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Effects of furadan in the brown mussel Perna perna and in the mangrove oyster Crassostrea rhizophorae
Marine Environmental Research 54 (2002) 241–245 www.elsevier.com/locate/marenvrev
Effects of furadan in the brown mussel Perna perna and in the mangrove oyster Crassostrea rhizophorae Sandra R.C. Alvesa, Patrı´cia C. Severinob, Dominique P. Ibbotsonc, Angela Z. da Silvad, Franklin R.A.S. Lopesa, Luis A. Sa´enza, Afonso C.D. Bainya,* a
Laborato´rio de Biomarcadores de Contaminac¸a˜o Aqua´tica e Imunoquı´mica, Universidade Federal de Santa Catarina, Floriano´polis, 88040-900, SC, Brazil b Laborato´rio de Interac¸a˜o DNA-Proteı´na, Depto. Bioquı´mica CCB, Universidade Federal de Santa Catarina, Floriano´polis, 88040-900, SC, Brazil c Laborato´rio de Cultivo de Moluscos Marinhos, Depto. Aquicultura CCA, Universidade Federal de Santa Catarina, Floriano´polis, 88040-900, SC, Brazil d CTTMar, Universidade do Vale do Itajaı´, Itajaı´, 88302-202, SC, Brazil
Abstract Furadan is a carbamate pesticide used widely to combat agricultural pests. However little information is available about the toxicity of furadan in aquatic macroinvertebrates. The in vivo effects of furadan were evaluated in mussels, Perna perna, and oysters, Crassostrea rhizophorae. Glutathione S-transferase (GST), catalase (CAT) and cholinesterase (ChE) activities were measured in the gills of both species exposed to furadan (100 mg/l) for 96 h. No changes were observed in GST activity in the exposed groups. CAT activity was higher (9%) in the oysters exposed to furadan. ChE activity was inhibited by 64 and 35%, respectively, in C. rhizophorae and P. perna exposed to furadan, suggesting that the former is more susceptible to the toxic effects of furadan. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Perna perna; Mussel; Crassostrea rhizophorae; Oyster; Furadan; Biomarker; Acetylcholinesterase; Biochemical effects; Antioxidant enzymes
Human health and aquatic life have been increasingly threatened by the use of organophosphates and carbamates in agriculture and in pest control. The pesticide runoff associated with agricultural operations adjacent to estuaries and coastal * Corresponding author. Fax: +55-48-3319672. E-mail address: [email protected] (A.C.D. Bainy). 0141-1136/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0141-1136(02)00138-1
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environments may compromise the health of marine organisms (Bocquene´, Roig, & Fournier, 1997). Some of these chemicals are known to act as acetylcholinesterase (AChE) inhibitors in aquatic organisms, particularly in bivalves (Medler, Thompson, Dietz, & Silverman, 1999). AChE terminates neurotransmission by catalyzing hydrolysis of the neurotransmitter acetylcholine (Medler et al., 1998). Acetylcholine activates muscle contraction in the gills of bivalves, an important mechanism for osmotic regulation and feeding in these organisms (Medler et al., 1998). Antioxidant and conjugating enzymes are important defense systems during xenobiotic biotranformation to protect the cells against oxidative stress and to yield more polar conjugates to facilitate their excretion (Ecobichon, 1996). Furadan, the comercial name of carbofuran, is a broad spectrum carbamate that kills insects, mites and nematodes on contact or after ingestion. This compound is considered to be very toxic to fish (Kidd & James, 1991), but very little information is available about the toxicity of furadan to aquatic macroinvertebrates. The in vivo effects of furadan on mussels, Perna perna, and oysters, Crassostrea rhizophorae, were investigated. The activity of cholinesterase (ChE), catalase (CAT) and glutathione S-transferase (GST) were measured in gills of these species exposed and unexposed to furadan. Mussels, P. perna, (average length 72.2 2.8 mm) and oysters, Crassostrea rhizophorae, (average length 58.5 5.0 mm) were collected at Sambaqui beach, Floriano´polis, SC, Brazil (27 280 3000 S/48 330 4000 W), in the ‘‘Laborato´rio de Cultivo de Moluscos Marinhos, UFSC’’. Both mussels and oysters were separated, respectively, into four 7-l-tanks each, containing five specimens per tank. The organisms were acclimated to lab conditions (temperature 16 C, pH 7.8, salinity 34 %) for 48 h when they were fed with Chautoceros mulleri and the water was partially renewed daily. Before exposure to furadan, water from all tanks was completely changed. Furadan (nominal concentration of 100 mg/l dissolved in filtered salt water, salinity 34 %) was added to the two treatment tanks, for each species (n=10 for each