Andrés M. Attademo

Toxicity of Four Herbicide Formulations with Glyphosate on Rhinella arenarum (Anura: Bufonidae) Tadpoles: B-esterases and Glutathione S-transferase Inhibitors

In this study, amphibian tadpoles Rhinella arenarum were exposed to different concentrations of Roundup Ultra-Max (ULT), Infosato (INF), Glifoglex, and C-K YUYOS FAV. Tadpoles were exposed to these commercial formulations with glyphosate (CF-GLY) at the following concentrations (acid equivalent [ae]): 0 (control), 1.85, 3.75, 7.5, 15, 30, 60, 120, and 240xa0mg ae/L for 6–48xa0h (short-term). Acetylcholinesterase (AChE), butyrylcholinesterase (BChE), carboxylesterase (CbE), and glutathione S-transferase (GST) activities were measured among tadpoles sampled from those treatments that displayed survival rates >85%. Forty-eight-hour LC50 for R. arenarum tadpoles exposed to CF-GLY in the static tests ranged from ULTxa0=xa02.42 to FAVxa0=xa077.52xa0mg ae/L. For all CF-GLY, the LC50 values stabilized at 24xa0h of exposure. Tadpoles exposed to all CF-GLY concentrations at 48xa0h showed decreases in the activities of AChE (controlxa0=xa017.50xa0±xa02.23xa0nmol/min/mg/protein; maximum inhibition INF 30xa0mg ae/L, 71.52%), BChE (controlxa0=xa06.31xa0±xa00.86xa0nmol/min/mg/protein; maximum inhibition INF 15xa0mg ae/L, 78.84%), CbE (controlxa0=xa04.39xa0±xa00.46xa0nmol/min/mg/protein; maximum inhibition INF 15xa0mg ae/L, 81.18%), and GST (controlxa0=xa04.86xa0±xa00.49xa0nmol/min/mg/protein; maximum inhibition INF 1.87xa0mg ae/L, 86.12%). These results indicate that CF-GLY produce a wide range of toxicities and that all enzymatic parameters tested may be good early indicators of herbicide contamination in R. arenarum tadpoles.

Diversity of Anurans Across Agricultural Ponds in Argentina

We examined the anuran diversity of 31 ponds (30 located on the border of soybean cropland and one within a protected forest) in mid-western Entre Ríos Province (Argentina). Moreover, each species found was characterised with respect to its vertical location. Using principal component (PCA) and canonical correspondence analyses (CCA) we quantified associations between species diversity and habitat and spatial variables. A total of 21 anuran species belonging to four families (Microhylidae, Bufonidae, Leptodactylidae and Hylidae) were detected in ponds surrounded by soybean croplands. PCA generated three principal components, which together explained variation in anuran diversity across the agricultural ponds and control site. Negative values of PC-1 described the smaller ponds with narrower hedgerow and monospecific shore vegetation. PC-2 had high loading on pond depth, and PC-3 had negative loading on air temperature. CCA showed a very strong association between the two data sets. We found all guilds related with pond area. Indeed, we found that arboreal species were recorded in large ponds with higher values of shore vegetation index and presence of wider hedgerow. Moreover, a higher number of terrestrial species was found to relate to large pond areas and greater shore vegetation diversity. Finally, aquatic species were related to pond area, shore vegetation index and depth. Anuran diversity across agricultural ponds of mid-western Entre Ríos Province can be affected by local habitat factors such as reduction in pond size and depth, shore vegetation richness, width of hedgerow and air temperatures. Management of anurans to reverse recent declines will require defining high-quality habitat for individual species or group of species, followed by efforts to retain or restore these aquatic habitat. The maintenance of shore vegetation of ponds and hedgerows may increases the number of species and diversity of anurans within agricultural landscapes.

Activity levels of B-esterases in the tadpoles of 11 species of frogs in the middle Paraná River floodplain: Implication for ecological risk assessment of soybean crops

