Marine Pollution Bulletin, Volume 27, pp. 285-292, 1993. 0025-326X/93 $6.00+0.00 Printed in Great Britain. C 1993 Pergamon Press Ltd

Analyses for Petroleum-related Contaminants in Marine Fish and Sediments Following the Gulf Oil Spill MARGARET M. KRAHN, GINA M. YLITALO, JON BUZITIS, JENNIE L. BOLTON, CATHERINE A. WIGREN, SIN-LAM CHAN and USHA VARANASI Environmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Boulevard East, Seattle, Washington 98112, USA

Approximately 1 year after Kuwaiti crude oil was spilled and burned during the Gulf War, scientists on board the NOAA R/V Mt Mitchell sampled sediment and biota in the Gulf to look for evidence of petroleum contamina- tion. Subtidal sediments and fish bile were analysed for petroleum-related aromatic compounds (ACs) on board the Mt Mitchell using cost-effective and rapid high- performance liquid chromatographic (HPLC) screening methods. Parent ACs and their alkyl homologs were measured in sediments. However, because fish extensively metabolize ACs in their livers to form polar metabolites that are concentrated in bile for excretion, metabolites of ACs were measured in bile. HPLC screening analyses found ACs in many of the sediment and fish bile samples collected. The results of HPLC screening were confirmed in selected samples by gas chromatography/mass spectrometry (GC/MS) analyses that identified petroleum-related ACs (e.g., alkyl phenanthrenes or dibenzothiophenes) in sediments and their metabolites in bile.

During the Gulf War, millions of barrels of Kuwaiti crude oil (KCO) were released into the waters of the Gulf. In addition, hundreds of oil wells were set on fire and aromatic compounds (ACs) resulting from the combustion of the petroleum may also have been deposited into the waters of the Gulf. To evaluate the impact of these petroleum-related hydrocarbons on the biota in these waters, the Regional Organization for the Protection of the Marine Environment (ROPME) in collaboration with the Intergovernmental Oceano- graphic Commission and the United Nations, mounted a joint scientific study which included 130 scientists from 20 nations. As part of this study, the US National Oceanic and Atmospheric Administration (NOAA) sent the R / V Mt Mitchell to the Gulf to serve as a platform from which to conduct studies and to collect samples of sediment and biota. Scientists from the United States, the ROPME countries and from other parts of the world joined together to study the impact of this poten- tial environmental disaster.

Sediments and marine organisms are often sampled after an oil spill to assess contamination. Usually, oil contamination in these samples is determined by measuring petroleum-related ACs by gas chromato- graphy/mass spectrometry (GC/MS). However, GC/ MS analyses are expensive and time-consuming. Alternatively, rapid high-performance liquid chromato- graphic (HPLC) screening methods, developed to measure ACs in sediments (Krahn et al., 1991) and AC metabolites in fish bile (Krahn et al., 1986a), were tested and validated on samples collected following the Alaskan oil spill (Krahn et al., 1992, 1993a; Varanasi et al., 1990). In addition, chromatographic patterns from the HPLC screening analyses provided information about possible contaminant sources, e.g., crude oils or pyrogenic contaminants (Krahn et al., 1993a). In contrast to sediments, contamination of fish by petrogenic or pyrogenic ACs is often more difficult to assess because these organisms extensively metabolize most ACs in their livers and then the metabolites are concentrated in bile for elimination (Statham et al., 1976; Varanasi et aL, 1979, 1989). A screening method, employing reverse-phase HPLC with fluorescence detection to measure AC metabolites in bile, has proven useful in estimating the exposure of fish to petroleum- related or pyrogenic ACs (Krahn et al., 1986b; 1992; Varanasi et al., 1990).

