How do different concentrations of aluminum and zinc affect the survival, body size, morphology and immune system of Physalaemus cuvieri (Fitzinger, 1826) tadpole?

The assessment of amphibian responses as bioindicators of exposure to chemical pollutants is an important tool for conservation of native species. This study aimed to investigate the effects of chronic aluminum (Al) and zinc (Zn) exposure on survival, body size, morphology (malformations), and immune system (leukocyte profile) in P. cuvieri tadpoles. Ecotoxicological analyses were performed utilizing chronic toxicity tests in which 210 tadpoles at the 25th Gosner developmental stage were exposed to Al and Zn. Individuals of P. cuvieri were maintained in glass containers containing various concentrations of aluminum sulfate (0.1, 0.2, or 0.3 mg/L) and zinc sulfate (0.18, 0.27 or 0.35 mg/L), and tests were performed in triplicate. After 14 days, amphibians were weighed, measured and survival rate, malformations in the oral and intestine apparatus, leukocyte profile, and ratio between neutrophils and lymphocytes determined. The differing concentrations of Al and Zn did not produce lethality in P. cuvieri where 95% of the animals survived 326 hr following metal exposure. Individuals exposed to Zn achieved greater body growth and weight gain compared to controls. Aluminum increased weight gain compared controls. These metals also produced malformations of the oral and intestine apparatus and enhanced occurrence of hemorrhages, especially at the highest doses. Lymphocytes were the predominant cells among leukocytes, with lymphopenia and neutrophilia observed following Al and Zn treatment, as evidenced by elevated neutrophil/lymphocyte ratio, an important indicator of stress in animals. Data suggest that further studies need to be carried out, even with metal concentrations higher than those prescribed by CONAMA, to ensure the conservation of this species.

2,4-D-based herbicide underdoses cause mortality, malformations, and nuclear abnormalities in Physalaemus cuvieri tadpoles.

Amphibians are considered bioindicators of the environment due to their high sensitivity and involvement in terrestrial and aquatic ecosystems. In the last two decades, 2,4-D has been one of the most widely used herbicides in Brazil and around the world, as its use has been authorized for genetically modified crops and therefore has been detected in surface and groundwater. Against this background, the aim of this work was to investigate the effects of environmentally relevant concentrations of 2,4-D-based herbicides on survival, malformations, swimming activity, presence of micronuclei and erythrocyte nuclear abnormalities in Physalaemus cuvieri tadpoles. The amphibians were exposed to six concentrations of 2,4-D-based herbicides: 0.0, 4.0, 30.0, 52.5, 75.0, and 100 µg L-1, for 168 h. At concentrations higher than 52.5 µg L-1, significantly increased mortality was observed from 24 h after exposure. At the highest concentration (100 µg L-1), the occurrence of mouth and intestinal malformations was also observed. The occurrence of erythrocyte nuclear abnormalities at concentrations of 30.0, 52.5, 75.0 and 100 µg L-1 and the presence of micronuclei at concentrations of 52.5, 75.0, and 100 µg L-1 were also recorded. These effects of 2,4-D in P. cuvieri indicate that the ecological risk observed at concentrations above 10.35 µg L-1 2,4-D may represent a threat to the health and survival of this species, i.e., exposure to 2,4-D at concentrations already detected in surface waters in the species' range is toxic to P. cuvieri.

Mortality and toxicity of a commercial formulation of cypermethrin in Physalaemus gracilis tadpoles.

This study evaluated the lethal, sublethal, and toxic of a commercial formulation of cypermethrin in the anuran species Physalaemus gracilis. In the acute test, concentrations of 100-800 µg L-1 were tested over 96 h. In the chronic test, cypermethrin concentrations recorded in nature (1, 3, 6, and 20 µg L-1) were tested for mortality and then used for the micronucleus test and erythrocyte nuclear abnormalities over a 7-days period. The LC50 determined for P. gracilis for the commercial cypermethrin formulation was 273.41 µg L-1. In the chronic test, a mortality of more than 50% was observed at the highest concentration (20 µg L-1), as it caused half of the tadpoles studied to die. The micronucleus test showed significant results at concentrations of 6 and 20 µg L-1 and recorded the presence of several nuclear abnormalities, indicating the genotoxic potential of the commercial cypermethrin formulation for P. gracilis. Cypermethrin presented a high risk to the species, indicating that it has the potential to cause several problems in the short and long term and to affect the dynamics of this ecosystem. Therefore, it can be concluded that the commercial formulation of cypermethrin had toxicological effects on P. gracilis.

Toxicity and genotoxicity of imidacloprid in the tadpoles of Leptodactylus luctator and Physalaemus cuvieri (Anura: Leptodactylidae).

