- Email: [email protected]
Effects of the herbicide Roundup™ on the ultrastructural pattern of hepatocytes in carp (Cyprinus carpio)
Marine Environmental Research 50 (2000) 263±266 www.elsevier.com/locate/marenvrev
Eects of the herbicide RoundupTM on the ultrastructural pattern of hepatocytes in carp (Cyprinus carpio) J. Szarek a,*, A. Siwicki b, A. Andrzejewska c, E. Terech-Majewska c, T. Banaszkiewicz d a
Department of Forensic and Administration Veterinary Medicine, Warmia and Masuria University in Olsztyn, Oczapowskiego St.13, 10-717 Olsztyn, Poland b Department of Epizootiology with Clinic of Infectious Diseases, Warmia and Masuria University in Olsztyn, 10-717 Olsztyn, Poland c Department of Pathological Anatomy, Medical Academy of Bialystok, 15-269 Bialystok, Poland d Department of Plant Protection, Warmia and Masuria University in Olsztyn, 10-717 Olsztyn, Poland Received 29 April 1999; received in revised form 9 December 1999; accepted 11 February 2000
Abstract Experimental studies were performed on healthy, 80±100 g carp (Cyprinus carpio). Fish were exposed by emersion in RoundupTM (205 mg of glyphosate/l or 410 mg of glyphosate/l) in concentrations of 40- to 20-fold lower than those used in practice. Electron microscopy revealed that the herbicide caused appearance of myelin-like structures in carp hepatocytes, swelling of mitochondria and disappearance of internal membrane of mitochondria in carp at both exposure concentrations. It means that Roundup was harmful to carp when used in applied concentrations. Results of these studies enhance our knowledge of ultrastructural pathomorphology of ®sh organs following exposure to Roundup. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Cyprinus carpio; Hepatocytes; Lesions; Pollution eects; RoundupTM toxicity
1. Introduction Many scienti®c experiments have examined the use of herbicides in terrestrial ecosystems and the toxicity of herbicides to animals (Medina, Lopata & Bacila, 1994; Piska & Waghray, 1997). However, less is known about their toxicity to aquatic * Corresponding author. Tel.: +48-89-523-3252; Fax: +48-89-523-3252. E-mail address: [email protected] (J. Szarek). 0141-1136/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0141-1136(00)00088-X
264
J. Szarek et al. / Marine Environmental Research 50 (2000) 263±266
ecosystems, including ®sh. The presence of herbicides in water is a consequence of weed control in terrestrial ecosystems and water reservoirs. The eects of herbicides will depend upon depth of water reservoir, rate of ¯owing water, ®sh densities and ®sh stock composition (Goldsborough & Beck, 1989). Hardness of water, pH and temperature may also be key factors (Mitchell, Chapman & Long, 1987). RoundupTM has recently become a popular herbicide used for the control of aquatic vegetation (Anton, Labora & De Ariz, 1994; Herrchen, Klein & Klein, 1993). The toxicity of herbicide to plants is high, but its active substance, a glyphosate, decomposes rapidly and is characterized by a very speci®c action, which blocks the synthesis of certain amino acids (LeÁvesque & Rahe, 1992). Thus, the herbicide is believed to be environmentally safe (Anton et al., 1994; Franz, Mao & Sikorski, 1997). However, glyphosate may inhibit microorganisms, thereby extending the duration of its decomposition and eects on various components of the soil micro¯ora (Ghosh & Konar, 1983; LeÁvesque & Rahe, 1992). Some evidence of glyphosate's environmental toxicity is presented by Cox (1998). Studies concerning small ponds with static water, showed that the rate of glyphosate dissipation/degradation ranged from 1.5 to 3.5 days, with ®rst order half-lives (Goldsborough & Beck, 1989). Therefore toxicity studies at relatively high glyphosate concentrations are environmentally relevant, particularly when ®sh are acutely exposed immediately after glyphosate application. The aim of this study was to examine the eect of Roundup upon the morphological cell substructure of carp hepatocytes, at concentrations 40- to 20-fold lower than the standard application. Triebskorn, KoÈhler, Honnen, Schramm, Adams and MuÈller (1997) suggested ultrastructural investigations of hepatocytes are a useful response for such studies. 2. Materials and methods Experimental exposures using the herbicide RoundupTM (Monsanto Europe, Brussels) were performed on 72 carp (Cyprinus carpio) with individual body weights of 80±100 g. The ®sh were divided into three groups: Group I, a control group with no herbicide exposure; Group II, ®sh held for 1 h to 0.05% aqueous solution of Roundup containing 205 mg of glyphosate/l; and Group III, ®sh exposed for 0.5 h to 0.1% of herbicide (410 mg of glyphosate/l). The experiment was carried out in two series and ®sh from each group were randomly selected for postmortem examinations (12 ®sh per group). The water conditions were as follows: temperature, 18 C; oxygen level, 8.0 mg/l; pH, 8.0. Fish were kept in 30-l open tanks. Dead carp were examined macroscopically and small pieces of hepatopancreas (ca. 1 mm3) were ®xed in a mixture of paraformaldehyde and 2% glutaraldehyde buered in 0.2 M phosphate buer (pH 7.4) for 2 h at 4 C and then post®xed in 2% osmium tetroxide for 1 h. After dehydration in alcohol, samples were washed in propylene oxide and embedded in Epon 812. Ultra-thin sections, from 12 ®sh of each group, were obtained with an LKB ultramicrotome, were double contrasted with uranyl acetate and lead citrate and examined with an Opton 900 TEM. For statistical analysis of ®sh mortality Student's t test was used.