The Mt Mitchell was equipped with an analytical laboratory capable of conducting HPLC analyses to screen for petroleum contamination in sediments and fish bile. The shipboard laboratory conducted sediment analyses in support of a project studying the persistence of intertidal oil in sediments after 1 year of natural weathering and the transport of intertidal oil to subtidal sediments (Michel et al., 1993). The study sites selected in the Tanaqib and Abu Ali areas (Fig. 1) were in the path of the spill and sections of these embayments were heavily impacted. More than 100 sediment samples were screened on board the ship to evaluate the extent and magnitude of oil contamination within these regions. In addition, bile samples from more than 100 fish from several fish species were screened on board the ship to determine if these animals showed evidence of petroleum contamination more than a year after the

285

spill. However, sheiry (Lethrinus kallopterus) was the only species captured in sufficient numbers at multiple sites so that intersite comparisons could be made. Two of the sheiry sampling sites (Karan Island and Rennic Shoals) were within the path of the spill and one site (Qatar) was beyond the spill (Fig. 1 ).

In this paper, we present some of the initial results for the analyses of sediment and fish bile samples collected from the Gulf. A number of the sediments showed elevated concentrations of fluorescent ACs by HPLC screening. In addition, screening of bile from sheiry indicated that some of these fish had been exposed to ACs. However, HPLC screening provided semiquantitative concentrations of ACs in sediments or AC metabolites in bile. Accordingly, the most contami- nated samples were located by screening and then, GC/MS analyses were conducted to verify contaminant concentrations in selected sediment and bile samples. Petroleum-based ACs, including alkylated phenan- threnes and dibenzothiophenes in proportions charac- teristic of crude oils from the Gulf region, were found in many of the sediments and, as metabolites, in the bile samples.

Experimental Field collection

Sheiry (Lethrinus kallopterus), a species of epi- benthic fish, were captured in the Gulf by hook and line in April and May 1992 (Fig. 1). Bile was collected from the gall bladders of fish within 5 min of sacrifice and the bile samples were frozen immediately. Subtidal sediments from a relatively limited area of the Gulf--Ra's Tanaqib (Tanaqib area) and Dawhat AI

Marine Poiiullon gulie~m

Musaltamiyah and Dawhat Ad Daffi (the Abu All area) (Fig. 2)--were collected by divers in March and Aprii, 1992; details of collection are reported by Michel eta/. (1993). The sediment cores were subsampled and the resulting samples were frozen. Analytical results reported in this paper are for the surface subsample (//-5 cm). The sample of fresh KCO (used as an analytical standard in this paper) was an American Petroleum Institute reference oil. Weathered KCO was collected from a near-shore trench, near Abu All, Saudi Arabia in May 1991 and a highly-weathered (asphalt- like) sample of KCO was collected from AI Arabiya, Saudi Arabia, in May 1992.

Screening of sediment and bile The method used to screen sediments for ACs

features sonic extraction and size exclusion HPLC chromatography with fluorescence detection as described by Krahn et al. (1991). The size exclusion column separated the analytes according to their molecular size and shape, so the chromatogram showed a molecular size distribution of the analytes (Krahn et al., 1993b). Fluorescence was recorded at wavelengths characteristic of ACs from weathered crude oil (e.g., phenanthrenes at 260/380 nm and naphthalenes at 290/335 nm) and of pyrogenic origin (e.g., pyrenes at 380/430 nm). Compounds that fluoresce at a particular wavelength pair include the parent AC, alkyl homologs of the parent and metabolites of the parent and homologs (Krahn et al., 1984). The chromatographic areas of the AC fraction (> 8.2 min) were integrated and equivalent concentrations of KCO were calculated (Krahn et al., 1993a). The method for bile analysis by reverse-phase HPLC with fluorescence detection was

048°E 050°E 052°E 054°E OS6°E 058°E

048oE 050°E 052°E 054°E 056°E 058°E

Fig. I Locations of sites where sheiry were captured (Karma Island, Rennie Shoals and Qatar). The sites of sediment collection (the Abu Ali mad Tanaqib areas) are also indicated; see Fig. 2 for more detail.

