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The agricultural impact of pesticides on Physalaemus cuvieri tadpoles (Amphibia: Anura) ascertained by comet assay

ABSTRACT

Amphibians inhabiting agricultural areas are constantly exposed to large amounts of chemicals, which reach the aquatic environment during the rainy season through runoff, drainage, and leaching. We performed a comet assay on the erythrocytes of tadpoles found in the surroundings of agricultural fields (soybean and corn crops), where there is an intense release of several kinds of pesticides in different quantities. We aimed to detect differences in the genotoxic parameters between populations collected from soybeans and cornfields, and between them and tadpoles sampled from non-agricultural areas (control group). Tadpoles collected from ponds located at soybean fields had significantly more DNA damage, followed by tadpoles collected from cornfields. In contrast, animals sampled from non-agricultural areas had the lowest incidence of DNA damage. In addition, we found a negative correlation between the parameters of the comet assay and the area of the ponds surrounding soybean. This correlation indicates a possible dilution effect in the concentration of pesticides. Finally, Physalaemus cuvieri Fitzinger, 1826 seems to be a good bioindicator for detecting the genotoxic effects of field agricultural insecticides; therefore, we suggest that this species should be used in environmental biomonitoring studies, since it is common and abundant where it occurs.

KEY WORDS:
Amphibians; bioindicators; exposure; genotoxicity; pesticides

INTRODUCTION

In Brazil, agriculture is an important economic activity, and in view of this, the country has developed a large-scale commercial agricultural system. Brazil accounts for approximately 50% of the agricultural pesticides consumed in Latin America (ANVISA 2005ANVISA (2005) Nota tecnica sobre livre comercio de agrotoxicos e impactos a saude humana. Available online at: Available online at: http://www.anvisa.gov.br [Accessed: 14/7/2015]
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). However, the success of this sector has been associated with the widespread destruction of Brazilian ecosystems, especially the Cerrado. In 2009, the world’s soybean production was 216.8 million tons, which is almost twice as much as in 1990. In Brazil, soybean covers the largest cropped area (23 million ha), followed by corn (12 million ha) (SoyStats 2015SoyStats (2015) A reference guide to important soybean facts and figures. The American Soybean Association. http://www.soystats.com [Accessed: 14/07/15]
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, OECD/FAO 2015OECD/Food and Agriculture Organization of the United Nations (2015) OECD-FAO Agricultural Outlook 2015. Paris, OECD Publishing. http://www.oecd-ilibrary.org/agriculture-and-food/oecd-fao-agricultural-outlook-2015_agr_outlook-2015-en [Accessed: 16/03/2016], org/agriculture-and-food/oecd-fao-agricultural-outlook-2015_agr_outlook-2015-en [Accessed: 16/03/2016], https://doi.org/10.1787/agr_outlook-2015-en
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). After the implementation of financing programs for agriculture in Brazil, the use of pesticides such as insecticides and herbicides has increased (Waissmann 2007Waissmann W (2007) Agrotóxicos e doenças não transmissíveis. Ciência & Saúde Coletiva 12: 20-21. https://doi.org/10.1590/S1413-81232007000100005
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) at an unprecedented rate (3 to 9.6 l/ha). Of special concern is the fact that several pesticides that have been banned from use in most countries are still allowed in Brazil (Cançado et al. 2006Cançado JED, Saldiva PHN, Pereira LAA, Lara LB, Artaxo P, Martinelli LA, Arbex MA, Zanobetti A, Braga AL (2006) The Impact of Sugar Cane-Burning Emissions on the Respiratory System of Children and the Elderly. Environmental Health Perspectives 114: 725-729. https://doi.org/10.1289/ehp.8485
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).

