Surface Chemistry Shapes Nanoplastic Mixture Responses in Developing Amniote Embryos

Nanoplastics (NPs) are an emerging environmental concern, yet their developmental impacts on terrestrial amniotes and their mixture effects are only beginning to be understood. Using a chicken embryo model, we compared the developmental and molecular effects of negatively charged (PS-COOH), positively charged (PS-NH2), and their equal-component mixture (50 nm). No significant mortality was observed across 0.1 to 10 ppm, whereas the lowest tested concentration (0.1 ppm) impaired growth and brain development without significantly increasing mortality. An IA-informed reference-response comparison showed that mixture effects were generally attenuated relative to reference responses derived from the experimentally tested single-particle treatments. Brain transcriptomic and proteomic analyses revealed distinct surface-chemistry-dependent responses: PS-COOH primarily affected extracellular matrix and glycan-related processes, whereas PS-NH2 altered synaptic, immune, and protein-quality-control pathways. The mixture induced the greatest number of DEGs; this broader omics response does not translate into higher phenotypic toxicity. DIABLO identified treatment-associated RNA-protein covariance, while reference-response comparisons indicated predominantly attenuated molecular responses among statistically supported deviations. Our findings demonstrate that coexposure to oppositely functionalized nanoplastics can reshape developmental and molecular responses during embryonic development.