Evaluation of the developmental effects of a glyphosate-based herbicide complexed with copper, zinc, and manganese metals in zebrafish.

The use of glyphosate-based herbicides (GBH) has increased dramatically, being currently the most used herbicides worldwide. Glyphosate acts as a chelating agent, capable of chelate metals. The synergistic effects of metals and agrochemicals may pose an environmental problem as they have been shown to induce neurological abnormalities and behavioural changes in aquatic species. However, as their ecotoxicity effects are poorly understood, evaluating the impacts of GBH complexed with metals is an ecological priority. The main objective of the study was to evaluate the potentially toxic effects caused by exposure to a GBH (1 µg a.i. mL-1), alone or complexed with metals (Copper, Manganese, and Zinc (100 µg L-1)), at environmentally relevant concentrations, during the early period of zebrafish (Danio rerio) embryo development (96 h post-fertilization), a promising model for in vivo developmental studies. To clarify the mechanisms of toxicity involved, lethal and sublethal development endpoints were assessed. At the end of the exposure, biochemical and cell death parameters were evaluated and, 24 h later, different behavioural responses were assessed. The results showed that metals induced higher levels of toxicity. Copper caused high mortality, low hatching, malformations, and changes in biochemical parameters, such as decreased Catalase (CAT) activity, increased Glutathione Peroxidase (GPx), Glutathione S-Transferase (GST), reduced Glutathione (GSH) and decreased Acetylcholinesterase (AChE) activity, also inducing apoptosis and changes in larval behaviour. Manganese increased the activity of SODs enzymes. Zinc increased mortality, reactive oxygen species (ROS) levels, superoxide dismutase activity (SODs) and caused a decrease in AChE activity. Embryos/larvae exposed to 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 the mechanisms of metal toxicity at the biochemical, physiological, and behavioural levels of zebrafish.

Behavioural impairment and oxidative stress by acute exposure of zebrafish to a commercial formulation of tebuconazole.

Tebuconazole is a systemic follicular fungicide known to cause diverse problems in non-target organisms namely associated to the pure active ingredient. As such, the objective of this work was to evaluate developmental changes induced by a tebuconazole commercial formulation to a non-target animal model. Zebrafish embryos at ± 2 h post-fertilization were exposed to tebuconazole wettable powder concentrations (0.05, 0.5 and 5 mg L-1) for 96 h with developmental toxicity assessed throughout the exposure period and biochemical parameters evaluated at the end of the exposure. Behavioural assessment (spatial exploration and response to stimuli) was conducted 24 h after the end of the exposure. While no developmental and physiological alterations were observed, exposure to tebuconazole resulted in an increased generation of reactive oxidative species at the 0.05 and 0.5 mg L-1 concentrations and a decreased GPx activity at the 0.5 mg L-1 concentration suggesting a potential protection mechanism. There was also a change in the avoidance-escape behaviour supporting an anxiolytic effect suggesting possible alterations in the central nervous system development demanding further studies.

Embryonic zebrafish response to a commercial formulation of azoxystrobin at environmental concentrations.

Azoxystrobin is a broad-spectrum strobilurin fungicide for use on a wide range of crops available to end-users as formulated products. Due to its extensive application, it has been detected in aquatic ecosystems, raising concerns about its environmental impact, which is still poorly explored. The objective of this work was to study the effects of a commercial formulation of azoxystrobin in the zebrafish embryo model. Sublethal and lethal effects were monitored during the exposure period from 2 h post fertilisation (hpf) to 96 hpf after exposure to azoxystrobin concentrations (1, 10 and 100 µg L-1). The responses of antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR)) as well as detoxifying enzymes (glutathione-s-transferase (GST) and carboxylesterase (CarE)) were evaluated at 96 hpf. Similarly, glutathione levels (reduced (GSH) and oxidised (GSSG) glutathione), neurotransmission (acetylcholinesterase (AChE)) and anaerobic respiration (lactate dehydrogenase (LDH)) -related enzymes were assayed. At 120 hpf, larvae from each group were used for behaviour analysis. Results from this study showed concentration-dependent teratogenic effects, particularly by increasing the number of malformations (yolk and eye), with a higher prevalence at the highest concentration. However, it was found that the lowest concentration induced a high generation of reactive oxygen species (ROS) and increased activity of SOD, GST, and CarE. In addition, GR and GSSG levels were decreased by the lowest concentration, suggesting an adaptive response to oxidative stress, which is also supported by the increased AChE activity and absence of behavioural changes. These findings advance the knowledge of the azoxystrobin developmental and environmental impacts, which may impose ecotoxicological risks to non-target species.

Toxic effects of a mancozeb-containing commercial formulation at environmental relevant concentrations on zebrafish embryonic development.