Amphibians, especially anurans, are broadly used as test animals and bioindicators in evaluating the effects of pollutants in aquatic and agricultural ecosystems (Linder and Grillitsch 2000Linder G, Grillitsch B (2000) Ecotoxicology of metals. In: Sparling DW, Linder G, Bishop CA (Eds) Ecotoxicology of Amphibians and Reptiles . Pensacola, Society of Environmental Toxicology and Chemistry, 325-459., Camargo and Alonso 2006Camargo JA, Alonso Á (2006) Ecological and toxicological effects of inorganic nitrogen pollution in aquatic ecosystems: A global assessment. Environment International 32: 831-849. https://doi.org/10.1016/j.envint.2006.05.002
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). Amphibians are among the most sensitive organisms to environmental changes, mainly due to their behavioral and physiological characteristics, such as a highly permeable skin, little mobility, complex life cycle, and simultaneous dependence on aquatic and terrestrial environments (Pollet and Bendell-Young 2000Pollet I, Bendell-Young LI (2000) Amphibians as indicators of wetland quality in wetlands formed from oil sands effluent. Environmental Toxicology Chemistry 19: 2589-2597. https://doi.org/10.1002/etc.5620191027
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). All of these characteristics facilitate the accumulation of environmental contaminants in their bodies (Carey and Bryant 1995Carey C, Bryant CJ (1995) Possible interrelations among environmental toxicants, amphibian development, and decline of amphibian populations. Environmental Health Perspectives 103: 13-17. https://doi.org/10.2307/3432406
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, Linder and Grillitsch 2000Linder G, Grillitsch B (2000) Ecotoxicology of metals. In: Sparling DW, Linder G, Bishop CA (Eds) Ecotoxicology of Amphibians and Reptiles . Pensacola, Society of Environmental Toxicology and Chemistry, 325-459., Henry 2000Henry PFP (2000) Aspects of amphibian anatomy and physiology. In: Sparling DW, Linder G, Bishop CA (Eds) Ecotoxicology of Amphibians and Reptiles. Pensacola, Society of Environmental Toxicology and Chemistry, 71-110.). In addition, pollution in the water and in the air, are the main causes of mortality in amphibian populations, often contributing to local and global extinction of species (Stuart et al. 2004Stuart SN, Chanson JS, Cox NA, Young BE, Rodrigues ASL, Fischman DL, Waller RW (2004) Status and trends of amphibian declines and extinctions worldwide. Science 306: 1783-1786. https://doi.org/10.1126/science.1103538
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).

Over the past decade, the comet assay or single-cell gel electrophoresis (SCGE) has become one of the standard methods for assessing DNA damage, with applications in genotoxicity testing, biomonitoring and molecular epidemiology, as well as fundamental research in ecogenotoxicology (Collins 2004Collins AR (2004) The comet assay for DNA damage and repair: principles, applications, and limitations. Molecular Biotechnology 26: 249-261. https://doi.org/10.1385/MB:26:3:249
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). The comet assay is now considered one of the most promising genotoxicity tools to detect a broad spectrum of DNA lesions, with very high sensitivity in aquatic species (Jha 2008Jha AN (2008) Ecotoxicological applications and significance of the comet assay. Mutagenesis 23: 207-221. https://doi.org/10.1093/mutage/gen014
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, Frenzilli et al. 2009Frenzilli G, Nigro M, Lyons B (2009) The Comet assay for the evaluation of genotoxic impact in aquatic environments. Mutation Research/reviews in Mutation Research 681: 80-92. https://doi.org/10.1016/j.mrrev.2008.03.001
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, Bolognesi and Cirillo 2014Bolognesi C, Cirillo S (2014) Genotoxicity biomarkers in aquatic bioindicator. Current Zoology 60: 273-284.).

Physalaemus cuvieri Fitzinger, 1826, known commonly as barker frog, belongs to Leptodactylidae. The reproductive activity of the species begins in late September and extends through March (Bastos et al. 2003Bastos RP, Motta JAO, Lima LP, Guimarães LD (2003) Anfíbios da Floresta Nacional de Silvânia, Estado de Goiás. Goiânia, R.P. Bastos.). Individuals lay eggs in foam nests directly on the water (Mijares et al. 2010Mijares A, Rodrigues MT, Baldo D (2010) Physalaemus cuvieri. The IUCN Red List of Threatened Species, version 2014.3. http://www.iucnredlist.org [Accessed: 09/01/2015]
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). This species is widely spread in the east-central region of South America, from northeastern Brazil to eastern Paraguay and northern Argentina, including several protected areas (Mijares et al. 2010Mijares A, Rodrigues MT, Baldo D (2010) Physalaemus cuvieri. The IUCN Red List of Threatened Species, version 2014.3. http://www.iucnredlist.org [Accessed: 09/01/2015]
http://www.iucnredlist.org...
). The aim of the present study was to evaluate the potential genotoxicity of the environmental matrix (agricultural areas associated to pesticide use) using P. cuvieri tadpoles as bioindicators. We selected this species as a sentinel organism (bioindicator) in view of its wide geographic distribution, dependence on the aquatic environmental and ease of handling.

