Ecotoxicological Methodologies to Evaluate Biomarkers at Different Scales in Neotropical Anurans.

The new questions in ecotoxicology highlight the importance of applying a battery of biomarkers, as this results in ecotoxicological predictions that improve not only the interpretation of the effects of environmental stressors on organisms but also the determination of their possible impact. It is well known that the use of ecotoxicological biomarkers at different levels of organization allows for the prediction of the biological responses of organisms to environmental stressors, which is useful in environmental risk assessment. Nevertheless, it is necessary to consider the optimization of basic procedures, to generate historical data in control groups, and to employ specific bioassays to evaluate responses in organs and tissues in order to elucidate the nature and variation of the effects observed. Therefore, the present work aims to describe several ecotoxicological methodologies employed in all stages of neotropical anurans at different ecological levels and to validate them as useful biomarkers to be used both in wildlife and in laboratory conditions. In this work, these biomarkers were applied at the individual/organismic level (body condition index), histological/physiological level (histopathology, histometric, and pigmentary analyses), biochemical level (oxidative stress enzymes), and genetic level (direct and oxidative damage in DNA by comet assay). Although these methodologies have small variations or modifications depending on the species, these techniques provide effective biomarkers for evaluating the effect of xenobiotics on anurans, which possess certain characteristics that make them useful indicator species of aquatic and terrestrial ecosystems. In conclusion, the battery of biomarkers employed in the present study has proven to be adequate for estimating toxic responses in Neotropical anurans and can be further recommended as bioindicators for identifying the impact of pollutants on the aquatic ecosystems of the region. Finally, it is recommended to achieve the standardization of these important biomarkers for anurans in specific regions as well as to possibly include them in risk assessments and decision-making.

Do environmentally relevant concentrations of the neonicotinoid insecticide imidacloprid induce DNA damage and oxidative stress on Cnesterodon decemmaculatus (Jenyns, 1842)?

Lethal and sublethal effects of imidacloprid (IMI) were assessed on Cnesterodon decemmaculatus (Pisces: Poeciliidae) by acute exposure to environmentally relevant concentrations of the commercial formulation Punto 35® (Gleba S.A.) under laboratory conditions. Specimens were exposed for 96 h to 1, 10, 20, 25, 35, 75, 100, 125, 150 and 175 mg IMI L-1 from which an LC50 96 h value of 35.59 mg IMI L-1 was calculated. Moreover, sublethal concentrations 0.175, 0.35 and 1 mg IMI L-1 for 96 h were employed for the evaluation of the comet assay and the variation of activities of catalase (CAT) and glutathione content (GSH). Result demonstrated an increased genetic damage index and activity of CAT was observed. Conversely, no significant variation was observed in GSH activity. Total protein content decreased in treated organisms. These results represent the first report of acute effects induced by IMI on C. decemmaculatus exposed under laboratory conditions.

Toxicity to Rhinella arenarum tadpoles (Anura, Bufonidae) of herbicide mixtures commonly used to treat fallow containing resistant weeds: glyphosate-dicamba and glyphosate-flurochloridone.

Glyphosate (GLY)-dicamba (DIC) and GLY-flurochloridone (FLC) are herbicide mixtures which are widely used for treating fallow containing glyphosate resistant weeds. The aim of this study was to evaluate the acute toxic effects and the prevailing interactions on stage 36 tadpoles of the anuran species Rhinella arenarum when exposed to equitoxic and non-equitoxic combinations of these herbicide combinations. Experiments were realized using the following combinations of commercial formulations: 48% GLY-based Credit® + 57.71% DIC-based Banvel® and 48% GLY-based Credit® + 25% FLC-based Twin Pack Gold®. GLY-DIC and GLY-FLC equitoxic mixtures were assayed mixing each constituent with an equivalent individual toxicity able to induce the same lethality effect. After 96 h of exposure, GLY-DIC and GLY-FLC equitoxic mixtures presented toxic unit 50 values (TU50 96h) of 1.74 (confidence interval: 1.58-1.92) and 1.54 (confidence interval: 1.46-1.62) respectively, indicating the presence of a weak antagonistic interaction as TU values were greater than 1. For their part, most non-equitoxic combinations of GLY-DIC and GLY-FLC tested did not significantly differ from additivity, the only exception being when DIC and FLC were fixed at 0.33 TUs, where a weak antagonism was observed. Overall, results indicate that the toxicity of both GLY-DIC and GLY-FLC mixtures to R. arenarum tadpoles vary from additive to slightly antagonistic, depending on the proportion of constituting herbicide formulations present in the mixture.

