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Flounder health status in the Seine Bay. A multibiomarker study
Marine Environmental Research 50 (2000) 373±377 www.elsevier.com/locate/marenvrev
Flounder health status in the Seine Bay. A multibiomarker study C. Minier a,*, F. Levy a, D. Rabel a, G. Bocquene b, D. Godefroy c, T. Burgeot b, F. Leboulenger a a
Laboratory of Ecotoxicology, University of Le Havre, 2BP540, 25 rue Philippe Lebon, F-76058 Le Havre, France b IFREMER, BP 21105, F-44311 Nantes Cedex 03, France c IFREMER, BP 32, F-14520, Port-en-Bessin France
Received 24 April 1999; received in revised form 8 December 1999; accepted 17 December 1999
Abstract The Seine Bay is used as a pilot area to assess the usefulness of monitoring programmes using a suite of biological measurements. These biomarkers included ethoxyresor®n-O-deethylase (EROD) and acetylcholinesterase (AChE) activities, multixenobiotic resistance (MXR) protein expression level assessment and gonad histopathology. Samples of European ¯ounder collected in three sites close to the Seine Estuary in late September 1998 showed that 8% of the males were intersex, i.e. had gonads with both male and female tissues. Another 10% of individuals, identi®ed as male by morphological observation during sampling, showed only female tissues on histological sections. These dramatic changes were associated with different patterns of EROD activity, MXR expression or AChE activity inhibition that might re¯ect shorter time eects of xenobiotics and constitute a starting point to integrate biological responses for the assessment of the health status of ¯ounder in the Seine Bay. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: P-glycoprotein; AchE; EROD; Intersex; Flounder
European ¯ounder (Platichthys ¯esus) is a species of special interest for environmental studies. widespread in western European waters, it lives in contact with the sediment where it buries itself, and spends most of its life in estuaries, going out to sea only to breed. Therefore, it has been chosen as a sentinel species for monitoring * Corresponding author. Tel. +33-2-32-744303. E-mail address: [email protected] (C. Minier). 0141-1136/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0141-1136(00)00059-3
374
C. Minier et al. / Marine Environmental Research 50 (2000) 373±377
biological eects in a highly contaminated site, the Seine Bay (Claisse, 1989). Biological indices have been chosen because of complementary information they could give regarding the health status of animals. Both ethoxyresoru®n-O-deethylase (EROD) and multixenobiotic resistance (MXR) proteins are biomarkers of exposure. EROD activity reveals the level of expression of the cytochrome P450-1A1 enzyme system responsible for the biotransformation of numerous contaminants including polychlorobiphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs), and for endogenous substances such as steroids. MXR proteins are membrane-bound transporters that can actively exclude xenobiotics out of the cell, thus maintaining their intracellular concentration below their toxic level. Acetylcholinesterase (AChE) activity is a biomarker of eect on the nervous system. In addition, histological examination of the gonad has been performed to investigate the occurrence of abnormalities within this organ. Flounder of 20±35 cm long were caught by beam trawling at three sites in the Seine Bay (®g. 1) in late September 1998 and immediately proceeded on board. Liver, muscle and gill tissues were dissected and frozen in liquid nitrogen. Gonads were cut into three parts and put into Bouin's medium for 24 h before transferring
Fig. 1. Sampling sites in the Seine Bay (English Channel). Site 1, Fosse Sud; Site 2, La Carosse; Site 3; Antifer.
