Biomarkers in Fish from Prince William Sound and the Gulf of Alaska

Aug 12, 2003 - Cloverdale, California 95425, and Battelle,. Waltham, Massachusetts 02451. To test the hypothesis that biomarker levels in fish collect...
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Environ. Sci. Technol. 2003, 37, 4043-4051

Biomarkers in Fish from Prince William Sound and the Gulf of Alaska: 1999-2000 R O B E R T J . H U G G E T T , * ,† JOHN J. STEGEMAN,‡ DAVID S. PAGE,§ KEITH R. PARKER,| BRUCE WOODIN,‡ AND JOHN S. BROWN⊥ Michigan State University, East Lansing, Michigan 48824, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543, Bowdoin College, Brunswick, Maine 04011, Data Analysis Group, Cloverdale, California 95425, and Battelle, Waltham, Massachusetts 02451

To test the hypothesis that biomarker levels in fish collected at Prince William Sound (PWS) sites impacted by the 1989 Exxon Valdez oil spill were higher than those collected at unimpacted sites, a 1999-2000 study collected five fish species and associated benthic sediments from 21 sites in PWS and the eastern Gulf of Alaska (GOA). PWS sites were divided in three oiling categories based upon 1989 shoreline assessments: nonspill path (NSP), spill path oiled (SPO), and spill path not oiled (SPNO). Rockfish (N ) 177), rock sole (N ) 30), and kelp greenling (N ) 49) were collected at near-shore locations (∼50500 m from shore); Pacific halibut (N ) 131) and Pacific cod (N ) 81) were collected further offshore (∼500-7000 m). Fish were assayed for bile fluorescent aromatic contaminants (FAC) and cytochrome P4501A (CYP1A) levels measured as liver ethoxyresorufin O-deethylase (EROD) activity and by immunohistochemistry (IHC) of various tissues. For all species studied at all sites, bile FAC concentrations and CYP1A levels were low and in the same range for fish collected at PWS SPO and SPNO sites relative to NSP sites in PWS and the GOA. Consequently, the hypothesis is rejected for the species studied. The bile FAC results further indicate a pervasive exposure of fish at all sites, including those in the GOA far removed from the effects of the spill, to low levels of polycyclic aromatic hydrocarbons. Analysis of the benthic sediments indicates that the probable sources of this exposure are petrogenic hydrocarbons derived from natural oil seeps and eroding sedimentary rocks in the eastern GOA.

Introduction The Exxon Valdez oil spill on March 24, 1989, discharged ∼258 000 barrels of North Slope crude oil into the waters of Prince William Sound (PWS), AK. It is estimated that * Corresponding author phone: (517)355-0306; fax: (517)432-1171; e-mail: [email protected]. † Michigan State University. ‡ Woods Hole Oceanographic Institution. § Bowdoin College. | Data Analysis Group. ⊥ Battelle. 10.1021/es0342401 CCC: $25.00 Published on Web 08/12/2003

