Elucidating nanoplastic aggregation behavior and ecotoxicological impacts via an HF5-UV coupled with PD-OES platform

Monitoring nanoplastic (NP) aggregation in bidisperse systems under environmentally relevant ionic conditions remains a significant challenge. Conventional techniques such as dynamic light scattering and transmission electron microscopy are hindered by matrix interference, low sensitivity, and difficulties in resolving polydispersity. Here, we addressed this gap by using a coupled system including hollow fiber flow field-flow fractionation (HF5), ultraviolet-visible detector (UV), and point-discharge optical emission spectroscopy (PD-OES) to investigate aggregation behavior of bidisperse polystyrene NPs (PSNPs, 50 nm and 100 nm) at ionic conditions below 1 mM, representative of freshwater ecosystems. We demonstrated that bidisperse PSNPs exhibit enhanced aggregation compared to monodisperse counterparts by using the HF5-UV/PD-OES platform for purification, separation, and carbon-specific quantification. This platform elucidated the critical roles of cation valence (Na + vs. Ca 2+ ) and low concentration ion gradients (0.01–1 mM) in modulating aggregation over extended scales (i.e., 24 h). Finally, toxicity assessments on Microcystis aeruginosa revealed that aggregation reduces PSNP ecotoxicity depending on cation valence, thereby alleviating growth inhibition, oxidative stress, and impairment of chlorophyll biosynthesis. These findings elucidate how environmentally relevant ionic conditions govern NP fate and effects in freshwater ecosystems, providing a critical link between physicochemical transformations and biological responses for ecological risk evaluation.