group). This concentration was used because it is lower than the 96-h LD50 for fish (240–380 mg/l; Kidd & James, 1991). Two other tanks which did not receive the chemical were used for the control groups for each species. Total water renewal was carried out after 48 h exposure in order to restore the nominal concentration of furadan. Temperature and pH were recorded during the experiment. At 96 h exposure, the animals were sacrificed and the gills were excised and homogenized (1:4 w/v for mussels and 1:2 w/v for oysters) in buffer containing 20 mM Tris(hydroxymethyl aminomethane) hydrochloride, 1 mM EDTA (ethylenediaminetetracetic acid), 0.5 M sucrose, 1 mM DTT (dl-dithiothreitol), 0.1 mM PMSF (phenylmethylsulfonylfluoride), pH 8.0 using a tissue tearor. The homogenate was centrifuged at 9000 g for 20 min and the supernatant was stored at 85 C. ChE activity was measured according to the method of Ellmann, Courtney, Andres, and Featherstone (1961), using 0.3 mM acetylthiocholine iodide as substrate. The activity of GST was measured according to Keen, Habig, and Jakoby (1976), but using 1-chloro, 2,4 dinitrobenzene and reduced glutathione, both at concentration of 2 mM in the assay, instead of 1 mM, as originally proposed, which was found to be the optimal GST assay conditions for these species. CAT activity was measured according to Beutler
S.R.C. Alves et al. / Marine Environmental Research 54 (2002) 241–245
243
Fig. 1. Activity of glutathione S-transferase (GST), catalase (CAT) and cholinesterase (ChE) in gills of the oyster Crassostrea rhizophorae and of the mussel Perna perna control and exposed to furadan 0.1 ppm for 96 h. Each group contained 10 individuals. **Indicates significant differences for P<0.0001 and * for P< 0.05, by ANOVA. Data are expressed as means standard deviations.
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(1975) but recorded at 240 nm. All enzymatic analyses were carried out at least in duplicate in an Ultrospec 3000 spectrophotometer. Protein content in the supernatant fraction was quantified according to Peterson (1977), using BSA as standard. The normality of the data was tested using a normal probability plot. The data were analyzed using ANOVA and were identified as statistically significant when P< 0.05. During the exposure period, no mortalities were observed in either species. No differences in all analyzed parameters were observed between the replicate tanks, thus the data were pooled for the statistical analysis. The salinity, pH and temperature of the tanks from all groups did not change significantly (salinity 34 %, pH 7.8+0.2 and temperature 15.7+1.3). No changes were observed in GST activity in gills from both bivalve species (Fig. 1). However, a slight (9%) but significant increase in CAT activity (P < 0.05) was observed only in oysters exposed to furadan. The etiology of this induction in not clearly understood, but it could be suggested that a pro-oxidant condition elicited by the presence of furadan could be triggering an increase in the activity of this antioxidant enzyme, as an adaptive response. Recently it was shown that carbofuran produces oxidative stress associated with a marked nitric oxide production in brain of intoxicated rats (Gupta, Milatovic, & Dettbarn, 2001). Crassostrea rhizophorae and P. Perna treated with furadan were significantly different from controls of the same species, and showed 64 and 35% of ChE activity inhibition in the gill, respectively, indicating that the osmotic regulation and feeding could be affected in these organisms. However, there were no significant differences between oyster and mussel control groups, and oyster and mussel treated groups, respectively.. From a study carried out by our group (Monserrat, Bianchini, & Bainy, 2002), it was observed that the cholinesterases present in both S9 and S9 Triton X-100 soluble fractions from gills of C. rhizophorae are acetylcholinesterases. Interestingly, preliminary results have indicated that besides the AChE activity, extracts of gills of P. perna have butyrylcholinesterase (BuChE) activity which is less sensitive to furadan (data not shown). If that is the case, this could partially explain the differences observed in the degree of inhibition of ChE between these species. Similar results were obtained by Bocquene´ et al. (1997) who detected only AChE activity in gills of Crassostrea gigas, while gill extracts of Mytilus edulis showed both AChE and BuChE activities. Acknowledgements The authors would like to thank Dr. Jaime Fernando Ferreira and M.Sc. Joa˜o Guzensky (Laborato´rio de Cultivo de Moluscos Marinhos) for providing the organisms.
