Mutagenicity, hepatotoxicity, and neurotoxicity of glyphosate and fipronil commercial formulations in Amazon turtles neonates (Podocnemis expansa).

Pesticides are considered one of the main causes of the population decline of reptiles worldwide, with freshwater turtles being particularly susceptible to aquatic contamination. In this context, we investigated the potential mutagenic, hepatotoxic, and neurotoxic effects in neonates of Podocnemis expansa exposed to substrate contaminated with different concentrations of glyphosate and/or fipronil during embryonic development. Eggs collected from the natural environment were artificially incubated in sand moistened with pure water, water added with glyphosate Atar 48® at concentrations of 65 and 6500 µg/L (groups G1 and G2, respectively), water added with fipronil Regent® 800WG at 4 and 400 µg/L (groups F1 and F2, respectively) and, water added with the combination of 65 µg/L glyphosate and 4 µg/L fipronil or with 6500 µg/L glyphosate and 400 µg/L fipronil (groups GF1 and GF2, respectively). For mutagenicity analysis, we evaluated the frequency of micronuclei (MN) and other erythrocyte nuclear abnormalities (ENAs), while for evaluation of hepatotoxicity and neurotoxicity, livers and encephalon were analyzed for histopathological alterations. Exposure to pesticides, alone or in combination, increased the frequency of erythrocyte nuclear abnormalities, particularly blebbed nuclei, moved nuclei, and notched nuclei. Individuals exposed to fipronil exhibited congestion and inflammatory infiltrate in their liver tissue, while, in the encephalon, congestion, and necrosis were present. Our study confirms that the incubation of eggs in substrate polluted with glyphosate and fipronil causes histopathological damage and mutagenic alteration in P. expansa, highlighting the importance of using different biomarkers to evaluate the ecotoxicological effects of these pesticides, especially in oviparous animals.

Eggshell composition of Amazon turtle (Podocnemis expansa) is altered after incubation in substrates containing glyphosate and fipronil formulations.

Besides its crucial role during embryo development, eggshells are an essential bioindicator of environmental contaminants. However, the effects of contaminant exposure during incubation on the eggshell composition remain poorly known for freshwater turtles. Accordingly, we tested the effects of incubating the eggs of Podocnemis expansa in substrates containing glyphosate and fipronil formulations on the eggshell's level of mineral and dry matter, crude protein, nitrogen, and ethereal extract. Eggs were incubated in sand moistened with water contaminated with glyphosate Atar 48 at concentrations of 65 or 6500 µg/L, fipronil Regent 800 WG at concentrations of 4 or 400 µg/L, or the combination of 65 µg/L glyphosate and 4 µg/L fipronil and 6500 µg/L glyphosate with 400 µg/L fipronil. Exposure to the tested pesticides, alone or in association, altered the chemical composition of the eggshell of P. expansa, reducing the eggshell's moisture and crude protein content and increasing levels of ethereal extract. These changes may cause significant deficiencies in the mobilization of water and nutrients to the embryo, affecting the development and reproductive success of P. expansa.

The exposure in ovo of embryos belonging to Amazonian turtle species Podocnemis expansa (Testudines) to commercial glyphosate and fipronil formulations impairs their growth and changes their skeletal development.

Pesticides are widely used in agricultural production; moreover, they can have direct and indirect effect on both flora and fauna. Aquatic organisms, among other animals, including reptiles, are mainly susceptible to contamination effects. Accordingly, the aim of the present study is to test the hypothesis that the incubation of Podocnemis expansa eggs in substrate added with glyphosate and fipronil formulations changes their viability, interferes with their growth and induces bone alterations. Eggs collected in natural environment were artificially incubated in sand moistened with water added with glyphosate Atar 48, at concentrations of 65 or 6500 µg/L (groups G1 and G2, respectively), and with fipronil Regent 800 WG at 4 or 400 µg/L (groups F1 and F2, respectively) or, yet, with the combination of 65 µg/L glyphosate and 4 µg/L fipronil, or with 6500 µg/L glyphosate and 400 µg/L fipronil (groups GF1 and GF2, respectively). The level of exposure to the herein assessed pesticides was quantified at the end of the incubation period; it was done by dosing its concentration in eggshells. Eggs exposed to the tested pesticides did not have their viability affected by it; however, all embryos exposed to the tested pesticides showed lowered body mass at hatch, as well as impaired development. In addition, bone malformation in the scleral ossicular ring was observed in individuals in groups F1, F2 and GF1. Pesticides accumulated in eggshells at concentrations related to exposure level. Thus, the recorded results have evidenced some remarkably relevant, and previously unknown, impacts associated with the exposure of a species listed as lower risk/conservation dependent, which spends most of its life in the water, to two widely used pesticides, at a very sensitive stage of its life, namely: egg incubation on land.

