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.

Toxicity of the neonicotinoid insecticides thiamethoxam and imidacloprid to tadpoles of three species of South American amphibians and effects of thiamethoxam on the metamorphosis of Rhinella arenarum.

The present study examined the acute and chronic toxicity of the neonicotinoid insecticides imidacloprid (IMI) and thiamethoxam (TIA) on the neotropical amphibian species Rhinella arenarum, Rhinella fernandezae and Scinax granulatus. The median lethal concentration after 96 hr exposure (96 hr-LC50) ranged between 11.28 and >71.2 mg/L amongst all species and development stages tested, indicating that these pesticides are not likely to produce acute toxicity in the wild. The subchronic toxicity was also low, with 21 day-LC50 values ranging between 27.15 and >71.2 mg/L. However, tadpoles of Rhinella arenarum exposed to thiamethoxam from stage 27 until completion of metamorphosis presented a significantly lower metamorphic success rate together with a smaller size at metamorphosis, starting from the lowest concentration tested. Although a number of studies previously examined the effects of neonicotinoids on amphibian tadpoles, these investigations focused on the time to metamorphosis and reported a variety of results including retardation, acceleration or lack of effect. Here, data demonstrated that thiamethoxam predominantly impacts metamorphosis through reduction of the transformation success and body weight, rather than by affecting the timings of metamorphosis. By closely monitoring progression of tadpoles through the different stages, impairment of metamorphosis was demonstrated to occur during the transition from stage 39 to 42, suggesting an effect on the thyroid system. An asymmetry in the length of the arms was also observed in metamorphs treated with thiamethoxam. Overall, these results indicate that thiamethoxam, and conceivably other neonicotinoids, have the potential to significantly impair metamorphosis of amphibians and diminish their performance and survival in the wild.

Comprehensive evaluation of the toxicity of the flame retardant (decabromodiphenyl ether) in a bioindicator fish (Gambusia affinis).

In recent years, concerns have increased about the adverse effects on health and the environment of polybrominated diphenyl ethers (PBDEs), especially BDE-209, the most widely PBDE used globally. These pollutants derive from e-waste and present different adverse effects on biota. In this work, a toxicological study on mosquitofish (Gambusia affinis) using BDE-209 (2,2',3,3',4,4',5,'5',6,6'-decabromodiphenyl ether) was carried out. Acute toxicity bioassays were conducted with daily renewal of solutions, using different concentrations of environmental relevance, ranged between 10 and 100 µg L-1 of BDE-209. At 48 and 96 h of exposure, several parameters were evaluated, such as mortality, individual activity (swimming), biochemical activity (catalase; thiobarbituric acid-reactive substances; and acetylcholinesterase), and cytotoxic responses (micronucleus frequencies). In addition, integrated biomarker response and multivariate analyses were conducted to study the correlation of biomarkers. The calculated Lethal Concentration-50 remained constant after all exposure times (24 to 96 h), being the corresponding value 27.79 µg L-1 BDE-209. Furthermore, BDE-209 induced effects on the swimming activity of this species in relation to acetylcholine, since BDE-209 increased, producing oxidative damage at the biochemical level and genotoxicity after 48 h of exposure to 10 and 25 µg L-1 BDE-209. The results indicate that BDE-209 has biochemical, cytotoxic, neurotoxic, and genotoxic potential on G. affinis. In addition, mosquitofish could be used as a good laboratory model to evaluate environmental stressors since they could represent a risk factor for Neotropical species.

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.

Biomarkers at different levels of organisation after atrazine formulation (SIPTRAN 500SC®) exposure in Rhinella schineideri (Anura: Bufonidae) Neotropical tadpoles.