 2003 American Chemical Society

approximately 40% of the spilled oil impacted 486 mi (∼16%) of PWS shorelines (1). Cleanup activities in 1989 and winter storms removed much of that oil, and by 1993 it is estimated that shoreline oiling had been reduced to ∼8.7 mi (2). Recently, the National Marine Fisheries Service estimated that approximately 20 acres of shoreline contained subsurface oil residues in 2001 (3). They further classify the oiling levels as in the “mostly light oil” category. Concerns for the safety of the commercial and subsistence fisheries immediately following the spill resulted in studies by NOAA (4-6) to assess exposure levels to the spill oil and the distribution of sites of exposure. These 1989 studies documented elevated levels of biomarkers in some fish collected along the spill path. These elevated biomarker levels were attributed to exposure to the spill oil. By 1990, fish biomarker levels at most spill path sites had dropped considerably and were comparable to those at nonspill path sites. Although levels were low, the sources of the continuing exposure were unclear and subject to conflicting interpretations (5, 6). One issue concerns the bioavailability of hydrocarbons from sources other than the spill, including the natural regional hydrocarbon background and anthropogenic hydrocarbons associated with local point sources in some PWS embayments. The current study was designed with the following major objective: to determine if biomarkers indicating exposure to hydrocarbons measured in fish collected in 1999 and 2000 at PWS sites impacted by the spill are elevated relative to sites that were not impacted. In 1999, fish and sea floor sediments were collected at PWS sites classified as oiled and unoiled in 1989 and at sites east of PWS in the northern Gulf of Alaska (GOA). In 2000, fish and sediments were collected only within PWS. The fish were subsequently analyzed for biomarker indicators of oil exposure. Biomarkers analyzed for this comparison were bile fluorescent aromatic contaminants (FACs) and liver cytochrome P4501A levels, measured as ethoxyresorufin Odeethylase (EROD) activity and by immunohistochemical (IHC) methods. The sea floor sediments were analyzed for total organic carbon (TOC), polycyclic aromatic hydrocarbons (PAH), saturated biomarkers, and triaromatic steroids. The field and laboratory methods as well as the results of the sediment analyses are reported elsewhere (7).

Experimental Section Field Methods. Sampling locations that had shorelines visibly contaminated with oil in 1989 (2) are categorized as spillpath oiled (SPO). Those locations that were in the spill path but where adjacent shorelines were not oiled (e.g., Sawmill Bay, which was protected by triple booms, and some southand west-facing embayments including Drier, Thumb, and Mummy Bays) are categorized as spill path not oiled (SPNO). Locations in PWS outside of the spill path and therefore not oiled in 1989 are categorized as nonspill path (NSP). Finally, locations from the Gulf of Alaska east of PWS are grouped as GOA. Table 1 lists the sites sampled by year and gives their oiling categories, and Figure 1 shows the geographic locations. In 1999, fish were collected between June 8 and June 23. In 2000, they were collected between June 10 and June 20. Five species were used to compare levels of biomarkers at nearshore (∼50-500 m) and offshore (∼500-7000 m) sites VOL. 37, NO. 18, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Prince William Sound and Eastern Gulf of Alaska Sites and Species Collected site Yakutat Johnston Creek Duktoth River Wingham Island Katalla Bay Port Etches Olsen Bay Sheep Bay West Twin Bay Ewan Bay Jackpot Bay Sawmill Bay Drier Bay Mummy Bay Bay of Isles Crafton Island Green Island Herring Bay Northwest Bay Snug Harbor Sleepy Bay

map no.

year

category

species collecteda

1 2 3 4 6 5 7 8 9 13 16 11 14 15 12 10

1999 1999 1999 1999 1999 2000 1999, 2000 2000 1999 2000 2000 1999 1999 1999 1999 2000 2000 1999, 2000 1999 2000 1999, 2000

GOA GOA GOA GOA GOA PWS NSP PWS NSP PWS NSP PWS NSP PWS SPNO PWS SPNO PWS SPNO PWS SPNO PWS SPNO PWS SPO PWS SPO PWS SPO PWS SPO PWS SPO PWS SPO PWS SPO

PH, PC PH, PC PH, PC PH, PC PH, PC PH, KG PH, PC, RF, RS PH, PC, KG RF, RS PH, RF, RS PH, PC, KG, RF, RS PH, KG, RF PH, PC, KG, RS PH, RF PH, RF, RS PH, RF, RS PH, KG, RF PH, PC, KG, RF, RS PH, KG, RF PH, RF PH, PC, KG, RF PH, KG, RF, RS

a PH, Pacific halibut; PC, Pacific cod; KG, kelp greenling; RF, copper and quillback rockfish combined; RS, rock sole.