Genotoxicity of mixtures of glyphosate with 2,4-dichlorophenoxyacetic acid chemical forms towards Cnesterodon decemmaculatus (Pisces, Poeciliidae).

Acute genotoxicity of commercial glyphosate (GLY) (Credit®)-, 2,4-D-acid (2,4-D) (Dedalo Elite)-, 2,4-D-amine (2,4-D DMA) (Weedar Full®)- and 2,4-D-ester (2,4-D BE) (Herbifen Super®)-based herbicide formulations alone and their combinations were analysed in Cnesterodon decemmaculatus. Mortality was evaluated as a lethal end-point and the single cell gel electrophoresis (SCGE) bioassay was used as a sublethal end-point. LC5096h values for Dedalo Elite was 0.46 mg/L and Herbifen Super® was 2.67 mg/L based on 2,4-D and 2,4-D BE, respectively. Results reveal a higher toxicity exerted on C. decemmaculatus after exposure to 2,4-D- rather than 2,4-D BE-based herbicide formulations. Overall, results demonstrated an enhancement in the genetic damage index committed to an enhancement of damaged erythrocytes of C. decemmaculatus when exposed to Credit®, Dedalo Elite, Weedar Full® and Herbifen Super® at 5% and 10% of LC5096h values alone as well as in their combinations. Overall, the combination of GLY plus 2,4-D or GLY plus 2,4-D DMA showed a synergistic pattern whereas the combination of GLY plus 2,4-D BE was antagonic. Furthermore, this research is pioneer in the assessment of lethality and genotoxicity induced by 2,4-D-, 2,4-D DMA- and 2,4-D BE-based formulations when combined with GLY-based formulated herbicides in fish after they are acutely exposed.

Auxinic herbicides induce oxidative stress on Cnesterodon decemmaculatus (Pisces: Poeciliidae).

Pesticides might increase the production of reactive oxygen species (ROS). Dicamba (DIC) and 2,4-dichlorophenoxyacetic acid (2,4-D) are auxinic herbicides commonly applied in agroecosystems to control unwanted weeds. We analysed the oxidative damage exerted on the fish Cnesterodon decemmaculatus by an acute exposure to DIC- and 2,4-D-based herbicides formulations Banvel® and DMA®, respectively. The Endo III- and Fpg-modified alkaline comet assay was employed for detecting DNA damage caused by oxidative stress, whereas enzymatic and non-enzymatic biomarkers such as the activities of catalase (CAT), glutathione-S-transferase (GST), acetylcholinesterase (AChE), and glutathione content (GSH) were used to assess antioxidant response to these two herbicides. At the DNA level, results demonstrate that both auxinic herbicides induce oxidative damage at purines level. An increase on CAT and GST activities were detected in 48 h- and 96 h-treated specimens with both auxinics. GSH content decreased in fish exposed to DIC during 48 h and to 2,4-D after 96 h of exposure. Additionally, a diminished AChE activity in specimens treated with DIC and 2,4-D was observed only after 96 h. Total protein content decreased in fish exposed to both auxinics during 96 h. These results represent the first evaluation of oxidative damage related to DIC and 2,4-D exposure on a fish species as the Neotropical freshwater teleost C. decemmaculatus.

DNA damage exerted by mixtures of commercial formulations of glyphosate and imazethapyr herbicides in Rhinella arenarum (Anura, Bufonidae) tadpoles.

