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.

Are the damaging effects induced by the imazethapyr formulation Pivot® H in Boana pulchella (Anura) reversible upon ceasing exposure?

In the present study, the damage recovery capabilities of Boana pulchella tadpoles after acute exposure (96h) to 0.39mg/L concentration of the imazethapyr (IMZT)-based herbicide formulation Pivot® H (25% IMZT LC50 value) were assessed during a period of 7 to -21 days. To appraise the recovery capabilities, frequency of micronuclei (MNs), other nuclear abnormalities and DNA single-strand breaks evaluated by single cell gel electrophoresis assay on circulating blood cells were employed as endpoints for genotoxicity. Growth, development, body mass, and morphological abnormalities were also employed as individual endpoints in the recovery assay. Results demonstrated that IMZT induced sublethal effects at both the individual (i.e., loss of keratodonts) and cytogenetic levels (e.g., increase of MN frequency, other nuclear abnormalities and DNA single-strand breaks). At 11 days of the exposure phase, tadpoles recovered their basal levels of frequency of MNs, other nuclear abnormalities, and comets. However, loss of keratodonts, observed at the end of the exposure period, was present up to 21 days thereafter. Finally, axial abnormalities and delay in development stage were observed only during the postexposure phase in IMZT-exposed tadpoles at 18 and 25 days, respectively and were observed until the end of the experiment. This is the first evidence of use the comet assay as cytogenetic biomarker of genotoxicity in evaluating the recovery capabilities of amphibians in general and also those of B. pulchella after exposure to IMZT.

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.

Ecotoxicological evaluation of foundry sands and cosmetic sludges using new earthworm biomarkers.

The management and final disposal of industrial wastes are a matter of considerable human concern. The present study evaluates the cyto/genotoxic effects and changes of the coelomic cell formulas exerted by aqueous leachates and solid waste (SW) of two industrial residues using coelomocytes extruded from Eisenia fetida. The assayed wastes corresponded to industrial foundry and cosmetic activities. After 14 days of exposure, we obtained a group of endpoints that reflect the toxicity/genotoxicity, coelomocyte formula and indexes; and the mortality classical value (LC50-14d). Among the variables measured, total coelomocytes formula (eleocytes + amebocytes + granulocytes) appears as a single and easy parameter to assess the toxicity of eluates at short exposure times. We applied a set of assays using earthworms as test organism that would allow evaluating SW as well as its aqueous leachates. It is easy to run trials combining exposures of 1 h to 14 days, which can be integrated into the implementation of the traditional test for evaluating acute toxicity.

Pirimicarb-based formulation-induced genotoxicity and cytotoxicity in the freshwater fish Cnesterodon decemmaculatus (Jenyns, 1842) (Pisces, Poeciliidae).

We analyzed the aspects of lethality, genotoxicity, and cytotoxicity in the ten spotted live-bearer exposed under laboratory conditions to the pirimicarb-based formulation Patton Flow® (50% active ingredient (a.i.)). Acute effects were evaluated using different end points for lethality, genotoxicity, and cytotoxicity. Median lethal concentration (LC50) estimation was employed as a bioassay for lethality, whereas micronucleus (MN) induction and alterations in erythrocyte/erythroblast frequency were used as end points for genotoxicity and cytotoxicity, respectively. Results demonstrated an LC5096h value of 88 mg/L. Patton Flow® increased the MN frequency in fish erythrocytes after 48 h of exposure at a concentration of 66 mg/L, whereas a concentration range of 22-66 mg/L was able to exert the same genotoxic effect at 96 h of treatment. Furthermore, cytotoxicity was also observed by alterations in erythrocyte/erythroblast frequencies within the concentration range of 22-66 mg/L, regardless of the exposure time. Our current observations provide evidence that Patton Flow® (50% a.i.) should be considered a clear lethal, cytotoxic, and genotoxic agent on Cnesterodon decemmaculatus. Thus, repeated applications of this carbamic insecticide can enter the aquatic environment and exert deleterious effects on aquatic organisms other than the evaluated species C. decemmaculatus.

Flurochloridone-based herbicides induced genotoxicity effects on Rhinella arenarum tadpoles (Anura: Bufonidae).