J. Szarek et al. / Marine Environmental Research 50 (2000) 263±266
265
3. Results and discussion Two applied herbicide concentrations, ie. 205 mg of glyphosate/l and 410 mg of glyphosate/l appeared to be lethal for all ®sh after 1 and 0.5 h exposure, respectively. The results were signi®cant at P=0.01. Macroscopic examination of ®sh from all groups did not reveal any morphological lesions. Ultrastructural morphology of the hepatocytes in all control ®sh was normal with a well-developed rough endoplasmatic reticulum (RER) and numerous round or oval mitochondria (MIT) with matrix, a few crista, and some mitochondrial bodies. A few primary lysosomes were usually present in the hepatocytes, while secondary lysosomes and dense bodies were rare. Myelin-like structures were observed in the cytoplasm of hepatocytes in almost always ®sh from Group II. Swollen MIT dominated in this pattern. Disappearance of the MIT crista was fairly frequent. The external membrane of MIT was missing in some ®sh specimens. A few cells contained enlarged Golgi complexes. Quite frequently, hepatocytes contained vacuoles of dierent sizes (Fig. 1). The majority of the examined ®sh had livers (including pancreases) with mononuclear in®ltration cells. Ultrastructural examination of ®sh from Group III revealed presence of myelinlike structures in the hepatocytes cytoplasm in all cases. MIT with noticeable degeneration and disintegration of the external membrane were observed. Quite often these structures were swollen (Fig. 2). RER canals were enlarged. Groups of hepatocytes with reduced level of glycogen were also observed (Fig. 2). Analyses of the ultrastructural changes observed in this study showed that RoundupTM in two concentrations (205 mg of glyphosate/l and 410 mg of glyphosate/l), had pathogenic eects in carp, which lead to death of ®sh after one or even
Fig. 1. Fragment of carp hepatocyte from Group II. Note the vacuoles (V) in the cytoplasm (magn. 12 500).
266
J. Szarek et al. / Marine Environmental Research 50 (2000) 263±266
Fig. 2. Fragment of carp hepatocyte from Group III. Note the swollen appearance of the mitochondria (arrows) and low concentration of glycogen (magn.21 500).
half an hour of exposition. It means that despite low toxicity of glyphosate to ®sh, its low rate of dissipation/degradation in water may induce toxic eects shortly after application. The presence of Roundup herbicide in water may therefore result in the following lesions in carp hepatocytes: myelin-like structures, MIT swelling and disappearance of MIT internal membrane. References Anton, F. A., Labora, E., & De Ariz, M. (1994). Chemosphere, 82, 745±753. Cox, C. (1998). Journal of Pesticide Reform, 3, 3±17. Franz, J. E., Mao, K. M., & Sikorski, J. A. (1997). Glyphosate: a unique global herbicide. ACS Monograph, 189, Washington, DC. Ghosh, T. K., & Konar, S. K. (1983). Geobios, 10, 104±107. Goldsborough, L. G., & Beck, A. E. (1989). Archives of Environmental Contamination and Toxicology, 18, 537±544. Herrchen, M., Klein, W., & Klein, A. W. (1993). Science of the Total Environment, 2, 1689±1699. LeÁvesque, A., & Rahe, J. E. (1992). Annual Review of Phytopathology, 30, 579±602. Medina, H. S. G., Lopata, M. E., & Bacila, M. (1994). Arquivos de Biologia e Tecnologia, 37, 895±906. Mitchell, D. G., Chapman, P. M., & Long, T. J. (1987). Bulletin of Environmental Contamination and Toxicology, 39, 1028±1035. Piska, M. B., & Waghray, S. (1997). Journal of Environmental Health, 33, 126±127. Triebskorn, R., KoÈhler, H. R., Honnen, W., Schramm, M., Adams, S. M., & MuÈller, E. F. (1997). Journal of Aquatic Ecosystem Stress and Recovery, 6, 57±73.
