Genotoxic effects of glyphosate on Physalaemus tadpoles

https://doi.org/10.1016/j.etap.2020.103516Get rights and content

Highlights

  • Glyphosate based formulation induced micronucleus formation in Physalaemus species.

  • All tested concentrations of Glyphosate caused erythrocyte nuclear abnormalities.

  • Glyphosate concentration permit by the Brazilian legislation for clean water can be genotoxic.

Abstract

Genotoxicity studies have revealed that pesticides bind to genetic material in non-target vertebrates, thereby impairing the genetic integrity of these animals. The main objective of this study was to determine the genotoxic damage in erythrocytes of two native South American amphibian Physalaemus cuvieri and Physalaemus gracilis, both species exposed to a glyphosate-based herbicide. We evaluated the presence of micronuclei (MN) and erythrocyte nuclear abnormalities (ENA) as biomarkers for potential genotoxic compounds. Tadpoles were exposed to doses permitted by Brazilian legislation and concentrations found naturally in Brazilian and Argentinian waters (500, 700 and 1000 μg/L). Glyphosate-based herbicide caused micronuclei formation and several types of erythrocyte nuclear abnormalities in both Physalaemus species. The total frequency of MN and ENA demonstrated the occurrence of cell damage at all tested concentrations. Glyphosate herbicide can be considered a genotoxic that may impact the genetic integrity of native populations of P. cuvieri and P. gracilis.

Introduction

Anuran amphibians are recognized as the most endangered vertebrates in the world, facing extinction or population decline (González-del-Pliego et al., 2019). Aquatic residual contaminants are among the causes for declines in amphibian populations due to of their physiological and toxicological effects (Egea-Serrano et al., 2012). Pesticides are thought to be the aquatic pollutants that contribute most to the worldwide decline of amphibian populations (Hayes et al., 2010; Lavorato et al., 2013), mainly because many species live in association with temporary ponds and small streams adjacent to agricultural areas (Howe et al., 2004; Hayes et al., 2010; Muniswamy et al., 2012).

Amphibians remain in the water during their most critical development stages (eggs and early life stages). Their eggs are unprotected, and both tadpole and adult skins are highly permeable (Hayes et al., 2006). In this way, they are susceptible to any aquatic contaminant (Bouhafs et al., 2009; Sparling and Fellers, 2009; Brühl et al., 2011). For these reasons, amphibians are recognized as a group of vertebrates suitable for investigating the genotoxic potential of pesticide formulations in the aquatic environment (Howe et al., 2004; Gonçalves et al., 2019).

Micronucleus assessment has been widely used as the most reliable evaluation for demonstrating mutagenicity, hazard identification and risk assessment (Hayashi, 2016). Micronucleus (MN) is a small chromatin containing round-shaped body visible in the cytoplasm of cells, and appears as a result of inducing different types of DNA damage and is also considered an indicator of chromosomal instability (Sommer et al., 2020). The MN are associated with the occurrence of both structural and numerical chromosome mutations induced by clastogenic agents and chromosomal segregation errors due to the action of chemicals (Russo and Degrassi, 2018).

Micronucleus techniques along with other categories of erythrocyte nuclear abnormalities (ENA), are extensively applied methods among currently available assays and act as early iindicators of pollution alert (Benvindo-Souza et al., 2020). The erythrocites of amphibians are nucleated cells and are considered models for studying cellular abnormalities. Nuclear abnormalities include: anucleated cells, apoptosis, binucleated cells, notched nucleus, lobed nucleus and nuclei with other morphological changes (Carrasco et al., 1990; Benvindo-Souza et al., 2020).

The ENA suggest cytotoxic, genotoxic, mutagenic or carcinogenic activity by the tested substances (Attademo et al., 2011; Gökalp-Muranli and Güner, 2011; Rocha et al., 2012). Thus, MN and ENA are being used to assess the initial effects of chronic exposure to xenobiotics, since these responses can be detected prior causing damage to amphibians populations (Adams et al., 2000, 2001; Furnus et al., 2014).

Glyphosate is the most extensively used active ingredient in pesticides worldwide, mainly because transgenic plants are resistant to this herbicide (Myers et al., 2016). The major concern about the intensive use of glyphosate is the increasingly ubiquitous presence in the environment which can cause severe damage to a wide variety of animals in the larger ecosystem (Richmond, 2018). Glyphosate is classified as an organophosphate herbicide; it is readily dissolve in bodies of water and they may enter rivers and streams through run-off, affecting non-target organism (Gill et al., 2016). Recent studies have analyzed the effects of glyphosate on amphibians and highlight that this herbicide is toxic to a high number of species (e.g., Agostini et al., 2020; Bolis et al., 2020; Herek et al., 2020). However, there is a lack of available information about genotoxic and mutagenic effects in this animals group exposed to glyphosate (Benvindo-Souza et al., 2020).