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49*00' E 49*30' E

27030' N- ' 27030 . N

49*00. E 49*30' E

Tanao lb

1 SC-16 2 SC-11 3 Trsnsect-27B

Abu All

4 $C-$0 11 Transect 16-03 S IIC-27 12 SC-21 6 SC-38 13 $C-04 7 SC-2S 14 SC-24 8 8C-37 15 Transect 13-01 9 Transect 16-01 16 Transect 04-01 10 Transect 16-02 17 T r snuc t 02-02

Fig. 2 Locations of sediment sampling sites (Tanaqib and Abu Ali areas).

described in detail by Krahn et al. (1984; 1986a). Fluorescence was recorded at the same wavelengths used for sediment analyses. The metabolite-containing portion of the chromatogram (>9.5 min) was integrated and concentrations were calculated by normalization to an AC standard, e.g., a phenanthrene standard for chromatograms recorded at 260/380 nm.

GC/MS analyses of sediment and bile Sediments were analysed for ACs according to the

procedure of Krahn et al. (1993a). The procedure included extraction of analytes by tumbling with methylene chloride, cleanup of the extract by size- exclusion HPLC and determination of ACs by GC/MS with sequenced selected ion monitoring. Bile samples were composited (n= 1-5; by species and by concen- trations of ACs as determined by HPLC screening) to obtain enough bile (minimum 50 ~tl) for the GC/ MS determination. Bile was enzymatically hydrolyzed and the hydrolyzed AC metabolites were extracted, separated from biogenic interferences by size-exclusion HPLC and determined by GC/MS (Krahn et al., 1992).

Quality assurance Each set of sediment or bile analyses (both HPLC

and GC/MS procedures) included the following quality assurance samples: a method blank, a duplicate, and a control material (reference material) or spiked matrix.

Resul ts

HPLC screening for aromatic compounds in sediment and bile

Because results from the HPLC screening of sediments at phenanthrene and naphthalene wave- lengths were highly correlated (r=0.994; n = 105) and thus, provided redundant information, only results from phenanthrene wavelengths will be presented in this paper. In addition, HPLC results are presented only for those sediments also analysed by GC/MS. Equivalent concentrations of KCO in extracts of sediments from the Abu Ali and Tanaqib areas of the Gulf ranged widely (Table 1). Sediments were divided into three groups by KCO concentration for graphing: (1) > 500 ng g-l; (2) 100-500 ng g-l; and (3) <100 ng g-~.

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TABLE I

Equivalents of Kuwaiti crude oil determined by HPLC screening (area > 8.2 min) at phenanthrene wavelengths (260/380 nm) in sediments (0-5 cm depth of the core) collected from the Abu Ali area of the Gulf

about I year after the 1991 oil spill.

Kuwaiti crude oil equivalents Sediment sample (~tg g-~, we! wl)

Transecl 02.(}2 < 1 Transect 04.01 42 Transect 13.(}1 450 Transect 16.01 360 Transect 16.02 321} Transect 16,03 < I Transect 27B 81 SC 04 ~2 SC 11 58 SC 16 120 SC 21 67 SC 24 1 t0 SC 25 480 SC 27 40() SC 30 200 SC 37 860 SC 38 68O0

Sediments with concentrations of KCO> 100 rig g-J came from the Abu Ali a r e a . Other sediments--those from Tanaqib and a few from Abu Ali--were found to have low concentrations of ACs (KCO concentrations < 100 ng g-~).

Phenanthrene equivalents in the bile composite samples from sheiry in this study also displayed a wide range of concentrations (860-60 000 ng g-l), even in fish collected from the same site (Table 2). For example, both the highest and lowest concentrations of ACs were found in the sheiry composite samples 03 and E) from Karan Island (Fig. 1). Although the mean of the bile composite samples for the site nearest the spill (Karan Island) was higher (20000+21 000; n = 6 ) than the means from the other two sites (Qatar: 17 000+ 13 000; n--2; and Rennie Shoals; 10 000+ 2200; n~3) , no significant differences were found due to large variances and small sample sizes.

