A multi-technique analytical approach to support (eco)toxicological investigation of zinc oxide nanoparticles

Understanding the mechanism of toxicity of nanoparticles and their behavior in biological environments is crucial for designing materials with reduced side effects and improved performance. Among the factors influencing nanoparticle behavior in biological environments, the release and bioavailability of potentially toxic metal ions can alter equilibria and cause adverse effects. In this study, we applied two on-line Field-Flow Fractionation (FFF) strategies and compared the results with off-line benchmarking centrifugal ultrafiltration to assess a key descriptor, namely the solubility of zinc oxide (ZnO) nanoparticles. We found that, at the highest nanoparticle concentrations, the nanoparticle-ion ratio quickly reaches equilibrium, and the stability is not significantly affected by the separation technique. However, at lower concentrations, dynamic, non-equilibrium behavior occurs, and the results depend on the method used to separate the solid from the ionic fraction, where FFF yielded a more representative dissolution pattern. To support the (eco)toxicological profiling of the investigated nanoparticles, we generated experimental data on colloidal stability over typical (eco)toxicological assay durations. The Zeta Potential vs pH curves revealed two distinct scenarios typical of surfaces that have undergone significant modification, most likely due to pH-dependent dissolution and re-precipitation of surface groups. Finally, to enhance hazard assessment screening, we investigated ion-dependent toxicity and the effects of exposure to fresh water. Using an in vitro human skin model, we evaluated the cytotoxicity of fresh and aged ZnO nanoparticles (exposed for 72 h in M7), revealing time-dependent, dose-dependent, and nanoparticledependent cytotoxicity, with lower toxicity observed in the case of aged samples.