C. Minier et al. / Marine Environmental Research 50 (2000) 373±377
375
them to 70% ethanol. In the laboratory, frozen samples were thawed and homogenised. EROD measurements were performed according to the method of Galgani and Payne (1991). AChE activity was measured in the muscle of ¯ounder according to the method adapted to the microplate reader by Bocquene and Galgani (1998). MXR protein levels of gill tissues were assessed by slot blot analysis as described in Minier and Moore (1996). Gonad tissues were embedded in paran wax and sectioned at 5 mm. Sections were made in three dierent regions of each gonad, stained with haematoxylin and eosin, then examined by light microscopy. Results on EROD activity showed that this enzyme activity was signi®cantly elevated in samples caught at Site 1, Fosse Sud, when compared to the two other sites (analysis of variance, ANOVA: F=6.04; P=0.005). Despite a large variation among individuals, the activity at Fosse Sud was on average three times higher than at Antifer (Site 3) and more than twice that observed at La Carrosse (Site 2) (table 1). AChE activity and MXR protein expression level showed a dierent pattern. Samples caught at Antifer expressed a large amount of proteins involved in multixenobiotic transport in their gill tissues. Indeed, gill MXR protein content was 13 and 25% higher at Antifer than at, respectively, sites 2 and 1 (ANOVA: F=4.87; P=0.012). In a similar manner, the AChE activity was inhibited by 45% at Antifer and by 40% at La Carosse when compared to Fosse Sud (ANOVA: F=4.77; P=0.017). These results suggest that exposure to some xenobiotics varies according to the dierent locations. Although it cannot be stated that ¯ounder are not migrating from one site to another, these results may re¯ect the in¯uence of dierential contamination levels present in these three sites on a short time scale. Indeed, major currents are known to run north±north/east from the Seine Estuary (in direction of Sites 2 and 3) which results, for instance, in a signi®cantly elevated PAH content in the sediments at Antifer when compared to sediments from Fosse Sud (Burgeot, 1994). In addition, Antifer is directly impacted by xenobiotics that are dredged from the estuary and disposed close to this site, and also by contaminants coming from the petroleum port located nearby (the port of Antifer). Table 1 Results of biological measurements in ¯ounder from the Seine Baya Biomarkers
EROD AChE MXR Intersex a
Sites 1±Fosse Sud
2±La Carosse
3±Antifer
76.96 (36.9) * 556 (104) 2.68 (0.2) 1/8
32.29 (9.2) 332 (108) * 3.04 (0.4) 1/12
23.78 (12.2) 307 (141) * 3.34 (0.2) * 0/4
Ethoxyresoru®n-O-deethylase (EROD) activity is expressed as pmol minÿ1 mg proteinÿ1. Acetylcholinesterase (AChE) activity is expressed as nmoles minÿ1 mg proteinÿ1. Multixenobiotic resistance (MXR) protein expression level is expressed as milliOD mg proteinÿ1. Con®dence intervals (a=0.05) are given in brackets. The incidence of intersex is presented as a ratio of the number of male ®sh displaying both male and female tissues within their testes to the total number of male ®sh examined. *Signi®cantly dierent from the lowest response; P<0.05; n56.
376
C. Minier et al. / Marine Environmental Research 50 (2000) 373±377
In view of these results, the higher EROD activity found in samples from Fosse Sud seems peculiar. One possible explanation for these data could involve the differential food availability encountered by the ®sh. Indeed, conversely to what happened at the other sites, Site 1 is characterised by a large biodiversity and ¯ounder caught at Fosse Sud had well-®lled stomachs. Thus, bioaccumulated contaminants or nutrients could have contributed to the induction of the expression of cytochrome P450 enzymes (Jùrgensen, Bye & Jobling, 1999). Gonad histopathology showed the incidence of intersex in male ¯ounder. Two individuals had both male and female tissues within their testes (Fig. 2). Moreover, three other individuals, identi®ed as male during sampling based on gonad morphological appearance, showed no male tissue, leaving only developing or formed oocytes in all sections analysed. This percentage of intesex ®sh (8%) is half the one obtained in ¯ounder from the Mersey river, the only British estuary out of ®ve studied that showed such impairment in previous work (Allen, Scott, Matthiessen, Haworth, Thain & Feist, 1999). Although ®sh with major histological changes were caught at Fosse Sud and La Carosse, no conclusion can be drawn on contamination with endocrine disrupters among the three sampling sites. Indeed, these eects were probably initialised during the larval stage of the ®sh when they were arguably at the most sensitive stage of their development to endocrine disrupters. Furthermore, exposure to xenoestrogens might have been important in the River Seine where these ¯at®sh spend most of their lives after metamorphosis. Nevertheless, contaminants concentrated in the food present in the coastal area might be of importance for this phenomenon and further studies should be performed to investigate this hypothesis. In conclusion, this work shows that the health status of the European ¯ounder is impaired in the Seine bay. The EROD activities found in this study are higher than
Fig. 2. Histology of intersex male ¯ounder showing developing primary oocytes among testicular tissue. Bar represents 100 pm.
