA4 were also similar to those for mollusc samples from CHE1, CHE10, WNB1, and WNB3. However, the presence of naphthalenes in the patterns was more prominent in the samples from KOD3 and OHA4 than in the Chenega and Windy Bay samples. This finding implies exposure of the KOD3 and OHA4 molluscs to a less weathered source of ACs. Therefore, the ACs in molluscs from KOD3 and OHA4 are suspected to be from a local continuing source of petroleum. Additional support for this conclusion includes: 1) KOD3 and OHA4 were near active boat harbors which could be a source of ACs, 2) the spilled oil was not observed to impact these areas, and 3) the concentrations in molluscs at KOD3 did not continually decline over four samplings during 1990. Based on patterns, PBCO was probably a minor source of the ACs in mollusc samples from station CHE7. The pattern of ACs implied that the source of these compounds in selected molluscs from CHE7 was due to exposure to creosote (perhaps from the creosoted pilings located near the sampling station) and/or products of combustion. Based on the patterns of ACs in selected samples, PBCO was probably not a source, or only a minor source of the ACs in molluscs from Tatitlek station T1. More likely, the source of the ACs in mollusc samples from T1 was products of combustion processes.

Interestingly, of the ACs found in fish muscle, unsubstituted ACs predominated, which was probably 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 patterns of ACs in molluscs more closely resembled that for petroleum components. Furthermore, in the blubber samples from harbor seals with elevated concentrations of ACs, the concentrations of alkyl-substituted ACs were similar to or greater than the concentrations of the corresponding nonsubstituted ACs. This pattern is similar to what was found in PBCO and molluscs, and generally the opposite of what was observed in fish.

In conclusion, the finding of elevated concentrations of FACs in some bile samples from fish and marine mammals was clear evidence of their exposure to petroleum. Generally, ACs were not found in muscle tissue of fish, harbor seals, and sea lions. Some harbor seal blubber samples did contain ACs; however, the concentration of ACs in most blubber samples was less than 100 ng/g. Smoked salmon contained higher concentrations of ACs (8,000 to 20,000 ng/g) than any of the untreated subsistence samples. The concentrations of ACs were less than 100 ng/g in approximately 90% of the more than 1,000 mollusc samples from 80 sampling beaches. The concentrations of ACs were elevated in some mollusc samples (as high as 18,000 ng/g), and the concentrations of ACs exceeded 1,000 ng/g in 24 samples.

The results to date provide important information on the level of contamination of subsistence fish, shellfish, and marine mammals from fishing areas of native Alaskan villages in and near Prince William Sound. In an advisory opinion, the Food and Drug Administration has indicated that little risk is involved in the consumption of the nonsmoked subsistence foods studied. Subsistence food gatherers were advised not to collect or consume food if oil was observed to be present. The results also show that in future oil spills, shellfish tissues should be given the highest priority for analysis, whereas rapid screening of bile from fish and marine mammals should be sufficient to provide information on level of exposure.

INTRODUCTION

The spreading of spilled oil raised concerns among native Alaskans that their subsistence seafood (fish, marine mammals, and invertebrate organisms) was contaminated by the spilled petroleum. In response to their concerns, NOAA entered into memoranda of understanding (MOU) with Exxon and subsequently with the Alaska Department of Fish and Game (ADF&G) to analyze subsistence seafoods sampled from sites used by native Alaskans, and from reference areas for selected aromatic compounds (ACs) found in spilled oil (Varanasi et al. 1990).

