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Field application of biochemical markers and a physiological index in the mussel, Mytilus galloprovincialis: transplantation and biomonitoring studies in the lagoon of Venice (NE Italy)
Marine Environmental Research 54 (2002) 811–816 www.elsevier.com/locate/marenvrev
Field application of biochemical markers and a physiological index in the mussel, Mytilus galloprovincialis: transplantation and biomonitoring studies in the lagoon of Venice (NE Italy) C. Nasci*, N. Nesto, R.A. Monteduro, L. Da Ros Consiglio Nazionale delle Ricerche, Istituto di Biologia del Mare, Castello 1363/a, Venice, Italy
Abstract A number of biochemical markers and a physiological index were measured in mussels, Mytilus galloprovincialis, transplanted or native to five different contaminated sites in the lagoon of Venice. Mussels from Pellestrina, a reference site in the adjacent Adriatic Sea, were transplanted for 6 weeks to areas of the lagoon where indigenous mussels were also collected. As biochemical indices, superoxide dismutase (SOD), catalase, aldehyde dehydrogenase (ADH) and NADPH cytochrome c reductase (NADPHcred) were measured in mussel digestive gland; survival in air as a physiological index was also determined. Biomarker responses varied among sites and between indigenous and transplanted animals. Significant induction of catalase and SOD was shown in animals transplanted to the urban sites of Salute and Chioggia, respectively. In indigenous mussels, induction of SOD and NADPHcred was seen in animals from the polluted site of Treporti and the heavily contaminated industrial area of Marghera. The overall biochemical data indicate significantly higher activity for ADH in transplanted animals in comparison with indigenous ones which, in contrast, present an increase in SOD. As regard survival in air, control mussels did not seem to be healthier in comparison either with transplanted or indigenous ones, suggesting that pollution has no effect on this parameter. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Mytilus galloprovincialis; Biomarkers; Biomonitoring; Lagoon of Venice
* Corresponding author. Tel.: +390-41-5207-622; fax: +390-41-5204-126. E-mail address: [email protected] (C. Nasci). 0141-1136/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0141-1136(02)00122-8
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C. Nasci et al. / Marine Environmental Research 54 (2002) 811–816
1. Introduction In the last few decades, several studies have highlighted the importance of the biomarker integrated approach in environment quality assessment, for better understanding of the pathways and mechanisms by which chemicals exert their toxicity in marine organisms. However, field studies pose great difficulties due to the complex and fluctuating nature of the environment and interactions among organisms. Animals ‘‘in situ’’ are exposed to a variety of natural stressors including salinity, hypoxia and temperature stress, and these factors, together with genetic differences in susceptibility to stress, result in increased variability of biomarker responses (Astley, Meigh, Glegg, Braven, & Depledge, 1999; Forbes & Depledge, 1996). In recent years, a relatively new field approach in monitoring programmes, called ‘‘active biomonitoring’’, has been proposed. It consists of the collection of samples from a population at one location and their transport to the monitoring sites, and has the advantage of ensuring comparable biological samples and therefore reducing their natural variability (De Kock & Kramer, 1994). In this study we measured a suite of five biomarkers (four biochemical, one physiological) indicative of pollution exposure in the mussel, Mytilus galloprovincialis, a well-known and extensively used ‘‘sentinel’’ organism in environmental monitoring programs. Mussels were collected from or transplanted to, several lagoon sites in order to evaluate the usefulness of the selected stress indices and to compare the two monitoring approaches as potentially useful tools in ecosystem quality projects in the Lagoon.