Imidacloprid is a neonicotinoid insecticide used to control agricultural pests around the world. This pesticide can have adverse effects on non-target organisms, especially in aquatic environments. The present study evaluated the toxicity of an imidacloprid-based insecticide in amphibians, using Leptodactylus luctator and Physalaemus cuvieri tadpoles as study models. Spawning of both species were collected within less than 24 h of oviposition from a non-agricultural land at Erechim, Rio Grande do Sul state, Brazil. Survival, swimming activity, body size, morphological malformations, and genotoxic parameters were analyzed at laboratory conditions. A short-term assay was conducted over 168 h (7 days) with five different concentrations of imidacloprid (3-300 µg L-1) being tested. The insecticide did not affect survival, although the tadpoles of both species presented reduced body size, malformed oral and intestine structures, and micronuclei and other erythrocyte nuclear abnormalities following exposure to this imidacloprid-based compound. Exposure also affected swimming activity in L. luctator, which reflected the greater sensitivity of L. luctator to imidacloprid in comparison with P. cuvieri. The swimming activity, body size, and malformations observed in L. luctator and the morphological malformations found in P. cuvieri indicated that even the lowest tested concentration of the insecticide were harmful to amphibians. At concentrations of over 3 µg L-1, P. cuvieri presents a smaller body size, and both species are affected by genotoxic cell damage. This demonstrates that imidacloprid is potentially toxic for the two study species at environmentally relevant concentrations.

Morphological, behavioral and genotoxic effects of glyphosate and 2,4-D mixture in tadpoles of two native species of South American amphibians.

Pesticide contamination is an important factor in the global decline of amphibians. The herbicides glyphosate and 2,4-D are the most applied worldwide. These herbicides are often found in surface waters close to agricultural areas. This study aims at evaluating the chronic effects caused by glyphosate + 2,4-D mixture in Boana faber and Leptodactylus latrans tadpoles. The combined solution of the glyphosate and 2,4-D, in 5 different concentrations, was applied for 168 h. Herbicide mixtures did not affect the survival of the exposed tadpoles but growth and swimming activity were altered; besides causing several damages in the mouth and intestine. The erythrocytes showed micronuclei and other nuclear abnormalities. There is an ecological risk in the exposure of tadpoles of B. faber and L. latrans from the mixture of glyphosate + 2,4-D. Therefore, the approach used in this study provides important information on how commonly used pesticides can affect non-target organisms.

Cypermethrin- and fipronil-based insecticides cause biochemical changes in Physalaemus gracilis tadpoles.

Insecticides used for agricultural pest control, as cypermethrin-based insecticide (CBI) and fipronil-based insecticide (FBI), are constant threats to non-target aquatic organisms. This study aimed to investigate the effect of different concentrations of cypermethrin and fipronil on neurotoxicity and oxidative stress in Physalaemus gracilis. Physalaemus gracilis tadpoles were exposed to five insecticide concentrations and a control treatment, with six replicates. During the experimental period, the tadpole mortality rate was evaluated and after 168 h, the neurotoxic enzyme activity and metabolite quantification related to the antioxidant system were measured. Tadpoles reduced acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities when exposed to 20 µg L-1 CBI and at all FBI concentrations, respectively. Glutathione S-transferase (GST) and superoxide dismutase (SOD) activities showed an increase from concentrations of 6 µg L-1 and 20 µg L-1 of CBI, respectively. After exposure of P. gracilis tadpoles to FBI, inhibitions of AChE and BChE were observed at the highest concentrations evaluated (500 and 1500 µg L-1). SOD activity decreased from 50 µg L-1 of FBI; however, catalase (CAT) and GST activities and carbonyl protein levels increased, regardless of the evaluated dose. We observed that both insecticides promoted oxidative stress and neurotoxic effects in P. gracilis tadpoles. These results suggest that biochemical biomarkers can be used for monitoring toxicity insecticides for the purpose of preservation of P. gracilis.

Morphological and biochemical traits and mortality in Physalaemus gracilis (Anura: Leptodactylidae) tadpoles exposed to the insecticide chlorpyrifos.

Organophosphate insecticides such as chlorpyrifos are commonly detected in surface waters around the world, where they are highly toxic to many organisms. The frog Physalaemus gracilis uses water sources located in open fields as reproductive sites, where it is exposed to insecticides. The study aimed to evaluate the lethal and sublethal effect of a commercial chlorpyrifos formulation on P. gracilis tadpoles (Anura: Leptodactylidae). In acute toxicity tests, five chlorpyrifos concentrations between 750 and 2,000 µg L-1 were tested. Chronic toxicity, swimming activity, morphological and enzymatic changes, as well as levels of non-protein thiols (NPSH), carbonyl proteins and lipid peroxidation were evaluated at five insecticide concentrations between 11 and 500 µg L-1. The highest mortality rate of P. gracilis tadpoles occurred at 24 and 48 h, with an LC50 of 893.59 µg L-1. At all chlorpyrifos concentrations, tadpoles displayed reduced mobility and spasms. Morphological anomalies were observed in the mouth and intestine, especially at the highest concentrations used. Acetylcholinesterase activity decreased at 250 and 500 µg L-1, catalase activity increased at all concentrations, and superoxide dismutase and glutathione S-transferase increased from 90 µg L-1 to 30 µg L-1, respectively. We also observed increases in NPSH levels at chlorpyrifos concentration starting at 30 µg L-1 and increases in carbonyl proteins from 90 µg L-1 of pesticide. Taken together, these data suggest that the insecticide chlorpyrifos presents acute and chronic risks for P. gracilis, causing neurotoxic effects and oxidative damage, culminating in high risk for this species.