Human exposure to nanoplastics (NPs) is an emerging health concern, yet the biological effects of NPs sizes and exposure routes remain poorly understood. To address this gap, we used zebrafish (Danio rerio) embryos, a 3Rs-compliant vertebrate model aligned with European regulatory directives. Zebrafish embryos are ethically acceptable for experimentation with high translational value. Their optical transparency and rapid development enable real-time in vivo visualization of NP biodistribution and cellular responses at single-cell resolution.

This study evaluated the biodistribution of 50 nm and 1 µm polystyrene nanoplastics (PS-NPs) at environmentally and human-relevant concentrations administered through four distinct exposure routes: bath immersion dermal deposition, nutrient uptake, and gametal transmission. Using the embryo stage, we observed that particle accumulation was strongly dependent on exposure route: The 50 nm nanoplastics preferentially localized in yolk-intestines and vasculature, whereas the 1 µm particles were predominantly retained in dermal and pericardial regions. Gametal exposure resulted in widespread distribution during early development, highlighting a potential transgenerational transfer. Functional analyses with transgenic fish lines revealed that macrophages were preferentially recruited to NP- laden tissues (below 6 %), while neutrophil activation was modest with minimal colocalization.

This study evaluated the biodistribution of 50 nm and 1 µm polystyrene nanoplastics (PS-NPs) at environmentally and human-relevant concentrations administered through four distinct exposure routes: bath immersion dermal deposition, nutrient uptake, and gametal transmission. Using the embryo stage, we observed that particle accumulation was strongly dependent on exposure route: The 50 nm nanoplastics preferentially localized in yolk-intestines and vasculature, whereas the 1 µm particles were predominantly retained in dermal and pericardial regions. Gametal exposure resulted in widespread distribution during early development, highlighting a potential transgenerational transfer. Functional analyses with transgenic fish lines revealed that macrophages were preferentially recruited to NP- laden tissues (below 6 %), while neutrophil activation was modest with minimal colocalization.

Vascular transgenics demonstrated that the PS-NPs tested induced subtle alterations in vessel branching and intersegmental vessels. Altogether, these findings provide evidence that both NPs and exposure route determine biodistribution, immune cell responses, and vascular development. Zebrafish embryos provide a powerful, 3Rs-aligned system to dissect NPs toxicology in vivo, offering mechanistic insights into immune and vascular interactions to advance the development of functional new approach methodologies (NAMs) for European risk assessment frameworks.

"This study was funded by the HORIZON-WIDERA-2024-TALENTS-02-01 (ENLIVEN project; 101244206) and the Slovenian Research and Innovation Agency (ARIS) through the Strategic Project (NanoBreak; STR-0001) and program P1-0245.“