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. | Instituto de Recursos Biológicos | Fil: Bahl, María Florencia. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Química. Centro de Investigaciones del Medio Ambiente; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. | Fil: Brodeur, Celine Marie Julie. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Recursos Bilológicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. | Fil: Salgado Costa, Carolina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Química. Centro de Investigaciones del Medio Ambiente; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. | Fil: D'Andrea, María Florencia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Recursos Bilológicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. | Fil: Sansiñena, Jesica Alejandra. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Química. Centro de Investigaciones del Medio Ambiente; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. | Fil: Marino, Damián José. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Química. Centro de Investigaciones del Medio Ambiente; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. | Fil: Natale, Guillermo S. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Química. Centro de Investigaciones del Medio Ambiente; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.

Herbicides improve the productivity of a monoculture by eliminating weeds, although they may also be toxic and have negative effects on non-target organisms, such as amphibians. The present study evaluated the genotoxic, mutagenic, and histopathological hepatic responses of Dendropsophus minutus tadpoles to acute exposure (96 h) to the herbicide glyphosate (GLY, 65, 130, 260 and 520 μg/L) and the surfactant polyoxyethylene amine (POEA, 1.25, 2.5, 5 and 10 μg/L). On average, 174 % more genomic damage was observed in the tadpoles exposed to all concentrations of POEA in comparison with the control, while up to seven times more micronuclei were recorded, on average, at a concentration of 5 μg/L of POEA. All the individuals exposed to 10 μg/L of POEA died. The tadpoles exposed to GLY presented 165 % more DNA damage than the control, on average, at the highest concentrations (260 and 520 μg/L), and up to six times more micronuclei at 520 μg/L. The Erythrocyte Nuclear Abnormality test (ENA) detected a relatively high frequency of cells with lobed nuclei in the tadpoles expose to POEA at 5 μg/L and binucleated cells in those exposed to GLY at 520 μg/L. The hepatic histopathological observations revealed several types of lesions in the tadpoles exposed to both GLY and POEA. Overall, then, the results of the study indicate that both GLY and POEA have potential genotoxic, mutagenic, and hepatotoxic effects in D. minutus tadpoles. We emphasize the need for further studies to monitor the amphibian populations, such as those of D. minutus, which breed in aquatic environments associated with agricultural areas. The release of pollutants into natural habitats may have significant long-term impacts on the survival of anuran tadpoles.

Amphibian larvae are commonly used as indicators of aquatic ecosystem health because they are susceptible to contaminants. However, there is limited information on how species characteristics and trophic position influence contaminant loads in larval amphibians. Importantly, there remains a need to understand whether grazers (frogs and toads [anurans]) and predators (salamanders) provide comparable information on contaminant accumulation or if they are each indicative of unique environmental processes and risks. To better understand the role of trophic position in contaminant accumulation, we analyzed composite tissues for 10 metals from larvae of multiple co-occurring anuran and salamander species from 20 wetlands across the United States. We examined how metal concentrations varied with body size (anurans and salamanders) and developmental stage (anurans) and how the digestive tract (gut) influenced observed metal concentrations. Across all wetlands, metal concentrations were greater in anurans than salamanders for all metals tested except mercury (Hg), selenium (Se), and zinc (Zn). Concentrations of individual metals in anurans decreased with increasing weight and developmental stage. In salamanders, metal concentrations were less correlated with weight, indicating diet played a role in contaminant accumulation. Based on batches of similarly sized whole-body larvae compared to larvae with their digestive tracts removed, our results indicated that tissue type strongly affected perceived concentrations, especially for anurans (gut represented an estimated 46–97% of all metals except Se and Zn). This suggests the reliability of results based on whole-body sampling could be biased by metal, larval size, and development. Overall, our data shows that metal concentrations differs between anurans and salamanders, which suggests that metal accumulation is unique to feeding behavior and potentially trophic position. To truly characterize exposure risk in wetlands, species of different life histories, sizes and developmental stages should be included in biomonitoring efforts.

Naphthenic acids (NA) are used in a variety of commercial and industrial applications, and are primary toxic components of oil sands wastewater. We investigated developmental and metabolic responses of tadpoles exposed to sub-lethal concentrations of a commercial NA blend throughout development. We exposed Lithobates pipiens tadpoles to 1 and 2 mg/L NA for 75 days and monitored growth and development, condition factor, gonad and liver sizes, and levels of liver glucose, glycogen, lipids and cholesterol following exposure. NA decreased growth and development, significantly reduced glycogen stores and increased triglycerides, indicating disruption to processes associated with energy metabolism and hepatic glycolysis. Effects on liver function may explain reduced growth and delayed development observed in this and previous studies. Our data highlight the need for greater understanding of the mechanisms leading to hepatotoxicity in NA-exposed organisms, and indicate that strict guidelines may be needed for the release of NA into aquatic environments.