C. Minier et al. / Marine Environmental Research 50 (2000) 373±377
377
those found in the Somme Estuary further north along the French coast (10 pmol minÿ1 mg proteinÿ1, Burgeot, unpublished data). Furthermore, although no references are available on esterase activity or MXR expression levels for this species, previous data obtained on Mytulis edulis showed increased MXR-like protein expression level in the Seine Estuary (Minier, Akcha & Galgani, 1993). Further work, on a larger scale, should then de®ne more precisely the ``normal range'' of biomarker responses. Nevertheless, the most important data and matter of concern arise from the fact that 8% of the males caught in this study were intersex. This, in combination with the incidence of neoplasmas and carcinomas found in this species during previous years of sampling (Cachot, 1998), shows that ¯ounder from the Seine Bay are being subject to a major threat to their health status. Acknowledgements This study was supported by the Conseil ReÂgional de Haute-Normandie and the Agence de l'eau Seine-Normandie. References Allen, Y., Scott, A. P., Matthiessen, P., Haworth, S., Thain, J. E., & Feist, S. (1999). Environ. Toxicol. Chem., 18, 1790±1800. BocqueneÂ, G., & Galgani, F. (1998i). ICES Tech. Mar. Environ. Sci., 22, 1±12. Burgeot, T. PhD thesis, University of Nantes, Nantes, France. Cachot, J. (1998). PhD thesis. University of Marseille, Marseille, France. Claisse, D. (1989). Mar. Pollut. Bull, 20, 523±528. Galgani, F., & Payne, J. (1991). ICES Tech. Mar. Environ. Sci., 13, 1±11. Jùrgensen, E. H., Bye, B. E., & Jobling, M. (1999). Aquatic Tox, 44, 233±244. Minier, C., Akcha, F., & Galgani, F. (1993). Comp. Biochem. Physiol, 106, 1029±1036. Minier, C., & Moore, M. N. (1996). Mar. Ecol. Prog. Ser., 142, 165±173.
Flounder health status in the Seine Bay. A multibiomarker study C. Minier a,*, F. Levy a, D. Rabel a, G. Bocquene b, D. Godefroy c, T. Burgeot b, F. Leboulenger a a
Laboratory of Ecotoxicology, University of Le Havre, 2BP540, 25 rue Philippe Lebon, F-76058 Le Havre, France b IFREMER, BP 21105, F-44311 Nantes Cedex 03, France c IFREMER, BP 32, F-14520, Port-en-Bessin France
Received 24 April 1999; received in revised form 8 December 1999; accepted 17 December 1999
Abstract The Seine Bay is used as a pilot area to assess the usefulness of monitoring programmes using a suite of biological measurements. These biomarkers included ethoxyresor®n-O-deethylase (EROD) and acetylcholinesterase (AChE) activities, multixenobiotic resistance (MXR) protein expression level assessment and gonad histopathology. Samples of European ¯ounder collected in three sites close to the Seine Estuary in late September 1998 showed that 8% of the males were intersex, i.e. had gonads with both male and female tissues. Another 10% of individuals, identi®ed as male by morphological observation during sampling, showed only female tissues on histological sections. These dramatic changes were associated with different patterns of EROD activity, MXR expression or AChE activity inhibition that might re¯ect shorter time eects of xenobiotics and constitute a starting point to integrate biological responses for the assessment of the health status of ¯ounder in the Seine Bay. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: P-glycoprotein; AchE; EROD; Intersex; Flounder
European ¯ounder (Platichthys ¯esus) is a species of special interest for environmental studies. widespread in western European waters, it lives in contact with the sediment where it buries itself, and spends most of its life in estuaries, going out to sea only to breed. Therefore, it has been chosen as a sentinel species for monitoring * Corresponding author. Tel. +33-2-32-744303. E-mail address: [email protected] (C. Minier). 0141-1136/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0141-1136(00)00059-3
374
C. Minier et al. / Marine Environmental Research 50 (2000) 373±377