2. Material and methods In April 1999, specimens of M. galloprovincialis were collected from a clean farming area, Pellestrina, and were transplanted to several contaminated lagoon sites: Marghera (industrial pollution), CVE (urban and industrial pollution), Treporti (anthropic and motorboat traffic pollution), Salute and Chioggia (urban pollution). At the same sites, animals from the indigenous populations were subsequently sampled. Biomarker responses were measured in both ‘‘transplanted ’’ and ‘‘indigenous’’ sets of organisms and the results compared. As biochemical parameters, the activities of aldehyde dehydrogenase (ADH), catalase and superoxide dismutase (SOD) in cytosol, were measured, together with NADPH-cytochrome c reductase (NADPHcred) in the microsomal fraction of pooled mussel digestive glands obtained by standard procedures. ADH activity was determined spectophotometrically at 340 nm by the reduction of NAD+ (Nasci, Da Ros, & Livingstone, 1999). Catalase and SOD activities were performed as described in Livingstone, Lips, Garcia Martinez, and Pipe (1992). NADPHcred was determined spectrophotometrically on digestive gland microsomes, following Livingstone (1988). As regards the physiological index, survival in air was performed on 30 animals placed in a box at a constant room temperature of 18 C and 100% humidity, the number of dead specimens being recorded in order to calculate the LT50 (lethal time
C. Nasci et al. / Marine Environmental Research 54 (2002) 811–816
813
for 50% of sample; Eertman & De Zwaan, 1994). Biochemical data was statistically compared by one-way analysis of variance (ANOVA) and physiological values by the Wilcoxon and Gehan test (Gehan, 1965).
3. Results and discussion The biomarker responses of transplanted and indigenous animals are shown in Figs. 1 and 2, respectively.
Fig. 1. Transplanted mussels. Enzymatic activities expressed as mean S.E., n=5. Statistical comparison (ANOVA) between control and lagoon sites: *=P<0.05; **=P <0.01; ***=P <0.001. Survival curves determined on 30 mussels. Statistical comparison (Wilcoxon & Gehan) between control and lagoon sites: *=P <0.05; **=P<0.01; ***=P <0.001.
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C. Nasci et al. / Marine Environmental Research 54 (2002) 811–816
In mussels transplanted to the lagoon sites, there were no significant changes in most of the biochemical markers, in comparison with the reference zone, Pellestrina, with the exception of antioxidant enzymes, which showed a significant increase in catalase (P < 0.01) at Salute and in SOD (P < 0.01) at Chioggia, both urban sites. Instead, catalase and ADH activities were depressed at Marghera, the industrial area (P < 0.05), and ADH activity was extremely significantly inhibited at CVE (P < 0.001), the mixed urban and industrial polluted site. SOD and NADPHcred activities were also depressed at Salute (P < 0.05). In indigenous mussels, Catalase and ADH activities were significantly decreased (P < 0.003) at Marghera and Salute. Meanwhile a significant induction (P < 0.05) of
Fig. 2. Indigenous mussels. Enzymatic activities expressed as meanS.E., n=5. Statistical comparison (ANOVA) between control and lagoon sites: *=P<0.05; **=P <0.01; ***=P <0.001. Survival curves determined on 30 mussels. Statistical comparison (Wilcoxon & Gehan) between control and lagoon sites: *=P <0.05; **=P<0.01; ***=P <0.001.