Petroleum, such as PBCO, contains many hundreds of organic compounds, generally classified into groups including aliphatic and polycyclic aromatic hydrocarbons and compounds that contain sulfur, oxygen, and nitrogen. Aromatic hydrocarbons and dibenzothiophenes comprise the group of ACs that are known for their toxicity, and these compounds can be monitored in biota as indicators of exposure to petroleum-related ACs (Table 1). Previous laboratory studies have shown that fish and mammals efficiently biotransform ACs to polar derivatives that are concentrated in their bile for excretion (Statham et al. 1976, Varanasi and Gmur 1981, Stein et al. 1984, Varanasi et al. 1989b). As a result of this biotransformation, ACs may not readily accumulate in the edible flesh of fish or marine mammals. Thus, to quickly measure the exposure of fish and marine mammals to ACs, a sensitive method for screening bile for the presence of fluorescent aromatic compounds (FACs) which are characteristic of petroleum ACs was utilized. This semiquantitative procedure for measuring FACs utilizes high performance liquid chromatography (HPLC) with fluorescence detection (Krahn et al. 1984, 1986a). This method has been employed previously to evaluate exposure of fish to ACs from an oil spill on the Columbia River (Krahn et al. 1986b) and in a variety of other laboratory and field studies. The results of biliary FACs analyses were used to prioritize corresponding samples of edible fish tissue for analyses of individual ACs.

Individual ACs in selected tissue samples from invertebrates, fish, marine mammals, and other tissue types (e.g., herring roe on kelp) were analyzed using gas chromatography/mass spectrometry (GC/MS). This procedure is much more labor and time intensive than the measurement of FACs in bile. However, recent important improvements in, and automation of, the extract cleanup procedure (Krahn et al. 1988a) and the use of a special sequenced selected-ion monitoring (SSIM) GC/MS program (Burrows et al. 1990) enabled us to provide high quality analytical data for low concentrations (<1 ng/g wet weight) of ACs in tissue samples quickly and efficiently. The GC/MS method also detects sulfur-containing ACs such as the dibenzothiophenes, which can be useful markers for oil contamination.

In addition to organic compounds, petroleum contains certain metals (e.g., nickel and vanadium). Metals were not analyzed as part of the study because of very low concentrations of these metals relative to hydrocarbon components in PBCO and the lack of information about the bioavailability processes of metals in various marine biota (including those sampled in this study).

This report presents the results of analyses of bile for FACs and tissue samples for ACs from subsistence species collected in 1989, 1990, and 1991. The results were evaluated both in terms of geographical distribution and temporal changes.

This report is divided into two volumes, with the second volume containing supplemental data too voluminous to be included as Appendices to the primary report. It was felt that because everyone reading the primary report might not be interested in the supplementary data, it would be preferable to publish it as a separate volume available on request. Volume II, Supplemental Information Concerning a Survey of Alaskan Subsistence Fish, Marine Mammal, and Invertebrate Samples Collected 1989-91 for Exposure to Oil Spilled from the Exxon Valdez, includes the following sections: A - Concentrations of Metabolites of Fluorescent Aromatic Compounds in Fish Bile; B - Concentrations of Aromatic Compounds in Edible Tissue of Fish; C- Concentrations of Metabolites of Fluorescent Aromatic Compounds in Bile from Marine Mammals; D - Concentrations of Aromatic Compounds in Edible Tissues of Marine Mammal Samples; E - Concentrations of Aromatic Compounds in Invertebrate Samples; and F - Quality Assurance.

METHODS

Details of protocols for the field sampling, chemical analyses, and statistical evaluation are outlined below.

Sample Collection

Fish and Shellfish

In 1990, all of the above-mentioned areas were sampled at least once and the number of samples for each species from each sampling area was increased compared to 1989. In addition, samples were collected from other areas in southwestern PWS near the village of Chenega Bay and from areas on the Kenai Peninsula as requested by villagers (Port Chatham, Sadie Cove, Point Bede, Port Dick, Chugach Bay) (Fig. 4, Fig. 5, Fig. 6), and from four village areas on the Alaska Peninsula (Chignik, Ivanof Bay, Kashvik, and Perryville) (Fig. 1, Fig. 14, Fig. 15). The focus during the second year was on collecting larger numbers of invertebrate species at each sampling station.

In 1991, the project focused primarily on areas in southwestern PWS and Lower Cook Inlet (areas near Chenega Bay and Windy Bay). Some shorelines in both of these areas were heavily oiled in 1989 and thus would be areas at which temporal changes in levels of ACs in invertebrate subsistence species could possibly be evaluated over the 3-year sampling period.

