Developmental effect of parental or direct chronic exposure to environmental concentration of glyphosate on the larvae of rainbow trout, Oncorhynchus mykiss

Highlights

• Glyphosate exposure did not increase the mortality rate of trout early stages

• Direct exposure to glyphosate induced biometric changes

• Intergenerationally exposed larvae showed behavioural changes

• Parental exposure to Roundup modified markers of energetic metabolism

• No correlation found between cellular and individual effects

Abstract

The environmental safety profile of glyphosate, the most commonly used herbicide worldwide, is still a subject of debate and little is known about the generational toxicity of this active substance (AS) and the associated commercial formulations called ”glyphosate-based herbicides” (GBHs). This study investigated the impact of parental and direct exposure to 1$\mu$gL${}^{-1}$ of glyphosate using the AS alone or one of two GBH formulations (i.e. Roundup Innovert® and Viaglif Jardin®) in the early developmental stages of rainbow trout. Three different modes of exposure on the F1 generation were studied: (1) intergenerational (i.e. fish only exposed through their parents); (2) direct (i.e. fish exposed only directly) and (3) multigenerational (i.e. fish both exposed intergenerationally and directly). The impact of chemical treatments on embryo-larval development (survival, biometry and malformations), swimming behaviour, biochemical markers of oxidative stress equilibrium (TBARS and catalase), acetylcholine esterase (AChE) and energy metabolism (citrate synthase, CS; cytochrome-c oxidase, CCO; lactate dehydrogenase, LDH; glucose-6-phosphate dehydrogenase, G6PDH) was explored. Chemical exposure did not affect the survival of F1 embryos or malformation rates. Direct exposure to the AS induced some biometric changes, such as reduction in head size (with a 10% decrease in head length), independently of co-formulants. Intergenerational exposure to the AS or the Roundup GBH increased swimming activity of the larvae, with increase of between 78 and 102% in travel speeds. Viaglif co-formulants appear to have counteracted this behavioural change. The minor changes detected in the assayed biochemical markers suggested that observed effects were not due to oxidative damage, AChE inhibition or alterations to energy metabolism. Nonetheless, multi- and intergenerational exposure to Roundup increased CS:CCO and LDH:CS ratios by 46% and 9%, respectively, with a potential modification of the aerobic-to-anaerobic energy production balance. These biochemical effects were not correlated with those observed on individual level of biological organization. Therefore, further studies on generational toxicity of glyphosate and its co-formulants are needed to identify the other mechanisms of glyphosate toxicity at the cellular level.

Introduction

Glyphosate is the most commonly used herbicidal active substance (AS) in the world (Benbrook, 2016). Due to its efficiency in controlling weed development, this agrochemical rapidly became vital to the agricultural sector, which has adapted its practices to this herbicide, particularly with the development of genetically modified glyphosate-resistant crops (Székács, Darvas, 2018, Torretta, Katsoyiannis, Viotti, Rada, 2018). The constant increase in the use of this herbicide throughout the world over the past years has made it ubiquitous in the environment (Bruggen et al., 2018). Naturally, this widespread use has raised issues on the ecotoxicity of glyphosate, and some studies have evaluated the risk posed by this substance for different environmental compartments ((EFSA), 2015, Folmar, Sanders, Julin, 1979, Giesy, Dobson, Solomon, 2000).

Glyphosate, with a mean half-life from 2.8 to 500.3 days in soils and 6.8 to 21.8 days in the water phase of water-sediment system (EFSA) (the variability of these values is due to the different biotic and abiotic parameters influencing its degradation (Giesy et al., 2000)), does not have a very high level of persistence in the environment (Matozzo, Munari, Masiero, Finos, Marin, 2019, Myers, Antoniou, Blumberg, Carroll, Colborn, Everett, Hansen, Landrigan, Lanphear, Mesnage, Vandenberg, vom Saal, Welshons, Benbrook, 2016). In surface water, its occurrence and concentrations depend on the climate, the agricultural region and the frequencies and the AS doses used (Coupe et al., 2011). In French surface water, a maximum concentration of 70.2$\mu$gL${}^{-1}$ was detected among the 21,561 sampling points followed in a 10-year period going from 2007 to 2017 (Anses, 2019). This was the unique analysis that overwhelmed the reported value of Predicted No Effect Concentration (PNEC of 60$\mu$gL${}^{-1}$, determined using both acute and chronic toxicity values) in 10 years. However lower concentrations were detected in 49.7% of the sampling point in 2017. Also, mean concentration in french surface water reported by Ineris (2020) was 0.22$\mu$gL${}^{-1}$ of active substance. Furthermore, several studies have provided evidence that glyphosate associated with co-formulants in glyphosate-based herbicides (GBHs) is toxic to aquatic wildlife (de Brito Rodrigues, Costa, Thá, da Silva, de Oliveira, Leme, Cestari, Grisolia, Valadares, de Oliveira, 2019, Folmar, Sanders, Julin, 1979, Giesy, Dobson, Solomon, 2000). Nevertheless, ecotoxicological research comparing the effect of long-term glyphosate exposure through pure AS or in GBHs in fish are lacking (Smith et al., 2019).