Brazil is an important consumer of herbicides. In sugarcane cultivation-the country's most extensive agricultural crop-atrazine-based formulations are the principal form of weed control. Several studies have investigated adverse effects of atrazine or their formulations on anurans, but not specifically on Brazilian species. Our aim was therefore to investigate the lethal and sublethal effects of an atrazine-based herbicide in Rhinella schneideri tadpoles and, in particular, effects on the pigmentation system as a new endpoint in ecotoxicological studies. Rhinella schneideri tadpoles at the Gosner-30 stage were exposed to the atrazine-based herbicide formulation, SIPTRAN 500 SC®, in acute bioassays at concentrations of 1.5-25 mg/L. The lethal and sublethal effects induced were analysed at different ecotoxicological levels: organismal level (alterations in behaviour, growth, development, and body mass; morphologic abnormalities), histological level (liver histopathology), the pigmentation system (melanomacrophages and dermal-melanophores), and cellular level (erythrocyte micronucleus formation and other nuclear-abnormalities). This herbicide induced sublethal effects at the organismal level with alterations in swimming and growth and morphologic abnormalities. These results demonstrated that, in anuran tadpoles, the atrazine-based agrochemical increased the frequency of micronucleus formation and other nuclear-abnormalities in erythrocytes and caused liver damage. In addition, we demonstrated for the first time effects of an atrazine-based formulation on the pigmentation system of anuran tadpoles, specifically an increase in the number of melanomacrophages and dermal melanophores. This study is the first to use several widely differing endpoints at different ecotoxicological levels in a comprehensive manner for assessment of the effects of environmental stressors in order to determine the health status of Neotropical anuran species. In doing so, this study establishes a foundation for future ecological assessments.

Effect on the growth and development and induction of abnormalities by a glyphosate commercial formulation and its active ingredient during two developmental stages of the South-American Creole frog, Leptodactylus latrans.

We evaluated the acute lethal and sublethal effects of technical-grade glyphosate (GLY) and the GLY-based commercial formulation Roundup ULTRA MAX® (RU) on two Gosner stages (Gss) 25 and 36 of the South-American Creole frog, Leptodactylus latrans. Bioassays were performed following standardized methods within a wide range of concentrations (0.0007-9.62 mg of acid equivalents per liter-a.e./L-of RU and 3-300 mg/L of GLY). The endpoints evaluated were mortality, swimming activity, growth, development, and the presence of morphologic abnormalities, especially in the mouthparts. No lethal effects were observed on larvae exposed to GLY during either Gs-25 or Gs-36. The concentrations inducing 50 % lethality in RU-exposed larvae at different exposure times and Gss ranged from 3.26 to 9.61 mg a.e./L. Swimming activity was affected by only RU. Effects on growth and development and the induction of morphologic abnormalities-like oral abnormalities and edema-were observed after exposure to either GLY or RU. Gs-25 was the most sensitive stage to both forms of the herbicide. The commercial formulation was much more toxic than the active ingredient on all the endpoints assessed. Effects on growth, development, and the induction of morphologic abnormalities observed in the range of environmental concentrations reported for agroecosystems of Argentina constitute an alert to the potential detrimental effects of the herbicide that could be affecting the fitness and survival of anurans in agroecosystems.

Effects of glyphosate on hepatic tissue evaluating melanomacrophages and erythrocytes responses in neotropical anuran Leptodactylus latinasus.

Glyphosate (GLY) is the most used herbicide worldwide and its effects on anurans are well known. Pollutants can cause physiological and morphological effects. Therefore, this study evaluated the effects of GLY on hepatic melanomacrophages as a response to environmental stressors. Three treatments were exposed to different concentrations of pure GLY (100, 1000, and 10,000 µg g(-1), respectively), and there was also a control group. After the experimental time, liver and blood were analyzed. Melanomacrophages (MMCs) were located between the hepatocyte cordons, close to sinusoids. GLY increased the melanin area in MMCs of Leptodactylus latinasus exposed since lowest concentration until highest concentration. GLY also changed the occurrence of hepatic catabolism pigments into melanomacrophages and erythrocyte nuclear abnormalities; therefore, it can interfere with the hepatic metabolism. In conclusion, GLY promotes alterations in the hepatic tissue and erythrocyte nuclear abnormalities. Furthermore, MMCs may be useful as morphological responses of GLY effects.