FBCB

B-esterase (BChE: butyrylcholinesterase and CbE: carboxylesterase) and glutathione S-transferase (GST) activity were measured in the plasma of Chaunus schneideri collected in rice fields and surrounding environments and in a reference pristine forest. The chemical criterion based on in-vitro reactivation of BChE activity using pyridine-2-aldoxime methochloride (2-PAM) was also determined. Mean values of plasma BchE, CbE, and GST activity for samples from agricultural areas were different from those for samples from pristine forest. Plasma samples from the two agricultural areas showed positive reactivation of BChE activity after incubation with 2-PAM. Based on our experimental evidence we suggest B-esterases and gluthatione S-transferases can be used in field monitoring as biomarkers of exposure of wildlife to pesticides, because the analysis in non-destructive and is sensitive to anti-ChE agrochemicals. Chemical reactivation of BChE is also a complementary method for assessing the effects of pesticides on toads inhabiting rice fields. Further studies are urgently needed to investigate adverse effects of massive exposure to pesticides experienced by native populations of anurans.

We determined the normal levels of butyrylcholinesterase (BChE), carboxylesterase (CbE), and glutathione S-transferases (GST) activities in three South American toad species in order to establish reference values for field pesticide monitoring purposes. Interspecies variations in B-esterase and GST activities were examined according to body mass. In addition, comparative inhibition of BChE and CbE activities using malaoxon, and chemical reactivation of malaoxon-inhibited BChE activity using pyridine-2-aldoxime methochloride (2-PAM) were investigated. Bufo fernandezae had average activity values for BChE: 17.31 mmol min−1 ml−1; CbE: 621.49 nmol min−1 ml−1 and GST: 1.94 mmol min−1 ml−1 while B. arenarum enzymatic average activities were BChE: 9.51 mmol min−1 ml−1; CbE: 270.07 nmol min−1 ml−1, and GST: 1.59 mmol min−1 ml−1; finally Bufo schneideri had enzymatic mean values of BChE: 2.08 mmol min−1 ml−1; CbE: 301.95 nmol min−1 ml−1, and GST: 1.60 mmol min−1 ml−1. Moreover, we found an allometric relationship between plasma BChE and CbE activities and body size for the three toad species. We suggest that B. fernandezae would be the species with a higher tolerance capacity to organophosphorous insecticides compared to the other toad species, while B. schneideri may be the most vulnerable toad species to field pesticide exposure, although some other factors (e.g., brain AChE sensitivity or pesticide metabolism by phosphotriesterases) should be also taken into account. The malaoxon-inhibited BChE activity of the three toad species was reactivated in the presence of 2-PAM, and it is recommended as a specific and sensitive methodology in the assessment of field exposure to OP insecticides together to compare BChE activity levels between OP-exposed and nonexposed individuals.

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 7 1.6 to 68.2 7 4.7 nmol min À 1 mg À 1 protein (PT). BChE activity also varied significantly, ranging from 3.3 7 0.4 to 7.5 7 0.4 nmol min À 1 mg À 1 PT. Both measures of CbE activities varied widely (CbE a-NA: 2.1 7 0.5-12.4 7 1.1 nmol min À 1 mg À 1 PT; CbE-4NPV: 21.8 7 1.8-102.6 7 7.9 nmol min À 1 mg À 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.

Environmental contaminants can disrupt interactions between aquatic species by altering community structure. We explored predator-prey interactions between marbled swamp juvenile eels (Synbranchus marmoratus; predator) and anuran tadpoles (Hypsiboas pulchellus; prey) in relation to two aspects: the importance of lateral line in the predator and whether the absence of light modifies predation rates; and the effect of a sub-lethal concentration of fenitrothion on both predator and prey. Eels were tested under two sensory conditions (lateral line intact and lateral line blocked by cobalt chloride) in dark conditions. Predation rates were evaluated using different treatments that combined predator and prey exposed or not to insecticide. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were also measured in muscle samples of eels and tadpoles to explore whether fenitrothion affects predator and prey differentially. Marbled swamp eels were more efficient in feeding on tadpoles during the night than during the day, showing that lateral line makes an important contribution to prey detection and capture. Regarding pesticide effects, short-term (6 h) exposure to an ecologically relevant fenitrothion dose of 2.5 mg L À1 altered the predator-prey relationship by changing prey behaviour, reducing prey detection and therefore increasing tadpole survival. At this concentration, the outcome of the predator-prey relationship appears biased in favor of the exposed tadpoles, which were released from predation risk, despite their altered behaviour and the higher inhibition percentages of tail BChE (70%) and AChE (51%) than in control individuals. Our study involving these model species and agrochemicals demonstrates that fenitrothion affected the outcome of a predator-prey relationship. Further studies are needed, in these species and other native amphibians, to investigate the nature of the mechanisms responsible for the adverse effects of pesticides on antipredator behaviour and predation efficiency.