The bioaccumulation potential of glyphosate in animal tissues is low (Duke, 2020, (EFSA), 2015), but some studies have revealed that glyphosate and GBHs can affect physiological processes in fish (e.g. oxidative stress, mitochondrial physiology, immune function, energy metabolism), with severity depending on the life stage and the species considered (Glusczak, dos Santos Miron, Moraes, Simões, Schetinger, Morsch, Loro, 2007, Kreutz, Barcellos, Marteninghe, dos Santos, Zanatta, 2010, Pereira, Jaramillo, Remor, Latini, Davico, da Silva, Müller, Ammar, Nazari, 2018, Webster, Santos, 2015, Weeks Santos et al., 2019). Most studies have focused on non-environmentally relevant doses with high toxicity, which trigger unspecific physiological responses, revealing general dysfunctions rather than perturbations directly related to the mode of action of glyphosate. Nonetheless, several ecotoxicological studies have pointed out the toxicity of glyphosate, alone or co-formulated, at lower doses that are more in line with environmental concentrations (Ayanda, 2018, Faria, Wu, Luja-Mondragón, Prats, Gómez-Oliván, Piña, Raldúa, 2020, Marchand, Tanguy, Charrier, Quiniou, Plee-Gauthier, Laroche, 2006, Zhang et al., 2017). At these environmentally relevant doses, glyphosate alone induces effects at different levels of biological organization in fish: e.g. at the cellular level, the inhibition of certain enzymes such as acetylcholine esterase (AChE) (Glusczak et al., 2007), changes in parameters related to the oxidant/antioxidant equilibrium (Ayanda, 2018, Persch, Weimer, Freitas, Oliveira, 2017), modifications in energy metabolism (Ayanda, Oniye, Auta, Ajibola, Bello, 2015, Glusczak, dos Santos Miron, Moraes, Simões, Schetinger, Morsch, Loro, 2007, Marchand, Tanguy, Charrier, Quiniou, Plee-Gauthier, Laroche, 2006) and at the individual level, disruptions in early development (Fiorino et al., 2018, Zebral, Costa, de Castro Knopp, Lansini, Zafalon-Silva, Bianchini, Robaldo, 2017) and behavioural changes (Bridi, Altenhofen, Gonzalez, Reolon, Bonan, 2017, Faria, Wu, Luja-Mondragón, Prats, Gómez-Oliván, Piña, Raldúa, 2020, Giaquinto, de Sá, Sugihara, Gonçalves, Delício, Barki, 2017, Zhang et al., 2017).

Toxicity can potentially be transmitted vertically through generations, thereby modulating the toxicity of glyphosate and its co-formulants in the natural environment (Hanson and Skinner, 2016). The generational toxicity of glyphosate has not been intensively studied, principally due to the complexity of the experimental design which must include several generations of fish. Intergenerational toxicity is defined as the toxicity transmitted from the F0 genitor generation to the F1 generation, with the F1 generation being directly contaminated via the germinal cells produced in the parental bodies (Best, Ikert, Kostyniuk, Craig, Navarro-Martin, Marandel, Mennigen, 2018, Stenz, Schechter, Serpa, Paoloni-Giacobino, 2018). Multigenerational toxicity corresponds to direct exposure, at the level of the entire individual, of both genitors and future generations (Hanson and Skinner, 2016). In Danio rerio, the exposure of the F0 generation to both glyphosate and a GBH at a concentration of 10mgL${}^{-1}$ increased the F1 susceptibility to these pesticides during embryogenesis (i.e. increased mortality rate and premature hatching) (Webster et al., 2014). Another study (Smith et al., 2019), showed that a reduction in hatching success and an increase in developmental abnormalities may be associated with epigenetic effects detected in the parental generation following exposure during their early life stages to glyphosate, co-formulated or not, at the concentration of 0.5mgL${}^{-1}$.

In this study, we evaluated the ability of a low environmental concentration of glyphosate and two GBHs to induce direct and/or generational toxic effects in an F1 generation of rainbow trout (Oncorhynchus mykiss). Potential toxicity was investigated at the individual level, with analyses of malformation frequencies and behavioural changes, and at the cellular level, focusing on energy metabolism and oxidative stress.