Publication
Evaluation of the effects of glyphosate association with copper, zinc, and manganese metals on zebrafish development
| Summary: | The use of herbicides with glyphosate as an active ingredient (a.i.) has increased dramatically in recent years being currently the most used in the world. Due to its inappropriate use, its residues have often been found in soil and water, raising concerns about its potential harmful effects on ecosystems. Recent data indicate that glyphosate can cause adverse effects in vertebrates, although these have been attributed to the presence of adjuvants in commercial formulations and not to the active ingredient. In any case, the underlying ecotoxicity mechanisms are not fully understood. Thus, the evaluation of the toxic effects of glyphosate in the environment and its impact on humans is a priority in ecotoxicology research. The studies developed in this thesis used the zebrafish (Danio rerio) as an animal model. This species has been frequently used in the evaluation of the toxicity profile of compounds in vivo. Early stages of zebrafish development offer a model that fits within the scope of the 3Rs policy, being an alternative to animal experimentation in biomedical and ecotoxicological research. Thus, in the introduction to this thesis (Chapter I), a literature review of the evidence regarding the toxic effects of pure glyphosate or glyphosate-based herbicide (GBH) on embryonic zebrafish stages was elaborated. There are differences in the literature regarding the toxic effects associated with exposure to glyphosate versus GBHs. Additionally, the impact of exposure to GBHs in the early stages of zebrafish development remains unclear. In order to investigate the lethal and sublethal effects of a GBH exposure on developing zebrafish embryos, a 72 h exposure to Roundup® Ultramax (RU) at concentrations of 2, 5, 8.5 μg a.i. mL−1 was performed. The results of this work are presented in chapter II, in which a 72 h LC50 of 8.53 μg a.i. mL−1 and an increased mortality of embryos exposed to concentrations greater than 8.5 μg a.i. mL−1 were reported, as well as an increase in malformations, changes in heart rate and hatching. At concentrations of 2 and 5 μg a.i. mL−1 , no changes were observed in the histological evaluation, namely teratogenic effects, nor in the biochemical parameters analysed, such as generation of reactive oxygen species (ROS), changes in oxidative stress enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), reduced glutathione (GSH), glutathione oxidized (GSSG)), induction of lipid peroxidation (LPO), alteration of acetylcholinesterase (AChE) or lactate dehydrogenase (LDH) activity. The results obtained are suggestive that the toxicological effects on zebrafish are concentration dependent. In chapter III the impact of 72 h exposure to RU at environmentally relevant concentrations (1, 2 and 5 μg a.i. mL−1 ) on different behavioural patterns of zebrafish embryos is reported. Zebrafish embryos were exposed for 72 h and maintained until 144 hpf, when larval behaviour was assessed through general exploratory motility, escape responses, anxiety-related behaviours, social interactions and through the measurement of cortisol levels. Cortisol levels remained similar between treatments, but larvae exposed to 5 μg a.i. mL−1 did not react to the presence of an aversive stimulus, suggesting that RU can induce changes in sensorimotor coordination during zebrafish development, possibly indicative of its neurotoxic effect. Chapter IV presents a study that aimed to evaluate the effects of acute exposure (4 h 30 min) to a commercial formulation of glyphosate, Roundup ® Flex (RF), at concentrations of 1, 5 and 10 μg a.i. mL−1 in Tg(mpxGFP)i114 transgenic and wild-type (WT) zebrafish larvae, with 72 hpf, by evaluating the inflammatory response, oxidative stress, and cell death. None of the concentrations tested induced changes in the number of neutrophils and nitric acid levels. No changes were observed in the activity of antioxidant enzymes SOD, CAT and glutathione peroxidase (GPx), GST, nor in the levels of GSH and GSSG. The results regarding LPO, LDH and AChE were also similar between the groups. However, at a concentration of 10 μg a.i. mL−1 , an increase in ROS levels and cell death was observed. Evidence suggests that toxicological effects are concentration dependent, albeit at concentrations that are not routinely found in the environment. Glyphosate has chelating properties, forming complexes with metal ions, including Cu, Mn and Zn. The synergistic effect between GBH and metals is believed to have a negative impact on ecosystems, however, these mechanisms are currently unclear. To clarify the mechanisms of toxicity involved, in Chapter V the impact of exposure to a GBH (Roundup® Flex) complexed with metals (Copper, Manganese and Zinc) were assessed, during the initial period of zebrafish embryo development (96 hpf). The endpoints of lethal and sublethal development were evaluated. The results showed that metals induced higher levels of toxicity in embryos/larvae, compared to GBH. Cu was able to induce teratogenic effects in the larvae as well as a decrease in CAT activity, an increase in GPx and GST, a reduction in GSH levels and AChE activity, induction of apoptosis and changes in larval behaviour. Zinc increased mortality, ROS levels and SOD activity, and caused a decrease in AChE activity. Manganese increased the activity of SODs enzymes. The combination of GBH/metal also showed teratogenic effects during their development but in smaller proportions than the metal alone. Although more studies are needed, the results suggest that GBH may interfere with mechanisms of metal toxicity in zebrafish. Finally, in Chapter VI, the general conclusions of the studies performed in this thesis on the toxic effects associated with exposure to GBHs in zebrafish embryos and their potential impacts on ecosystems are explored. Possible directions for future research in this area are also proposed. |
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| Main Authors: | Lanzarin, Germano Augusto Bacin |
| Subject: | Behaviour Glyphosate |
| Year: | 2023 |