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SOD and NADPHcred was shown in animals from Treporti exposed to heavy traffic. The overall data comparison of biochemical responses between transplanted and indigenous mussels showed markedly higher activity of SOD in indigenous population (P < 0.002) and of ADH in transplanted animals (P < 0.001). These results suggest that antioxidant enzymes Catalase and SOD may be considered as good markers of urban pollution, with a relatively short time response to environmental stressors, as suggested by the transplant experiment. NADPHcred activity confirmed its usefulness as an indicator of chronic organic pollution, and transplantation seemed to have little effect on it. Low antioxidant and ADH activities in the polluted industrial area of Marghera may be due to the presence of pollutant mixtures with possible antagonist effects on animals’ response, as indicated in other studies (Fossato, Campesan, Craboledda, Dolci, & Stocco, 2000; Livingstone et al., 1995). Contaminant levels determined in indigenous mussels (Monteduro, 2000) were generally highest at the industrial site of Marghera with PCBs and DDT decreasing from Marghera> CVE > Salute > Chioggia > Treporti > Pellestrina and PAHs diminishing from Marghera> Salute> CVE> Treporti> Chioggia=Pellestrina. Data from previous researches on ADH activity in mussels indicated an induction by pro-oxidant conditions and organic pollutants but with lower values at Marghera than at Salute and other urban sites, or only slight differences among them (Nasci, Da Ros, Campesan, & Fossato, 1998; Nasci et al., 1999). As regards the physiological index, survival in air did not seem to be negatively affected in the transplanted or indigenous polluted site mussels in comparison with the respective control. The fact that the mussels collected at the farming area of Pellestrina, considered as control site, did not seem healthier than the lagoon samples, this may be due more to their varied diet in the Adriatic, than to the impact of contaminant (Lowe & Da Ros, 2000) and to the positive effects of increased food availability inside the Lagoon resulting from eutrophication (Widdows & Nasci, 2000). Indeed, further studies should be undertaken to ascertain its suitability as reference site for future studies.
Acknowledgements This work was partly supported by the Consortium for the Management of the Research Coordination Centre in the Venetian Lagoon System (CORILA).
References Astley, K. N., Meigh, H. C., Glegg, G. A, Braven, J., & Depledge, M. H. (1999). Marine Pollution Bulletin, 39(1–2), 145–154. De Kock, W. C., & Kramer, K. J. M. (1994). In K. J. M. Kramer (Ed.), Biomonitoring of coastal waters and estuaries (pp. 51–78). Boca Raton: CRC. Eertman, R. H. M., & De Zwaan, A. (1994). In K. J. M. Kramer (Ed.), Biomonitoring of coastal waters and estuaries (pp. 269–284). Boca Raton: CRC.
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C. Nasci et al. / Marine Environmental Research 54 (2002) 811–816
Forbes, V. E., & Depledge, M. H. (1996). In D. J. Baird, L. Maltby, P. W. Greig-Smith, & P. E. T. Douben (Eds.) Ecotoxicology: ecotoxicological dimension (pp. 71–86). London: Chapman and Hall. Fossato, V. U., Campesan, G., Craboledda, L., Dolci, F., & Stocco, G. (2000). In P. Lassere, & A. Marzollo (Eds.), The Venice lagoon ecosystem (Vol. 25; pp. 97–126), Man and the Biosphere Series. New York: Parthenon Publishing. Gehan, E. A. (1965). Biometrica, 52, 203–223. Livingstone, D. R. (1988). Marine Ecology Progress Series, 91, 141–153. Livingstone, D. R., Lemaire, P., Matthews, A., Peters, L. D., Porte, C., Fitzpatrick, P. J., Forlin, L., Nasci, C., Fossato, V. U., Wotton, N., & Goldfarb, P. (1995). Marine Environmental Research, 39, 235– 240. Livingstone, D. R., Lips, F., Garcia Martinez, P., & Pipe, R. K. (1992). Marine Biology, 112, 265–276. Lowe, D. M., & Da Ros, L. (2000). In P. Lassere, & A. Marzollo (Eds.), The Venice lagoon ecosystem (Vol. 25; pp. 375–385) Man and the Biosphere Series. New York: Parthenon Publishing. Monteduro, R. A. (2000). Valutazione degli effeti della contaminazione chimica mediante l’uso di bioindicatori di stress in Mytilus galloprovincialis della laguna di Venezia. Degree thesis, University of Venice, Ca` Foscari. Nasci, C., Da Ros, L., Campesan, G., & Fossato, V. U. (1998). Marine Environmental Research, 46, 279– 282. Nasci, C., Da Ros, L., & Livingstone, D. R. (1999). Bollettino del Museo Civico di Storia naturale Venezia, 49, 125–133. Widdows, J., & Nasci, C. (2000). In P. Lassere, & A. Marzollo (Eds.), The Venice lagoon ecosystem (Vol. 25; pp. 387–396). Man and the Biosphere Series. New York: Parthenon Publishing.