References Beutler, E. (1975). Red cell metabolism: a manual of biochemical methods. New York: Grune & Straton. Bocquene´, G., Roig, A., & Fournier, D. (1997). FEBS Letters, 407, 261–266.
S.R.C. Alves et al. / Marine Environmental Research 54 (2002) 241–245
245
Ecobichon, D. J. (1996). Toxic effects of pesticides. In C. D. Klaassen (Ed.), Casarett & Doull’s toxicology. The basic science of poisons (pp. 1111). New York: McGraw-Hill. Ellman, G. L., Courtney, K. O., Andres, V., & Featherstone, R. M. (1961). Biochemical Pharmacology, 7, 88–95. Gupta, R. C., Milatovic, D., & Dettbarn, W. D. (2001). Archives of Toxicology, 75, 346–356. Keen, J. H., Habig, W. H., & Jakoby, W. B. (1976). Journal of Biological Chemistry, 251, 6183–6188. Kidd, H., & James, D. R. (1991). The agrochemicals handbook. Cambridge, UK: Royal Society of Chemistry Information Services. Medler, S., Thompson, C. C., Dietz, T. H., & Silverman, H. (1999). Comprehensive Biochemistry and Physiology Part A, 122, 163–172. Monserrat, J. M., Bianchini, A., & Bainy, A. C. D. (2002). Marine Environmental Research (this issue). Peterson, G. L. (1977). Analytical Biochemistry, 83, 346–356.
Effects of furadan in the brown mussel Perna perna and in the mangrove oyster Crassostrea rhizophorae Sandra R.C. Alvesa, Patrı´cia C. Severinob, Dominique P. Ibbotsonc, Angela Z. da Silvad, Franklin R.A.S. Lopesa, Luis A. Sa´enza, Afonso C.D. Bainya,* a
Laborato´rio de Biomarcadores de Contaminac¸a˜o Aqua´tica e Imunoquı´mica, Universidade Federal de Santa Catarina, Floriano´polis, 88040-900, SC, Brazil b Laborato´rio de Interac¸a˜o DNA-Proteı´na, Depto. Bioquı´mica CCB, Universidade Federal de Santa Catarina, Floriano´polis, 88040-900, SC, Brazil c Laborato´rio de Cultivo de Moluscos Marinhos, Depto. Aquicultura CCA, Universidade Federal de Santa Catarina, Floriano´polis, 88040-900, SC, Brazil d CTTMar, Universidade do Vale do Itajaı´, Itajaı´, 88302-202, SC, Brazil
Abstract Furadan is a carbamate pesticide used widely to combat agricultural pests. However little information is available about the toxicity of furadan in aquatic macroinvertebrates. The in vivo effects of furadan were evaluated in mussels, Perna perna, and oysters, Crassostrea rhizophorae. Glutathione S-transferase (GST), catalase (CAT) and cholinesterase (ChE) activities were measured in the gills of both species exposed to furadan (100 mg/l) for 96 h. No changes were observed in GST activity in the exposed groups. CAT activity was higher (9%) in the oysters exposed to furadan. ChE activity was inhibited by 64 and 35%, respectively, in C. rhizophorae and P. perna exposed to furadan, suggesting that the former is more susceptible to the toxic effects of furadan. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Perna perna; Mussel; Crassostrea rhizophorae; Oyster; Furadan; Biomarker; Acetylcholinesterase; Biochemical effects; Antioxidant enzymes
Human health and aquatic life have been increasingly threatened by the use of organophosphates and carbamates in agriculture and in pest control. The pesticide runoff associated with agricultural operations adjacent to estuaries and coastal * Corresponding author. Fax: +55-48-3319672. E-mail address: [email protected] (A.C.D. Bainy). 0141-1136/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0141-1136(02)00138-1
242
S.R.C. Alves et al. / Marine Environmental Research 54 (2002) 241–245