| Country: | Portugal |
| Document type: | doctoral thesis |
| Access type: | restricted access |
| Associated institution: | Universidade de Trás-os-Montes e Alto Douro |
| Language: | English |
| Origin: | Repositório da UTAD |
| Summary: | The use of herbicides with glyphosate as an active ingredient (a.i.) has increased dramatically in recent years being currently the most used in the world. Due to its inappropriate use, its residues have often been found in soil and water, raising concerns about its potential harmful effects on ecosystems. Recent data indicate that glyphosate can cause adverse effects in vertebrates, although these have been attributed to the presence of adjuvants in commercial formulations and not to the active ingredient. In any case, the underlying ecotoxicity mechanisms are not fully understood. Thus, the evaluation of the toxic effects of glyphosate in the environment and its impact on humans is a priority in ecotoxicology research. The studies developed in this thesis used the zebrafish (Danio rerio) as an animal model. This species has been frequently used in the evaluation of the toxicity profile of compounds in vivo. Early stages of zebrafish development offer a model that fits within the scope of the 3Rs policy, being an alternative to animal experimentation in biomedical and ecotoxicological research. Thus, in the introduction to this thesis (Chapter I), a literature review of the evidence regarding the toxic effects of pure glyphosate or glyphosate-based herbicide (GBH) on embryonic zebrafish stages was elaborated. There are differences in the literature regarding the toxic effects associated with exposure to glyphosate versus GBHs. Additionally, the impact of exposure to GBHs in the early stages of zebrafish development remains unclear. In order to investigate the lethal and sublethal effects of a GBH exposure on developing zebrafish embryos, a 72 h exposure to Roundup® Ultramax (RU) at concentrations of 2, 5, 8.5 μg a.i. mL−1 was performed. The results of this work are presented in chapter II, in which a 72 h LC50 of 8.53 μg a.i. mL−1 and an increased mortality of embryos exposed to concentrations greater than 8.5 μg a.i. mL−1 were reported, as well as an increase in malformations, changes in heart rate and hatching. At concentrations of 2 and 5 μg a.i. mL−1 , no changes were observed in the histological evaluation, namely teratogenic effects, nor in the biochemical parameters analysed, such as generation of reactive oxygen species (ROS), changes in oxidative stress enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), reduced glutathione (GSH), glutathione oxidized (GSSG)), induction of lipid peroxidation (LPO), alteration of acetylcholinesterase (AChE) or lactate dehydrogenase (LDH) activity. The results obtained are suggestive that the toxicological effects on zebrafish are concentration dependent. In chapter III the impact of 72 h exposure to RU at environmentally relevant concentrations (1, 2 and 5 μg a.i. mL−1 ) on different behavioural patterns of zebrafish embryos is reported. Zebrafish embryos were exposed for 72 h and maintained until 144 hpf, when larval behaviour was assessed through general exploratory motility, escape responses, anxiety-related behaviours, social interactions and through the measurement of cortisol levels. Cortisol levels remained similar between treatments, but larvae exposed to 5 μg a.i. mL−1 did not react to the presence of an aversive stimulus, suggesting that RU can induce changes in sensorimotor coordination during zebrafish development, possibly indicative of its neurotoxic effect. Chapter IV presents a study that aimed to evaluate the effects of acute exposure (4 h 30 min) to a commercial formulation of glyphosate, Roundup ® Flex (RF), at concentrations of 1, 5 and 10 μg a.i. mL−1 in Tg(mpxGFP)i114 transgenic and wild-type (WT) zebrafish larvae, with 72 hpf, by evaluating the inflammatory response, oxidative stress, and cell death. None of the concentrations tested induced changes in the number of neutrophils and nitric acid levels. No changes were observed in the activity of antioxidant enzymes SOD, CAT and glutathione peroxidase (GPx), GST, nor in the levels of GSH and GSSG. The results regarding LPO, LDH and AChE were also similar between the groups. However, at a concentration of 10 μg a.i. mL−1 , an increase in ROS levels and cell death was observed. Evidence suggests that toxicological effects are concentration dependent, albeit at concentrations that are not routinely found in the environment. Glyphosate has chelating properties, forming complexes with metal ions, including Cu, Mn and Zn. The synergistic effect between GBH and metals is believed to have a negative impact on ecosystems, however, these mechanisms are currently unclear. To clarify the mechanisms of toxicity involved, in Chapter V the impact of exposure to a GBH (Roundup® Flex) complexed with metals (Copper, Manganese and Zinc) were assessed, during the initial period of zebrafish embryo development (96 hpf). The endpoints of lethal and sublethal development were evaluated. The results showed that metals induced higher levels of toxicity in embryos/larvae, compared to GBH. Cu was able to induce teratogenic effects in the larvae as well as a decrease in CAT activity, an increase in GPx and GST, a reduction in GSH levels and AChE activity, induction of apoptosis and changes in larval behaviour. Zinc increased mortality, ROS levels and SOD activity, and caused a decrease in AChE activity. Manganese increased the activity of SODs enzymes. The combination of GBH/metal also showed teratogenic effects during their development but in smaller proportions than the metal alone. Although more studies are needed, the results suggest that GBH may interfere with mechanisms of metal toxicity in zebrafish. Finally, in Chapter VI, the general conclusions of the studies performed in this thesis on the toxic effects associated with exposure to GBHs in zebrafish embryos and their potential impacts on ecosystems are explored. Possible directions for future research in this area are also proposed. |
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