MATERIAL AND METHODS

This study was conducted during the rainy season in the Brazilian Cerrado biome from November 2013 to January 2014, in the municipalities of Bela Vista (16°58’24”S, 48°57’35”W), Bonfinópolis (16°37’2”S, 48°57’36”W), Caldazinha (16°42’17”S, 48°59’43”W), Leopoldo de Bulhões (16°42’17”S, 48°59’43”W), and Silvânia (16°38’35”S, 48°36’15”W), all of which are situated in the state of Goiás (Fig. 1). We sampled 177 tadpoles (Table 1) of P. cuvieri in stage 37 (sensu Gosner 1960Gosner KL (1960) A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica 16: 183-190.). All specimens were placed in plastic bags containing water samples from the same pond from which they had been captured. They were kept alive until they were brought to the laboratory. The permission for collecting was granted by ICMBio, a Brazilian Environmental Institute linked to the Ministry of Environment (code 18163-1). In all experiments, animal care was performed following the guidelines of the Ethical Committee on Animal Use (CEUA-UFG), in accordance with the National Council for Animal Experiments Control (CONCEA). Voucher specimens were deposited in the zoological collection of the Universidade Federal de Goiás (ZUFG). For the test group, 83 tadpoles were collected from nine permanent ponds from agricultural lands (four in soybean and five in corn). To improve the accuracy of the sampling design, areas where there was only soybean or corn were selected. All samples were performed during pesticide application campaigns. For the control group (non-agricultural areas) we sampled from eleven permanent ponds (94 tadpoles) in areas where no agricultural activities had taken place in the last six months. Table 1 represents the sampled areas for tadpoles, and the pesticides used according to information provided by the farmers, from November 2013 to January 2014. We quantified the percentage of non-natural areas around each pond with a buffer radius of 480m, using the ArcGIS (10).

Figure 1
Spatial distribution of the ponds sampled at five municipalities in the state of Goiás.

Table 1
Sample areas of the Physalaemus cuvieri tadpoles (pond, city), number of individuals (n) and type of pesticides (related by the farmers), from November/2013 to January/2014.

Tadpoles were not fed, and four hours after the sampling they were anesthetized for approximately two minutes in a 5% benzocaine solution. Blood samples were obtained by a transversal cut in the tail. We performed the alkaline comet assay method described by Singh et al. (1988Singh NP, McCoy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Experimental Cell Research 175: 184-191. https://doi.org/10.1016/0014-4827(88)90265-0
https://doi.org/10.1016/0014-4827(88)902...
) with a few modifications. Fifteen µl erythrocytes mixed with 0.5% LMP agarose were placed on normal 1.5% agarose microscope slides. The essential steps of comet assay involved at least three hours of cell lysis by detergent at a high salt concentration (1% Triton X-100, 10% DMSO, Stock Lysis Solution pH = 10; at 4 °C). Electrophoresis under alkaline conditions (300 mM NaOH, 1 mM EDTA, pH > 13, 25 min unwinding, 25 min electrophoresis at 300 mA and 25V, at 4 °C). Nucleoids were stained with 20 µg/ml ethidium bromide (EB). We analyzed 50 nucleoids per slide, totaling 100 nucleoids per sample. The analysis was performed by a fluorescence microscopy system called Axioplan-Imaging using the Isis software with an excitation filter of 510-560 nm and a barrier filter of 590 nm, with 20× objective.

For the evaluation of genomic damage, we used the TriTek Comet ScoreTM program, version 1.5. This software evaluated pixel intensity to provide corresponding values to estimate genomic damage, as arbitrary units (AU). We quantified genomic damages with tail length (TL), the percentage of DNA in the tail (% DNA), and the Olive tail moment (OTM) (Collins 2004Collins AR (2004) The comet assay for DNA damage and repair: principles, applications, and limitations. Molecular Biotechnology 26: 249-261. https://doi.org/10.1385/MB:26:3:249
https://doi.org/10.1385/MB:26:3:249...
).

