14C-Enabled Quantitative Fate Mapping of Orally Administered Polystyrene Nanoplastics in Laying Hens

Nanoplastics are emerging pollutants of global concern, yet their quantitative in vivo fate in terrestrial vertebrates remains poorly resolved owing to analytical limitations. Here, we constructed a quantitative whole-body fate map in laying hens orally exposed to backbone 14C-labeled polystyrene nanoplastics (PS-NPs) at 0.4 mg kg-1 day-1, a dose selected to represent an environmentally plausible upper-bound exposure scenario. The 14C-PS-NPs had an approximately 80 nm dry-state primary particle diameter, while the vacuum-dried dosing material showed micrometer-scale agglomeration upon standardized redispersion. Excreta represented the dominant compartment in the mass balance, accounting for 88.55 ± 0.54% and 90.06 ± 1.02% of the cumulative administered dose after 3- and 7-day exposures, respectively. Gastrointestinal contents constituted the largest residual nonexcreted compartment, whereas kidney, liver, and lung showed the most prominent 14C signals among internal organs. Trace brain-associated 14C was below the matrix-specific LOD at day 3 but quantifiable at day 7, and the overall egg-associated signal remained below 0.1% of the cumulative administered dose. In a complementary 21-day dietary study, laying hens were exposed to PS-NPs at 0.4 and 2 mg kg-1 day-1 to assess intestinal responses together with circulating and selected-organ readouts. The 21-day study showed intestinal histopathological changes, elevated permeability markers, and pro-inflammatory and oxidative-stress readouts, while cecal microbiota compositional shifts were most evident in the 2 mg kg-1 day-1 group. This study provides a quantitative whole-body fate map of PS-derived 14C together with an assessment of biological responses to repeated dietary PS-NP exposure in laying hens.