Charge Matters, but So Does Species: Amphibian Early Development under Exposure to Clean Dialyzed Nanoplastics

Nanoplastics (NPs) are emerging aquatic contaminants, yet their impacts on amphibians remain poorly resolved despite their ecological relevance and developmental sensitivity. We assessed developmental, behavioral, and transcriptomic responses in early life-stage Xenopus laevis exposed to clean, dialyzed polystyrene NPs bearing negative carboxyl (PS-COOH) or positive amine (PS-NH 2 ) surface charges, tested individually and in binary mixtures (0.1–10 ppm). Particle characterization indicated limited aggregation but medium-driven surface transformations, including charge shifts yielding net negative ζ-potentials for PS-NH 2 . Toxicity was charge-dependent, with elevated mortality in PS-COOH exposures and deformities observed across treatments. Morphometric analyses revealed reduced body length, head and trunk height, and eye area, with stronger effects in PS-NH 2 and mixture groups, while behavioral responses were modest. Transcriptomic profiling showed widespread downregulation of coding genes and long noncoding RNAs. PS-COOH suppressed oxidative phosphorylation, ribosomal function, and peptide metabolism pathways, whereas PS-NH 2 disrupted cytoskeletal organization, adhesion signaling, and extracellular interaction networks in a dose-responsive manner. Mixtures produced distinct signatures enriched in extracellular matrix and enzymatic processes. Integrated mixture modeling indicated predominantly additive responses with antagonistic deviations across biological levels. Collectively, amphibian early-life stages show high sensitivity to sublethal NP exposure and represent a promising New Approach Methodology platform for mechanistically informed nanoplastic risk assessment.