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PAH metabolites in bile and EROD activity in North Sea fish
Marine Environmental Research, Vol. 46, No. l-5, pp. 229-232, 0 PII:
1998 Published
SOlSl-1136(97)00035-4
1998 by Elsevier Science Ltd. All rights reserved Printed in Great Britain 0141-1136/98 $19.00+0.00
ELSEVIER
PAH Metabolites in Bile and EROD Activity in North Sea Fish Endre Aasa and Jarle Klungsaryrb “Rogaland Research, PO Box 2503 Ullandhaug, N-4004 Stavanger, Norway bInstitute of Marine Research, PO Box 1870 Nordnes, N-5024 Bergen, Norway
ABSTRACT Atlantic cod (Gadus morhua) , haddock (Melanogrammus aeglefinus) , and long rough dab (Hippoglossoides platessoides) were sampled from areas with oil production in the North Sea. Egersundbanken, an area without oil production, was used as a reference site. Ethoxyresorufin-O-deethylase (EROD) activity was measured in liver to estimate cytochrome P4SOlA levels. Bile was analysed by fixed wavelength fluorescence (FF) detection for polyaromatic hydrocarbon (PAH) metabolites. No increased levels of EROD activity were detected in$sh caught in the vicinity of oilproductionfields, when compared to the reference site, Egersundbanken. No dtrerences in levels of PAH metabolites in bile were detected between the stations for any of the species. Therefore, contamination from 25 ring PAH compounds in the vicinity of oilfields in the North Sea may not be of great concern for theseJish species. 0 1998 Published by Elsevier Science Ltd. All rights reserved
INTRODUCTION An environmental monitoring survey organised by Institute of Marine Research was made in January 1996, in the North Sea. One main purpose of the survey was to monitor possible negative biological effects in the vicinity of oil fields. Atlantic Cod (Gadus morhua), haddock (Melanogrammus aeglefinus), and long rough dab (Hippoglossoides platessoides) were captured by trawling. Four main sampling areas were chosen: Stattjord, Oseberg, Brent and Egersundbanken. The three first are oil fields, while the last has been chosen as a reference site. A number of samples were taken from three fish species for various analyses. In this study results from bile and liver analyses will be reported. Bile was sampled for fixed wavelength fluorescence (FF) detection of polyaromatic hydrocarbon (PAH) metabolites. Liver was sampled for ethoxyresorufinO-deethylase (EROD) analyses, to estimate cytochrome P4501A activity. Ten fish per species per station were analysed. FF analyses of fish bile have earlier not been carried 229
230
E. Aas, J. Klungs0yr
out on wild fish in the northern North Sea, and data on EROD activity is also sparse in these areas. Bile samples were frozen at -20°C and stored until analysis. They were diluted 1:1600 times in 48% ethanol, and FF was measured at the excitation/emission wavelengths 341/ 383nm, optimised for the detection of pyrene metabolites (see Aas et al., this volume). Some of the samples were also measured at 290/335 nm for naphthalene type metabolites and 380/430 nm for benzo[a]pyrene type metabolites (Lin et al., 1996). The concentration of the bile pigment biliverdin was measured in all samples according to the method of Larson et al. (1947) in order to estimate bile density. Biliverdin is primarily measured to check if differences in fluorescence intensity simply could be a result of differences in bile 30
Oil exposed cod
20
Oil z
10
Fig. 1. Synchronous fluorescence specter (AL = 42 nm) of individual cod, haddock and long rough dab caught at the Stattjord station. For comparison, experimentally oil exposed turbot and cod are shown. The turbot was chronically exposed to 0.125 ppm dispersed light oil in water for 24 h, while the cod was exposed to crude oil spread onto the water surface, 4 d exposure.
