Developmental toxicity of co-exposure of heavy metal and polystyrene microplastics in Xenopus laevis embryo

Microplastics are widespread pollutants that bioaccumulate and biomagnify across trophic levels. In freshwater ecosystems, the co-occurrence with heavy metals, resulting from anthropogenic activities, is frequent and raises significant environmental concerns. Although combined exposures generally produce more potent effects than single contaminants, the interactions remain poorly understood, as weaker, antagonistic, inhibitory, and sub- or infra-additive effects have also been reported. Therefore, this study investigated the developmental toxicity of simultaneous exposure to cadmium, copper, lead, and 5 μm polystyrene microbeads in the amphibian Xenopus laevis, a model considered equivalent to the later embryonic stages of higher vertebrates. Our multi-endpoint approach included a standard FETAX test to assess mortality, growth, and malformations, as well as histological and molecular analyses. We also quantified volatile organic compounds (VOCs) released from microbeads via gas chromatography-mass spectrometry, and analysed metal uptake using inductively coupled plasma-mass spectrometry. This allowed us to explore an additional, often overlooked route of toxicity and detect interactions not evident in single-pollutant studies. Microbeads released high levels of styrene, ethylbenzene, and xylene. Although no mortality was observed, exposure, particularly in the presence of cadmium, led to intestinal malrotation, mucosal damage, oxidative stress, and increased apoptosis. Moreover, we observed significant dysregulation of key developmental genes (otx2, pax6, sox3, sox9, egr2, bmp4, fgf8). Overall, our findings indicate that polystyrene microbeads can induce physiological stress during early development, and that their toxicity is significantly increased when combined with heavy metals. These results highlight the importance of assessing the effects of microplastics within complex environmental mixtures.