Statistical analyses were based on the average of TL parameters,% DNA, and OTM analyzed for each individual. Previously, we performed the Kolmogorov-Smirnov (K-S) test in order to verify the normality of the three comet parameters. To test the discriminative power of the comet parameters we performed a discriminant function analysis using agricultural and non-agricultural areas as grouping variables and the comet parameters TL,% DNA, and OTM as explanatory variables. We also performed an analysis of variance (ANOVA) among soybean, corn, and non-agricultural areas also considering the three comet parameters. The principal components analysis (PCA) was performed to observe the dispersal patterns of locations and their relationship to the three comet variables using the pond area (in square meters) and the percentage of non-natural area as covariates. Therefore, we used the method based on the correlation matrix. Finally, we also carried out a simple regression analysis in order to verify the relationship between genomic damage and the pond area occupied by the tadpoles. All statistical analyses were performed using the statistical package SPSS 23.0 and STATISTICA 10, with a 5% significance level.

RESULTS

All the points associated with the agricultural areas showed positive scores, in contrast with non-agricultural areas, which had negative scores. We found that the% DNA presented the highest contribution (F = 180.3, p = 8.82e-29), followed by OTM (F = 178.54, p = 1.59e-28) revealing DNA damage. In addition, statistically significant differences were found between the agricultural and non-agricultural areas for all parameters of the comet (Fig. 2).

Figure 2
Differences between the average scores of Soybean, Corn and Non-agricultural lands, generated by the discriminant function scores.

Tadpoles located in soybean areas had the highest stretches of DNA damage estimated by the TL parameter (9.39 ± 1.08), followed by tadpoles in corn fields (7.95 ± 0.23), differing significantly from the damage suffered by the tadpoles in the non-agricultural areas (7.25 ± 0.60) (Fig. 2). Regarding% DNA, we also found significant differences between sites. Tadpoles sampled from ponds next to soybean fields showed the highest values of this parameter (7.54 ± 0.47), while tadpoles occupying ponds surrounding corn fields presented an average of 6.73 ± 0.55, differing from the values found in the non-exposed areas (5.04 ± 1.39). Tadpoles occupying areas surrounding soybean presented the greatest genomic damage for OTM (35.67 ± 6.14). In corn, the average value was 26.92 ± 2.64, whereas in the non-agricultural areas the average was 18.90 ± 5.97.

We observed a separation between the points associated with the soybean, corn, and non-agricultural areas (Table 2, Fig. 3). The PCA 1 accounts for 57.4% of the total variance, whereas the PCA 2 explains 21.5%. Thus, there was a negative correlation between the three comet assay variables and the pond area. The OTM parameter had the higher contribution in the separation between points (Table 2, Fig. 3). On the second axis, the% DNA had a greater negative association with the area of the pond. In addition, in relation to the first axis we observed a negative association of genomic damage to the percentage of natural area within the buffer (Fig. 3). We observed that the points associated with soybean were clearly separated from the other points, as shown by the first canonical axis in Fig. 3.

Figure 3
Principal component analysis (PCA) of the descriptor variables and the covariate pond area. Relationship between the PCA 1 and PCA 2, points were grouped according to the soybean (white triangle), corn (white circles) and non-agricultural lands (black circles). (TL) Tail Length, (% DNA) percentage of DNA in Tail, (OTM) Olive tail moment, (RNV) Remnant native vegetation.

Table 2
Results from a principal component analysis of comet assay parameters, related to pond area and remnant native vegetation (RNV). The factors loading for all three comet assay parameters, pond area and RNV and those higher than 0.05 are in bold. (TL) Tail length, (% DNA) % DNA in tail, (OTM) Olive tail moment, (EV) eigenvalue, (V) variance (%), (CV) Cumulative variance (%).

Considering the soybean crop, we found a negative correlation between the TL and the ponds area (r = -0.75, p = 6.0e-05), i.e., the smaller the area of the pond, the greater the genomic damage. Conversely we observed a positive correlation in areas where corn was cultivated (r = 0.44, p = 4.0e-04) or the non-agricultural areas (r = 0.45; p = 6.0e-05) (Fig. 4a). In the association of the pond area with% DNA, we found that only in the soybean crops there was a negative correlation (r = -0.73, p = 4,0e-06), while the corn crop and non-agricultural areas had positive correlations (r = 0.28, p = 0.036 and r = 0.19, p = 0.092, respectively) (Fig. 4b). We still found a negative correlation between the OTM parameter and the area of the ponds located in soybean fields (r = -0.76, p = 3.0e-06), in contrast with corn areas and non-agricultural areas that showed a positive correlation (r = 0.35, p = 0.036 and r = 0.28, p = 0.015, respectively) (Fig. 4c).