Several amphibian species reproduce in regions associated with agricultural areas in South America and have a high potential for exposure to glyphosate; these species include Physalaemus cuvieri (dog frog) and Physalaemus gracilis (crying frog). These two species are tolerant to a wide range of habitats and occur in various locations in Argentina, Brazil, and Paraguay (Frost, 2020). They can be found in temporary water bodies such as puddles, often in open plant formations and anthropized environments (Achaval and Olmos, 2003; Lavilla et al., 2010). They are widely distributed and are usually abundant; nevertheless, P. cuvieri is considered locally threatened in Argentina because of habitat destruction caused by agriculture and logging, as well as land and water pollution caused by runoff (Mijares et al., 2010). P. cuvieri and P. gracilis can be considered good models for ecotoxicological assays (e.g., Macagnan et al., 2017; Wrubleswski et al., 2018; Herek et al., 2020).

This study aimed to evaluate the genotoxic potential of a glyphosate-based herbicide in two native South American amphibian species, Physalaemus cuvieri and Physalaemus gracilis, using the micronucleus assay as a biomarker.

Section snippets

Pesticide

We used a glyphosate-based herbicide, original Roundup Glyphosate® DI commercial formulation, a non-selective systemic herbicide. Its composition includes: N-(phosphonomethyl) glycine (glyphosate) Di-Ammonium Salt 445 g/L (44.5 % w/v), N-(phosphonomethyl) glycine (glyphosate) acid equivalent 370 g/L (37.0 % w/v) and other ingredients, 751 g/L (75.1 % w/v).

Glyphosate is included in the substituted glycine chemical group, considering its soluble formulation group (SL). According to the Original

Results

Analyses accounted for a total of 59850 erythrocytes for P. cuvieri and 62383 for P. gracilis. The occurrence of micronuclei (MN, Fig. 1B) in cells was significantly higher at 1000 μg/L of glyphosate-based herbicide (FPcuv (3, 54) = 4.23, p = 0.009, FPgra (3, 56) = 175.4, p < 0.0001; Fig. 2A-B). Neither species control showed micronuclei.

Regarding the eight types of erythrocyte nuclear abnormalities analyzed (ENA, Fig. 1C–J), eight of them were registered in both Physalaemus species, and the

Discussion

Glyphosate-based formulation induced micronucleus formation and various ENA in Physalaemus cuvieri and Physalaemus gracilis. Therefore, it can be considered genotoxic for both species. This finding corroborates with studies that show that glyphosate commercial formulations were genotoxic to different vertebrates, such as in fish (Cavalcante et al., 2008; Rodrigues et al., 2019), mice (Prasad et al., 2009), and humans (Koller et al., 2012). The herbicide formulation used in this study contains

Conclusions

All tested glyphosate concentrations induced nuclear abnormalities, representing cytotoxic and genotoxic damage in the two tested Physalaemus species. Considering the widespread use of commercial glyphosate formulations in crops, high concentrations of this herbicide represent a potential risk of genotoxicity, especially concerning the genetic integrity of native populations of P. cuvieri and P. gracilis.

Author contributions

Marilia T. Hartmann and Jéssica Samara Herek designed the research. Jéssica Samara Herek, Luana Vargas, Suélen Andressa Rinas Trindade, Camila Fatima Rutkoski and Natani Macagnan conducted the chronic assays, collected the samples and performed the micronucleus assay. Jéssica S. Herek and Marilia Hartmann analyzed the data. All authors discussed the data. Jéssica S. Herek wrote the manuscript with contributions from Marilia T Hartmann and Paulo A. Hartmann. Marilia T. Hartmann and Paulo A.

Compliance with ethical standards

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. This research was licensed by Brazilian Institute of Environment and Renewable Natural Resources (nº54939-1) and authorized by the Animal Use Ethics Committee of the Federal University of Southern Frontier (23205.003251/2016-11). This article does not contain studies with human participants performed by any of the authors.

Declaration of Competing Interest

The authors reported no declarations of interest.

Acknowledgements

The authors are grateful to the Federal University of Fronteira Sul—UFFS for providing logistical support. Luana Vargas and Suélen V. R. Trindade were supported by fellowship from Fundação de Amparo a Pesquisa do Estado do Rio Grande do Sul – FAPERGS. This study is financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior – Brazil (CAPES), finance code 001

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