biological eects in a highly contaminated site, the Seine Bay (Claisse, 1989). Biological indices have been chosen because of complementary information they could give regarding the health status of animals. Both ethoxyresoru®n-O-deethylase (EROD) and multixenobiotic resistance (MXR) proteins are biomarkers of exposure. EROD activity reveals the level of expression of the cytochrome P450-1A1 enzyme system responsible for the biotransformation of numerous contaminants including polychlorobiphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs), and for endogenous substances such as steroids. MXR proteins are membrane-bound transporters that can actively exclude xenobiotics out of the cell, thus maintaining their intracellular concentration below their toxic level. Acetylcholinesterase (AChE) activity is a biomarker of eect on the nervous system. In addition, histological examination of the gonad has been performed to investigate the occurrence of abnormalities within this organ. Flounder of 20±35 cm long were caught by beam trawling at three sites in the Seine Bay (®g. 1) in late September 1998 and immediately proceeded on board. Liver, muscle and gill tissues were dissected and frozen in liquid nitrogen. Gonads were cut into three parts and put into Bouin's medium for 24 h before transferring
Fig. 1. Sampling sites in the Seine Bay (English Channel). Site 1, Fosse Sud; Site 2, La Carosse; Site 3; Antifer.
C. Minier et al. / Marine Environmental Research 50 (2000) 373±377
375
them to 70% ethanol. In the laboratory, frozen samples were thawed and homogenised. EROD measurements were performed according to the method of Galgani and Payne (1991). AChE activity was measured in the muscle of ¯ounder according to the method adapted to the microplate reader by Bocquene and Galgani (1998). MXR protein levels of gill tissues were assessed by slot blot analysis as described in Minier and Moore (1996). Gonad tissues were embedded in paran wax and sectioned at 5 mm. Sections were made in three dierent regions of each gonad, stained with haematoxylin and eosin, then examined by light microscopy. Results on EROD activity showed that this enzyme activity was signi®cantly elevated in samples caught at Site 1, Fosse Sud, when compared to the two other sites (analysis of variance, ANOVA: F=6.04; P=0.005). Despite a large variation among individuals, the activity at Fosse Sud was on average three times higher than at Antifer (Site 3) and more than twice that observed at La Carrosse (Site 2) (table 1). AChE activity and MXR protein expression level showed a dierent pattern. Samples caught at Antifer expressed a large amount of proteins involved in multixenobiotic transport in their gill tissues. Indeed, gill MXR protein content was 13 and 25% higher at Antifer than at, respectively, sites 2 and 1 (ANOVA: F=4.87; P=0.012). In a similar manner, the AChE activity was inhibited by 45% at Antifer and by 40% at La Carosse when compared to Fosse Sud (ANOVA: F=4.77; P=0.017). These results suggest that exposure to some xenobiotics varies according to the dierent locations. Although it cannot be stated that ¯ounder are not migrating from one site to another, these results may re¯ect the in¯uence of dierential contamination levels present in these three sites on a short time scale. Indeed, major currents are known to run north±north/east from the Seine Estuary (in direction of Sites 2 and 3) which results, for instance, in a signi®cantly elevated PAH content in the sediments at Antifer when compared to sediments from Fosse Sud (Burgeot, 1994). In addition, Antifer is directly impacted by xenobiotics that are dredged from the estuary and disposed close to this site, and also by contaminants coming from the petroleum port located nearby (the port of Antifer). Table 1 Results of biological measurements in ¯ounder from the Seine Baya Biomarkers
EROD AChE MXR Intersex a
Sites 1±Fosse Sud
2±La Carosse
3±Antifer
76.96 (36.9) * 556 (104) 2.68 (0.2) 1/8
32.29 (9.2) 332 (108) * 3.04 (0.4) 1/12
23.78 (12.2) 307 (141) * 3.34 (0.2) * 0/4
Ethoxyresoru®n-O-deethylase (EROD) activity is expressed as pmol minÿ1 mg proteinÿ1. Acetylcholinesterase (AChE) activity is expressed as nmoles minÿ1 mg proteinÿ1. Multixenobiotic resistance (MXR) protein expression level is expressed as milliOD mg proteinÿ1. Con®dence intervals (a=0.05) are given in brackets. The incidence of intersex is presented as a ratio of the number of male ®sh displaying both male and female tissues within their testes to the total number of male ®sh examined. *Signi®cantly dierent from the lowest response; P<0.05; n56.