Figures 4-6
Relationship between comet assay parameters and the pond area according to soybean (gray squares), corn (white circles) and non-agricultural lands (black circles); 4) TL with pond area; 5)% DNA; 6) OTM with pond area. (TL) Tail Length, (% DNA) percentage of DNA in Tail, (OTM) Olive tail moment.

DISCUSSION

We found significant differences in DNA damage among specimens collected from soybean, corn and non-agricultural fields in all parameters of the comet assay. Tadpoles in ponds surrounded by soybeans presented more DNA damages, followed by tadpoles collected from ponds in corn fields. In contrast, the tadpoles sampled from non-agricultural areas had the lowest rates of DNA damage. In addition, in soybean fields alone, we found a negative correlation between the parameters of the comet assay and the area of the ponds. That is, the smaller the area of the pond the more extensive the genomic damage was. This correlation indicates that, since the concentration of pesticides is more diluted in larger pools, the number of genomic lesions they cause would be mitigated by dilution. In soybean fields, the pesticide more commonly used is glyphosate. A number of studies have demonstrated that this pesticide can cause the generation of free radicals and reactive oxygen species in bullfrogs (Costa et al. 2008Costa MJ, Monteiro DA, Oliveira-Neto AL, Rantin FT, Kalinin AL (2008) Oxidative stress biomarkers and heart function in bull frog tadpoles exposed to Round up original. Ecotoxicology 17: 153-163. https://doi.org/10.1007/s10646-007-0178-5
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) and other organisms, such as fish species (Nwani et al. 2013Nwani CD, Nagpuer NS, Kumar R, Kushwaha B, Lakra WS (2013) DNA damage and oxidative stress modulatory effects of glyphosate-based herbicide in fresh water fish, Channa punctatus. Environmental Toxicology and Pharmacology 36: 539-547. https://doi.org/10.1016/j.etap.2013.06.001
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), increasing DNA damage.

According to Giesy et al. (2000Giesy JP, Dobson S, Solomon KR (2000) Ecotoxicological Risk Assessment for Roundup Herbicide. Reviews of Environmental Contamination and Toxicology 167: 35-120. https://doi.org/10.1007/978-1-4612-1156-3_2
https://doi.org/10.1007/978-1-4612-1156-...
) an aggravating factor in the use of glyphosate, mainly in soybean fields, is its long life in water bodies, reaching up to 70 days. Due to its high solubility in water and extensive use in the environment, the harmful effects of glyphosate on aquatic organisms are of great concern (Cavas and Könen 2007Cavas T, Konen S (2007) Detection of cytogenetic and DNA damage in peripheral erythrocytes of goldfish (Carassius auratus) exposed to a glyphosate formulation using the micronucleus test and the comet assay. Mutagenesis 22: 263-268. https://doi.org/10.1093/mutage/gem012
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). However, several studies have indicated that the toxic effect of glyphosate is not only in its active principle, but mainly in polyoxyethylene amine surfactant (POEA), which is present in its most common commercial formulation, Roundup (Mann and Bidwell 1999Mann RM, Bidwell JR (1999) The toxicity of glyphosate and several glyphosate formulations to four species of southwestern Australian frogs. Archives of Environmental Contamination and Toxicology 36: 193-199. https://doi.org/10.1007/s002449900460
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, Perkins et al. 2000Perkins PJ, Boermans HJ, Stephenson GR (2000) Toxicity of glyphosate and triclopyr using the frog embryo teratogenesis assay-Xenopus. Environmental Toxicology and Chemistry 19: 940-945., Howe et al. 2004Howe CM, Berrill M, Pauli BD, Helbing CC, Werry K, Veldhoen N (2004) toxicity of glyphosate-based pesticides to four North american frog species. Environmental Toxicology and Chemistry 23: 1928. https://doi.org/10.1897/03-71
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The results found in our study justify the growing concern over the continued increase in the use of different classes of pesticides in agriculture (Cerejeira et al. 2003Cerejeira M, Viana P, Batista S, Pereira T, Silva E, Valério M, Silva A, Ferreira M, Silva-Fernandes A (2003) Pesticides in Portuguese surface and ground waters. Water Research 37: 1055-1063. https://doi.org/10.1016/S0043-1354(01)00462-6
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, Konradsen2007Konradsen F (2007) Acute pesticide poisoning - a global public health problem. Danish Medical Bulletin 54: 58-59., Alavanja 2009Alavanja MCR (2009) Pesticides Use and Exposure Extensive Worldwide. Reviews on Environmental Health 24: 303-309. https://doi.org/10.1515/REVEH.2009.24.4.303
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). Unfortunately, the indiscriminate use of a wide range of pesticides, in order to improve agricultural production, ends up generating a negative impact on non-target organisms, especially anurans that depend on the water until the adult stage (See review of Sparling et al. 2010Sparling D, Linder G, Bishop C, Krest S (2010) Ecotoxicology of Amphibians and Reptiles . CRC Press, 2nd ed. http://www.crcnetbase.com/doi/book/10.1201/EBK1420064162, https://doi.org/10.1201/EBK1420064162
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). It is worth mentioning that the main period of application of pesticides in the area usually occurs during the rainy season, coinciding with the breeding season of amphibians, from November to March (Mijares et al. 2010Mijares A, Rodrigues MT, Baldo D (2010) Physalaemus cuvieri. The IUCN Red List of Threatened Species, version 2014.3. http://www.iucnredlist.org [Accessed: 09/01/2015]
http://www.iucnredlist.org...
) which could cause developmental and reproductive failures, as described by many authors worldwide (Carey and Bryant 1995Carey C, Bryant CJ (1995) Possible interrelations among environmental toxicants, amphibian development, and decline of amphibian populations. Environmental Health Perspectives 103: 13-17. https://doi.org/10.2307/3432406
https://doi.org/10.2307/3432406...
, Lowcock et al. 1997Lowcock LA, Sharbel TF, Bonin J, Quellet M, Rodrigue J, DesGranges JL (1997) Flow cytometric assay for in vivo genotoxic effects of pesticides in Green frogs (Rana clamitans). Aquatic Toxicology 38: 241-255. https://doi.org/10.1016/S0166-445X(96)00846-6
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, Ralph and Petras 1997Ralph S, Petras M (1997) Genotoxicity monitoring of small bodies of water using two species of tadpoles and the alkaline single cell gel (comet) assay. Environmental and Molecular Mutagenesis 29: 418-430., Thompson et al. 2004Thompson DG, Wojtaszek BF, Staznik B, Chartrand DT, Stephenson GR (2004) Chemical and Biomonitoring to Assess Potential Acute Effects of Vision Herbicide on Native Amphibian Larvae in Forest Wetlands. Environmental Toxicology and Chemistry 23: 843. https://doi.org/10.1897/02-280
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).