Selection of sample stations and the subsistence resources to be sampled was done in cooperation with village representatives. Sampling at each village area usually included two beaches for intertidal invertebrates. Salmon and bottomfish were collected from stations designated by village fishermen as ones generally fished. Hence, sample collection represented the approach of subsistence collectors rather than random or statistical sampling. No attempt was made to avoid or seek out oiled areas for sampling of fish or invertebrates. If oil was observed on a beach or in the vicinity of a sample collection area, it was noted in the sampling log. Since the habitat requirements vary for different species, individual stations sometimes encompassed a relatively large beach area over a range of tidal elevations in order to collect several species. For example, mussels were collected from higher tidal elevations than clams, while chitons were collected from rocky, lower intertidal areas.

In most cases, an individual from each village participated in sample collections conducted in areas adjacent to his/her village, except for marine mammal samples. Field collection protocols were followed to minimize the possibility of external contamination of samples. Tools used in sampling (e.g., shovels) were washed with soap and water and a new pair of surgical gloves was worn between sample collections. Intertidal shellfish were collected by hand or shovel. A minimum of four to six individual animals of each invertebrate species was collected at each station as one sample. Gear used in the fish collections included hook-and-line and gill nets for salmon, and hook-and-line and longline for halibut and bottomfish. Bile taken from fish was placed in solvent rinsed 4-mL vials and frozen as soon as possible for analysis for FACs. Fillets or whole fish were double wrapped in aluminum foil (which had been pre-baked at 350 °F for 1 hour), placed in Zip-Loc® freezer bags and placed in ice coolers. Samples were shipped frozen and stored at -80°C at the laboratory until analyzed. Bile and tissue samples from fish were sampled and maintained as individual samples, whereas the invertebrate organisms that comprised a sample were packaged together in the field. Chain-of-custody forms were used and were signed by both parties when samples were transferred from sample collectors to laboratory personnel.

The number of fish and mollusc samples collected by site and year is shown in Table 2.

Marine Mammals

Bile Analyses

The total integrated area from each detector was then converted to corresponding equivalents of either NPH or PHN standards that would give the same integrated response. Concentrations of FACs in bile are reported on the basis of bile weight and biliary protein. The levels of protein in bile samples were determined by the method of Lowry et al. (1951).

Analyses of Aromatic Compounds in Tissue (Edible Flesh)

Edible flesh of fish, marine mammals, and invertebrate samples were analyzed for the ACs listed in Table 1 using the procedures of Krahn et al. (1988a) and Burrows et al. (1990). Summaries of the analytical protocols are given below and consisted of four major steps: a) extraction; b) HPLC cleanup; c) analyte determination by GC/MS; and d) quality assurance. In addition, samples of petroleum and related products were weighed, dissolved in methylene chloride, and analyzed, starting with the HPLC cleanup.

Extraction of Aromatic Compounds

Fractionation of Extract to Isolate Aromatic Compounds

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 it. The helium was delivered via a regulator equipped with a stainless-steel diaphragm and was passed through an in-line charcoal filter (200-cc hydrocarbon trap, Alltech Assoc., Deerfield, Illinois), then through a high-purity heated trap (Supelco Inc., Bellefonte, Pennsylvania), and an oxygen indicating trap (Alltech Assoc.) to eliminate compounds 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 collected according to Krahn et al. (1988b). The solvent in the HPLC fraction was exchanged into hexane as the volume was reduced by evaporation to approximately 1 mL. Further evaporation, and then the addition of standards, brought the final volume to ca. 120 µL for analysis by capillary column GC with mass spectrometric quantification.

Aromatic Compound Determinations by Gas Chromatography/Mass Spectrometry-Sequenced Selected Ion Monitoring

Statistical Methods

The advantage of using comparison intervals is that they provide a graphical way of depicting significant differences between means (e.g., when comparison intervals overlap they are not significantly different (p <= 0.05)). When the number of samples for the means of interest are unequal, it is desirable to use the GT2 method (Gabriel 1978, Sokal and Rohlf 1981) for calculating the comparison intervals because the span of a comparison interval depends on the within-group variability in the entire data set and on the number of samples in each category.