PAH metabolites in bile and EROD activity in North Sea fish
231
densities. In this paper the PAH metabolite results are not expressed normal&d to biliverdin. The reason for this is that such a normalisation may add extra error to the data. Therefore, PAH metabolites are expressed here as relative values. Synchronous fluorescence spectra, achieved by scanning simultaneously on both excitation and emission wavelengths (AA = 42 nm), were made for a representative selection of bile samples from all sites. Liver samples were frozen in liquid nitrogen, and transferred to -80°C before homogenising and processing of S-9 supernatant. EROD activity in liver was measured according to a modified Burke and Mayer (1974) method, using a fluorometric plate reader as described by Eggens and Galgani (1992). Protein concentrations were measured according to Bradford (1976). The results from the FF measurements at wavelength pair 341/383 nm did not reveal differences in PAH content between the areas for any of the fish species. Nor were any differences found at the wavelength pairs 2901335 nm or 380/430 nm. Synchronous
T
Sn
Sf
0
B
E
Station Fig. 2. EROD activity (pmol resorufin mine1 mg protein-‘)
with standard deviation (n = 10 except for long rough dab at Egersundbanken where n = 7). Sn and Sf: Statfjord oil field, 0: Oseberg oil field, B: Brent oilfield and E: Egersundbanken.
232
E. Aas, J. Klungstiyr
fluorescence spectra (Ahh= 42 nm) are shown for one individual bile from each species (from the Statfjord station), and compared to bile from turbot and cod experimentally exposed to oil. The turbot was chronically exposed to a concentration of 0.125 ppm dispersed light oil (BAL 150) in water for 24 h, and the cod was exposed for 4 d to crude oil spread onto the water surface of a tank (Fig. 1). Bile from the North Sea fish showed weak fluorescence with profiles similar to the background fluorescence from the solvent ethanol, and unlike profiles for oil exposed fish. Analyses of PAH metabolites in fish bile by fluorescence, have shown to be a sensitive method for detection of PAH contamination both from pyrolytic and petroleum origin (Krahn et al., 1986; Ariese et al. 1993; Lin et al. 1996). Therefore, PAH metabolites should presumably have been detected by FF in bile, if present in the fish. Another monitoring project, carried out in 1994, has also shown that levels of PAHs in North Sea fish, detected by GC/MS, are not higher in the vicinity of oil and gas fields compared to remote reference areas (Johnsen et al. 1996). The sex, stage of maturation and liver somatic index (LSI) were noted for each fish. For cod and haddock the sex distribution was more or less even between males and females, and the stage of maturity was also similar between the stations, around stage 1 to 2 on a scale where stage 3 is fully matured. For long rough dab, most of the fish sampled from all groups were females and fully matured. At Egersundbanken three males at maturation stage 1 were caught. These are excluded from the presented data in order to get comparable groups. The LSI was similar between the stations. The bottom temperature was similar between the oil field stations, ranging from 8 to 9°C. At Egersundbanken the bottom temperature was 7.O”C. EROD activity did not show different levels for cod and haddock between the stations. However, long rough dab showed higher EROD levels at Egersundbanken compared to the other stations (Fig. 2). One possible explanation for this may be the somewhat lower sea temperature at Egersundbanken, which could lead to temperature compensation, as reported in Sleiderink et al. (1993). In summary, EROD activity did not show increased levels in fish caught in the vicinity of oil production fields, when compared to Egersundbanken. No differences in levels of PAH metabolites (2-5 ring structures) in bile were detected between the stations in any of the fish sampled. This could indicate that contamination from these PAH compounds near oil fields in the North Sea, should not be of great concern in these fish species.
REFERENCES Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. Burke, M. D and Mayer, R. T. (1974) Drug Metab. Disposis. 2, 583-588. Eggens, M. and Galgani, F. (1992) Marine Environmental Research 33, 213-221. Johnsen, S., Restucci, R. and Klungsqr, J. (1996) SPE paper 35909. Society of Petroleum Engineers. Krahn, M. M., Kittle, L. J. and MacLeod, W. D. (1986) Marine Environmental Research 20, 291298. Larson, E. A., Evans G. T. and Watson C. J. (1947) J. Lab. Clin. Med. 32,481488. Lin, E. L. C., Cormier, S. M. and Torsella, J. A. (1996) Ecotoxicology and Environmental Safety 35, 16-23. Sleiderink, H. M, Beyer, J., Scholtens, E., Gokseyr, A., Nieuwenhuize, J., Van Liere, J. M., Everaarts, J. M. and Boon, J. P. (1995) Aquat. Toxicol. 32, 189-209.
