Assessment of multiple biomarkers in Lithobates catesbeianus (Anura: Ranidae) tadpoles exposed to zinc oxide nanoparticles and zinc chloride: integrating morphological and behavioral approaches to ecotoxicology.

The ecotoxicological risk to vertebrates posed by zinc oxide nanoparticles (ZnO NPs) is still poorly understood, especially in animals with a biphasic life cycle, which have aquatic and terrestrial phases, such as amphibians. In the present study, we investigated whether acute exposure (7 days) to ZnO NPs and zinc chloride (ZnCl2) at three environmentally relevant concentrations (0.1, 1.0, and 10 mg L-1) induces changes in the morphology, chondrocranium, and behavior of the tadpoles of Lithobates catesbeianus (Anura: Ranidae). Tadpoles exposed to both forms of Zn did not undergo any morphological or behavioral changes at the lowest concentrations (0.1 and 1.0 mg L-1). However, the animals exposed to the highest concentration (10 mg L-1) lacked oral disc structures, were smaller in size, had a longer tail, and presented changes in the position and coiling of the intestine and malformations of the chondrocranium in comparison with the control group. This indicates that ZnO NPs and ZnCl2 altered the development of the tadpoles, causing delays in their metamorphosis and even reducing individual fitness. The tadpoles exposed to both forms of Zn at 10 mg L-1 also had reduced mobility, especially in the presence of conspecifics. Based on these findings, we emphasize the importance of studying morphological, skeletal, and behavioral biomarkers to evaluate the toxic effects of metal-based nanoparticles in amphibians.

Titanium dioxide nanoparticles as a risk factor for the health of Neotropical tadpoles: a case study of Dendropsophus minutus (Anura: Hylidae).

The production and use of titanium dioxide (TiO2) nanoparticles are increasing worldwide. The release of this substance into the environment can induce toxic effects in aquatic invertebrates and vertebrates, although the exact nature of its impacts on Neotropical amphibians is still poorly understood. In this context, the present study evaluated the toxicity of TiO2 nanoparticles and their counterpart-dissolved titanium dioxide (TiO2)-in the tadpoles of Dendropsophus minutus. The biometric parameters, DNA damage, and behavioral changes were verified in tadpoles exposed to three different concentrations (0.1 mg·L-1, 1.0 mg·L-1, and 10 mg·L-1) of TiO2 nanoparticles and dissolved TiO2 for 7 days. We verified significant DNA damage in the D. minutus tadpoles exposed to both forms of Ti, in comparison with the control group. We also identified a reduction in total size, body length, and width, and the height of the musculature of the tail of the tadpoles exposed to all concentrations of both substances in comparison with the control. In the behavioral test, the tadpoles exposed to nanoparticles and dissolved TiO2 presented reduced mobility and a tendency to be less aggregated than normal. Here, the simultaneous use of multiple biomarkers was fundamental for the reliable assessment of the adverse effects of nanomaterials on anuran amphibians and the establishment of a systematic approach to the biomonitoring of aquatic ecosystems. The present study expands our understanding of the genotoxic, morphological, and behavioral effects of TiO2 nanoparticles and dissolved TiO2 on anuran amphibians, and contributes to the establishment of further research for the more systematic assessment of the environmental risk of nanomaterials.

Ecotoxicity of nanomaterials in amphibians: A critical review.

Nanomaterials (NMs) have been used in a growing number of commercial products, and their rapid expansion could lead to their release into the aquatic environments. However, there is limited knowledge about the impact of NMs in the biota, especially the amphibians. The present study revised the historical use of amphibian species as a model system for nanoecotoxicological studies and summarized the data available in the scientific literature about the genotoxic, mutagenic, histopathological, embryotoxic and reproductive effects of NMs in different groups of amphibians. The interaction, bioaccumulation, mode of action (MoA) and ecotoxicity of NMs on amphibians were also revised. The nanoecotoxicological studies were conducted with 11 amphibian species, being eight species of the order Anura and three species of the order Caudata. Xenopus laevis was the most studied species. The studies were conducted mainly with inorganic NMs (72%) compared to organic ones. The nanoecotoxicity depends on NM behavior and transformation in the environment, as well as the developmental stages of amphibians. The known effects of NMs in amphibians were mainly reported with reactive oxygen species (ROS) production, oxidative stress, and genotoxic effects. Results emphasize the need for further studies testing the ecotoxicity of different NMs, concentrations and exposure periods at environmentally relevant approaches. Furthermore, standard protocols for nanoecotoxicological tests using amphibians are required. Revised data showed that amphibians are suitable organisms to assess the environmental impact of NMs and indicated significant research gaps concerning the ecotoxicity of NMs on freshwater ecosystems and recommendations for future researches.