Field application of biochemical markers and a physiological index in the mussel, Mytilus galloprovincialis: transplantation and biomonitoring studies in the lagoon of Venice (NE Italy) C. Nasci*, N. Nesto, R.A. Monteduro, L. Da Ros Consiglio Nazionale delle Ricerche, Istituto di Biologia del Mare, Castello 1363/a, Venice, Italy
Abstract A number of biochemical markers and a physiological index were measured in mussels, Mytilus galloprovincialis, transplanted or native to five different contaminated sites in the lagoon of Venice. Mussels from Pellestrina, a reference site in the adjacent Adriatic Sea, were transplanted for 6 weeks to areas of the lagoon where indigenous mussels were also collected. As biochemical indices, superoxide dismutase (SOD), catalase, aldehyde dehydrogenase (ADH) and NADPH cytochrome c reductase (NADPHcred) were measured in mussel digestive gland; survival in air as a physiological index was also determined. Biomarker responses varied among sites and between indigenous and transplanted animals. Significant induction of catalase and SOD was shown in animals transplanted to the urban sites of Salute and Chioggia, respectively. In indigenous mussels, induction of SOD and NADPHcred was seen in animals from the polluted site of Treporti and the heavily contaminated industrial area of Marghera. The overall biochemical data indicate significantly higher activity for ADH in transplanted animals in comparison with indigenous ones which, in contrast, present an increase in SOD. As regard survival in air, control mussels did not seem to be healthier in comparison either with transplanted or indigenous ones, suggesting that pollution has no effect on this parameter. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Mytilus galloprovincialis; Biomarkers; Biomonitoring; Lagoon of Venice
* Corresponding author. Tel.: +390-41-5207-622; fax: +390-41-5204-126. E-mail address: [email protected] (C. Nasci). 0141-1136/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0141-1136(02)00122-8
812
C. Nasci et al. / Marine Environmental Research 54 (2002) 811–816
1. Introduction In the last few decades, several studies have highlighted the importance of the biomarker integrated approach in environment quality assessment, for better understanding of the pathways and mechanisms by which chemicals exert their toxicity in marine organisms. However, field studies pose great difficulties due to the complex and fluctuating nature of the environment and interactions among organisms. Animals ‘‘in situ’’ are exposed to a variety of natural stressors including salinity, hypoxia and temperature stress, and these factors, together with genetic differences in susceptibility to stress, result in increased variability of biomarker responses (Astley, Meigh, Glegg, Braven, & Depledge, 1999; Forbes & Depledge, 1996). In recent years, a relatively new field approach in monitoring programmes, called ‘‘active biomonitoring’’, has been proposed. It consists of the collection of samples from a population at one location and their transport to the monitoring sites, and has the advantage of ensuring comparable biological samples and therefore reducing their natural variability (De Kock & Kramer, 1994). In this study we measured a suite of five biomarkers (four biochemical, one physiological) indicative of pollution exposure in the mussel, Mytilus galloprovincialis, a well-known and extensively used ‘‘sentinel’’ organism in environmental monitoring programs. Mussels were collected from or transplanted to, several lagoon sites in order to evaluate the usefulness of the selected stress indices and to compare the two monitoring approaches as potentially useful tools in ecosystem quality projects in the Lagoon.