Glyphosate (GLY) and imazethapyr (IMZT) are two herbicides commonly used worldwide, either alone or in mixtures. They represent key pesticides in modern agricultural management. The toxicity that results when employed as mixtures has not been characterized so far. Acute toxicity of the 48% GLY-based herbicide (GBH) Credit® and the 10.59% IMZT-based herbicide (IBH) Pivot® H alone and their binary combinations was analyzed in Rhinella arenarum tadpoles exposed in a semi-static renewal test. Lethal effects were determined using mortality as the end-point, whereas sublethal effects were determined employing the single-cell gel electrophoresis (SCGE) bioassay. Based on mortality experiments, results revealed LC5096 h values of 78.18 mg/L GBH and 0.99 mg/L IBH for Credit® and Pivot® H, respectively. An increase in the genetic damage index (GDI) was found after exposure to Credit® or Pivot® H at 5 and 10% of LC5096 h values. The combinations of 5% Credit®-5% Pivot® H LC5096 h and 10% Credit®-10% Pivot® H LC5096 h concentrations significantly enhanced the GDI in comparison with tadpoles exposed only to Credit® or Pivot® H. Thus, the effect of interaction between GBH and IBH inducing DNA damage in R. arenarum blood cells can be considered to be synergistic.

Genotoxicity by long-term exposure to the auxinic herbicides 2,4-dichlorophenoxyacetic acid and dicamba on Cnesterodon decemmaculatus (Pisces: Poeciliidae).

Long-term genotoxic effects of two auxinic herbicide formulations, namely, the 58.4% 2,4-dichlorophenoxyacetic acid (2,4-D)-based DMA® and the 57.7% dicamba (DIC)-based Banvel® were evaluated on Cnesterodon decemmaculatus. Primary DNA lesions were analyzed by the single-cell gel electrophoresis methodology. Two sublethal concentrations were tested for each herbicide corresponding to 2.5% and 5% of the LC5096h values. Accordingly, fish were exposed to 25.2 and 50.4 mg/L or 41 and 82 mg/L for 2,4-D and DIC, respectively. Fish were continuously exposed for 28 days with replacement of test solutions every 3 days. Genotoxicity was evaluated in ten individuals from each experimental point at the beginning of the exposure period (0 day) and at 7, 14, 21 and 28 days thereafter. Results demonstrated for first time that 2,4-D-based formulation DMA® induced primary DNA strand breaks after 7-28 days exposure on C. decemmaculatus regardless its concentration. On the other hand, DIC-based formulation Banvel® exerted its genotoxic effect after exposure during 7-14 days and 7 days of 2.5 and 5% LC5096h, respectively. The present study represents the first evidence of primary DNA lesions induced by two widely employed auxinic herbicides on C. decemmaculatus, namely 2,4-D and DIC, following long-term exposure.

Evaluation of imazethapyr-induced DNA oxidative damage by alkaline Endo III- and Fpg-modified single-cell gel electrophoresis assay in Hypsiboas pulchellus tadpoles (Anura, Hylidae).

Imazethapyr (IMZT) is a selective postemergent herbicide with residual action. Available data analyzing its effects in aquatic vertebrates are scarce. In previous studies, we demonstrated that IMZT induces lesions into the DNA of Hypsiboas pulchellus tadpoles using the single-cell gel electrophoresis (SCGE) assay as a biomarker for genotoxicity. Currently, this assay can be modified by including incubation with lesion-specific endonucleases, e.g., endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg), which detect oxidized pyrimidine and purine bases, respectively. The aim of this study was to evaluate the role of oxidative stress in the genotoxic damage in circulating blood cells of H. pulchellus tadpoles exposed to the IMZT-based Pivot H® formulation (10.59% IMZT) at a concentration equivalent to 25% of the LC50 (96h) value (0.39mg/L IMZT) during 48 and 96h. Our results demonstrate that the herbicide induces oxidative DNA damage on H. pulchellus tadpoles at purines bases but not at pyrimidines. Our findings represent the first evidence of oxidative damage caused by IMZT on anuran DNA using the alkaline restriction enzyme-modified SCGE assay.

Genotoxicity of the herbicide imazethapyr in mammalian cells by oxidative DNA damage evaluation using the Endo III and FPG alkaline comet assays.