Acute toxicity and genotoxicity of the flurochloridone (FLC)-containing commercial formulation herbicides Twin Pack Gold(®) (25 percent a.i.) and Rainbow(®) (25 percent a.i.) were evaluated on Rhinella arenarum (Anura: Bufonidae) tadpoles exposed under laboratory conditions. Lethal effect was evaluated as end point for lethality, whereas frequency of micronuclei (MN) and single cell gel electrophoresis (SCGE) were employed as end points for genotoxicity. Lethality studies revealed equivalent LC-5096 h values of 2.96 and 2.85 mg/L for Twin Pack Gold(®) and Rainbow(®), respectively. Twin Pack Gold(®) did not induce DNA damage at the chromosomal level, whereas Rainbow(®) increased the frequency of MN only when the lowest concentration (0.71 mg/L) was used. However, all concentrations of Twin Pack Gold(®) and Rainbow(®) increased the frequencies of primary DNA lesions estimated by alkaline SCGE. This study represents the first evidence of the acute toxic and genotoxic effects exerted by two FLC-based commercial formulations, Twin Pack Gold(®) and Rainbow(®), on tadpoles of an amphibian species native to Argentina under laboratory conditions. Finally, our findings highlight the importance of minimizing the impacts on nontarget living species exposed to agrochemicals.

Chlorpyrifos-based insecticides induced genotoxic and cytotoxic effects in the ten spotted live-bearer fish, Cnesterodon decemmaculatus (Jenyns, 1842).

Mortality, genotoxicity, and cytotoxicity of the 48% chlorpyrifos (CPF)-based formulations Lorsban* 48E(®) and CPF Zamba(®) were evaluated on Cnesterodon decemmaculatus (Jenyns, 1842) (Pisces, Poeciliidae) under laboratory conditions. Induction of micronucleus (MN) and alterations in the erythrocyte/erythroblast frequencies were employed as end points for genotoxicity and cytotoxicity, respectively. For Lorsban* 48E(®) , mean values of 0.13 and 0.03 mg/L were determined for LC50 at 24 and 96 h, respectively, and these concentrations reached mean values of 0.40 and 0.21 mg/L for CPF Zamba(®) . Mortality values increased as a positive linear function of the CPF Zamba(®) concentrations, but not for Lorsban* 48E(®) concentrations. There was no significant relationship between mortality and exposure time within the 0-96 h period for both formulations. LC50 values indicated that the fish were seven fold more sensitive to Lorsban* 48E(®) than to CPF Zamba(®) . Lorsban* 48E(®) within the concentration range of 0.008-0.025 mg/L increased MN frequency at both 48 and 96 h of treatment. Similar results were also observed when fish were exposed to 0.052-0.155 mg/L of CPF Zamba(®) , regardless of the exposure time. Cellular cytotoxicity was found after Lorsban* 48E(®) and CPF Zamba(®) treatments for all concentrations and time exposures, estimated by a decrease in the frequency of mature erythrocytes and a concomitant enhanced frequency of erythroblasts in circulating blood. Furthermore, our results demonstrated that Lorsban* 48E(®) and CPF Zamba(®) should be considered as CPF-based commercial formulations with marked genotoxic and cytotoxic properties.

Comparative evaluation in vitro of the herbicide flurochloridone by cytokinesis-block micronucleus cytome and comet assays.

The in-vitro effects of flurochloridone and its formulations Twin Pack Gold® (25% a.i.) and Rainbow® (25% a.i.) were evaluated in Chinese Hamster Ovary K1 (CHO-K1) cells. The cytokinesis-block micronucleus cytome (CBMN-cyt) and single-cell gel electrophoresis (SCGE) assays were used. The activities were tested within the range of final concentrations of 0.25-15 µg flurochloridone/mL. The results demonstrated that both the flurochloridone and Rainbow® were not able to induce micronuclei (MN). On the other hand, Twin Pack Gold® only increased the frequency of MN at 5 µg/mL. Furthermore, 10 and 15 µg/mL of both formulations resulted in a cellular cytotoxicity demonstrated by alterations in the nuclear division index and cellular death. SCGE assay appeared to be a more sensitive bioassay for detecting primary DNA strand breaks at lower concentrations of flurochloridone than MN did. A marked increase in the genetic damage index was observed when 5 and 15 µg/mL of both flurochloridone and Rainbow® but only when 15 µg/mL of Twin Pack Gold® were used. This is the first report demonstrating that flurochloridone and its two commercial formulations are able to induce single-strand DNA breaks in vitro on mammalian cells.

DNA damage kinetics and apoptosis in ivermectin-treated Chinese hamster ovary cells.