1998 Published
SOlSl-1136(97)00035-4
1998 by Elsevier Science Ltd. All rights reserved Printed in Great Britain 0141-1136/98 $19.00+0.00
ELSEVIER
PAH Metabolites in Bile and EROD Activity in North Sea Fish Endre Aasa and Jarle Klungsaryrb “Rogaland Research, PO Box 2503 Ullandhaug, N-4004 Stavanger, Norway bInstitute of Marine Research, PO Box 1870 Nordnes, N-5024 Bergen, Norway
ABSTRACT Atlantic cod (Gadus morhua) , haddock (Melanogrammus aeglefinus) , and long rough dab (Hippoglossoides platessoides) were sampled from areas with oil production in the North Sea. Egersundbanken, an area without oil production, was used as a reference site. Ethoxyresorufin-O-deethylase (EROD) activity was measured in liver to estimate cytochrome P4SOlA levels. Bile was analysed by fixed wavelength fluorescence (FF) detection for polyaromatic hydrocarbon (PAH) metabolites. No increased levels of EROD activity were detected in$sh caught in the vicinity of oilproductionfields, when compared to the reference site, Egersundbanken. No dtrerences in levels of PAH metabolites in bile were detected between the stations for any of the species. Therefore, contamination from 25 ring PAH compounds in the vicinity of oilfields in the North Sea may not be of great concern for theseJish species. 0 1998 Published by Elsevier Science Ltd. All rights reserved
INTRODUCTION An environmental monitoring survey organised by Institute of Marine Research was made in January 1996, in the North Sea. One main purpose of the survey was to monitor possible negative biological effects in the vicinity of oil fields. Atlantic Cod (Gadus morhua), haddock (Melanogrammus aeglefinus), and long rough dab (Hippoglossoides platessoides) were captured by trawling. Four main sampling areas were chosen: Stattjord, Oseberg, Brent and Egersundbanken. The three first are oil fields, while the last has been chosen as a reference site. A number of samples were taken from three fish species for various analyses. In this study results from bile and liver analyses will be reported. Bile was sampled for fixed wavelength fluorescence (FF) detection of polyaromatic hydrocarbon (PAH) metabolites. Liver was sampled for ethoxyresorufinO-deethylase (EROD) analyses, to estimate cytochrome P4501A activity. Ten fish per species per station were analysed. FF analyses of fish bile have earlier not been carried 229
230
E. Aas, J. Klungs0yr
out on wild fish in the northern North Sea, and data on EROD activity is also sparse in these areas. Bile samples were frozen at -20°C and stored until analysis. They were diluted 1:1600 times in 48% ethanol, and FF was measured at the excitation/emission wavelengths 341/ 383nm, optimised for the detection of pyrene metabolites (see Aas et al., this volume). Some of the samples were also measured at 290/335 nm for naphthalene type metabolites and 380/430 nm for benzo[a]pyrene type metabolites (Lin et al., 1996). The concentration of the bile pigment biliverdin was measured in all samples according to the method of Larson et al. (1947) in order to estimate bile density. Biliverdin is primarily measured to check if differences in fluorescence intensity simply could be a result of differences in bile 30
Oil exposed cod
20
Oil z
10
Fig. 1. Synchronous fluorescence specter (AL = 42 nm) of individual cod, haddock and long rough dab caught at the Stattjord station. For comparison, experimentally oil exposed turbot and cod are shown. The turbot was chronically exposed to 0.125 ppm dispersed light oil in water for 24 h, while the cod was exposed to crude oil spread onto the water surface, 4 d exposure.