TABLE 2

Equivalents of fluorescent aromatic compounds determined by HPLC screening at phenanthrene wavelengths (260/380 nm) in bile of sheiry

captured from the Gulf about 1 year after the 1991 oil spill.

Bile composite Phenanthrene equivalents sample Site (ng g-~, wet wt)

Sheiry-A Karan Island 19 000 Sheiry-B Karan Island 60 000 Sheiry-C Karan Island 8700 Sheiry-D Karan Island 16 000 Sheiry-E Karan Island 860 Sheiry-F1 Karan Island 18 000 Sheiry-F2 Karan Island 18 000 Sheiry-G Qatar 26 000 Sheiry-H Qatar 7500 Sheiry-I Rennie Shoals 11 000 Sheiry-J Rennie Shoals 12 000 Sheiry-K Rennie Shoals 7800

HPLC chromatographic patterns in petroleum products, sediment and bile

HPLC chromatographic patterns of crude oils showed visible differences in the lower molecular

weight portion ( > 8.2 min) of their chromatograms (i-ig 3A). For example, the highly-weathered KCO had a smaller proportion of lower molecular ~cigh~ ACs compared to the weathered KCO or Prudhoc Bay crude oil (PBCO). Diesel fuel, an intermediate distillate frac- tion of petroleum, contained a narrower molecular size range of the components than did the crude ~ils (Krahn ~'t al., 1993b). As a result, its HPLC chromatogram was very different from those of the crude oil.~ (Fig. 3A). The sediments from the Gulf (Fig. 3A) had HPLC chromatographic patterns that most resembled those of the weathered KCOs. For example, the chromato- graphic pattern of sediment SC-38 was nearly identical to that of weathered KCO, whereas SC-16 closely approximated the highly-weathered KCO (Fig. 3A). A pattern typical of sediments with a low background concentration of ACs is shown by Transect 02-02 in Fig. 3. Chromatograms of bile from ~wo sheiry composites, an English sole from an urban site, a halibut injected with crude oil and a control halibut (captured from an uncontaminated site) arc shown in Fig, 3.

GC/MS analyses of sediment and bile for individual aromatic compounds

Analyses of selected sediment samples were conducted by GC/MS. Numerous petroleum-related ACs were identified in these sediments (Table 3) by comparing retention times and mass spectra for these ACs to those from reference standards or from the mass spectral library. Several of the sediments contained high concentrations of ACs (e.g., alkylated phenanthrenes and dibenzothiophenes) that are known to resist weathering, but none of the sediments contained appreciable quantities of pyrogenic ACs (e.g., fluor- anthenes and pyrenes). All the sediments with summed concentrations of ACs > 100 ng g-i (Table 3) came from the Abu Ali area (Fig. 2). Other sediment samples--from the Abu Ali and Tanaqib areas (Transects 02.02, 04.01, 16.03, 27B and Sediment cores 04, 11, 16, 21; not shown in Table 3)--and the method blank were found to have low concentrations of summed ACs ( < 100 ng g-~; Fig. 2).

Sequenced selected ion monitoring GC/MS analyses were conducted on hydrolyzed bile composite samples from sheiry. Numerous metabolites of petroleum- related ACs were identified in these bile samples by comparing retention times and mass spectra for each metabolite to those from reference standards or from the mass spectral library. The library included spectra of metabolites from previous studies in which fish were injected with PBCO (Krahn et al., 1992) or with individual ACs (Krahn et al., 1987). Metabolites of petroleum-related ACs (e.g., phenanthrols or dibenzo- thiophenols) were found in these bile composites (Table 4). In addition, metabolites of pyrogenic ACs (e.g., pyrenols) were identified several composites (Sheiry-A, -B and -G). The highest summed concentration of metabolites was found in a composite 03) from Karan Island and the lowest in a composite (H) from Qatar.