Soybean fields provide habitats for many species of amphibians. However, the persistence and health of amphibian populations may be at risk from the increasing use of pesticides and other agricultural chemicals. We examined the activities of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and carboxylesterases (CbEs) in 11 syntopic species of larval anurans. In vitro effects of malaoxon causing 50% BChE inhibition (IC(50)) were also studied. In addition, we calculated a relative risk index (RI) based on the geographic distributions of the anurans, the phenology of soybean cultivation, and basal enzymatic values related to potential pesticide detoxification. Among the 11 species, AChE activity varied from 17.5 ± 1.6 to 68.2 ± 4.7 nmol min(-1) mg(-1) protein (PT). BChE activity also varied significantly, ranging from 3.3 ± 0.4 to 7.5 ± 0.4 nmol min(-1) mg(-1)PT. Both measures of CbE activities varied widely (CbE α-NA: 2.1 ± 0.5-12.4 ± 1.1 nmol min(-1) mg(-1) PT; CbE-4NPV: 21.8 ± 1.8-102.6 ± 7.9 nmol min(-1) mg(-1) PT). We also corroborate that lower BChE activity levels for the tadpoles were associated at minor IC(50) values. The results of this study demonstrate significant variation in enzymatic levels among several tadpole species and intermediate to high RI values for 7 species. Based on these results, it appears that a conversion of native ecosystems to soybean crops may lead to increased ecological risk for anuran amphibians.

Induction of micronuclei and nuclear abnormalities in tadpoles of the common toad (Rhinella arenarum) treated with the herbicides Liberty® and glufosinate-ammonium.

The assessment of micronucleated erythrocytes (ME) in blood represents a widely used method for the detection of chromosomal damage by chemical agents, such as herbicides that may occur as water contaminants. We investigated the changes in some circulating blood-cell parameters of tadpoles of the common toad (Rhinella arenarum) that were exposed during 48 or 96 h to three sub-lethal concentrations (3.75, 7.5, and 15 mg/L) of a commercial formulation of a glufosinate-ammonium (GLA)-based herbicide (Liberty(®), LY(®)) as well as to the corresponding active ingredient GLA. The frequency of ME and other erythrocyte nuclear abnormalities (ENA, i.e., lobed nuclei, binucleates or segmented nuclei, kidney-shaped nuclei, notched nuclei, and picnotic nuclei) were evaluated and compared with positive (cyclophosphamide, CP, 40 mg/L) and negative (de-chlorinated tap water) controls. The results indicate that the exposure of R. arenarum tadpoles to LY(®) induces a concentration-dependent increase in ME frequency. The ENA frequency at 48 h was also significantly higher than that in the negative control group for all the chemicals assayed (CP, LY(®) and GLA) whereas at 96 h, increases in ENA over the negative control group were found only for CP and GLA (7.5 mg/L). Our study demonstrates that the commercial formulation of a GLA-based herbicide induces micronucleus formation in R. arenarum tadpoles, in contrast to the active ingredient. According to these results, the inert ingredients of the commercial formulation played an important role in the production of genotoxic damage in erythrocytes of amphibian tadpoles.

Individual and Mixture Toxicity of Commercial Formulations Containing Glyphosate, Metsulfuron-Methyl, Bispyribac-Sodium, and Picloram on Rhinella arenarum Tadpoles

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 LC50) 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.

B-esterase activities and blood cell morphology in the frog Leptodactylus chaquensis (Amphibia: Leptodactylidae) on rice agroecosystems from Santa Fe Province (Argentina).

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.

Induction of micronuclei in tadpoles of Odontophrynus americanus (Amphibia: Leptodactylidae) by the pyrethroid insecticide cypermethrin

Cypermethrin (CY) is an active cyano pyrethroid effective against a wide range of pests encountered in agriculture and forestry. Although CY is not mutagenic in in vitro assays for gene mutation, in vivo assays showed conflicting results. In vivo genotoxicity of the synthetic pyrethroid CY in erythrocytes of Odontophrynus americanus tadpoles was examined. The frequency of micronuclei (MN) was recorded in blood smears obtained from tadpoles exposed in vivo to four different nominal concentrations 5, 10, 20 or 40u2009µgu2009L−1 of the compound and fixed at two sampling times 48 and 96u2009h. As a positive control larvae were exposed to 40u2009mgu2009L−1 of cyclophosphamide (CP). Tadpoles exposed to all CY treatments showed a significant increase in single small MN compared to the negative control group after 48u2009h and at 5 and 10u2009µgu2009L−1 of CY at 96u2009h. Results obtained here demonstrated the genotoxic effects of the commercial formulation CY in the anuran larvae analyzed. Thus, data suggest that measurements of MN and other erythrocytes morphological aberrations performed in circulating blood samples of O. americanus tadpoles is a method for detecting cytogenetic damage in other native species.