2. Material and methods In April 1999, specimens of M. galloprovincialis were collected from a clean farming area, Pellestrina, and were transplanted to several contaminated lagoon sites: Marghera (industrial pollution), CVE (urban and industrial pollution), Treporti (anthropic and motorboat traffic pollution), Salute and Chioggia (urban pollution). At the same sites, animals from the indigenous populations were subsequently sampled. Biomarker responses were measured in both ‘‘transplanted ’’ and ‘‘indigenous’’ sets of organisms and the results compared. As biochemical parameters, the activities of aldehyde dehydrogenase (ADH), catalase and superoxide dismutase (SOD) in cytosol, were measured, together with NADPH-cytochrome c reductase (NADPHcred) in the microsomal fraction of pooled mussel digestive glands obtained by standard procedures. ADH activity was determined spectophotometrically at 340 nm by the reduction of NAD+ (Nasci, Da Ros, & Livingstone, 1999). Catalase and SOD activities were performed as described in Livingstone, Lips, Garcia Martinez, and Pipe (1992). NADPHcred was determined spectrophotometrically on digestive gland microsomes, following Livingstone (1988). As regards the physiological index, survival in air was performed on 30 animals placed in a box at a constant room temperature of 18 C and 100% humidity, the number of dead specimens being recorded in order to calculate the LT50 (lethal time
C. Nasci et al. / Marine Environmental Research 54 (2002) 811–816
813
for 50% of sample; Eertman & De Zwaan, 1994). Biochemical data was statistically compared by one-way analysis of variance (ANOVA) and physiological values by the Wilcoxon and Gehan test (Gehan, 1965).
3. Results and discussion The biomarker responses of transplanted and indigenous animals are shown in Figs. 1 and 2, respectively.
Fig. 1. Transplanted mussels. Enzymatic activities expressed as mean S.E., n=5. Statistical comparison (ANOVA) between control and lagoon sites: *=P<0.05; **=P <0.01; ***=P <0.001. Survival curves determined on 30 mussels. Statistical comparison (Wilcoxon & Gehan) between control and lagoon sites: *=P <0.05; **=P<0.01; ***=P <0.001.
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C. Nasci et al. / Marine Environmental Research 54 (2002) 811–816
In mussels transplanted to the lagoon sites, there were no significant changes in most of the biochemical markers, in comparison with the reference zone, Pellestrina, with the exception of antioxidant enzymes, which showed a significant increase in catalase (P < 0.01) at Salute and in SOD (P < 0.01) at Chioggia, both urban sites. Instead, catalase and ADH activities were depressed at Marghera, the industrial area (P < 0.05), and ADH activity was extremely significantly inhibited at CVE (P < 0.001), the mixed urban and industrial polluted site. SOD and NADPHcred activities were also depressed at Salute (P < 0.05). In indigenous mussels, Catalase and ADH activities were significantly decreased (P < 0.003) at Marghera and Salute. Meanwhile a significant induction (P < 0.05) of
Fig. 2. Indigenous mussels. Enzymatic activities expressed as meanS.E., n=5. Statistical comparison (ANOVA) between control and lagoon sites: *=P<0.05; **=P <0.01; ***=P <0.001. Survival curves determined on 30 mussels. Statistical comparison (Wilcoxon & Gehan) between control and lagoon sites: *=P <0.05; **=P<0.01; ***=P <0.001.
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SOD and NADPHcred was shown in animals from Treporti exposed to heavy traffic. The overall data comparison of biochemical responses between transplanted and indigenous mussels showed markedly higher activity of SOD in indigenous population (P < 0.002) and of ADH in transplanted animals (P < 0.001). These results suggest that antioxidant enzymes Catalase and SOD may be considered as good markers of urban pollution, with a relatively short time response to environmental stressors, as suggested by the transplant experiment. NADPHcred activity confirmed its usefulness as an indicator of chronic organic pollution, and transplantation seemed to have little effect on it. Low antioxidant and ADH activities in the polluted industrial area of Marghera may be due to the presence of pollutant mixtures with possible antagonist effects on animals’ response, as indicated in other studies (Fossato, Campesan, Craboledda, Dolci, & Stocco, 2000; Livingstone et al., 1995). Contaminant levels determined in indigenous mussels (Monteduro, 2000) were generally highest at the industrial site of Marghera with PCBs and DDT decreasing from Marghera> CVE > Salute > Chioggia > Treporti > Pellestrina and PAHs diminishing from Marghera> Salute> CVE> Treporti> Chioggia=Pellestrina. Data from previous researches on ADH activity in mussels indicated an induction by pro-oxidant conditions and organic pollutants but with lower values at Marghera than at Salute and other urban sites, or only slight differences among them (Nasci, Da Ros, Campesan, & Fossato, 1998; Nasci et al., 1999). As regards the physiological index, survival in air did not seem to be negatively affected in the transplanted or indigenous polluted site mussels in comparison with the respective control. The fact that the mussels collected at the farming area of Pellestrina, considered as control site, did not seem healthier than the lagoon samples, this may be due more to their varied diet in the Adriatic, than to the impact of contaminant (Lowe & Da Ros, 2000) and to the positive effects of increased food availability inside the Lagoon resulting from eutrophication (Widdows & Nasci, 2000). Indeed, further studies should be undertaken to ascertain its suitability as reference site for future studies.
