Micronucleus Test in Post Metamorphic Odontophrynus Cordobae and Rhinella Arenarum (Amphibia: Anura) for Environmental Monitoring
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Journal of Toxicology and Environmental Health Sciences Vol. 3(6) pp. 155-163, June 2011 Available online at http://www.academicjournals.org/JTEHS ISSN 2006-9820 ©2011 Academic Journals Full Length Research Paper Micronucleus test in post metamorphic Odontophrynus cordobae and Rhinella arenarum (Amphibia: Anura) for environmental monitoring Beatriz Bosch 1*, Fernando Mañas 2,3, Nora Gorla 2,3 and Delia Aiassa 1 1Departamento de Ciencias Naturales, Facultad de Ciencias Exactas Físico-Químicas y Naturales (FCEFQN), Universidad Nacional de Río Cuarto (UNRC), Argentina. 2Facultad de Agronomía y Veterinaria (FAV) Universidad Nacional de Río Cuarto (UNRC), Argentina. 3CONICET, Argentina. Accepted 28 March 2011 The genotoxic effect of cyclophosphamide and glyphosate in a commercial formulation were determined using the micronucleus test in peripheral blood erythrocytes of Odontophrynus cordobae and Rhinella arenarum , amphibians widely distributed in the Province of Córdoba, Argentina. For this, the basal frequency of the micronucleated erythrocytes (MNE) was determined by: 0.40±0.18 MNE/1000 erythrocytes in Odontophrynus cordobae and 0.30±0.09 MNE/1000 erythrocytes in Rhinella arenarum . The frequency of MNE in Odontophrynus cordobae increased after 5 days of exposure to glyphosate (100 mg ai/L) and cyclophosphamide. After 2 and 5 days of exposure to glyphosate (200, 400 and 800 mg ai/L), the MNE frequency in Rhinella arenarum was higher than the basal frequency, as it occurred in the group exposed to cyclophosphamide. Regarding acute toxicity and genotoxicity, the results show that Odontophrynus cordobae is more sensitive to cyclophosphamide and glyphosate exposure than Rhinella arenarum . A correlation was detected between exposure concentration and MNE frequency in Rhinella arenarum . Key words: Genotoxicity, micronucleus test, roundup ®, glyphosate, amphibians. INTRODUCTION Many authors have reported that in the past 30 years Jones et al., 2010). Pesticides, including insecticides and there has been a significant decline in amphibian herbicides, are particularly detrimental to amphibians due populations in diverse parts of the world (Hayes et al., to its aquatic habitat, sensitive skin and unprotected eggs 2010). Multiple causes have been suggested to explain (Govindarajulu, 2008; Bouhafs et al., 2009). this decline and among them, environmental pollution Glyphosate based herbicides are the most widely non- due to chemicals is gaining attention. Amphibians, like selective, broad-spectrum herbicides used in the world. In other organisms inhabiting agroecosystems, are highly Argentina, glyphosate use on its wide variety of exposed to agrochemicals and a correlation has been commercial formulations has increased dramatically. With reported between the use of pesticides and the decline of the increased use of glyphosate-based herbicides amphibian populations (Beebee and Griffiths, 2005; containing the surfactant polyethoxylated tallowamine (POEA) increased concern about the potential impact that the formulations may have on amphibians’ populations. Some studies indicate that the toxic effect of *Corresponding author. E-mail: [email protected]. glyphosate herbicides containing surfactants is higher Tel/Fax: 54-358-4676230. than the active ingredient (ai) per se. Thus, the toxicity of 156 J. Toxicol. Environ. Health Sci. some formulated glyphosate products to amphibians is species as a diagnostic tool and monitoring of greater than that caused by the active ingredient (Relyea, environmental quality. 2005a; Mann et al., 2009; Bernal et al., 2009; Modesto and Martinez, 2010). Furthermore, there is a global MATERIALS AND METHODS concern regarding the adverse genetic response that the formulated herbicide may have on non-target organisms Animals (Clements et al., 1997; Hole čková, 2006; Çava ş and Könen, 2007; Cavalcante et al., 2008; Andreik ėnait ė et Two species of anuran amphibians in post-metamorphic stage, O. ė cordobae and R. arenarum were selected as bioassay organisms. al., 2007; Poletta et al., 2007; Baršien et al., 2008; These anurans have an extensive Neotropical distribution, and Poletta et al., 2009; Vera et al., 2010a). Recent studies inhabit natural areas, agricultural land and urban territories with have shown that amphibians are one of the most availability of temporary ponds (Leynaud et al., 2006). Both species sensitive vertebrate groups to the toxicological effects of are easily collected by hand and acclimated to laboratory this herbicide (Govindarajulu, 2008). conditions. Several end-points have been used to assess Specimens of O. cordobae were manually collected in temporary ponds in a pristine zone (Villa de