Microplastics and TiO2 nanoparticles mixture as an emerging threat to amphibians: A case study on bullfrog embryos.

Emerging contaminants can act as contributing factors to the decline of amphibian populations worldwide. Recently, scientists have drawn attention to the potential ecotoxicity of microplastics and nanomaterials in amphibians, however, their possible effects on embryonic developmental stages are still absent. Thus, the present study analyzed the developmental toxicity of environmentally relevant concentrations of polyethylene microplastics (PE MPs; 60 mg/L) and titanium dioxide nanoparticles (TiO2 NPs; 10 µg/L), isolated or in combination (Mix group) on bullfrog embryos, Aquarana catesbeiana, adapting the Frog Embryo Teratogenesis Assay (FETAX, 96h). Allied to the FETAX protocol, we also analyzed the heart rate and morphometric data. The exposure reduced the survival and hatching rates in groups exposed to TiO2 NPs, and to a lesser extent, also affected the Mix group. TiO2 NPs possibly interacted with the hatching enzymes of the embryos, preventing hatching, and reducing their survival. The reduced effects in the Mix group are due to the agglomeration of both toxicants, making the NPs less available for the embryos. PE MPs got attached to the gelatinous capsule of the chorion (confirmed by fluorescence microscopy), which protected the embryos from eventual direct effects of the microplastics on the hatching and survival rates. Although there were no cardiotoxic effects nor morphometric alterations, there was a significant increase in abdominal edemas in the hatched embryos of the PE MPs group, which indicates that osmoregulation might have been affected by the attachment of the microplastics on the embryos' gelatinous capsule. This study presents the first evidence of developmental toxicity of environmental mixtures of microplastics and nanoparticles on amphibians and reinforces the need for more studies with other amphibian species, especially neotropical specimens that could present bigger sensibility. Our study also highlighted several features of the FETAX protocol as useful tools to evaluate the embryotoxicity of several pollutants on amphibians.

The amphibian's spleen as a source of biomarkers for ecotoxicity assessment: Historical review and trends.

Amphibians are very sensitive to many environmental changes, so these animals are considered good bioindicator models for ecotoxicology. Given the importance of the amphibian spleen for hematopoietic and immune responses, this can be a key organ for the evaluation of biomarkers to monitor the health of individuals in nature or in captivity. In this systematic review, we searched databases and summarized the main findings concerning the amphibian spleen as a source of possible biomarkers applied in different scientific fields. The searches resulted in 83 articles published from 1923 to 2022, which applied the use of splenic samples to evaluate the effects of distinct stressors on amphibians. Articles were distributed in more than twenty countries, with USA, Europe, and Brazil, standing out among them. Publications focused mainly on anatomical and histomorphological characterization of the spleen, its physiology, and development. Recently, the use of splenic biomarkers in pathology and ecotoxicology began to grow but many gaps still need to be addressed in herpetological research. About 85 % of the splenic biomarkers showed responses to various stressors, which indicates that the spleen can provide numerous biomarkers to be used in many study fields. The limited amount of information on morphological description and splenic anatomy in amphibians may be a contributing factor to the underestimated use of splenic biomarkers in herpetological research around the world. We hope that this unprecedented review can instigate researchers to refine herpetological experimentation, using the spleen as a versatile and alternative source for biomarkers in ecotoxicology.