Clean nitrile laboratory gloves were worn during the handling and dissection of all fish. All utensils were cleaned by wiping with isopropyl alcohol wipes, rinsing with distilled water, and rinsing with methanol between the dissections. In addition, the dissection utensils were cleaned daily with soap and water, rinsed with distilled water and methanol, and stored in precleaned aluminum foil. The syringes used for bile sampling were rinsed with distilled water, methanol, and distilled water again (three times each) prior to use on each fish. Fish were killed immediately prior to sampling by blows to the head or by bleeding (cut through the gill connective tissue). The peritoneal cavity was opened, taking care not to pierce the gall bladder. Any observations of external or internal abnormalities (e.g., parasites, lesions, etc.) were documented. A bile sample was collected from each fish by piercing the gall bladder with a syringe and withdrawing up to 2 mL of bile. The bile was immediately transferred into a labeled, precleaned 4-mL screw cap glass vial and flash-frozen by placing the vial on dry ice. All bile samples were stored and shipped to the analytical laboratory on dry ice. Duplicate bile samples from the same fish were collected at random to evaluate the precision of laboratory procedures for FAC. A bile “field blank” consisting of distilled water drawn into a syringe and dispensed into a sample vial was collected each day bile sampling occurred. It was handled and stored in the same manner as the bile samples. A sample for liver EROD activity was collected from each fish by slicing several 1-2-cm pieces of the liver (∼0.5 cm in thickness) with scissors and transferring those pieces to a prelabeled Whirlpak bag. The sample was immersed in liquid nitrogen for approximately 30 s and then stored and shipped on dry ice. Three tissue types (liver, heart, and gill) were then collected for immunohistochemical (IHC) analysis using the same precleaned scissors. A 1-2-cm slice of each tissue type was sectioned and placed in a prelabeled 50-mL polyethylene bottle. The tissues were then fixed and preserved with approximately 35 mL of 10% buffered formaldehyde. The tissue samples were stored and shipped at ambient temperature. All data related to the fish sampling were recorded on fish sampling forms and/or in bound field notebooks. Chainof-custody forms were used to track sample shipment and receipt at the designated laboratories.

FIGURE 1. Prince William Sound and Gulf of Alaska sampling site locations. PWS numbers correspond to sites listed in Table 1. among major sampling location categories: kelp greenling (Hexagrammos decagrammus), Pacific cod (Gadus macrocephalus), Pacific halibut (Hippoglossus stenolepis), rockfish (copper and quillback rockfish combined: Sebastes caurinus and Sebastes maliger), and rock sole (Pleuronectes bilineatus). Fish were collected by hook and line with herring bait and/ or unbaited jigs. Fish were usually dissected within 1 h of collection; nontarget fish species were released. If dissection of fish could not be performed within 1 h of collection, they were kept alive in a holding tank supplied with clean seawater until sampling could be initiated, but no longer than several hours. The species, sex, weight, and length of each fish were recorded before sampling. 4044

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Laboratory Methods. Bile FAC. The analyses of bile samples for FAC were conducted by Texas A&M University’s Geochemical and Environmental Research Group (GERG) using the method described previously (8, 9). The quality assurance measurements include procedural blanks, duplicate samples, and laboratory standard reference material. The quality control criteria chosen for the relative difference between duplicate samples was less than 25% for analytes three times above the method detection limits (MDL). MDL were 0.6, 0.1, and 0.1 µg/g for naphthalene, phenanthrene, and benzo[a]pyrene, respectively. The GERG Standard Bile Reference material (Bile Reference Standard II) was analyzed prior to each batch of samples. Bile Reference Standard II is a fish bile composite with PAH metabolite concentrations of 380, 110, and 2.0 µg/g for naphthalene, phenanthrene, and benzo[a]pyrene metabolites, respectively. These concentrations were established by inter-laboratory calibration exercises among several laboratories. Thirty-seven replicate analyses of the standard by GERG for the period 1997-2001 gave the following averages and standard deviations: naphthalene ) 359 ( 58 µg/g,