Las Rosas, Province of Córdoba, genotoxicity in aquatic and terrestrial organisms. Among lat. S 31° 56'; leng. W 65° 4'; alt. 713 m.a.s.l.) during September them, the micronucleus test (MN) has been widely used and October, 2005. Specimens of R. arenarum were manually in various species to detect clastogenic or aneugenic collected in temporary ponds in a suburban area, in the east of Río effects, both under laboratory conditions or in situ studies Cuarto, (Province of Córdoba, 33°06’ lat. S, 64°25’ leng. O; alt. 438, (Udroiu, 2008). Micronuclei are chromosome fragments 62 m.a.s.l.) during November and December, 2008 (Figure 1). or whole chromosomes which were not incorporated into The average total size (snout-vent) was 10.45±0.15 cm for R. arenarum and 6.20± 0.35 cm for O. cordobae . The animals the main nucleus, in cells of any actively dividing tissue. collected were those considered young adults in reproductive age, Increasing frequency of MN is considered as a biomarker according to their body size. The animals were kept under of genotoxic effect at subcellular level and an early laboratory conditions at ambient temperature and natural response of chromosomal damage (Fenech, 2000; photoperiod, according to the time of the year in which each test (Cuenca and Ramírez, 2004; Garaj-Vrhovac et al., 2008). was performed, for acclimatization before starting the assays. They were placed in 10 L plastic containers that were lined with damp This biomarker has been measured in amphibian species paper towels to keep the animals hydrated. During this time, the that inhabit environments where large quantities of animals were fed on larvae of Tenebrio molitor (Coleoptera: pesticides and other chemical substances are periodically Tenebrionidae) bred in the laboratory. poured (Lajmanovich et al., 2005; Cabagna et al., 2006; Vera Candiotti et al., 2010b). Chemicals The micronucleus test has been frequently used to detect genotoxicity induced by clastogenic and aneugenic The following substances were used: lyophilized cyclophosphamide agents in pre-metamorphic anuran amphibians and monohydrate (CAS Nº 6055-19-2), Microsules ® and isopropilamine salt of N-phosphomethyl-glycine, glyphosate, (CAS Nº 38641-94-0) urodeles (Jaylet et al., 1986; Marty et al., 1989; Cabagna ® et al., 2006; Mouchet et al., 2006; Vera et al., 2010b), but Roundup (glyphosate 48%). All test solutions were freshly prepared before use and renewed every 48 h. few used in post-metamorphic amphibians (Zhuleva and Dubinin, 1994; Matson et al., 2005). The interest of the study of post-metamorphic anuran amphibians is related to Treatments the temporal and spatial preponderance of this stage in Hayashi (2007) recommendations for the treatments were followed agroecosystems, which added to the characteristics of this regarding the number of animals per treatment group, the route of group and turned them into the organisms that are exposure to the substances tested, the animal preparation and the recommended for developing of biomarkers for test conditions. After acclimatization the animals were assigned to environmental monitoring (Burggren et al., 2007). groups of equal size (n = 5) as follows: The objectives of this study were: (I) Negative control group (C). (ii) Positive control group (PC). i) to define the basal MN frequency in peripheral blood (iii) Groups exposed to glyphosate (G). erythrocytes of adult Odontophrynus cordobae (Anura: Cyclorhamphidae) and Rhinella arenarum Hensel, 1867 Each experimental group consisted of three males and two (Anura: Bufonidae), amphibians widely distributed in the females; sex was determined by the presence/absence of vocalization activity and position in amplexus, observed in the field. agroecosystems of the Province of Córdoba, Argentina. The negative control group was kept in water. ii) propose the variation of these species in MN frequency Cyclophosphamide, well known as a genotoxic substance as a biomarker of genotoxic effect using (clastogenic effect), was used as a positive control. The positive cyclophosphamide as positive control. control group was kept in 40 mg/L cyclophosphamide during 5 ® iii) to test the MN frequency with four different days. The groups exposed to the herbicide were kept in Roundup concentrations of glyphosate herbicide in a commercial solutions prepared in water, at the following concentrations: glyphosate 100 mg (G100), 200 mg (G200), 400 mg (G400) and formulation. 800 mg (G800) active ingredient (a.i.)/ L. Dermal exposure was iv) to compare the sensitivity of both amphibians to these achieved by keeping the animals in the corresponding solutions in a compounds and propose the micronucleus test in these volume sufficient to cover half of the distal humerus. Bosch et al. 157 Figure 1. Study localities. Blood samples were obtained from Days 2 and 5 of treatment, erythrocytes. through