A comet assay was used to analyze DNA damage kinetics in Chinese hamster ovary (CHO-K1) cells induced by antiparasitic ivermectin (IVM) and the IVM-containing technical formulation Ivomec® (IVO; 1% IVM). Cells were treated with 50 µg ml(-1) IVM and IVO for 80 min, washed and re-incubated in antiparasiticide-free medium for 0-24 h until assayed using the single-cell gel electrophoresis assay (SCGE). Cell viability remained unchanged up to 3 h of incubation. After 6 h of treatment, cell survival decreased up to 75% and 79% in IVM- and IVO-treated cultures, respectively, remaining unchanged within 12-24 h after treatment. For both anthelmintics, biphasic behavior in DNA damage occurred during the incubation time. A time-dependent increase of IVM- and IVO-induced DNA damage was observed within 0 to 3 h after pulse treatment, revealed by a progressive decrease of undamaged cells and an increase in slightly damaged and damaged cells. Finally, a time-dependent decrease in IVM- and IVO-induced DNA damage was revealed by a progressive decrease of slightly damaged cells and the absence of damaged cells simultaneously with an increase in the frequency of undamaged cells during the final 18 h of incubation. Flow cytometry analysis revealed that both compounds are able to induce a marked increase in early and late apoptosis. Based on our observations, we could conclude that the decrease in DNA lesions is mostly related to IVM-induced cytotoxicity rather than attributable to a repair process.

Evaluation of the genotoxic and cytotoxic effects of glyphosate-based herbicides in the ten spotted live-bearer fish Cnesterodon decemmaculatus (Jenyns, 1842).

Mortality, genotoxicity, and cytotoxicity of the 48% glyphosate-based formulations Panzer and Credit(®) were evaluated on Cnesterodon decemmaculatus (Jenyns, 1842) (Pisces, Poeciliidae) under laboratory conditions. Induction of micronuclei (MN) and alterations in the erythrocytes:erythroblasts ratio were employed as end points for genotoxicity and cytotoxicity, respectively. For Panzer(®), mean values of 16.70 and 15.68 mg/L were determined for LC(50) at 24 and 96 h, respectively, and these concentrations reached mean values of 98.50 and 91.73 mg/L for Credit(®). LC(50) values decreased as a negative linear function of Panzer(®) exposure time within the 0-96 h period, but not for Credit(®). LC(50) values indicated that the fish were more sensitive to Panzer(®) than to Credit(®). Both 3.9 and 7.8 mg/L of Panzer(®) increased MN frequency at 48 and 96 h of treatment. When fish were exposed to Credit(®), an increased frequency of MN over control values was found after 96 h for all concentrations assayed, but not after 48 h. No cellular cytotoxicity was found after Panzer(®) and Credit(®) treatment, regardless of both the concentration and the sampling time. Furthermore, our results demonstrated that Panzer(®) and Credit(®) should be considered as glyphosate-based commercial formulations with genotoxic but not cytotoxic effect properties.

Single-cell gel electrophoresis assay in the ten spotted live-bearer fish, Cnesterodon decemmaculatus (Jenyns, 1842), as bioassay for agrochemical-induced genotoxicity.

The ability of two 48 percent chlorpyrifos-based insecticides (Lorsban* 48E® and CPF Zamba®), two 50 percent pirimicarb-based insecticides (Aficida® and Patton Flow®), and two 48 percent glyphosate-based herbicides (Panzer® and Credit®) to induce DNA single-strand breaks in peripheral blood erythrocytes of Cnesterodon decemmaculatus (Jenyns, 1842) (Pisces, Poeciliidae) exposed under laboratory conditions was evaluated by the single-cell gel electrophoresis (SCGE) assay. In those fish exposed to Lorsban* 48E®, CPF Zamba®, Aficida®, Patton Flow®, Credit®, and Panzer®, a significant increase of the genetic damage was observed for all formulations regardless of the harvesting time. This genotoxic effect was achieved by an enhancement of Type II-IV comets and a concomitant decrease of Type 0-I comets over control values. A regression analysis revealed that the damage varied as a negative function of the exposure time in those Lorsban* 48E®- and Aficida®-treated fish. On the other hand, a positive correlation between damage increase and exposure time was achieved after Patton Flow® and Credit® treatment. Finally, no correlation was observed between increase in the genetic damage and exposure time after treatment with CPF Zamba® or Panzer®. These results highlight that all agrochemicals inflict primary genotoxic damage at the DNA level at sublethal concentrations, regardless of the exposure time of the aquatic organisms under study, at least within a period of 96 h of treatment.

Cytotoxic and genotoxic profiles of the pyrethroid insecticide lambda-cyhalothrin and its microformulation Karate® in CHO-K1 cells.