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.

To test the hypothesis that eutrophication ponds modulate some aspects of the health responses and survival of anuran tadpoles, we conducted field experiments using Scinax nasicus as sentinel organism to evaluate the quality of two ponds filled with agricultural runoff in a dominant agricultural landscape of Mid-Western Entre Rı´os Province (Argentina). The survival, growth and development rates, erythrocytes nuclei aberrations, parasite infection, and brain cholinesterase activity were monitored after seven days of exposure. Water samples from the ponds were also analyzed for physico-chemical variables and levels of pesticide residues. Residues of organochlorine pesticides and nutrients were higher in the agricultural ponds with respect to those from the control pond. We suggest that the interactions among washed-off nutrients and pesticides from agriculture and environmental factors account for deleterious effects on S. nasicus survival, growth and development rate, thereby compromising their health status. These effects can lead, in turn, to an increase in tadpole vulnerability to opportunistic parasites, erythrocytes nuclei aberrations or hemolysis. r

Purpose In this study, we determined normal serum butyrylcholinesterase (BChE) and carboxylesterase (CbE) activities in Tupinambis merianae in order to obtain reference values for organophosphorus pesticide monitoring. Methods Forty-two T. merianae individuals were grouped by sex and size to identify potential differences in their enzyme levels to allow for proper representation of normal values for females, males, juveniles, and hatchlings. Mean CbE was determined using two model substrates: alpha-naphtylacetate (α-NA) and p-nitrophenyl valerate (4-NPV). BChE and CbE sensitivity to malaoxon (Mx) was also evaluated as well as the possibility of BChE reactivation with pyridine-2-aldoxime methochloride (2-PAM). Results Mean adult females’ BChE was significantly higher than adult males, juveniles, and hatchlings. No significant differences were found between groups regarding CbE. CbE (4-NPV) activity showed slightly negative correlation with lizard snout–vent length, while BChE and CbE (α-NA) showed no correlation with body size. Apparent IC50 values for BChE and CbE (α-NA) suggested different sensitivities among groups. CbE (4-NPV) could not be inhibited. All Mx-inhibited groups treated with 2-PAM in a final concentration of 2.8 mM showed clear signs of reactivation. Conclusions In conclusion, the results demonstrate that (1) plasma esterase activity did not vary with age and sex, except for BChE activity, and (2) because biological and environmental variables could be confounding factors in the response of plasma cholinesterases, complementary biomarkers like CbE inhibition and oxime-induced reactivation of esterases are strongly recommended.

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 7 1.6 to 68.2 7 4.7 nmol min À 1 mg À 1 protein (PT). BChE activity also varied significantly, ranging from 3.3 7 0.4 to 7.5 7 0.4 nmol min À 1 mg À 1 PT. Both measures of CbE activities varied widely (CbE a-NA: 2.1 7 0.5-12.4 7 1.1 nmol min À 1 mg À 1 PT; CbE-4NPV: 21.8 7 1.8-102.6 7 7.9 nmol min À 1 mg À 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.

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.