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A Sediment Phonanthreno wavelongth= 260/3110 nm

[ 8.2 mln

\ \

I

A I

0.0

SC-38

SC-25

SC-16

B

Bile Phenanthrene w-volonglh= 2SO/~O nm

_,4L ~ o W - B

Karen Island

Sheiry-E Karan Island

Treme= 02-02

Fig. 3

Hlghly-weet hotod k . ~ Kuweltl ©rude

Weathered Prudhoo

~_ Bay crude o,

Dle=ol Fuel

English sole from urban site in Puget Sound

Prudhoe Bay crude oil-injected

halibut

Control halibut

12.0 0.0 Retention time (minutes)

(A) Chromatograms from the HPLC/fluorescence screening at 260/380 nm (phenanthrene wavelengths) of (A) bile from 2 sheiry captured from the Karan Island area of the Gulf after the oil spill, from an English sole (Parophrys vetulus) captured from an urban site in Puget Sound, Washington, USA, from a halibut injected with Prudhoe Bay crude oil; and from a halibut injected with carrier solvent (control). (B) sediments collected from the Gulf after the oil spill (SC-38, SC-25, SC-16 and Transect 02- 02) and from possible sources of contamination in the marine environment: weathered (for 3 months) and highly-weathered (for 15 months; asphalt-like) Kuwaiti crude oil, weathered (for 1 year) Prudhoe Bay crude oil; and diesel fuel. The retention time--8.2 min--at which integration of the AC fraction was begun is marked on the chromatograms and the peak at about 4.5 min is the polystyrene internal standard.

24.0

Statistical comparisons of HPLC screening and GC/MS results

To make a comparison between the semiquantitative results obtained with the HPLC screening methods and the quantitative results from detailed GC/MS analyses, concentrations of the phenanthrenes from the GC/MS analyses of sediment (Table 3) and the phenanthrols from bile (Table 4) were summed for each sample. The KCO equivalents at phenanthrene wavelengths from sediment screening (Table 1) were correlated with the summed concentrations of the phenanthrenes (n= 15;

r--0.93; p<0.0001). Similarly, a correlation was found for the phenanthrene equivalents (Table 2) and the sums of the phenanthrols in bile (n=12; r--0.77; p<0.003). Although the HPLC sediment screening results at naphthalene wavelengths are not reported in this paper, characteristic petroleum-related ACs--the naphthalenes and dibenzothiophenes--fluoresce at these wavelengths. HPLC sediment screening results at naphthalene wavelengths were correlated with the sum of dibenzothiophenes and naphthalenes from GC/MS analyses (n= 15; r---- 0.90; p<0.0001).

289

Marine ~'oli~lt~ora Bulkq!o

"IABLE 3

Sums of concentrations by sequenced selected ion monitoring GC/MS of aromatic compounds in selected sediments collected from the Abu Ali area in the Gulf in March and April, 1992, about 1 year after Kuwaiti crude oil was spilled into the Gulf*.

Concentration (ng g-L wet wt) Trans 13.01 Trans 16.01 Trans 16.t!2 S(" 24 SC 2~ S(7 27 S( 30 S ( 37 ~(7 3g

Naphthalenes C~ < 3 <4 < 3 < 3 < 3 < 3 < 3 <: 3 ~: 3 C~ <3 <4 <3 <3 <3 <3 <3 < 3 : 3 C 2 <3 <4 <3 < 3 <3 <3 < :~ <3 Y70 C 3 < 3 <4 < 3 < 3 13 <3 < 3 9 2000 C 4 <3 <4 <3 <:3 5 <3 .<3 23 2100

Fluorenes C 0 <3 < 4 <3 <3 <3 <3 <3 <3 26 C 1 <3 < 4 <3 <3 <3 <3 <3 <3 320 C.~ <3 < 4 <3 <3 <3 <3 <3 <3 980 C 3 <3 < 4 <3 <3 <3 <3 <3 <3 680

Dibenzothiophenes C 0 < 2 <3 <2 <3 <3 <2 <2 <3 370 Cj < 2 < 3 < 2 < 3 120 60 30 1.30 3800 C 2 30 < 3 170 12 690 470 240 1100 10 000 C 3 430 160 520 130 1100 830 450 2500 18 000 C4 370 61 350 5 520 330 160 1300 7800