Lambda-cyhalothrin (LCT) and its microformulation Karate® (25 % a.i.) were analysed for its genotoxicity and cytotoxicity on Chinese hamster ovary (CHO-K1) cells. Cytokinesis-block micronucleus cytome (CBMN-cyt) and alkaline single-cell gel electrophoresis (SCGE) bioassays were selected to test genotoxicity. Neutral red uptake (NRU), succinic dehydrogenase activity (MTT) and apoptogenic induction were employed for estimating cytotoxicity. Both compounds were analysed within a concentration range of 0.1-100 µg/mL. Only LCT produced a significant augment in the frequency of micronuclei (MNs) when the cultures were exposed to highest concentrations of 10 and 100 µg LCT/mL. A noticeable decrease in NDI was observed for cultures treated with LCT at 10 and 100 µg/mL. Karate® induced the inhibition of both the proportion of viable cells and succinic dehydrogenase activity and triggered apoptosis 24 h of exposition. Whilst an increased GDI in CHO-K1 cells was observed in the treatments with 1-100 µg Karate®/mL, the GDI was not modified in the treatments employing LCT at equivalent doses. SCGE showed that Karate® was more prone to induce genotoxic effects than LCT. Only 50 µg/mL of Karate® was able to increase apoptosis. Our results demonstrate the genomic instability and cytotoxic effects induced by this pyrethroid insecticide, confirming that LCT exposure can result in a severe drawback for the ecological equilibrium of the environment.

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.

Measuring DNA modifications with the comet assay: a compendium of protocols.

The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.

Cytogenetic and microtubule array effects of the zineb-containing commercial fungicide formulation Azzurro(®) on meristematic root cells of Allium cepa L.

Zineb [ethylene bis(dithiocarbamate) zinc] is a widely employed foliar fungicide for agricultural and industrial applications. Allium cepa L. is a reliable model for the assessment of xenobiotic genotoxicity and cytotoxicity. We evaluated the effects of the zineb-containing commercial formulation Azzurro(®) (70% zineb) in cell cycle stages of the meristem root cells of A. cepa. The mitotic index (MI), chromosomal aberrations at anaphase/telophase (CAs), micronuclei (MN), and abnormalities in immunodetected microtubule structures, e.g., preprophasic band (PPB), mitotic spindle (MS), and phragmoplast (Phrag), were used as end-points. Azzurro(®) (1 and 10µg/ml) induced a significant increase in the frequency of CAs (P<0.05), and the higher concentration inhibited the MI (P<0.05) compared to control values. The frequency of MN did not differ from control values at any concentration. Treatment with 1µg/ml Azzurro(®) induced a significant increase in the frequency of abnormal PPB (P<0.01), MS (P<0.001), and Phrag (P<0.01) and, at 10µg/ml, enhancements in the frequencies of abnormal MS (P<0.05) and Phrag (P<0.05) were seen. A tubulin immunodetection assay showed that exposure to Azzurro(®) interferes with normal assembly of microtubule structures during mitosis.

Genotoxic and cytotoxic effects of the formulated insecticide Aficida on Cnesterodon decemmaculatus (Jenyns, 1842) (Pisces: Poeciliidae).

The acute toxicity, genotoxicity, and cytotoxicity of the pirimicarb-containing commercial-formulation carbamate insecticide Aficida (50% pirimicarb) were evaluated on Cnesterodon decemmaculatus (Pisces, Poeciliidae) exposed under laboratory conditions. Micronucleus (MN) induction as well as alterations in the erythrocytes:erythroblasts ratios were employed as end-points for genotoxicity and cytotoxicity, respectively. Cr(VI) and cyclophosphamide were used as positive controls for the toxicity and geno-cytotoxicity assays, respectively. Mean values of 344.3 and 225.5mg Aficida(®)/L were determined for LC-50(24h) and LC-50(96h), respectively. In 48h-exposed fish, a MN increase was found in Aficida-treated fish in the 50-157mg/L concentration range. When fish were exposed to Aficida for 96h, only those animals treated at 50-100mg/L showed an increase in MN frequency. Cellular cytotoxicity, revealed by a decreased proportion of circulating erythrocytes and an enhancement of erythroblasts, was found after 48h of exposure in 50-157mg Aficida/L-treated fish, while, after 96h exposure, only 100-157mg Aficida/L induced the same effect. This species provides a useful experimental model for the biomonitoring of aquatic ecosystems.

Testing genotoxicity and cytotoxicity strategies for the evaluation of commercial radiosterilized fetal calf sera.