phenanthrene ) 113 ( 22 µg/g, benzo[a]pyrene ) 1.8 ( 0.5 µg/g. QA criteria for Reference Standard II is the range defined by two times the standard deviation of the calibrated values. Total protein content was determined by modified Lowry method (10, 11). Bovine serum albumin (BSA) was used as the calibration standard, and quantification was performed at 595 nm wavelength. Protein determinations were duplicated on 10% of the samples. Sites were tested for equality of mean protein-normalized phenanthrenes (pnp) and mean protein-normalized naphthalenes (pnn) concentrations. Results were similar for both pnp and pnn. Consequently, this paper reports only the pnp results since they were used in previous studies of PWS and allow us to compare our results with those reported for samples collected soon after the spill. EROD Activity and Immunohistochemistry. CYP1A levels were assessed at Woods Hole Oceanographic Institution by EROD analysis and IHC detection of protein using methods described elsewhere (12, 13). The analysis of EROD activity was carried out with post-mitochondrial supernatant preparations (PMS). Tissues that had been stored at -80 °C were thawed to 2 °C, homogenized in ice-cold buffer, and centrifuged at 60g for 10 min followed by centrifugation at 12000g for 10 min (14). EROD activities were determined for the resulting PMS using a Cytoflour fluorescence plate reader (13). Data were expressed in pmol min-1 (g of liver)-1. For IHC analysis, CYP1A was immunodetected in 5-µm sections of paraffin-embedded tissue using monoclonal antibody 1-12-13 to scup CYP1A. As described by Woodin et al. (15), a scalar score of occurrence on 0-15 intensity was determined for CYP1A staining in specific cell types. Statistical Methods. ANOVA was used to test the hypothesis of equal mean pnp concentrations, EROD activity, and IHC scores among sample site categories against the alternative hypothesis of a higher mean at the SPO category. If no fish were collected at the SPO category, the alternative hypothesis was for a higher mean at the SPNO category. Taxa and years were tested separately. For 1999 data, the four categories of sites were compared. GOA sites were not sampled in 2000. IHC scores for hepatocytes, gill vascular endothelium, atrial endothelium, and ventricle endothelium were tested. High numbers of zero scores precluded meaningful tests for liver vascular endothelium, liver bile duct epithelium, gill pillar cells, and gill epithelium. Because of the preponderance of zero scores for IHC, Poisson (P) and binomial error were used (16) to test hypotheses on mean IHC scores. Statistically significant results were found only for hepatocytes, and mean scores and P values are tabled only for this cell type. To attain sufficient sample size for testing rockfish, copper and quillback (S. caurinus and S. maliger) were combined. In evaluating assumption of equal levels of biomarkers between the two species, means of EROD, FAC, and IHC were compared among oiling categories in PWS; no rockfish were collected in GOA. For 1999, means were compared for the NSP category, the only category where both species were collected. For 2000, means were compared among SPO and SPNO categories; only copper rockfish were caught in the NSP category in 2000. Previous studies that used the bile FAC analytical procedure followed here (4-6, 17) report fish biomarker results for four of the species (Pacific halibut, Pacific cod, rockfish, and rock sole) used in this study, and sampling locations for these earlier studies can be grouped into categories similar to those used here. Where data geographically overlap, we compare results between this and previous studies. Because differences in size and sex could affect pnp concentrations and EROD levels, initial analyses were run with weight and sex as covariates. This reduced the effects of weight and sex differences prior to testing for effects of oiling. To conserve

TABLE 2. Bile FAC Summary Results (Means and 1 SE) for Protein-Normalized Phenanthrene Equivalentsa spill path spill path oiled not oiled Pacific halibut Pacific cod rockfishb rock sole Pacific halibut Pacific cod kelp greenling rockfishb rock sole

nonspill path

eastern GOA

P value

Year ) 1999 1.7 (0.5) 1.9 (0.3) 1.3 (0.3)

2.3 (0.4) 0.765

2.0 (1) 1.2 (0.2) 1.0 (0.0) 2.8 (0.8) 4.0 (2.5) 2.2 (1.0)

2.4 (0.7) 0.112 0.531

Year ) 2000 1.7 (0.2) 3.6 (0.6) 1.9 (0.4)

