Biodistribution and elemental co-localisation of europium-doped nanoplastics in Daphnia magna revealed by synchrotron-based nanoprobe X-ray fluorescence imaging.

Nanoplastic (NPl) particles are increasingly found in aquatic environments due to the long-term degradation of mismanaged plastic waste, and their uptake and accumulation in aquatic organisms are progressively reported. However, their biodistribution and spatial association with biologically relevant elements after ingestion remain poorly understood. In this study, europium-doped polystyrene NPls (Eu-doped NPls) were used as model particles to investigate their spatial distribution in Daphnia magna, a representative freshwater organism, using synchrotron-based nanoprobe X-ray fluorescence (nano-XRF). Daphnia magna neonates (<24 h old) were exposed to 5-20 mg L-1 Eu-doped NPls for 48 h and by using Eu as the tracer, their biodistribution was mapped using nano-XRF at multiple resolutions. No mortality was observed during exposure, although body length was significantly reduced relative to the control under all tested conditions (p < 0.05). Reactive oxygen species (ROS)-associated fluorescence also increased significantly at 10 and 20 mg L-1, indicating an organism-level oxidative-stress response at higher exposure concentrations. Toxicokinetic analysis revealed rapid uptake and efficient depuration, yielding a low bioconcentration factor (BCF = 0.982 L g-1). Two-dimensional nano-XRF maps showed that most Eu-associated signals were localised within gut-associated regions and spatially co-occurred with endogenous elements including Fe, Ca and K. Eu-derived signal metrics increased with external exposure concentration, with broader distribution at lower concentration and more pronounced hotspot formation at higher concentrations. ROI-based co-localisation analysis showed increasing spatial association between Eu and endogenous elements, particularly Fe, suggesting that Eu-associated signals were spatially structured within gut-associated elemental microenvironments rather than uniformly distributed. This study demonstrates that Eu-doped NPls combined with ICP-MS and synchrotron nano-XRF provide a complementary element-specific framework for linking quantitative body-burden analysis with spatially resolved biodistribution in aquatic organisms.