Recovery of Ceratophrys ornata tadpoles exposed to environmental concentrations of chlorpyrifos: evaluation of biomarkers of exposure.

Chlorpyrifos (CPF) is one of the most widely used insecticides worldwide despite the fact that many authors have warned about its effects in non-target biota. The effects of CPF on anurans are well known, but the process of recovery from these effects after exposure is less explored. The aim of this study was to evaluate the duration of sublethal effects induced by environmental concentrations of CPF on Ceratophrys ornata tadpoles after exposure. The experimental design consisted of an exposure phase (96 h) in which tadpoles were individually exposed to three concentrations of CPF (0, 0.01 and 0.02 mg CPF/L) and a post-exposure phase (72 h) in which exposed tadpoles were transferred to CPF-free media. Individuals that survived the exposure phase to CPF showed neither long-term lethal effects nor long-term swimming alterations and altered prey consumption after being transferred to CPF-free media. No morphological abnormalities were observed either. However, at the end of both phases, tadpoles emitted shorter sounds with a higher dominant frequency than the tadpoles in the control group, i.e., the tadpoles did not recover their normal sounds. Thus, for the first time in this species, we have shown that effects on sounds should be prioritized as biomarkers of exposure, as they not only provide longer detection times after cessation of exposure, but also involve non-destructive methods. The following order of priority could be established for the selection of biomarkers that diagnose the health status of individuals and precede irreversible responses such as mortality: alterations in sounds > swimming alterations > prey consumption.

Lethal and sublethal effects of the natural and healthy spinosad-based formulation Tracer™ on tadpoles of two neotropical species.

This paper presents the first acute toxicity data of the natural insecticide spinosad in amphibians. The sensitivity of two neotropical sympatric anuran species, Boana pulchella and Rhinella arenarum, to spinosad-based formulation Tracer™ was evaluated. Lethal effects are reported in tadpoles of B. pulchella stage 25 between 2.81 and 35.44 mg spinosad/L, while for the same concentration range no lethal effects were detected in tadpoles of R. arenarum of the same stage. In addition, Tracer™ produced sublethal effects at the individual level on the swimming activity, morphology (growth and presence of abnormalities), and development of B. pulchella from 2.81 to 5.78 mg spinosad/L, while in R. arenarum effects were only detected in the swimming activity and growth from 2.78 and 6.22 mg/L, respectively. At the biochemical level, Tracer™ produced inhibition of different enzymatic activities, among them, catalase activity at 2.81 mg spinosad/L, glutathione S- transferase activity from 2.81 to 2.98 mg spinosad/L, and acetylcholinesterase activity at 2.81 mg spinosad/L. These findings allow us to conclude that B. pulchella is more sensitive than R. arenarum to spinosad-based formulation Tracer™. The effects demonstrated here are not consistent with those expected since spinosad is supposed to be an environmental healthy alternative. This paper provides useful and necessary information to implement regulations on the use of new compounds entering the market and its associated risks.

Biomarker and hematological fieldwork with amphibians: is it necessary to sample all night?

In the context of the global amphibian crisis, biomonitoring constitutes a valuable assessment tool to provide critical up to date information on the status and health of amphibians worldwide. The aim of the current study was to evaluate the possible confounding effects of sex, size, and time since capture on enzymatic biomarkers and hematologic parameters of the South American frog Leptodactylus latrans. Frogs were collected by hand between 9 pm and 12 am on two consecutive nights. On the first night, captured frogs were transported for 2 h by car to laboratory installations, maintained overnight in plastic containers, and blood and tissue sampled on the next morning. In contrast, frogs collected on the second night were blood and tissue sampled in the field, immediately after the capture period. Hematological parameters were analyzed, and enzymatic activities of catalase, cholinesterase (ChE), and glutathione S-transferase (GST) were determined in the plasma, liver, kidney, and muscle. A sex difference was observed only for total white blood cell counts (WBC), females exhibiting significantly greater values than males. The packed cell volume (PCV), mean corpuscular hemoglobin concentration (MCHC), WBC, and muscle ChE activity were significantly correlated with snout-vent length (SVL). The correlation was inversed in the case of MCHC, WBC, and muscle ChE, while the correlation was positive between PCV and SVL. Most examined parameters presented similar values when frogs were sampled at night following capture or the next morning. Total red blood cells (RBCs) count, and plasma enzymatic activities of ChE and GST were the only parameters that presented significantly increased values in morning samplings compared with night samplings. Overall, the current study indicates that it is best to sample the frogs as soon as possible after capture if hematologic or plasmatic biomarkers are examined. Nevertheless, it is possible to sample on the next morning if tissular biomarkers are employed.