-

0.965

2.4 (0.3) 1.9 (0.2) 2.4 (0.3) 4.9 (0.6) 4.2 (1.0) 4.0 (1.2)

-

0.310 0.107

1.7 (0.2) 2.4 (0.7) 1.8 (0.23) 4.9 (1.1) 1.8 4.3 (2.1)

-

0.729 0.163

a mg of phenanthrene equivalent/g of biliary protein. (-) indicates that no fish were examined. b Copper and quillback rockfish combined

power, covariates were dropped if not significant at the R ) 0.05 level. Data were positively skewed, and log transformations were used to achieve additivity. Regression analyses were used to test for relationships between the concentration of pnp versus EROD activity and IHC score. Individual fish were used as replicates. Separate regressions were run for each species. The null hypothesis of no slope was tested against the alternative hypothesis of a positive slope. All tests were performed at R ) 0.05. Residuals were tested (Shapiro-Wilk) and visually examined for normality. The effect of laboratory procedures on the precision and accuracy of pnp concentrations was assessed in two ways. First, a variance components analysis was run to estimate the influence of laboratory imprecision on the overall variability of pnp concentrations. Laboratory variability was based on blind duplicate samples from the same fish. Data from both years were combined in the variance component analysis. Second, the bile reference standard was examined for trends over time and precision. Data from replicate analyses of the bile standard were available from 1997 to 2001. To test these data for accuracy trends over time, a regression was made against date. In addition, the coefficient of variation (CV) for the 37 bile reference samples was compared to median CV for FACs for taxa sampled in 1999 and 2000.

Results and Discussion Precision of the Data. Variance component analysis of replicate analyses of the laboratory standard for pnp shows that imprecision in the laboratory has little influence on the overall variance of pnp. Imprecision in the laboratory accounts for less than 10% of the overall variability. There is no relationship between the bile reference standard concentrations and time of sampling. Hence, over time, laboratory results obtained for the standard are precise. The CV of the bile reference sample is approximately 4%, which compares to median CV of approximately 38% for pnp among taxa for 1999 and 2000. Biomarkers. Pacific Halibut. Pacific halibut were collected at all four categories of sites in 1999 and the three categories of PWS sites in 2000. Bile pnp means for the four site categories sampled in 1999 are statistically indistinguishable (Table 2). The eastern GOA results indicate exposure to aromatic hydrocarbons that are unrelated to those associated with the Exxon Valdez oil spill or with human activities in PWS. Concentration ranges for pnp are similar among categories VOL. 37, NO. 18, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Halibut bile FAC and EROD activities. 1999 and 2000 data, this study. An asterisk (*) indicates NOAA data of 1989-1991 (4, 5). for 1999 and 2000 data combined (Figure 2). NOAA data (4, 5) from 1989 through 1991 are also presented in Figure 2. Samples collected in 1989 along the spill path were reported to be significantly different from those collected from PWS NSP sites and from samples collected in 1990 and 1991 including eastern GOA samples (6). The NOAA 1990 and 1991 results are indistinguishable from the 1999 and 2000 results reported in this study. NOAA samples collected near Angoon (the reference site selected by ref 6) show no evidence of exposure to aromatic hydrocarbons. No significant differences are observed in mean EROD activity and IHC scores among site categories for halibut collected in 1999 and 2000 (Tables 3 and 4, Figure 2). Furthermore, regression analyses reveal no statistically significant relationships between pnp concentration versus EROD activity and pnp concentrations versus IHC scores. Kelp Greenling. Kelp greenling were collected only in PWS. No significant differences are observed in mean pnp concentrations among categories in 2000 (Table 2). Greenling were not sampled for pnp in 1999. Figure 3 shows generally similar ranges of pnp concentrations among site categories for the 2000 samples. Mean EROD activity and IHC scores do not differ significantly among categories in 1999 and 2000 (Tables 3 and 4). The ranges in EROD activity among categories for 1999 and 2000 data combined are similar (Figure 3). ANOVA shows no significant difference in mean EROD activity because there is broad overlap for the SPO, SPNO, and NSP 4046