Phenanthrenes C o <1 <2 <2 <2 <2 <2 <2 <2 74 C~ < 1 <2 <2 <2 18 3 <2 14 790 C 2 32 < 2 28 < 2 88 64 27 160 2700 C 3 78 2 51 < 2 110 74 25 310 3100 C 4 <1 < 2 3 <2 <2 <2 <2 36 990

Fluoranthene C O < 1 <2 <2 <2 <2 < 1 < 1 <2 < i

Pyrene C. <1 <2 <1 <1 <1 <1 <1 <2 59

Fluoranthenes/pyrenes C~ <1 <2 <2 <2 <2 <1 < I <2 270

Sum of ACs 940 220 1120 150 2660 1830 930 5580 54 430

Recovery (%) of naphthalene-d8 90 83 79 88 90 9 l 89 88 86

*The limit of quantitation is indicated by a ' < ' preceeding the value. Trans -- transect where a sediment core was collected. SC= sediment core. Sums of ACs for the following samples were < 100 ng g-l: Transects 02.02, 04.01, 16.03, 27B and Sediment cores 04, 11, 16, 21.

TABLE 4

Sums of concentrations by sequenced selected ion m o a i t o ~ GC/MS of metabolites of aromatic compounds in composite bile samples of sheiry captured in the Gulf in April and May, 1992, about I year after Kuwaiti crude oil was spilled into the Gulf*.

Concentration (ng g-1 wet wt) Karan Island Qatar Rennie Shoals

A B C D E F 1 F 2 G H I J K

Naphthols C 0 34 66 17 39 <28 <30 <36 100 12 <32 48 <18 C~ 250 <94 <32 94 <36 <40 <46 160 22 <42 120 <23 C 2 320 690 63 150 70 260 280 380 43 92 130 74 C 3 460 1400 19 160 47 260 340 290 18 68 100 8

Fluorenols C O 410 530 280 410 230 280 370 200 110 100 130 79

Dibenzothiophenols C 1 1200 3300 230 440 230 830 1000 750 141) 450 570 220 C 2 1700 5600 310 320 260 1100 1000 750 83 1400 1.300 500 C 3 530 1200 32 120 57 79 300 370 7 730 600 300

Phenanthrols C O 670 < 110 180 < 36 < 32 100 110 280 55 100 110 < 20 C 1 1200 3200 240 500 120 400 500 1000 70 390 310 140 C 2 640 1800 150 270 30 270 330 490 33 550 530 200

Naphthols C 0 1100 1200 200 360 130 190 320 1300 69 890 370 140

Sum of metabolites 8514 18 990 1720 2860 1220 3770 4550 6070 660 4770 4320 1660

Recovery (%) of naphthalene-d8 77 81 100 85 92 83 75 80 140 94 100 100

*The limit of quantitation is indicated by a ' < ' preceeding the table.

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Discussion

HPLC screening of sediments, collected from a relatively limited area of the Gulf, revealed substantial concentrations of ACs in many of the samples (Table 1), particularly in those from the Abu Ali area (Fig. 2). High concentrations of petroleum-related ACs, e.g., alkylated phenanthrenes and dibenzothiophenes, were also found in many of the sediments by GC/MS, thus confirming the HPLC results. In particular, the alkylated dibenzothiophenes that are known to comprise a high proportion of KCO (Payne et al., 1984), were present in higher proportions than were other ACs (Table 3). However, pyrogenic ACs were present in low or nondetectable concentrations. Analogous results--high concentrations of petroleum- related ACs characteristic of PBCO--were found by HPLC and GC/MS analyses of sediments following the Exxon Valdez oil spill in Alaska (Krahn et al., 1993a).