Purpose In this study, we determined normal serum butyrylcholinesterase (BChE) and carboxylesterase (CbE) activities in Tupinambis merianae in order to obtain reference values for organophosphorus pesticide monitoring. Methods Forty-two T. merianae individuals were grouped by sex and size to identify potential differences in their enzyme levels to allow for proper representation of normal values for females, males, juveniles, and hatchlings. Mean CbE was determined using two model substrates: alpha-naphtylacetate (α-NA) and p-nitrophenyl valerate (4-NPV). BChE and CbE sensitivity to malaoxon (Mx) was also evaluated as well as the possibility of BChE reactivation with pyridine-2-aldoxime methochloride (2-PAM). Results Mean adult females’ BChE was significantly higher than adult males, juveniles, and hatchlings. No significant differences were found between groups regarding CbE. CbE (4-NPV) activity showed slightly negative correlation with lizard snout–vent length, while BChE and CbE (α-NA) showed no correlation with body size. Apparent IC50 values for BChE and CbE (α-NA) suggested different sensitivities among groups. CbE (4-NPV) could not be inhibited. All Mx-inhibited groups treated with 2-PAM in a final concentration of 2.8 mM showed clear signs of reactivation. Conclusions In conclusion, the results demonstrate that (1) plasma esterase activity did not vary with age and sex, except for BChE activity, and (2) because biological and environmental variables could be confounding factors in the response of plasma cholinesterases, complementary biomarkers like CbE inhibition and oxime-induced reactivation of esterases are strongly recommended.

We determined the normal levels of butyrylcholinesterase (BChE), carboxylesterase (CbE), and glutathione S-transferases (GST) activities in three South American toad species in order to establish reference values for field pesticide monitoring purposes. Interspecies variations in B-esterase and GST activities were examined according to body mass. In addition, comparative inhibition of BChE and CbE activities using malaoxon, and chemical reactivation of malaoxon-inhibited BChE activity using pyridine-2-aldoxime methochloride (2-PAM) were investigated. Bufo fernandezae had average activity values for BChE: 17.31 mmol min À1 ml À1 ; CbE: 621.49 nmol min À1 ml À1 and GST: 1.94 mmol min À1 ml À1 while B. arenarum enzymatic average activities were BChE: 9.51 mmol min À1 ml À1 ; CbE: 270.07 nmol min À1 ml À1 , and GST: 1.59 mmol min À1 ml À1 ; finally Bufo schneideri had enzymatic mean values of BChE: 2.08 mmol min À1 ml À1 ; CbE: 301.95 nmol min À1 ml À1 , and GST: 1.60 mmol min À1 ml À1 . Moreover, we found an allometric relationship between plasma BChE and CbE activities and body size for the three toad species. We suggest that B. fernandezae would be the species with a higher tolerance capacity to organophosphorous insecticides compared to the other toad species, while B. schneideri may be the most vulnerable toad species to field pesticide exposure, although some other factors (e.g., brain AChE sensitivity or pesticide metabolism by phosphotriesterases) should be also taken into account. The malaoxoninhibited BChE activity of the three toad species was reactivated in the presence of 2-PAM, and it is recommended as a specific and sensitive methodology in the assessment of field exposure to OP insecticides together to compare BChE activity levels between OP-exposed and nonexposed individuals.

In this study, amphibian tadpoles of Hypsiboas pulchellus were exposed to herbicide Liberty Ò , 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.

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.

Environmental contaminants can disrupt interactions between aquatic species by altering community structure. We explored predator-prey interactions between marbled swamp juvenile eels (Synbranchus marmoratus; predator) and anuran tadpoles (Hypsiboas pulchellus; prey) in relation to two aspects: the importance of lateral line in the predator and whether the absence of light modifies predation rates; and the effect of a sub-lethal concentration of fenitrothion on both predator and prey. Eels were tested under two sensory conditions (lateral line intact and lateral line blocked by cobalt chloride) in dark conditions. Predation rates were evaluated using different treatments that combined predator and prey exposed or not to insecticide. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were also measured in muscle samples of eels and tadpoles to explore whether fenitrothion affects predator and prey differentially. Marbled swamp eels were more efficient in feeding on tadpoles during the night than during the day, showing that lateral line makes an important contribution to prey detection and capture. Regarding pesticide effects, short-term (6 h) exposure to an ecologically relevant fenitrothion dose of 2.5 mg L À1 altered the predator-prey relationship by changing prey behaviour, reducing prey detection and therefore increasing tadpole survival. At this concentration, the outcome of the predator-prey relationship appears biased in favor of the exposed tadpoles, which were released from predation risk, despite their altered behaviour and the higher inhibition percentages of tail BChE (70%) and AChE (51%) than in control individuals. Our study involving these model species and agrochemicals demonstrates that fenitrothion affected the outcome of a predator-prey relationship. Further studies are needed, in these species and other native amphibians, to investigate the nature of the mechanisms responsible for the adverse effects of pesticides on antipredator behaviour and predation efficiency.

