Elsevier

Chemosphere

Volume 156, August 2016, Pages 37-44
Chemosphere

Effects of glyphosate and the glyphosate based herbicides Roundup Original® and Roundup Transorb® on respiratory morphophysiology of bullfrog tadpoles

https://doi.org/10.1016/j.chemosphere.2016.04.083Get rights and content

Highlights

  • We evaluated histological and respiratory as biomarkers of toxicity in tadpoles.

  • Glyphosate and Roundup formulations cause distinct skin alterations.

  • Glyphosate and Roundup formulations differently altered the respiratory function.

  • Bullfrog tadpoles' skin is very sensitive to glyphosate and Roundup formulations.

Abstract

Glyphosate-based herbicides are widely used in agriculture and are commonly found in water bodies. Roundup Original® (RO) contains an isopropylamine glyphosate (GLY) salt containing the surfactant POEA, while Roundup Transorb R® (RTR) contains a potassium salt of GLY with unknown surfactants. Both contain different compositions of so-called “inert” ingredients, more toxic than glyphosate. Amphibian tadpoles often experience variations in O2 availability in their aquatic habitats; an ability to tolerate hypoxia can condition their survival and fitness. We evaluated the impacts of sublethal concentrations of GLY (1 mg L−1), RO (1 mg L−1 GLY a.e) and RTR (1 mg L−1 GLY a.e) on metabolic rate (V·O2 – mLO2 Kg1 h−1) of bullfrog tadpoles during normoxia and graded hypoxia, and related this to morphology of their skin, their major site of gas exchange. In control (CT) V·O2 remained unaltered from normoxia until 40 mmHg, indicating a critical O2 tension between 40 and 20 mmHg. GLY significantly reduced V·O2, possibly due to epidermal hypertrophy, which increased O2 diffusion distance to O2 uptake. In contrast, RTR increased V·O2 during hypoxia, indicating an influence of “inert” compounds and surfactants. V·O2 of RO did not differ from CT, suggesting that any increase in V·O2 caused by exposure was antagonized by epidermal hypertrophy. Indeed, all herbicides caused marked alterations in skin morphology, with cell and epithelium wall presenting hyperplasia or hypertrophy and chromatid rupture. In summary, GLY, RO and RTR exert different effects in bullfrog tadpoles, in particular the surfactants and inert compounds appear to influence oxygen uptake.

Introduction

Glyphosate, N-(phosphonomethyl)glycin, is the broad-spectrum and post-emergent herbicide used most commonly worldwide (Duke and Powles, 2008). It acts specifically in plants and some microorganisms by inhibiting the enzyme 5-enolpyruvyl-shikimate- 3-phosphate synthase (EPSPS), which catalyzes the synthesis of aromatic amino acids that are essential for survival (Williams et al., 2000). In contrast, aromatic amino acids are obtained directly from the diet in animals, which consequently lack EPSPS such that glyphosate does not act directly on them (Cerdeira and Duke, 2007).

Several different formulations of glyphosate-based herbicides are currently in use, but Roundup® (Monsanto, Saint Louis, MO, USA) formulations have been increasingly utilized due to their close association with genetically modified glyphosate-resistant crop plants (Giesy et al., 2000). The different Roundup® formulations result from the association of glyphosate salts with surfactants, an important component of the herbicide that permits glyphosate to pass through the waxy cuticle of plant leaves (Modesto and Martinez, 2010).

Roundup Original® was the first commercially available formulation, it contains a glyphosate isopropylamine salt and polyethoxylated tallowamine (POEA) as the surfactant, which comprises about 15% of the herbicide formulation (Howe et al. 2004). The remaining 68.4% (m/v) of other components are generically termed “inert ingredients”. Many studies have indicated that POEA is the most toxic component in this formulation (Brausch et al., 2007, Perkins, 2000), while the inert ingredients may also harm humans and the environment (Cox and Surgan, 2006). In this sense, ecotoxicological studies on commercial glyphosate-based formulations are needed to provide information about their toxicity to non-target organisms (Piola et al., 2013).

Importantly, many new glyphosate-based herbicides composed of different blends of surfactants and “inert compounds” have been widely applied, although their toxic effects on non-target animals are poorly known. Unlike Roundup Original®, Roundup Transorb R® is based on a potassium-salt of glyphosate and a higher content of “inert ingredients” (82% m/v). According to the manufacturer, Roundup Transorb R® can be easily absorbed and translocated into the target weed, nevertheless, the surfactant and/or chemical compounds driving these properties in plants remain unidentified. According to Modesto and Martinez (2010), studies focusing on Roundup Transorb® herbicide, a predecessor of Roundup Transorb R®, suggest that this product is potentially more toxic than the original formulation (Dos Santos et al., 2005; Howe et al., 2004).