The toxicological knowledge of mancozeb (MZ)-containing commercial formulations on non-target species is scarce and limited. Therefore, the objective of this work was to represent a realistic application scenario by evaluating the toxicity of environmental relevant and higher concentrations of a commercial formulation of MZ using zebrafish embryos. Following determination of the 96-h LC50 value, the embryos at the blastula stage (~ 2 h post-fertilisation, hpf) were exposed to 0.5, 5, and 50 µg L-1 of the active ingredient (~ 40× lower than the 96-h LC50). During the exposure period (96 h), lethal, sublethal, and teratogenic parameters, as well as behaviour analysis, at 120 hpf, were assayed. Biochemical parameters such as oxidative stress-linked enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR)), reactive oxygen species (ROS) levels, and glutathione levels (GSH and GSSG), as well as the activity of degradation (glutathione S-transferase (GST) and carboxylesterase (CarE)), neurotransmission (acetylcholinesterase (AChE)), and anaerobic respiration (lactate dehydrogenase (LDH))-related enzymes, were analysed at the end of the exposure period. Exposed embryos showed a marked decrease in the hatching rate and many malformations (cardiac and yolk sac oedema and spinal torsions), with a higher prevalence at the highest concentration. A dose-dependent decreased locomotor activity and a response to an aversive stimulus, as well as a light-dark transition decline, were observed at environmental relevant concentrations. Furthermore, the activities of SOD and GR increased while the activity of GST, AChE, and MDA contents decreased. Taken together, the involvement of mancozeb metabolites and the generation of ROS are suggested as responsible for the developmental phenotypes. While further studies are needed to fully support the hypothesis presented, the potential cumulative effects of mancozeb-containing formulations and its metabolites could represent an environmental risk which should not be disregarded.

Behavioural toxicity of environmental relevant concentrations of a glyphosate commercial formulation - RoundUp® UltraMax - During zebrafish embryogenesis.

The use of herbicides with glyphosate as an active ingredient (a.i.) has increased dramatically in recent years, with its residues often being found in either soil or water. Nevertheless, concerns have arisen about its harmful side effects for both ecosystems and wildlife health. Therefore, the objective of this work was to assess the effects of a commercial formulation of glyphosate (RoundUp® UltraMax), at environmentally relevant concentrations on zebrafish embryos through a set of behavioural patterns. Zebrafish embryos were exposed to 0, 1, 2 and 5 µg a.i. mL-1 concentrations of the glyphosate formulation for 72 h (from 2.5 to 75 h post-fertilization (hpf)). After exposure, larvae were washed and allowed to develop until 144 hpf. At this point, the larvae behaviour was evaluated using a battery of tests to assess the general exploratory motility, escape-like responses, anxiety-related behaviours and social interactions. In addition, cortisol levels were assessed. No significant changes were observed relative to the exploratory behaviour in the standard open field. The anxiety-related behaviours were similar among groups, and no social interference was observed following exposure to these glyphosate concentrations. Likewise, cortisol levels remained similar among treatments. Still, the larvae exposed to 5 µg a.i. mL-1 did not react to the presence of an aversive stimulus, supporting glyphosate-induced changes in the sensory-motor coordination during development. In general, these results indicate a possible neurotoxic effect of this glyphosate-based formulation that should be further evaluated. In addition, the results obtained could impose a risk for wildlife sensitive species that should not be neglected.

Dose-dependent effects of a glyphosate commercial formulation - Roundup® UltraMax - on the early zebrafish embryogenesis.

The use of herbicides with glyphosate as an active ingredient, the so-called glyphosate-based herbicides (GBH), has increased dramatically in recent years currently being the most widely used in the world. Therefore, glyphosate residues have been detected in water and soils near the application sites. Recent studies indicate that GBH may cause adverse effects on vertebrates although these have been attributed to the presence of adjuvants in the commercial formulations rather than to the sole compound. Accordingly, the objective of this work was to investigate the lethal and sub-lethal developmental effects, neurotoxic potential and oxidative stress responses of zebrafish embryos to Roundup® Ultramax (RU) exposure. Embryos were exposed during 72 h to 0, 2, 5, 8.5 µg a.i. mL-1 of RU. Increased mortality was observed in embryos exposed to concentrations above 8.5 µg a.i. mL-1 as well as increased number of malformations. Decreased heart rate and hatchability were also observed. By contrast, exposure to concentrations that do not evoke teratogenic outcomes induced a dose-dependent decrease of heart rate although not inducing significant developmental changes. However, histological changes were not observed in the larvae exposed to these concentrations. Moreover, the generation of reactive oxygen species, the antioxidant enzymes activities (SOD and CAT), the GST biotransformation activity, the glutathione levels (GSH and GSSG), the oxidative damage (MDA) and the acetylcholinesterase and lactate dehydrogenase were similar among groups following exposure. Overall, available evidence suggests a dose-dependent toxicological effect of this formulation at concentrations that are not routinely detected in the environment. However, additional studies should be performed to better understand the underlying molecular mechanisms in favor of this formulation.