We present the first compilation and analysis of cases of morphologically abnormal anurans from the mideastern region of Argentina (Córdoba, Santa Fe, and Entre Ríos Provinces). We sampled for abnormal individuals at 51 sites in agricultural, suburban, and forest settings between January 2000 and December 2009. We recorded 71 abnormal individuals, including 16 types of abnormalities in 15 anuran species. In agricultural sites, we found 12 types of abnormalities, with ectromelia being the most abundant. In suburban sites, we recorded seven types of abnormalities, with brachygnathia and ectromelia being the most common. In forest sites, we found three types of abnormalities, with ectromelia again being the most abundant. This study is the first catalog of anurans with morphological abnormalities in Argentina, thus expanding the geographic range of observed abnormalities in amphibians and illustrating the ubiquity of this phenomenon.

We determined normal plasma butyrylcholinesterase (BChE), carboxylesterase (CbE using α-NA substrate), and glutathione S-transferase (GST) activities in Caiman latirostris and Phrynops hilarii to obtain reference values for organophosphorus (OP) pesticide monitoring. BChE and CbE sensitivity to malaoxon was also evaluated. C. latirostris (N012; six males and six females) and P. hilarii (N012; seven males and five females) were obtained from the programs Yacaré (Entre Ríos Province, Argentina) and Zoo of Córdoba (Córdoba Province, Argentina). Mean total (female and male) plasma BChE activity was significantly different between reptile species, ranging between 0.337±0.085 μmol min −1 ml −1 of plasma for C. latirostris and 0.251±0.070 μmol min −1 ml −1 of plasma for P. hilarii. However, plasma CbE (α-NA) and GST activities were significantly higher in P. hilarii (4.81 ±1.00 and 0.145±0.045 μmol min −1 ml −1 of plasma, respectively) than in C. latirostris (0.57±0.20 and 0.059 ±0.013 μmol min −1 ml −1 of plasma, respectively). No significant differences in B-esterase and GST activities were detected between sexes, except CbE (α-NA) for C. latirostris. IC 50 values for BChE and CbE (α-NA) suggested different sensitivity levels between species and between sexes. The results demonstrate that plasma esterase activity varied between species, but not between sexes (except CbE for C. latirostris). The in vitro inhibition tests indicated that CbE (α-NA) is more sensitive to inhibition than BChE. C. latirostris may be the reptile species most vulnerable to field pesticide exposure because this reptile presents the lowest CbE activity levels and its B-esterase levels seem more sensitive to OP.

Activity of B-esterases (BChE: butyrylcholinesterase and CbE: carboxylesterase using two model substrates: a-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-2aldoxime 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.

To test the hypothesis that eutrophication ponds modulate some aspects of the health responses and survival of anuran tadpoles, we conducted field experiments using Scinax nasicus as sentinel organism to evaluate the quality of two ponds filled with agricultural runoff in a dominant agricultural landscape of Mid-Western Entre Rı´os Province (Argentina). The survival, growth and development rates, erythrocytes nuclei aberrations, parasite infection, and brain cholinesterase activity were monitored after seven days of exposure. Water samples from the ponds were also analyzed for physico-chemical variables and levels of pesticide residues. Residues of organochlorine pesticides and nutrients were higher in the agricultural ponds with respect to those from the control pond. We suggest that the interactions among washed-off nutrients and pesticides from agriculture and environmental factors account for deleterious effects on S. nasicus survival, growth and development rate, thereby compromising their health status. These effects can lead, in turn, to an increase in tadpole vulnerability to opportunistic parasites, erythrocytes nuclei aberrations or hemolysis. r