Eects of the herbicide RoundupTM on the ultrastructural pattern of hepatocytes in carp (Cyprinus carpio) J. Szarek a,*, A. Siwicki b, A. Andrzejewska c, E. Terech-Majewska c, T. Banaszkiewicz d a
Department of Forensic and Administration Veterinary Medicine, Warmia and Masuria University in Olsztyn, Oczapowskiego St.13, 10-717 Olsztyn, Poland b Department of Epizootiology with Clinic of Infectious Diseases, Warmia and Masuria University in Olsztyn, 10-717 Olsztyn, Poland c Department of Pathological Anatomy, Medical Academy of Bialystok, 15-269 Bialystok, Poland d Department of Plant Protection, Warmia and Masuria University in Olsztyn, 10-717 Olsztyn, Poland Received 29 April 1999; received in revised form 9 December 1999; accepted 11 February 2000
Abstract Experimental studies were performed on healthy, 80±100 g carp (Cyprinus carpio). Fish were exposed by emersion in RoundupTM (205 mg of glyphosate/l or 410 mg of glyphosate/l) in concentrations of 40- to 20-fold lower than those used in practice. Electron microscopy revealed that the herbicide caused appearance of myelin-like structures in carp hepatocytes, swelling of mitochondria and disappearance of internal membrane of mitochondria in carp at both exposure concentrations. It means that Roundup was harmful to carp when used in applied concentrations. Results of these studies enhance our knowledge of ultrastructural pathomorphology of ®sh organs following exposure to Roundup. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Cyprinus carpio; Hepatocytes; Lesions; Pollution eects; RoundupTM toxicity
1. Introduction Many scienti®c experiments have examined the use of herbicides in terrestrial ecosystems and the toxicity of herbicides to animals (Medina, Lopata & Bacila, 1994; Piska & Waghray, 1997). However, less is known about their toxicity to aquatic * Corresponding author. Tel.: +48-89-523-3252; Fax: +48-89-523-3252. E-mail address: [email protected] (J. Szarek). 0141-1136/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0141-1136(00)00088-X
264
J. Szarek et al. / Marine Environmental Research 50 (2000) 263±266
ecosystems, including ®sh. The presence of herbicides in water is a consequence of weed control in terrestrial ecosystems and water reservoirs. The eects of herbicides will depend upon depth of water reservoir, rate of ¯owing water, ®sh densities and ®sh stock composition (Goldsborough & Beck, 1989). Hardness of water, pH and temperature may also be key factors (Mitchell, Chapman & Long, 1987). RoundupTM has recently become a popular herbicide used for the control of aquatic vegetation (Anton, Labora & De Ariz, 1994; Herrchen, Klein & Klein, 1993). The toxicity of herbicide to plants is high, but its active substance, a glyphosate, decomposes rapidly and is characterized by a very speci®c action, which blocks the synthesis of certain amino acids (LeÁvesque & Rahe, 1992). Thus, the herbicide is believed to be environmentally safe (Anton et al., 1994; Franz, Mao & Sikorski, 1997). However, glyphosate may inhibit microorganisms, thereby extending the duration of its decomposition and eects on various components of the soil micro¯ora (Ghosh & Konar, 1983; LeÁvesque & Rahe, 1992). Some evidence of glyphosate's environmental toxicity is presented by Cox (1998). Studies concerning small ponds with static water, showed that the rate of glyphosate dissipation/degradation ranged from 1.5 to 3.5 days, with ®rst order half-lives (Goldsborough & Beck, 1989). Therefore toxicity studies at relatively high glyphosate concentrations are environmentally relevant, particularly when ®sh are acutely exposed immediately after glyphosate application. The aim of this study was to examine the eect of Roundup upon the morphological cell substructure of carp hepatocytes, at concentrations 40- to 20-fold lower than the standard application. Triebskorn, KoÈhler, Honnen, Schramm, Adams and MuÈller (1997) suggested ultrastructural investigations of hepatocytes are a useful response for such studies. 2. Materials and methods Experimental exposures using the herbicide RoundupTM (Monsanto Europe, Brussels) were performed on 72 carp (Cyprinus carpio) with individual body weights of 80±100 g. The ®sh were divided into three groups: Group I, a control group with no herbicide exposure; Group II, ®sh held for 1 h to 0.05% aqueous solution of Roundup containing 205 mg of glyphosate/l; and Group III, ®sh exposed for 0.5 h to 0.1% of herbicide (410 mg of glyphosate/l). The experiment was carried out in two series and ®sh from each group were randomly selected for postmortem examinations (12 ®sh per group). The water conditions were as follows: temperature, 18 C; oxygen level, 8.0 mg/l; pH, 8.0. Fish were kept in 30-l open tanks. Dead carp were examined macroscopically and small pieces of hepatopancreas (ca. 1 mm3) were ®xed in a mixture of paraformaldehyde and 2% glutaraldehyde buered in 0.2 M phosphate buer (pH 7.4) for 2 h at 4 C and then post®xed in 2% osmium tetroxide for 1 h. After dehydration in alcohol, samples were washed in propylene oxide and embedded in Epon 812. Ultra-thin sections, from 12 ®sh of each group, were obtained with an LKB ultramicrotome, were double contrasted with uranyl acetate and lead citrate and examined with an Opton 900 TEM. For statistical analysis of ®sh mortality Student's t test was used.