Toxicity of the fungicide trifloxystrobin on tadpoles and its effect on fish–tadpole interaction

Contamination of aquatic systems is a major environmental stress that can interfere with predator-prey interactions, altering prey or predator behavior differentially. We determined toxicity parameters of the fungicide trifloxystrobin (TFS) and examined its effects on predation rate, using a fish predator (Synbranchus marmoratus) and four anuran tadpole species as prey (Rhinella arenarum, Physalaemus santafecinus, Leptodactylus latrans, and Elachistocleis bicolor). TFS was not equally toxic to the four tadpole species, E. bicolor being the most sensitive species, followed by P. santafecinus, R. arenarum, and L. latrans. Predation rates were evaluated using different treatments that combined predator and prey exposed or not to this fungicide. TFS would alter the outcome of eel-tadpole interaction by reducing prey movements; thus, prey detection would decrease and therefore tadpole survival would increase. In addition, eels preyed selectively upon non-exposed tadpoles avoiding the exposed ones almost all throughout the period evaluated. Predation rate differed among prey species; such differences were not due to TFS exposure, but to interspecific differences in behavior. The mechanism that would explain TFS-induced reduction in predation rates remains unclear; however, what is clear is that sublethal TFS concentrations have the potential to alter prey behavior, thereby indirectly altering predator-prey interactions. In addition, we consider that predator-prey relationships are measurable responses of toxicant exposure and provide ecological insight into how contaminants modify predator-prey interactions.

Cholinesterase activities and behavioral changes in Hypsiboas pulchellus (Anura: Hylidae) tadpoles exposed to glufosinate ammonium herbicide

In this study, amphibian tadpoles of Hypsiboas pulchellus were exposed to herbicide Liberty®, which contains glufosinate ammonium (GLA), for 48xa0h to the following concentrations: 0 (control), 3.55, 4.74, 6.32, 8.43, 11.25, 15, 20, 26.6, and 35.5xa0mg 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 >85xa0%. 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 (50.86xa0%) and BChE (53.02xa0%) were registered in tadpoles exposed to 15xa0mg GLAxa0L−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.

Biological endpoints, enzyme activities, and blood cell parameters in two anuran tadpole species in rice agroecosystems of mid-eastern Argentina

Different biological variables of tadpoles, including survival, development and growth rates, and biomarkers [cholinesterases, glutathione-S-transferases (GST), and blood cell morphology] were evaluated in two anuran species, Scinax squalirostris (Hylidae) and Leptodactylus mystacinus (Leptodactylidae), using in situ experimental chambers in a rice field (RF) sprayed with insecticide Lambda-cyhalothrin (LTC) by aircraft in Santa Fe Province, Argentina. We found a significant decrease in body weight (0.62u2009±u20090.04xa0g) of L. mystacinus and an increased development rate of S. squalirostris in individuals from RF (41u2009±u20091; Gosner) with respect to individuals from the reference site (RS: 0.93u2009±u20090.04xa0g and 37u2009±u20090; respectively). In S. squalirostris, individuals from RF mean values of butyrylcholinesterase activities decreased at 48 (4.09u2009±u20090.32xa0nmolxa0min-1xa0mg-1 of TP) and 96xa0h (3.74u2009±u20090.20xa0nmolxa0min-1xa0mg-1 of TP), whereas inhibition of acetylcholinesterase was observed at 96xa0h (47.44u2009±u20092.78xa0nmolxa0min-1xa0mg-1 of TP). In L. mystacinus from RF, an induction of acetylcholinesterase activity was observed at 96xa0h (36.01u2009±u20091.09xa0nmolxa0min-1xa0mg-1 of TP). Glutathione-S-transferase levels varied between species, being higher in L. mystacinus individuals but lower in S. squalirostris from RF at 48 (272.29 ±11.78 and 71.87u2009±u20091.70xa0nmolxa0min-1xa0mg-1 of TP; respectively) and 96xa0h (279.25u2009±u200913.06 and 57.62u2009±u20094.58xa0nmolxa0min-1xa0mg-1 of TP, respectively). Blood cell parameters revealed a lower number of mitotic cells (MC: 0.36u2009±u20090.31%o for S. squalirostris and 0.08u2009±u20090.05 %o for L. mystacinus) and higher number of eosinophils (E: 3.45u2009±u20091.75 %o for S. squalirostris and 7.64u2009±u20090.98 %o for L. mystacinus) in individuals from the RF than in individuals from the RS (MC: 2.55u2009±u20090.74 %o for S. squalirostris and 1.87u2009±u20090.72%o for L. mystacinus; and E: 0.13u2009±u20090.09 for S. squalirostris and 3.20u2009±u20090.80 for L. mystacinus). Overall, our results demonstrate the existence of apparent differences in sensitivity between species in a series of sublethal responses to short-term exposure in RF after the application of Lambda-cyhalothrin. We suggest that the integral use of biological endpoints (development and growth) together with biomarkers (cholinesterase, GST, and blood cell parameters) may be a promising integral procedure for investigating pesticide exposure in wild frog populations.