Effects of 18 commercial lots of fetal calf serum (FCS) after gamma-irradiation and their non-irradiated counterparts were comparatively analyzed on CHO-K1 and MDBK MDL1 cells for genotoxicity [sister chromatid exchange (SCE), micronuclei (MNi), and single cell gel electrophoresis (SCGE)], cytotoxicity [cell-cycle progression (CCP), proliferative replication index (PRI), mitotic index (MI), growth promotion (GP), and plating efficiency (PE)], and microbiological properties (mycoplasma and bovine viral diarrhea virus contamination). SCE and SCGE were the most informative end-points for genotoxicity since significant differences were found in 44.4% (P<0.05-0.001, Student's t-test) and 61.1% (P<0.05-0.001, chi(2) test) samples, respectively. MI was the cytotoxicity assay revealing the greatest variation, showing differences in 66.7% (P<0.05-0.001, chi(2) test) samples. Thus, these three end-points for screening bioproducts such as FCS were found most suitable for detecting potential geno-cytotoxicants in biological samples; their simultaneous use could be strongly recommended.

Cytotoxic and genotoxic assessments of 2,4-dichlorophenoxyacetic acid (2,4-D) in in vitro mammalian cells.

A combined approach employing alkaline single cell gel electrophoresis (SCGE) and cytokinesis-blocked micronucleus (MNs) cytome bioassays was adopted to assess the deleterious properties of the auxinic 2,4-dichlorophenoxyacetic acid (2,4-D) and its microparticulated low volatility product Dedalo Elite (30% a.i.) on Chinese hamster ovary (CHO-K1) cells. Cytotoxicity was estimated by neutral red uptake (NRU), succinic dehydrogenase activity (MTT) and apoptosis assessment. Both compounds were assayed at 0.1-10 µg/ml concentration range. Whereas exposed CHO-K1 cells revealed a statistically significant enhancement of MNs when 10 µg 2,4-D/ml was assayed, MNs were only achieved in cells treated with 2 µg Dedalo Elite/ml. A diminution in the nuclear division index was only achieved after exposure to Dedalo Elite within the 1-10 µg/ml concentration range. Whereas increased genetic damage index was achieved when 6 and 10 µg 2,4-D/ml were assayed, GDI induction was observed in treatments employing 4 µg Dedalo Elite/ml. Both compounds induced cytotoxicity by inhibition of both lysosomal and MTT activities by enhancing the frequencies of early and late apoptotic cells. Our results not only indicate the genotoxic and cytotoxic potential of 2,4-D and its microparticulated marketplace formulation, but also highlight the risk of these agrochemicals present towards the biota and human health.

An imazethapyr-based herbicide formulation induces genotoxic, biochemical, and individual organizational effects in Leptodactylus latinasus tadpoles (Anura: Leptodactylidae).

Genotoxic, biochemical, and individual organizational effects on Leptodactylus latinasus tadpoles were evaluated after exposure to an imazethapyr (IMZT)-based commercial herbicide formulation, Pivot® H (10.59% IMZT). A determination of the value of the lethal concentration (LC50) was determined as a toxicological endpoint. Alterations in animal behavior and morphological abnormalities as well as cholinesterase (ChE), catalase (CAT), and glutathione S-transferase (GST) activities were employed as individual sublethal endpoints. Micronuclei frequencies (MNs), binucleated cells (BNs), blebbed nuclei (BLs), lobed nuclei (LBs), notched nuclei (NTs), erythroplastids (EPs), and evaluation of DNA strand breaks were employed as genotoxic endpoints. All biomarkers were evaluated after 48 and 96 h of exposure to concentrations of IMZT within 0.07-4.89 mg/L. LC5096h values of 1.01 and 0.29 mg/L IMZT were obtained for Gosner stages 25 and 36, respectively. Irregular swimming, diamond body shape, and decreased frequency of keratodonts were detected at both sampling times. Results showed that IMZT increased GST activity and MN frequency at 48 and 96 h of exposure. Other nuclear abnormalities were also observed in the circulating erythrocytes of tadpoles, i.e., NT and BL values after 48 h, and LN, BL, and EP values after 96 h. Finally, results showed that IMZT within 0.07-0.22 mg/L increased the genetic damage index in tadpoles exposed for both exposure times (48 and 96 h). This study is the first to report the sublethal biochemical effects of IMZT in anurans and is also the first report using L. latinasus tadpoles as a bioindicator for ecotoxicological studies.

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.