HPLC chromatographic patterns of sediments can provide additional evidence for the types of contami- nants present, e.g., crude oil or diesel fuel. For example, analytes elute from the size-exclusion HPLC column according to molecular size, with compounds of 'largest volume' eluting first, to form a characterisic size- distribution pattern (Fig. 3A). The sediments from the Gulf had chromatographic patterns that closely resembled those of the weathered KCOs (Fig. 3A). Similarly, after the Exxon Valdez oil spill, a number of sediments screened by HPLC were found to have chromatographic patterns nearly identical to that of weathered PBCO (Krahn et aL, 1983a). Although the patterns from the weathered crude oils were generally similar (Fig. 3A), the patterns of these crudes were different from those of diesel fuel (Fig. 3A) or other petroleum fractions (Krahn et at , 1993b) that are possible sources of contamination in areas of maritime activity. Thus, both HPLC screening and GC/MS analyses provided evidence for the petroleum contami- nation in sediments from the Gulf.

HPLC screening analyses of sheiry bile composites (Table 2) showed elevated concentrations of AC metabolites in several samples, thus indicating recent exposure of these fish to ACs (Krahn et al., 1992). Although the site at Qatar was out of the path of the spill and fish from that site may not have been exposed to oil, moderate concentrations of AC metabolites were measured in one composite (Sheiry-G). Conversely, at the Karan Island site in the path of the spill, where the majority of the bile composites had elevated concentra- tions of AC metabolites, one composite (Sheiry-E) had very low levels of contaminants, similar to concentra- tions of ACs found in fish from uncontaminated sites in Alaska (compared to control halibut, Fig. 3B) (Krahn et a/., 1992). Because these epibenthic fish can move rapidly over relatively large distances, a range of concentrations of AC metabolites is likely to be found in the bile of fish from a particular site (Krahn et al., 1986a). As a result of large variances and small sample sizes, no statistical differences were found in bile metabolite concentrations for the three sites at which sheiry were captured for this study.

Confirmation of the exposure of the sheiry to petroleum-related ACs was provided by GC/MS analyses of hydrolyzed bile that showed high propor- tions of certain metabolic products (e.g., alkylated phenanthrols and dibenzothiophenols; Table 4) of degradation-resistant ACs (Atlas et al., 1981; Boehm et al., 1981). Furthermore, concentrations of alkylated ACs identified in the sheiry composites were similar to those found in pollock sampled from the Gulf of Alaska more than a year after the Exxon Valdez oil spill (Krahn et al., 1992). In addition, a few of the composites (e.g., Sheiry-A, -B, -G) from the current study had concentra- tions of the pyrogenic fluoranthenols/pyrenols (Table 4) typical of fish from urban sites (Krahn et al., 1987). Because the fish were sampled over a relatively wide area of the Gulf (Fig. 1), the fish may have been exposed to spilled crude oil, to pyrogenic ACs from the burning oil wells or to petroleum previously deposited into the Gulf. Alternatively, the petrogenic and pyrogenic ACs could have entered the Gulf from other anthropogenic activities.

As found in previous studies (Krahn et al., 1991; 1992; 1993a), concentrations of ACs in sediments or AC metabolites in bile measured by HPLC screening were correlated with the sums of individual compounds determined by GC/MS. Therefore, the important role that HPLC screening methods play in environmental analysis has again been demonstrated. Screening methods allow samples to be ranked and selected on the basis of contaminant concentrations, so fewer GC/MS analyses need to be conducted. As a result, the overall costs of the analyses have been reduced, while essential GC/MS data is provided in a timely fashion. Furthermore, HPLC screening analyses can be con- ducted "on-site", (e.g., in a shipboard laboratory) to allow modification of the sampling strategy based on the screening results. Thus, additional samples from a particular fish species or sediment site can be taken before the ship moves to another site.