Thermal regime effects on the resting metabolic rate of rattlesnakes depend on temperature range.

While ectothermic organisms often experience considerable circadian variation in body temperature under natural conditions, the study of the effects of temperature on metabolic rates are traditionally based on subjecting animals to constant temperature regimes. Whether data resulting from constant-temperature experiments accurately predicts temperature effects under more natural fluctuating temperature regimes remains uncertain. To address such possibility, we measured the resting metabolic rates of the South American rattlesnakes (Crotalus durissus) under constant and circadian fluctuating thermal regimes in a range of temperatures. Metabolic rates measured at constant 20 °C and 25 °C did not differ from the rates measured at fluctuating regimes with corresponding mean temperatures. However, the difference between thermal regimes increased with temperature, with the metabolic rate measured at constant 30 °C being greater than that measured at the fluctuating thermal regime with corresponding mean temperature. Therefore, our results indicate that thermal regime effects on rattlesnakes' metabolism is dependent on temperature range. Broadly, our results highlight the importance of considering multi-factorial attributes of temperature variation in the exam of its effects over functional traits. Such approach provides a more solid support for inferences about temperature effects on the life history, ecology and conservation of ectothermic organisms.

Temperature and dehydration effects on metabolism, water uptake and the partitioning between respiratory and cutaneous evaporative water loss in a terrestrial toad.

Terrestrial anurans often experience fluctuations in body temperature and hydration state, which are known to influence evaporative water loss through the skin (EWLSkin) and lungs (EWLResp). These effects arise from associated changes in skin permeability, metabolism and lung ventilation. Herein, we determined the rates of EWLSkin and EWLResp in the terrestrial toad Rhinella diptycha at different temperatures and hydration states. We measured oxygen uptake rates to verify whether alterations in the partitioning between EWLSkin and EWLResp were associated with metabolism-induced changes in pulmonary gas exchange. We also measured the influence of hydration and temperature on water uptake (WU) through the skin. Finally, as estimates of skin resistance to evaporation (Rs) are usually inferred from total evaporative water loss (EWLTotal), under the assumption of negligible EWLResp, we calculated the potential error in accepting this assumption for different temperature and hydration states. EWLSkin and EWLResp increased with temperature, but this response was greater for EWLResp, which was attributed to the temperature-induced elevation in metabolism and lung ventilation. Dehydration caused a decrease in the relative contribution of EWLSkin to EWLTotal, mirrored by the concurrent increase in the contribution of EWLResp, at all temperatures. Thus, Rs increased with dehydration. WU rates were dictated by dehydration with little influence of temperature. The partitioning between EWLSkin and EWLResp was affected by both temperature and hydration state and, under some conditions, considering EWLResp as negligible led to significant errors in the assessment of skin resistance to evaporation.

Seasonal variation in the thermal biology of a terrestrial toad, Rhinella icterica (Bufonidae), from the Brazilian Atlantic Forest.

As ectotherms, amphibians may exhibit changes in their thermal biology associated with spatial and temporal environmental contingencies. However, our knowledge on how amphibian´s thermal biology responds to seasonal changes in the environment is restricted to a few species, mostly from temperate regions, in a marked contrast with the high species diversity found in the Neotropics. We investigated whether or not the seasonal variation in climatic parameters from a high-montane ombrophilous forest in the Brazilian Atlantic Forest could lead to concurrent adjustments in the thermal biology of the terrestrial toad Rhinella icterica. We measured active body temperature (Tb) in the field, and preferred body temperature (Tpref) and thermal tolerance (critical thermal minimum, CTmin, and maximum, CTmax) in the laboratory, for toads collected at two distinct seasons: warm/wet and cold/dry. We also measured operative environmental temperatures (Te) using agar toad models coupled with dataloggers distributed in different microhabitats in the field to estimate accuracy (db) and effectiveness (E) of thermoregulation of the toads for both seasons. Toads had higher Tpref in the warm/wet season compared to the cold/dry season, even though no seasonal change occurred in field Tb's. In the warm/wet season, toads decreased the accuracy of thermoregulation and avoided thermally favorable microhabitats, while in the cold/dry season they increased the accuracy of thermoregulation and exhibited high degree of thermoconformity. This result may encompass thermoregulatory adjustments to seasonal changes in Te's, but may also reflect seasonal differences in compromises between Tb regulation and other ecologically relevant activities (reproduction, foraging). Toads did not exhibit changes in CTmin or CTmax, which indicates a low risk of exposure to extreme temperatures in this particular habitat, at both seasons, possibly combined with a low flexibility of this trait. Overall, our study shows seasonal acclimatization in some aspects of the thermal biology of the toad, R. icterica.