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TABLE 3. EROD Activity Summary Results (Means and 1 SE)a taxa

spill path oiled

spill path not oiled

nonspill path

eastern GOA

P value

Year ) 1999 Pacific halibut 157 (35) 155 (48) 119 (13) 128 (15) 0.182 Pacific cod 37.7 23.2 (2.7) 41.4 (4.5) 0.327 kelp greenling 966 (209) 1562 (246) 0.955 rockfishb 175 (24) 350 (91) 275 (44) 0.997 rock sole 1175 (282) 1232 (704) 424 (151) 0.442

Year ) 2000 Pacific halibut 149 (18) 155 (27) 172 (39) Pacific cod 92 (25) 25 (8.5) 42 (7.4) kelp greenling 1354 (224) 497 (82) 1167 (17) rockfishb 363 (33) 273 (45) 328 (75) rock sole 1211 (451) 1160 143 (126) b

-

0.649 0.205 0.331 0.128 0.013

a pmol min-1 (g of liver)-1. (-) indicates that no fish were examined. Copper and quillback rockfish combined.

site categories. The highest value (3580 pmol min-1 (g of liver)-1) obtained for PWS SPO sites is from one (of six) greenling collected in 2000 at Snug Harbor. However, bile FAC levels in this sample were not elevated. Consequently, the elevated EROD activity for this fish appears to be unrelated to PAH exposure. There are no statistically significant relationships between pnp concentration versus EROD activity and pnp concentrations versus IHC scores. Pacific Cod. Pacific cod (2 fish) were collected and analyzed for FAC only from the eastern GOA in 1999. In 2000, Pacific

TABLE 4. Mean Scores for Immunochemistry (Hepatocyte) Stain Index and the Numbers of Fish Tested (Ntested) by Site Categorya SPO

SPNO

Pacific halibut Pacific cod kelp greenling rockfishc rock sole

0.35 (13) 0.00 (3) 0.00 (11) 1.16 (8)

0.09 (8) 1.50 (1) 0.00 (4) 0.00 (10) 2.00 (3)

Pacific halibut Pacific cod kelp greenling rockfishc rock sole

1.50 (14) 3.75 (6) 4.26 (19) 2.35 (37) 6.33 (9)

0.00 (9) 0.00 (4) 1.13 (8) 0.029 (21) 0.00 (1)

NSP

GOA

P value

Year ) 1999 0.00 (7) 0.05 (70) 0.00 (4) 0.01 (51) 0.35 (10) 0.60 (5) -

Year ) 2000 1.80 (5) 1.91 (11) 3.68 (11) 1.50 (11) 1.00 (3)