The composition of the weathered crude oil found in sediments from the Gulf differed from that in sediments collected following the Exxon Valdez oil spill. GC/MS analyses of sediments from the Alaskan spill showed substantial proportions of C3-C4 naphthalenes, C2-C4 phenanthrenes and C1-C3 dibenzothiophenes. (Krahn et al., 1993a). In contrast, the Gulf sediments contained only a low proportion of naphthalenes, a somewhat larger proportion of C2-C4 phenanthrenes and a high proportion of C1-C4 dibenzothiophenes. The composi- tions of the weathered oils would be expected to differ because fresh KCO and PBCO are substantially differ- ent (Payne et al., 1984), i.e., the proportion of dibenzo- thiophenes is much higher in KCO than in PBCO. However, a greater proportion of the more volatile ACs (e.g., naphthalenes) were lost from KCO compared to the proportion lost from PBCO, due to conditions of weathering (e.g. amount of sunlight, water temperature, microbial activity, and currents) that were very different in the Gulf compared to those in Alaskan waters.

In summary, following the Gulf oil spill, an inter- national group of scientists on board the NOAA R/V Mt Mitchell collected and analysed samples of sediment

291

and biota from the Gulf to assess damage to these resources. Cost-effective and rapid HPLC screening methods found evidence of petroleum contamination in many of the sediments and fish collected. The HPLC screening results were confirmed by GC/MS analyses that identified proportions of parent ACs in sediments and metabolites in bile that were characteristic of KCO. This approach--combining HPLC screening for AC:s with confirmation of contaminant concentrations in selected sediment or bile samples by GC/MS--has proven invaluable in this and previous oil spill studies.

We thank Richard Permenter, captain of the NOAA R/V Mt Mitchell, along with the other officers and crew, for their able support in the field during sample collection. We appreciate the assistance of Robert Clark, Chief Scientist on the Mt Mitchell, in collecting samples of bile, sediment and the weathered Kuwaiti crude oil. We also thank the other scientists aboard the Mt Mitchell who assisted in the collection and neocropsy of fish and in the laboratory analyses: Nahida Badr Ak-Majed, Fuad Al-Saffar, Salem AI-Shubhy, Abdul Hadi Bu-Olayan, Abdul-Rahman Al-Obaidly, Ahmed Buqais, Ali Al-Baz, Ali AI- Sumaiti, Ali Basaham, Bahgat Habashi, Hassan Abdul Hamed Jastania, Johnny Beyer, Mahmoud Al-Khabbaz, Mohsen At-Ansi, Sami Mohammad Al-Yakoob, Zahra Sadiq, Ahmed Mal Allah, Mohammed A. R. Hassan, Ibtesam Khalaf and Ibrahim Saleh. We also value the technical assistance or advice of Donald Brown, Ronald Pearce, Daryle Boyd, Susan Pierce, Nicolaus Adams, Thomas Merculief and James Boulter. We appreciate the financial support of John Robinson who arranged funding from the National Oceanic and Atmospheric Administration, the Department of State and the Marine Spill Response Corporation.

Atlas, R. M., Boehm, R D. & Calder, J. A. (1981). Chemical and biological weathering of oil, from the Amoco Cadiz spillage, within the littoral zone. Estuar. Coast. ShelfSci. 12,589-608.

Boehm, R D., Fiest, D. L. & Elskus, A. (1981). Comparative weather- ing patterns of hydrocarbons from the Amoco Cadiz oil spill observed at a variety of coastal environments. In International Symposium on the Amoco Cadiz: Fates and Effects of the Oil Spill, pp. 159-173. Centre Oceanologique de Bretagne. Centre National pour I'Exploitation des Oceans.

Krahn, M. M., Myers, M. S., Burrows, D. G. & Malins, D. C. (1984). Determination of metabolites of xenobiotics in the bile of fish from polluted waterways. Xenobiotica, 14,633-646.

Krahn, M. M., Rhodes, L. D., Myers, M. S., Moore, L. K., MacLeod, W. D., Jr. & Matins, D. C. (1986a). Associations between metabolites of aromatic compounds in bile and the occurrence of hepatic lesions in English sole (Parophrys vetulus) from Puget Sound, Washington. Arch. Environ. Contain. Toxicol. 15, 61-67.

Krahn, M. M., Kitfle, L. J., Jr. & MacLeod, W. D. Jr. (1986b). Evidence

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