Acknowledgements This work was partly supported by the Consortium for the Management of the Research Coordination Centre in the Venetian Lagoon System (CORILA).
References Astley, K. N., Meigh, H. C., Glegg, G. A, Braven, J., & Depledge, M. H. (1999). Marine Pollution Bulletin, 39(1–2), 145–154. De Kock, W. C., & Kramer, K. J. M. (1994). In K. J. M. Kramer (Ed.), Biomonitoring of coastal waters and estuaries (pp. 51–78). Boca Raton: CRC. Eertman, R. H. M., & De Zwaan, A. (1994). In K. J. M. Kramer (Ed.), Biomonitoring of coastal waters and estuaries (pp. 269–284). Boca Raton: CRC.
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C. Nasci et al. / Marine Environmental Research 54 (2002) 811–816
Forbes, V. E., & Depledge, M. H. (1996). In D. J. Baird, L. Maltby, P. W. Greig-Smith, & P. E. T. Douben (Eds.) Ecotoxicology: ecotoxicological dimension (pp. 71–86). London: Chapman and Hall. Fossato, V. U., Campesan, G., Craboledda, L., Dolci, F., & Stocco, G. (2000). In P. Lassere, & A. Marzollo (Eds.), The Venice lagoon ecosystem (Vol. 25; pp. 97–126), Man and the Biosphere Series. New York: Parthenon Publishing. Gehan, E. A. (1965). Biometrica, 52, 203–223. Livingstone, D. R. (1988). Marine Ecology Progress Series, 91, 141–153. Livingstone, D. R., Lemaire, P., Matthews, A., Peters, L. D., Porte, C., Fitzpatrick, P. J., Forlin, L., Nasci, C., Fossato, V. U., Wotton, N., & Goldfarb, P. (1995). Marine Environmental Research, 39, 235– 240. Livingstone, D. R., Lips, F., Garcia Martinez, P., & Pipe, R. K. (1992). Marine Biology, 112, 265–276. Lowe, D. M., & Da Ros, L. (2000). In P. Lassere, & A. Marzollo (Eds.), The Venice lagoon ecosystem (Vol. 25; pp. 375–385) Man and the Biosphere Series. New York: Parthenon Publishing. Monteduro, R. A. (2000). Valutazione degli effeti della contaminazione chimica mediante l’uso di bioindicatori di stress in Mytilus galloprovincialis della laguna di Venezia. Degree thesis, University of Venice, Ca` Foscari. Nasci, C., Da Ros, L., Campesan, G., & Fossato, V. U. (1998). Marine Environmental Research, 46, 279– 282. Nasci, C., Da Ros, L., & Livingstone, D. R. (1999). Bollettino del Museo Civico di Storia naturale Venezia, 49, 125–133. Widdows, J., & Nasci, C. (2000). In P. Lassere, & A. Marzollo (Eds.), The Venice lagoon ecosystem (Vol. 25; pp. 387–396). Man and the Biosphere Series. New York: Parthenon Publishing.