-

0.061 0.209 b 0.858 0.553 0.920 0.103 0.520 0.091 800 pmol min-1 (g of liver)-1) are from fish that do not have correlatively elevated bile FAC and all but one are from embayments (SPNO and NSP) associated with human activity impact (7). Regression analyses reveal no statistically significant relationships between pnp concentration versus EROD activity and pnp concentrations versus IHC scores. Rock Sole. There are no significant differences in mean pnp concentrations among PWS site categories for fish collected in 1999 and 2000 (Table 2). Figure 6 shows generally similar ranges of pnp concentrations for the 1999 and 2000 results combined. NOAA (5) data show higher mean values and wider ranges for fish collected at spill path sites in 1989 and again in 1990-1991. High values are for fish collected in 1989 in Snug Harbor, an oiled embayment also having a history of human activity (18). High values for 1990-1991 samples are for fish collected in Snug Harbor, Sleepy Bay, and Squirrel Bay. A single sample from Squirrel Bay has a pnp value of 171 mg. If that value is excluded, the mean pnp for 1990-1991 spill path sites is 4.28 mg pnpsa value not different from the means reported in this study for PWS sites. Mean EROD activity and IHC scores do not differ significantly among PWS site categories for fish collected in 1999 (Tables 3 and 4). The significant differences in EROD among categories in 2000 (Table 3) are due to one of five rock sole collected in Sleepy Bay (SPO) (∼4500 pmol min-1 (g of liver)-1, Figure 6). In addition, mean IHC scores for VOL. 37, NO. 18, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. Pacific cod bile FAC and EROD activities. 1999 and 2000 data, this study. An asterisk (*) indicates NOAA data of 1989-1991 (4, 5). hepatocytes were significantly higher (Table 4) at SPO sites in 2000 due to two fish with relatively high scores of 12 (out of 15). Both fish, one male and one female, were from Sleepy Bay. The male was the same fish with the highest EROD activity. However, its bile pnp concentration is low. Consequently, the cause of elevated EROD and IHC is uncertain in this fish. Although the site was heavily oiled in 1989, relatively small amounts (80-125 ng/g of TPAH) of spill residues were detected in benthic sediments sampled in 1999 and 2000. The likely sources of exposure are combustion product PAH that form the dominant PAH component (up to 560 ng/g of TPAH) in Sleepy Bay sea floor sediments (7). Regression analyses reveal no statistically significant relationships between pnp concentration versus EROD activity and pnp concentrations versus IHC scores. Examination of the IHC data revealed that hepatocytes gave the most nonzero scores for all species (Table 4). This suggests that PAH exposure is via ingestion rather than through the gills. Otherwise, one would expect gill tissues to have elevated scores as well. Site Fidelity. Fish biomarker responses resulting from exposure to PAH are indicative of relatively recent exposure, i.e., within 2-4 weeks (19). Consequently, it is important to understand the probable range of movement of the fish collected in this study during the sampling period (AugustSeptember). If the range of movement is small, then it may be assumed that any exposure detected was induced close to the site of collection. One study of the site fidelity of copper rockfish involved tagging and subsequent re-sighting of tagged fish over a 2-yr period (20). In this study re-sighted fish constituted 51.6% of the tagged fish. For high relief reefs (up to 5 m above the 4048

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bottom), 99% of the re-sighted fish had a home range size of e90 m2. For low relief reefs (1-2 m), the range size increased to 1500 m2 for 68-80% of the fish. These findings indicate a strong site fidelity for copper rockfish, and the chances that a large number of the ones collected in this study had migrated from SPO to SPNO and NSP sites is small. Another study by the same author (21) also found strong site fidelity for quillback rockfish. Pacific halibut conduct seasonal migrations. In the GOA, they migrate to the shallow waters of the Continental Shelf where they disperse during the summer feeding (22). One tagging study (22) conducted during the summer months off Yakutat, an important halibut spawning ground, found that 96% of the recoveries were made in the release area. Consequently, it seems unlikely that Pacific halibut caught in the GOA ∼300 km east of PWS would have migrated from the PWS spill zone within 2-4 weeks during the summer months. Pacific cod also conduct seasonal migrations (23). Their pre-spawn summer distributions are in shallow waters of the shelf. Winter month distributions are in the deep offshelf waters where they spawn. Little is known about their range of movement during the summer. Summary. Studies of fish collected in 1989 at some PWS spill path sites showed evidence of exposure to petroleum hydrocarbons when the results are compared to those for similar species of fish collected at reference sites (4-6). Subsequent studies in 1990 and 1991 show that fish exposure levels at these oiled sites had decreased significantly and were comparable to those for nonoiled sites. However, those exposure levels were not zero.

