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We investigated the effects of four commercial formulations of herbicides (glyphosate [GLY], metsulfuron-methyl [MET], bispyribac-sodium [BIS], and picloram [PIC]) individually, and in three 50:50 mixtures (GLY-MET, GLY-BIS, GLY-PIC) on the common toad Rhinella arenarum (Anura: Bufonidae) tadpoles. Enzymatic parameters such as, glutathione S-transferase (GST), butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) activities, as well as erythrocyte nuclear abnormalities (ENA) were studied. Interactions between herbicides in mixtures were evaluated and classified as additive, synergistic, or antagonistic. Toxicity results (48-h LC 50 ) showed that PIC was the most toxic herbicide, followed by BIS, GLY, and MET, while GLY-PIC was the most toxic mixture, followed by GLY-BIS, and GLY-MET. All commercial herbicide formulations and their mixtures significantly inhibited BChE activity in exposed tadpoles. The AChE activity was also inhibited by all herbicides and their mixtures, except by GLY-BIS. The inhibition of GST activity was only significant for GLY, MET, PIC, and GLY-MET. A significant increase in the frequency of ENA was found for tadpoles exposed either to commercial herbicide formulations or to mixtures, except for GLY. All the mixtures showed synergism for BChE activity while for AChE only the GLY-MET and GLY-PIC mixtures acted synergistically. GLY-MET showed synergism for GST, whereas for ENA, the mixture GLY-BIS was antagonistic. This study with R. arenarum tadpoles demonstrates that the interactions between three of the most intensively used herbicides in soybean crops results in synergistic effects on mortality and neurotoxicity and synergistic or additive effects in genotoxicity.

In this study, amphibian tadpoles of Hypsiboas pulchellus were exposed to herbicide Liberty®, which contains glufosinate ammonium (GLA), for 48 h to the following concentrations: 0 (control), 3.55, 4.74, 6.32, 8.43, 11.25, 15, 20, 26.6, and 35.5 mg GLA L(-1). Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities, as well as swimming capabilities (swimming speed and mean distance) were measured in tadpoles whose concentrations displayed survival rates > 85 %. Our results reveal that sublethal concentrations of GLA significantly inhibited both AChE and BChE activities in tadpoles with respect to the control, showing a concentration-dependent inhibitory effect. The highest inhibition percentages of AChE (50.86%) and BChE (53.02%) were registered in tadpoles exposed to 15 mg GLA L(-1). At this concentration, a significant increase of the swimming speed and mean distance were found in exposed tadpoles with respect to the control, as well as a negative and significant correlation between swimming speed and BChE activity, thus suggesting that this enzyme inhibition is related to an increase in swimming speed. Therefore, exposure of tadpoles to GLA in the wild at concentrations similar to those tested here may have adverse consequences at population level because neurotransmission and swimming performance are essential for tadpole performance and survival.

Activity of B-esterases (BChE: butyrylcholinesterase and CbE: carboxylesterase using two model substrates: α-naphthyl acetate and 4-nitrophenyl valerate) in a native frog, Leptodactylus chaquensis from rice fields (RF1: methamidophos and RF2: cypermethrin and endosulfan sprayed by aircraft) and non-contaminated area (pristine forest) was measured. The ability of pyridine-2-aldoxime methochloride (2-PAM) to reactivate BChE levels was also explored. In addition, changes in blood cell morphology and parasite infection were determined. Mean values of plasma BChE activities were lower in samples from the two rice fields than in those from the reference site. CbE (4-nitrophenyl valerate) levels varied in the three sites studied, being highest in RF1. Frog plasma from RF1 showed positive reactivation of BChE activity after incubation with 2-PAM. Blood parameters of frogs from RF2 revealed morphological alterations (anisochromasia and immature erythrocytes frequency). Moreover, a major infection of protozoan Trypanosoma sp. in individuals from the two rice fields was detected. We suggest that integrated use of several biomarkers (BChE and CBEs, chemical reactivation of plasma with 2-PAM, and blood cell parameters) may be a promising procedure for use in biomonitoring programmes to diagnose pesticide exposure of wild populations of this frog and other native anuran species in Argentina.