Although we did not measure the concentration of pesticides in the ponds, we sampled the tadpoles during the pesticide application campaigns, in all sites. Our study did not attempt to sort out which pesticide and/or concentrations are responsible for genomic damage; the purpose was to ascertain the stress caused by the agricultural activities associated with the use of complex mixtures of pesticides, as those we observed, including herbicides, insecticides and fungicides. Farmers use this combination to decrease the total time of insecticide applications (Pedlowski et al. 2012Pedlowski MA, Canela MC, da Costa Terra MA, Ramos de Faria RM (2012) Modes of pesticides utilization by Brazilian smallholders and their implications for human health and the environment. Crop Protection 31: 113-118. https://doi.org/10.1016/j.cropro.2011.10.002
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). Among the pesticides applied during the sample collection were glyphosate - Roundup (herbicide), alto-100 (fungicide); dimethoate, MD (insecticide and acaricide); atrazine (herbicide), carbofuran - Furadan (insecticide), Lannate (insecticide) and malathion (pesticide). Indeed, there are several studies with tadpoles, under controlled laboratory conditions, that have drawn attention to the cytotoxic, mutagenic and genotoxic effects of these pesticides and their susceptibility may vary depending on the studied species (Howe et al. 2004Howe CM, Berrill M, Pauli BD, Helbing CC, Werry K, Veldhoen N (2004) toxicity of glyphosate-based pesticides to four North american frog species. Environmental Toxicology and Chemistry 23: 1928. https://doi.org/10.1897/03-71
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, Relyea 2005Relyea RA (2005) The Lethal Impact of Roundup on Aquatic and Terrestrial Amphibians. Ecological Applications 15: 1118-1124. https://doi.org/10.1890/04-1291
https://doi.org/10.1890/04-1291...
, Relyea and Jones 2009Relyea RA, Jones DK (2009) The toxicity of Roundup Original Max to 13 species of larval amphibians. Environmental Toxicology Chemistry 28: 2004-2008. https://doi.org/10.1897/09-021.1
https://doi.org/10.1897/09-021.1...
, Bernal et al. 2009Bernal MH, Solomon KR, Carrasquilla G (2009). Toxicity of formulated glyphosate (glyphos) and cosmo-flux to larval and juvenile colombian frogs 2. Field and laboratory microcosm acute toxicity. Journal of Toxicology and Environmental Health. A 72: 966-73. https://doi.org/10.1080/15287390902929717
https://doi.org/10.1080/1528739090292971...
, Bosch et al. 2011Bosch B, Mañas F, Gorla N, Aiassa D (2011) Micronucleus test in post metamorphic Odontophrynus cordobae and Rhinella arenarum (Amphibia: Anura) for environmental monitoring. Journal of Toxicology and Environmental Health Sciences 3: 155-163., Meza-Joya et al. 2013Meza-Joya FL, Ramírez-Pinilla MP, Fuentes-Lorenzo JL (2013) Toxic, cytotoxic, and genotoxic effects of a glyphosate formulation (RoundupSL-Cosmoflux411F) in the direct-developing frog Eleutherodactylus johnstonei. Environmental and Molecular Mutagenesis 54: 362-73. https://doi.org/10.1002/em.21775
https://doi.org/10.1002/em.21775...
,; Yadav et al. 2013Yadav SS, Giri S, Singha U, Boro F, Giri A (2013) Toxic and genotoxic effects of Roundup on tadpoles of the Indian skittering frog (Euflictis cyanophlyctis) in the presence and absence of predator stress. Aquatic Toxicology 132-133: 1-8. https://doi.org/10.1016/j.aquatox.2013.01.016
https://doi.org/10.1016/j.aquatox.2013.0...
). Our study, however, differs from those in that it measured genomic damages in animals exposed in their natural habitat, in real conditions of contamination.