environments may compromise the health of marine organisms (Bocquene´, Roig, & Fournier, 1997). Some of these chemicals are known to act as acetylcholinesterase (AChE) inhibitors in aquatic organisms, particularly in bivalves (Medler, Thompson, Dietz, & Silverman, 1999). AChE terminates neurotransmission by catalyzing hydrolysis of the neurotransmitter acetylcholine (Medler et al., 1998). Acetylcholine activates muscle contraction in the gills of bivalves, an important mechanism for osmotic regulation and feeding in these organisms (Medler et al., 1998). Antioxidant and conjugating enzymes are important defense systems during xenobiotic biotranformation to protect the cells against oxidative stress and to yield more polar conjugates to facilitate their excretion (Ecobichon, 1996). Furadan, the comercial name of carbofuran, is a broad spectrum carbamate that kills insects, mites and nematodes on contact or after ingestion. This compound is considered to be very toxic to fish (Kidd & James, 1991), but very little information is available about the toxicity of furadan to aquatic macroinvertebrates. The in vivo effects of furadan on mussels, Perna perna, and oysters, Crassostrea rhizophorae, were investigated. The activity of cholinesterase (ChE), catalase (CAT) and glutathione S-transferase (GST) were measured in gills of these species exposed and unexposed to furadan. Mussels, P. perna, (average length 72.2 2.8 mm) and oysters, Crassostrea rhizophorae, (average length 58.5 5.0 mm) were collected at Sambaqui beach, Floriano´polis, SC, Brazil (27 280 3000 S/48 330 4000 W), in the ‘‘Laborato´rio de Cultivo de Moluscos Marinhos, UFSC’’. Both mussels and oysters were separated, respectively, into four 7-l-tanks each, containing five specimens per tank. The organisms were acclimated to lab conditions (temperature 16 C, pH 7.8, salinity 34 %) for 48 h when they were fed with Chautoceros mulleri and the water was partially renewed daily. Before exposure to furadan, water from all tanks was completely changed. Furadan (nominal concentration of 100 mg/l dissolved in filtered salt water, salinity 34 %) was added to the two treatment tanks, for each species (n=10 for each group). This concentration was used because it is lower than the 96-h LD50 for fish (240–380 mg/l; Kidd & James, 1991). Two other tanks which did not receive the chemical were used for the control groups for each species. Total water renewal was carried out after 48 h exposure in order to restore the nominal concentration of furadan. Temperature and pH were recorded during the experiment. At 96 h exposure, the animals were sacrificed and the gills were excised and homogenized (1:4 w/v for mussels and 1:2 w/v for oysters) in buffer containing 20 mM Tris(hydroxymethyl aminomethane) hydrochloride, 1 mM EDTA (ethylenediaminetetracetic acid), 0.5 M sucrose, 1 mM DTT (dl-dithiothreitol), 0.1 mM PMSF (phenylmethylsulfonylfluoride), pH 8.0 using a tissue tearor. The homogenate was centrifuged at 9000 g for 20 min and the supernatant was stored at 85 C. ChE activity was measured according to the method of Ellmann, Courtney, Andres, and Featherstone (1961), using 0.3 mM acetylthiocholine iodide as substrate. The activity of GST was measured according to Keen, Habig, and Jakoby (1976), but using 1-chloro, 2,4 dinitrobenzene and reduced glutathione, both at concentration of 2 mM in the assay, instead of 1 mM, as originally proposed, which was found to be the optimal GST assay conditions for these species. CAT activity was measured according to Beutler
S.R.C. Alves et al. / Marine Environmental Research 54 (2002) 241–245
243
Fig. 1. Activity of glutathione S-transferase (GST), catalase (CAT) and cholinesterase (ChE) in gills of the oyster Crassostrea rhizophorae and of the mussel Perna perna control and exposed to furadan 0.1 ppm for 96 h. Each group contained 10 individuals. **Indicates significant differences for P<0.0001 and * for P< 0.05, by ANOVA. Data are expressed as means standard deviations.