376
C. Minier et al. / Marine Environmental Research 50 (2000) 373±377
In view of these results, the higher EROD activity found in samples from Fosse Sud seems peculiar. One possible explanation for these data could involve the differential food availability encountered by the ®sh. Indeed, conversely to what happened at the other sites, Site 1 is characterised by a large biodiversity and ¯ounder caught at Fosse Sud had well-®lled stomachs. Thus, bioaccumulated contaminants or nutrients could have contributed to the induction of the expression of cytochrome P450 enzymes (Jùrgensen, Bye & Jobling, 1999). Gonad histopathology showed the incidence of intersex in male ¯ounder. Two individuals had both male and female tissues within their testes (Fig. 2). Moreover, three other individuals, identi®ed as male during sampling based on gonad morphological appearance, showed no male tissue, leaving only developing or formed oocytes in all sections analysed. This percentage of intesex ®sh (8%) is half the one obtained in ¯ounder from the Mersey river, the only British estuary out of ®ve studied that showed such impairment in previous work (Allen, Scott, Matthiessen, Haworth, Thain & Feist, 1999). Although ®sh with major histological changes were caught at Fosse Sud and La Carosse, no conclusion can be drawn on contamination with endocrine disrupters among the three sampling sites. Indeed, these eects were probably initialised during the larval stage of the ®sh when they were arguably at the most sensitive stage of their development to endocrine disrupters. Furthermore, exposure to xenoestrogens might have been important in the River Seine where these ¯at®sh spend most of their lives after metamorphosis. Nevertheless, contaminants concentrated in the food present in the coastal area might be of importance for this phenomenon and further studies should be performed to investigate this hypothesis. In conclusion, this work shows that the health status of the European ¯ounder is impaired in the Seine bay. The EROD activities found in this study are higher than
Fig. 2. Histology of intersex male ¯ounder showing developing primary oocytes among testicular tissue. Bar represents 100 pm.
C. Minier et al. / Marine Environmental Research 50 (2000) 373±377
377
those found in the Somme Estuary further north along the French coast (10 pmol minÿ1 mg proteinÿ1, Burgeot, unpublished data). Furthermore, although no references are available on esterase activity or MXR expression levels for this species, previous data obtained on Mytulis edulis showed increased MXR-like protein expression level in the Seine Estuary (Minier, Akcha & Galgani, 1993). Further work, on a larger scale, should then de®ne more precisely the ``normal range'' of biomarker responses. Nevertheless, the most important data and matter of concern arise from the fact that 8% of the males caught in this study were intersex. This, in combination with the incidence of neoplasmas and carcinomas found in this species during previous years of sampling (Cachot, 1998), shows that ¯ounder from the Seine Bay are being subject to a major threat to their health status. Acknowledgements This study was supported by the Conseil ReÂgional de Haute-Normandie and the Agence de l'eau Seine-Normandie. References Allen, Y., Scott, A. P., Matthiessen, P., Haworth, S., Thain, J. E., & Feist, S. (1999). Environ. Toxicol. Chem., 18, 1790±1800. BocqueneÂ, G., & Galgani, F. (1998i). ICES Tech. Mar. Environ. Sci., 22, 1±12. Burgeot, T. PhD thesis, University of Nantes, Nantes, France. Cachot, J. (1998). PhD thesis. University of Marseille, Marseille, France. Claisse, D. (1989). Mar. Pollut. Bull, 20, 523±528. Galgani, F., & Payne, J. (1991). ICES Tech. Mar. Environ. Sci., 13, 1±11. Jùrgensen, E. H., Bye, B. E., & Jobling, M. (1999). Aquatic Tox, 44, 233±244. Minier, C., Akcha, F., & Galgani, F. (1993). Comp. Biochem. Physiol, 106, 1029±1036. Minier, C., & Moore, M. N. (1996). Mar. Ecol. Prog. Ser., 142, 165±173.