FIGURE 5. Copper and quillback rockfish bile FAC and EROD activities. 1999 and 2000 data, this study. An asterisk (*) indicates NOAA data for undifferentiated rockfish 1989-1991 (4). Results reported here for fish collected in 1999 and 2000 show that by some measure (bile FAC or EROD) most fish collected at the three categories of PWS sites and the GOA site show exposure to low levels of aromatic hydrocarbons or to other enzyme inducers. In addition, individual fish collected in PWS embayments, oiled or not, may have high biomarker readings. Bile FAC levels for Pacific halibut are most revealing because this fish species is the only one common to the present study and previous NOAA studies where the four site categories are represented. Results for 1999-2000 show that halibut from all site categories have measurable bile FAC levels. For halibut collected in 2000, pnp concentrations differ significantly among categories. Specifically, PWS SPNO are significantly higher than the two other PWS categories. However, PWS SPO sites are not elevated and do not differ from PWS NSP sites in mean pnp concentrations. Although the low levels of EROD activity measured here are difficult to assess, levels for the same fish are consistent with the FAC dataslow levels at all site categories and no increment at PWS spill path sites (oiled and unoiled) that might be attributed to spilled oil. Consequently, there is no evidence for continuing exposure to oil from the spill on this species of fish. Furthermore, comparison with the 1989-1991 NOAA data reveals no significant differences in bile FAC levels for halibut collected at spill path sites in 1990-1991 and those reported here. These comparisons indicate that, after 1989,

spill oil exposure to halibut was no longer detectable above other regional sources. Bile FAC levels and EROD activities for Pacific cod are consistent with those of the Pacific halibut. Fish collected at all categories of sites in 1999-2000 have equally low levels of FAC and EROD that can be attributed to regional sources. Fish collected at PWS SPO and SPNO sites show no increment to FAC or EROD levels that might be attributed to spill oil. Furthermore, the NOAA data (4, 5) indicate that there were no significant exposures of either halibut or cod to spill oil in 1990-1991. Similar results for FAC and EROD were found for kelp greenling, copper rockfish, and rock sole, i.e., there is no evidence of exposure to Exxon Valdez spill oil residues. Although none of these fish were collected from sites outside of PWS, by comparison with the cod and halibut results, it is likely that the measured levels of bile FAC and EROD activities are induced by regional sources. Individual samples with unusually high FAC and EROD results are generally from embayments not impacted by the spill. Some appear to be associated with anthropogenic activities. The fish biomarker results reported here do not support the hypothesis of continuing spill oil exposure at PWS spill path sites. The fact that biomarker levels for PWS fish are not zero and are similar to exposure levels in fish collected in the eastern GOA indicates that regional hydrocarbon sources may be involved. VOL. 37, NO. 18, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 6. Rock sole bile FAC and EROD activities. 1999 and 2000 data, this study. An asterisk (*) indicates NOAA data of 1989-1991 (4, 5).

Acknowledgments This study was supported, in part, by the ExxonMobil Corporation. We thank captain Glen Hodge and crew of theM/V Pacific Star for assistance in the field. The assistance of William Cooper is gratefully appreciated.

Note Added after ASAP This paper was released on 08/12/2003 with incorrect version of Figures 1 and 3-5. The correct version was posted on 09/15/2003.

Literature Cited (1) Wolfe, D. A.; Hameedi, M. J.; Galt, J. A.; Watabayashi, G.; Short, J.; O’Claire, C.; Rice, S.; Michel, J.; Payne, J. R.; Braddock, J.; Hanna, S.; Dale, D. Environ. Sci. Technol. 1994, 28, 561A. (2) Neff, J. M.; Owens, E. H.; Stoker, S. W.; McCormick, D. M. Shoreline oiling conditions in Prince William Sound following the Exxon Valdez oil spill. In Exxon Valdez Oil Spill: Fate and Effects in Alaskan Waters; Wells, P. G., Butler, J. N., Hughes, J. S., Eds.; ASTM STP 1219; ASTM: Philadelphia, PA, 1995; p 312. (3) Alaska Fisheries Science Center Quarterly Report for JulyAugust-September 2001. The Exxon Valdez Oil Spill: How Much Oil Remains?; NOAA NMFS: Auke Bay, AK, 2001; pp 1-5. 4050

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Received for review March 18, 2003. Revised manuscript received June 9, 2003. Accepted June 16, 2003. ES0342401

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