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S.R.C. Alves et al. / Marine Environmental Research 54 (2002) 241–245
(1975) but recorded at 240 nm. All enzymatic analyses were carried out at least in duplicate in an Ultrospec 3000 spectrophotometer. Protein content in the supernatant fraction was quantified according to Peterson (1977), using BSA as standard. The normality of the data was tested using a normal probability plot. The data were analyzed using ANOVA and were identified as statistically significant when P< 0.05. During the exposure period, no mortalities were observed in either species. No differences in all analyzed parameters were observed between the replicate tanks, thus the data were pooled for the statistical analysis. The salinity, pH and temperature of the tanks from all groups did not change significantly (salinity 34 %, pH 7.8+0.2 and temperature 15.7+1.3). No changes were observed in GST activity in gills from both bivalve species (Fig. 1). However, a slight (9%) but significant increase in CAT activity (P < 0.05) was observed only in oysters exposed to furadan. The etiology of this induction in not clearly understood, but it could be suggested that a pro-oxidant condition elicited by the presence of furadan could be triggering an increase in the activity of this antioxidant enzyme, as an adaptive response. Recently it was shown that carbofuran produces oxidative stress associated with a marked nitric oxide production in brain of intoxicated rats (Gupta, Milatovic, & Dettbarn, 2001). Crassostrea rhizophorae and P. Perna treated with furadan were significantly different from controls of the same species, and showed 64 and 35% of ChE activity inhibition in the gill, respectively, indicating that the osmotic regulation and feeding could be affected in these organisms. However, there were no significant differences between oyster and mussel control groups, and oyster and mussel treated groups, respectively.. From a study carried out by our group (Monserrat, Bianchini, & Bainy, 2002), it was observed that the cholinesterases present in both S9 and S9 Triton X-100 soluble fractions from gills of C. rhizophorae are acetylcholinesterases. Interestingly, preliminary results have indicated that besides the AChE activity, extracts of gills of P. perna have butyrylcholinesterase (BuChE) activity which is less sensitive to furadan (data not shown). If that is the case, this could partially explain the differences observed in the degree of inhibition of ChE between these species. Similar results were obtained by Bocquene´ et al. (1997) who detected only AChE activity in gills of Crassostrea gigas, while gill extracts of Mytilus edulis showed both AChE and BuChE activities. Acknowledgements The authors would like to thank Dr. Jaime Fernando Ferreira and M.Sc. Joa˜o Guzensky (Laborato´rio de Cultivo de Moluscos Marinhos) for providing the organisms.
References Beutler, E. (1975). Red cell metabolism: a manual of biochemical methods. New York: Grune & Straton. Bocquene´, G., Roig, A., & Fournier, D. (1997). FEBS Letters, 407, 261–266.
S.R.C. Alves et al. / Marine Environmental Research 54 (2002) 241–245
245
Ecobichon, D. J. (1996). Toxic effects of pesticides. In C. D. Klaassen (Ed.), Casarett & Doull’s toxicology. The basic science of poisons (pp. 1111). New York: McGraw-Hill. Ellman, G. L., Courtney, K. O., Andres, V., & Featherstone, R. M. (1961). Biochemical Pharmacology, 7, 88–95. Gupta, R. C., Milatovic, D., & Dettbarn, W. D. (2001). Archives of Toxicology, 75, 346–356. Keen, J. H., Habig, W. H., & Jakoby, W. B. (1976). Journal of Biological Chemistry, 251, 6183–6188. Kidd, H., & James, D. R. (1991). The agrochemicals handbook. Cambridge, UK: Royal Society of Chemistry Information Services. Medler, S., Thompson, C. C., Dietz, T. H., & Silverman, H. (1999). Comprehensive Biochemistry and Physiology Part A, 122, 163–172. Monserrat, J. M., Bianchini, A., & Bainy, A. C. D. (2002). Marine Environmental Research (this issue). Peterson, G. L. (1977). Analytical Biochemistry, 83, 346–356.