We evaluated the role of oxidative stress in the genotoxic damage induced by imazethapyr (IMZT) and its formulation Pivot® in mammalian CHO-K1 cell line. Using the alkaline comet assay, we observed that a concentration of 0.1 µg/mL of IMZT or Pivot® was able to induce DNA damage by increasing the frequency of damaged nucleoids. To test whether the DNA lesions were caused by oxidative stress, the DNA repair enzymes endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg), which convert base damage to strand breaks, were used. Our results demonstrate that after treatment of CHO-K1 cells with the pure active ingredient as well as the commercial formulation Pivot®, an increase in DNA strand breaks was observed after incubation of both Endo III and Fpg enzymes, indicating that both compounds induce DNA damage involving both pyrimidine and purine-based oxidations, at least in CHO-K1 cells. Our findings confirm the genotoxic potential of IMZT and suggest that this herbicide formulation must be employed with great caution, especially not only for exposed occupational workers but also for other living species.

Genotoxic effect of a binary mixture of dicamba- and glyphosate-based commercial herbicide formulations on Rhinella arenarum (Hensel, 1867) (Anura, Bufonidae) late-stage larvae.

The acute toxicity of two herbicide formulations, namely, the 57.71 % dicamba (DIC)-based Banvel(®) and the 48 % glyphosate (GLY)-based Credit(®), alone as well as the binary mixture of these herbicides was evaluated on late-stage Rhinella arenarum larvae (stage 36) exposed under laboratory conditions. Mortality was used as an endpoint for determining acute lethal effects, whereas the single-cell gel electrophoresis (SCGE) assay was employed as genotoxic endpoint to study sublethal effects. Lethality studies revealed LC5096 h values of 358.44 and 78.18 mg L(-1) DIC and GLY for Banvel(®) and Credit(®), respectively. SCGE assay revealed, after exposure for 96 h to either 5 and 10 % of the Banvel(®) LC5096 h concentration or 5 and 10 % of the Credit(®) LC5096 h concentration, an equal significant increase of the genetic damage index (GDI) regardless of the concentration of the herbicide assayed. The binary mixtures of 5 % Banvel(®) plus 5 % Credit(®) LC5096 h concentrations and 10 % Banvel(®) plus 10 % Credit(®) LC5096 h concentrations induced equivalent significant increases in the GDI in regard to GDI values from late-stage larvae exposed only to Banvel(®) or Credit(®). This study represents the first experimental evidence of acute lethal and sublethal effects exerted by DIC on the species, as well as the induction of primary DNA breaks by this herbicide in amphibians. Finally, a synergistic effect of the mixture of GLY and DIC on the induction of primary DNA breaks on circulating blood cells of R. arenarum late-stage larvae could be demonstrated.

Influence of existing site contamination on sensitivity of Rhinella fernandezae (Anura, Bufonidae) tadpoles to Lorsban®48E formulation of chlorpyrifos.

Effects of the widely employed insecticide Lorsban(®)48E formulation of chlorpyrifos (CPF) was studied on Rhinella fernandezae tadpoles, a native species of Argentina, Brazil, Paraguay and Uruguay, under the hypothesis of a differential response of organisms from ponds of two sites with different degree of anthropogenic disturbance: S1 an unpolluted area, and S2 area with high degree of antropogenic disturbance. To collect a representative sample of the genotypic variability of each population, small portions from six clutches were taken randomly from each site when the period of clutching was finished. Embryos and tadpoles were maintained under controlled laboratory conditions. Toxicity tests were conducted under standardized conditions to study acute and chronic lethal (mortality) and sublethal effects (behavior, growth, and abnormalities), within the range of concentrations of 0.010 to 5 mg/L. Chronic effects were assessed with organisms from one of the demes (S1). CPF showed high toxicity on the tadpoles, inducing lethal and sublethal effects at 96 h exposure within a narrow range of concentrations from 0.066 to 0.887 mg/L. Results indicate that R. fernandezae tadpoles are below the 30th percentile in the species sensitivity distribution of existing data. The acute LC50, NOEC, and LOEC values were 0.151, 0.066, and 0.133 mg/L for S1, and 0.293, 0.177, and 0.266 mg/L for S2, respectively. Considering all acute end-points evaluated, the effects of CPF showed no significant differences (p = 0.3484) between the studied populations. CPF has more severe effects at higher concentrations than at higher times of exposure. Contaminants in S2 do not seem to induce local adaptation. Sublethal effects data and measured environmental concentrations indicate potential risk for populations inhabiting agroecosystems.