To examine the link between corn agriculture and the observed decline of the endangered southern bell frog (SBF), the effects of two corn crop pesticides on larval growth and development were investigated. Tadpoles were exposed to terbufos sulfone (10 μg/L), a major breakdown product of the insecticide terbufos, and the herbicide atrazine (25 μg/L) individually and as a mixture until the completion of metamorphosis. Atrazine did not interact synergistically with terbufos sulfone or result in significant effects on growth and development alone, although there was some indication of accelerated metamorphosis in the pilot study. Terbufos sulfone alone and as a mixture (terbufos/atrazine) significantly slowed larval development and ultimately delayed metamorphosis. The observed developmental effects from an environmentally relevant concentration of terbufos sulfone indicates a risk posed by this persistent degradation product to the endangered SBF, which breeds and develops in the rice bays adjacent to corn fields treated with pesticides.

The ongoing decline in anuran populations is linked primarily to the effects of stressor agents such as pathogens, pesticides, alterations of natural landscapes, and the introduction of exotic species. Most studies that have evaluated the effects of these stressors have focused on a single component, which is the opposite of the reality of most natural environments, where anuran populations tend to suffer the influence of multiple agents simultaneously. Studies of the effects of the interaction between these components are extremely important, given that one agent may potentialize (synergistic effect) or weaken another (antagonistic effect) or, in some cases, have a neutral effect. The present study is based on the scientometric analysis of three bibliographic databases (ISI Web of Science, Scopus, and PubMed), which identified 1376 papers that reported on the global decline of anuran populations, although only 172 of these studies focused on the interactive effects of environmental stressors. Synergistic effects were the most frequent type of interaction, followed by antagonistic effects, and a small number of studies that found no clear interaction between the stressors. Pathogens and pesticides were the classes of stressor studied most frequently, while climate-pathogen and pathogen-pesticide interactions were the combinations that featured in the largest number of studies. Overall, we would recommend a more systematic focus on the dynamics of the interactions among the stressors that impact anuran populations, in particular for the elaboration of conservation programs, given that these agents tend to have complex combined effects.

PURPOSE: Heavy metals have been detected in water and sediments from the Embalse La Florida, an artificial lake in the arid region of San Luis province, Argentina, representing one of the few sources of permanent water for reproduction of native anuran species. This study assesses lead (Pb) and cadmium (Cd) concentrations in the anuran species found in this water reservoir as well as differences between compounds, species and sites of collection. METHODS: Adult anuran amphibians were collected on the north and south shores of the Embalse La Florida and Pb and Cd concentrations were measured in whole body homogenates digested using wet ashing techniques. RESULTS: All individuals of the six species assayed had detectable levels of Pb and Cd that ranged from 1.19 to 5.57 μg/g dry mass and from 1.09 to 6.86 μg/g dry mass, respectively. Anuran amphibians collected in the more contaminated south shore accumulated 21% more Cd and 40% more Pb than individuals from the less altered north shore. Cd and Pb accumulation was not significantly correlated with the concentration in water at the site of collection. CONCLUSIONS: Amphibians of the Embalse La Florida accumulate Cd and Pb. Between and within species, differences were detected in Cd and Pb concentrations. Differences in metal concentrations between species, metals, and individuals collected on shores of the Embalse La Florida with different contamination, were detected. Therefore, it is crucial to implement adequate policies to protect amphibians from the accelerated urban development experienced in this location.

PURPOSE: Organophosphate pesticides (OPs) are among the most used insecticides in agriculture, causing the inhibition of esterases like acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and carboxylesterase (CbE). Pesticides can reach the aquatic environment, posing risks to non-target organisms, including tadpoles. METHODS: In this work, we characterized the activities of AChE, BChE and CbE in tadpoles of the snouted treefrog Scinax fuscovarius, and verified their in vitro sensibility to different inhibitors [phenylmethane sulfonyl fluoride (PMSF), tetra-isopropylpyrophosphamide (iso-OMPA) and the OP diazinon]. In vivo effects of diazinon and esterase recovery after 2-pyridine-aldoxime (2-PAM) treatment of the protein extract were also studied in tadpoles with distinct stages of development exposed to 1 and 3 mg/l for 2 and 7 days. RESULTS: Optimal conditions were established for AChE and CbE; BChE activity was negligible. PMSF affected esterase activities and is not recommended for homogenization buffers. Iso-OMPA treatment caused no changes in AChE and CbE activities, but diazinon inhibited these enzymes in a dose-responsive manner. In vivo, CbE activity was insensitive to diazinon in younger tadpoles, but inhibited after 2 days of exposure in more developed tadpoles. AChE activity was inhibited after 2 and 7 days of exposure, in a dose-responsive manner. Esterase reactivation by 2-PAM was obtained both in vitro and in vivo. CONCLUSIONS: (1) Tadpoles can be adequate sentinel organisms in biomonitoring studies of OP contamination; (2) AChE was more sensitive than CbE to diazinon; (3) tadpoles from earlier developmental stages seems to be less responsive to OPs; (4) AChE activity was sensitive to diazinon in both development stages, being a better OP biomarker.