Once these agrochemicals have been extensively applied in modern agriculture, risks of environmental contamination are aggravated (Wagner and Lötters, 2013). These chemicals can reach water bodies, such as lakes, streams, ponds or marshy areas, by agricultural runoff and leaching processes, as well as by direct applications to control aquatic weeds (Giesy et al., 2000, Thompson et al., 2004). These environments may be important microhabitats for survival, reproduction and development of many amphibians. As a consequence, their contamination could jeopardize these sensitive components of aquatic biota (Johansson et al., 2006). Indeed, data indicate that environmental contamination by pesticides is one of the main factors causing global amphibian population declines (Mann et al., 2009, Smith et al., 2011, Sparling and Fellers, 2009).

LC50 (96-h) values for different species of tadpoles exposed to glyphosate-based herbicides with POEA ranged around 1.8–12 mg L−1 a.e, showing that anuran larvae are among the most sensitive vertebrates (Moore et al., 2012; Wojtaszek et al., 2004; Relyea, 2005). Additionally, several laboratory, field and mesocosm toxicological studies with different species of tadpole have demonstrated that theses formulations also cause distinct impacts on mortality, growth and development that can be exacerbated in natural environments (Edge et al., 2014, Edge et al., 2013, Edge et al., 2012, Lanctôt et al., 2013) Furthermore, physiological, biochemical (Costa et al., 2008) and morphological (Howe et al., 2004; Orton and Routledge, 2011) alterations have been reported as a result of sublethal exposure of tadpoles to this herbicide, which could impair their development and/or survival (Relyea, 2005).

Tadpole skin is an extremely thin and permeable cutaneous epithelium, and is the major gas exchange surface for several species (Tattersall and Ultsch, 2008). Bullfrog tadpoles (Lithobates catesbeianus, Shaw, 1802), for instance, obtain around 70% of their O2 uptake through the skin (Martin and Warren, 1985). Acting as an aquatic respiratory surface, like gills, the tadpole cutaneous epithelium is also the first target for xenobiotic molecules. Due to its direct contact with water and its delicate structure, which is essential to ensure a short diffusion distance for gases between water and blood, the tadpole cutaneous integument is very susceptible to damage. The goal of this study was to investigate possible effects of sublethal concentrations of the active ingredient (pure glyphosate) and of two different commercial formulations of Roundup® (Original® and Transorb R®), on the capacity for oxygen uptake and the skin morphology of bullfrog tadpoles. The ability to regulate aerobic metabolism during progressive aquatic hypoxia was used as an indicator of capacity for oxygen uptake. Once the two formulations of herbicide present distinct compositions of surfactants and other unknown “inert compounds” that may add different proprieties in comparison to pure glyphosate, the analysis of their histopathological and respiratory effects on tadpoles can be a useful tool to access the harmful effects of each formulation.

Section snippets

Animals

Newly hatched Lithobates catesbeianus tadpoles (n = 40) were obtained at Santa Rosa breeding colony, Santa Barbara d’Oeste, São Paulo State, Brazil. They were housed in 500 L holding tanks equipped with a continuous supply (1.2 L/h) of well-aerated and dechlorinated artesian wellwater, filtered through sand and sterilized by UV, under natural photoperiod (∼12 h light/dark cycle) until they reached 25 Gosner (1960) developmental stage (∼1 week), with a body mass of 3.1 ± 0.1 g (mean ± SD). The

Results

The effects of graded hypoxia onV·O2 of the different experimental groups are shown in Fig. 1(A–C). The CT group maintained a constant V·O2 from normoxia down to a PinO2 of 40 mm Hg (42.7 ± 1.0 mLO2 Kg−1 h−1), decreasing significantly at 20 mmHg (13.9 ± 3.1 mLO2 Kg−1 h−1). In the GLY group (Fig. 1A), VO2 values were constant over the PinO2 interval from 140 to 80 mm Hg (33.9 ± 1.9 mLO2 Kg−1 h−1). Below this, V·O2 decreased progressively and significantly with further reductions in the PinO2,

Discussion

In the present study, histological analysis of tadpole dorsal skin revealed that the delicate epidermis was affected differently by each herbicide formulation. The increase in epidermis thickness in GLY group was also described by Bueno-Guimarães et al. (2001), who reported epithelial skin and gill hyperplasia in bullfrog tadpoles exposed to formaldehyde at concentrations of 0.5 and 2.0 mg L−1 for 24, 48, and 96 h. As with formaldehyde, glyphosate is extremely hydrophilic and has a reduced

Ethics in animal experimentation

This study was conducted in accordance with the Brazilian legislation (Law #11.794, October 8, 2008). All capture, holding and experimental techniques were performed under the approval of the Federal University of São Carlos Committee of Ethics in Animal Experimentation (CEUA – approval #022/2010) and Committee of Environmental Ethics (CEA – approval #023/2009).

Acknowledgments

This study was supported by São Paulo State Research Foundation (FAPESP, Proc. #10/03607-5) and National Institute for Science and Technology in Comparative Physiology (INCT-FisC/FAPESP, Proc. #08/57712-4). We are also grateful to Mr. Angelo Canevarolo and Mr. Fernando Urban Gamero for technical assistance and Catherine Williams for the text review.

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