PAH metabolites in bile and EROD activity in North Sea fish
231
densities. In this paper the PAH metabolite results are not expressed normal&d to biliverdin. The reason for this is that such a normalisation may add extra error to the data. Therefore, PAH metabolites are expressed here as relative values. Synchronous fluorescence spectra, achieved by scanning simultaneously on both excitation and emission wavelengths (AA = 42 nm), were made for a representative selection of bile samples from all sites. Liver samples were frozen in liquid nitrogen, and transferred to -80°C before homogenising and processing of S-9 supernatant. EROD activity in liver was measured according to a modified Burke and Mayer (1974) method, using a fluorometric plate reader as described by Eggens and Galgani (1992). Protein concentrations were measured according to Bradford (1976). The results from the FF measurements at wavelength pair 341/383 nm did not reveal differences in PAH content between the areas for any of the fish species. Nor were any differences found at the wavelength pairs 2901335 nm or 380/430 nm. Synchronous
T
Sn
Sf
0
B
E
Station Fig. 2. EROD activity (pmol resorufin mine1 mg protein-‘)
with standard deviation (n = 10 except for long rough dab at Egersundbanken where n = 7). Sn and Sf: Statfjord oil field, 0: Oseberg oil field, B: Brent oilfield and E: Egersundbanken.
232
E. Aas, J. Klungstiyr
fluorescence spectra (Ahh= 42 nm) are shown for one individual bile from each species (from the Statfjord station), and compared to bile from turbot and cod experimentally exposed to oil. The turbot was chronically exposed to a concentration of 0.125 ppm dispersed light oil (BAL 150) in water for 24 h, and the cod was exposed for 4 d to crude oil spread onto the water surface of a tank (Fig. 1). Bile from the North Sea fish showed weak fluorescence with profiles similar to the background fluorescence from the solvent ethanol, and unlike profiles for oil exposed fish. Analyses of PAH metabolites in fish bile by fluorescence, have shown to be a sensitive method for detection of PAH contamination both from pyrolytic and petroleum origin (Krahn et al., 1986; Ariese et al. 1993; Lin et al. 1996). Therefore, PAH metabolites should presumably have been detected by FF in bile, if present in the fish. Another monitoring project, carried out in 1994, has also shown that levels of PAHs in North Sea fish, detected by GC/MS, are not higher in the vicinity of oil and gas fields compared to remote reference areas (Johnsen et al. 1996). The sex, stage of maturation and liver somatic index (LSI) were noted for each fish. For cod and haddock the sex distribution was more or less even between males and females, and the stage of maturity was also similar between the stations, around stage 1 to 2 on a scale where stage 3 is fully matured. For long rough dab, most of the fish sampled from all groups were females and fully matured. At Egersundbanken three males at maturation stage 1 were caught. These are excluded from the presented data in order to get comparable groups. The LSI was similar between the stations. The bottom temperature was similar between the oil field stations, ranging from 8 to 9°C. At Egersundbanken the bottom temperature was 7.O”C. EROD activity did not show different levels for cod and haddock between the stations. However, long rough dab showed higher EROD levels at Egersundbanken compared to the other stations (Fig. 2). One possible explanation for this may be the somewhat lower sea temperature at Egersundbanken, which could lead to temperature compensation, as reported in Sleiderink et al. (1993). In summary, EROD activity did not show increased levels in fish caught in the vicinity of oil production fields, when compared to Egersundbanken. No differences in levels of PAH metabolites (2-5 ring structures) in bile were detected between the stations in any of the fish sampled. This could indicate that contamination from these PAH compounds near oil fields in the North Sea, should not be of great concern in these fish species.
REFERENCES Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. Burke, M. D and Mayer, R. T. (1974) Drug Metab. Disposis. 2, 583-588. Eggens, M. and Galgani, F. (1992) Marine Environmental Research 33, 213-221. Johnsen, S., Restucci, R. and Klungsqr, J. (1996) SPE paper 35909. Society of Petroleum Engineers. Krahn, M. M., Kittle, L. J. and MacLeod, W. D. (1986) Marine Environmental Research 20, 291298. Larson, E. A., Evans G. T. and Watson C. J. (1947) J. Lab. Clin. Med. 32,481488. Lin, E. L. C., Cormier, S. M. and Torsella, J. A. (1996) Ecotoxicology and Environmental Safety 35, 16-23. Sleiderink, H. M, Beyer, J., Scholtens, E., Gokseyr, A., Nieuwenhuize, J., Van Liere, J. M., Everaarts, J. M. and Boon, J. P. (1995) Aquat. Toxicol. 32, 189-209.