It is worth mentioning that if genomic damages are not repaired, the DNA damage may be fixed, after, at least, one cell cycle. In this case, mutations arise and may impact the survival of the affected animals. It is known that the effects of pesticides are especially concerning in aquatic environments, which are particularly vulnerable as they have several exposure routes for the influx of chemicals. These effects are of particular concern as biodiversity loss reaches unprecedented rates. This includes recent declines in amphibian populations and loss of amphibian species (Makkimane and Krishnamurthy 2013Makkimane BN, Krishnamurthy SV (2013) Exposure of tadpoles of Fejervarya limnocharis (Anura: Ranidae) to combinations of carbaryl and cypermethrin. Toxicological and Environmental Chemistry 95: 1408-1415. https://doi.org/10.1080/02772248.2014.881828
https://doi.org/10.1080/02772248.2014.88...
).

Finally, our results indicate that the tadpoles of P. cuvieri are good bioindicators when the alkaline comet assay is used, and that the combination of the two can be used for biomonitoring studies of agricultural areas. However, the methodology for field studies needs to be standardized, so that the results of different surveys can be compared. In summary, genotoxicity studies involving amphibian tadpoles may be more informative and applied routinely to assess the impact of anthropogenic environments and/or exposure to pesticides.

ACKNOWLEDGMENTS

The authors acknowledged S. Quail for proofreading this manuscript. We also thank Projeto Girinos do Brasil (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Edital SISBIOTA, Process CNPq 563075/2010-4 and FAPESP 2010/52321-7) and to Fundação de Amparo à Pesquisa de Goiás (Process: 201210267001094, Universal/2012 and 201210767000812, Pronex). ADC, DMS, NMM and RPB thank the CNPq fellowship, and MWG, PG and FRG thank the individuals and organizations that have granted their scholarships. We thank T.S. Nascimento and A. Morais for the suggestions to improve the paper.

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  • Editorial responsibility:

    Carolina Arruda Freire
  • Zoobank:

    http://zoobank.org/A65FFC07-75B6-4DE4-BE59-8CE6BB2D4448

Data availability

Data citations

Mijares A, Rodrigues MT, Baldo D (2010) Physalaemus cuvieri The IUCN Red List of Threatened Species, version 2014.3. http://www.iucnredlist.org [Accessed: 09/01/2015]

Publication Dates

  • Publication in this collection
    2017

History

  • Received
    17 Apr 2016
  • Reviewed
    28 Nov 2016
  • Accepted
    04 Jan 2017
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