Sub-lethal effects induced by a mixture of different pharmaceutical drugs in predicted environmentally relevant concentrations on Lithobates catesbeianus (Shaw, 1802) (Anura, ranidae) tadpoles.

The increasing consumption of medications by humans has negative effects such as the increased disposal of these compounds in the environment. Little is known about how the disposal of a "drug mix" (DM) in aquatic ecosystems can affect their biota. Thus, we evaluated whether the exposure of Lithobates casteibeianus tadpoles to a DM composed of different medication classes (antibiotic, anti-inflammatory, antidepressant, anxiolytic, analgesic, and antacid drugs)-at environmentally relevant concentrations-may change their oral morphology, trigger behavioral disorders, and have mutagenic effects on erythrocyte cells. Based on our data, animals exposed to the DM showed changes in mandibular sheath pigmentation, dentition, and swimming activity, as well as atypical behavior in the social aggregation test [with co-specific and interspecific (Physalaemus cuvieri) individuals] and antipredatory defensive response deficit (chemical stimulus from Odonata larvae), after 15 exposure days. The mutagenic analysis revealed higher frequency of nuclear abnormalities in the erythrocytes of tadpoles exposed to the DM (e.g., multilobulated, blebbed, kidney-shaped, notched nucleus, binuclear, and micronucleated erythrocytes). Given the chemical complexity of the DM, we assumed that several organic functions may have been affected, either by the isolated, synergistic, antagonistic, or additive action of DM compounds. Finally, our study confirms the toxicological potential of DM in L. catesbeianus tadpoles, with emphasis to impacts that can affect the fitness of individuals and their natural populations. Thus, we suggest that more attention should be given to the disposal of medications in the environment and reinforce the need of improving water and sewage treatment systems.

Behavioral and mutagenic biomarkers in tadpoles exposed to different abamectin concentrations.

It is known that pesticides such as abamectin (ABA) present cytotoxic effects on target organisms; however, the effects from ABA on non-target organisms such as amphibians are poorly understood. The aim of the current study is to investigate whether the exposure of Lithobates catesbeianus tadpoles to different abamectin concentrations [12.5, 25, and 50% of the median lethal concentration (LC50)] leads to behavioral and morphological changes and/or generates possible cytotoxic effects. The aggregation test showed that tadpoles exposed to the highest ABA concentrations did not respond to the stimulus from non-familial and unrelated co-specific species. On the other hand, there was no difference in the total number of crossings in the central line of the herein adopted apparatus between groups; it suggests that ABA did not affect animal locomotion in the aforementioned test, although changes in the normal swimming pattern of tadpoles exposed to the pesticide were recorded in the swimming activity test. In addition, the herein exposed animals did not respond to the predatory stimulus in the antipredator response test; this result suggests defensive response deficit caused by the pesticide. With respect to their oral morphology, tadpoles exposed to ABA presented the lowest scores for mandibular pigmentation and structures, as well as for dentition condition. Finally, it was possible seeing that the exposure to ABA, even at the lowest concentration (12.5% of the LC50), resulted in nuclear changes in the erythrocytes of the animals; these changes became evident in the increased number of micronuclei and in other nuclear abnormalities. Thus, besides confirming the cytotoxic potential of ABA in amphibians, the current study corroborates the hypothesis that the exposure to the herein investigated pesticide leads to behavioral and morphological changes in tadpoles, fact that may negatively reflect on the survival, as well as on natural populations of these individuals.