J. Szarek et al. / Marine Environmental Research 50 (2000) 263±266
265
3. Results and discussion Two applied herbicide concentrations, ie. 205 mg of glyphosate/l and 410 mg of glyphosate/l appeared to be lethal for all ®sh after 1 and 0.5 h exposure, respectively. The results were signi®cant at P=0.01. Macroscopic examination of ®sh from all groups did not reveal any morphological lesions. Ultrastructural morphology of the hepatocytes in all control ®sh was normal with a well-developed rough endoplasmatic reticulum (RER) and numerous round or oval mitochondria (MIT) with matrix, a few crista, and some mitochondrial bodies. A few primary lysosomes were usually present in the hepatocytes, while secondary lysosomes and dense bodies were rare. Myelin-like structures were observed in the cytoplasm of hepatocytes in almost always ®sh from Group II. Swollen MIT dominated in this pattern. Disappearance of the MIT crista was fairly frequent. The external membrane of MIT was missing in some ®sh specimens. A few cells contained enlarged Golgi complexes. Quite frequently, hepatocytes contained vacuoles of dierent sizes (Fig. 1). The majority of the examined ®sh had livers (including pancreases) with mononuclear in®ltration cells. Ultrastructural examination of ®sh from Group III revealed presence of myelinlike structures in the hepatocytes cytoplasm in all cases. MIT with noticeable degeneration and disintegration of the external membrane were observed. Quite often these structures were swollen (Fig. 2). RER canals were enlarged. Groups of hepatocytes with reduced level of glycogen were also observed (Fig. 2). Analyses of the ultrastructural changes observed in this study showed that RoundupTM in two concentrations (205 mg of glyphosate/l and 410 mg of glyphosate/l), had pathogenic eects in carp, which lead to death of ®sh after one or even
Fig. 1. Fragment of carp hepatocyte from Group II. Note the vacuoles (V) in the cytoplasm (magn. 12 500).
266
J. Szarek et al. / Marine Environmental Research 50 (2000) 263±266
Fig. 2. Fragment of carp hepatocyte from Group III. Note the swollen appearance of the mitochondria (arrows) and low concentration of glycogen (magn.21 500).
half an hour of exposition. It means that despite low toxicity of glyphosate to ®sh, its low rate of dissipation/degradation in water may induce toxic eects shortly after application. The presence of Roundup herbicide in water may therefore result in the following lesions in carp hepatocytes: myelin-like structures, MIT swelling and disappearance of MIT internal membrane. References Anton, F. A., Labora, E., & De Ariz, M. (1994). Chemosphere, 82, 745±753. Cox, C. (1998). Journal of Pesticide Reform, 3, 3±17. Franz, J. E., Mao, K. M., & Sikorski, J. A. (1997). Glyphosate: a unique global herbicide. ACS Monograph, 189, Washington, DC. Ghosh, T. K., & Konar, S. K. (1983). Geobios, 10, 104±107. Goldsborough, L. G., & Beck, A. E. (1989). Archives of Environmental Contamination and Toxicology, 18, 537±544. Herrchen, M., Klein, W., & Klein, A. W. (1993). Science of the Total Environment, 2, 1689±1699. LeÁvesque, A., & Rahe, J. E. (1992). Annual Review of Phytopathology, 30, 579±602. Medina, H. S. G., Lopata, M. E., & Bacila, M. (1994). Arquivos de Biologia e Tecnologia, 37, 895±906. Mitchell, D. G., Chapman, P. M., & Long, T. J. (1987). Bulletin of Environmental Contamination and Toxicology, 39, 1028±1035. Piska, M. B., & Waghray, S. (1997). Journal of Environmental Health, 33, 126±127. Triebskorn, R., KoÈhler, H. R., Honnen, W., Schramm, M., Adams, S. M., & MuÈller, E. F. (1997). Journal of Aquatic Ecosystem Stress and Recovery, 6, 57±73.