From Pristine to Laboratory-weathered Micro- and Nanoplastics: Interaction with Environmental Contaminants and Biological Effects

Plastic pollution is a major global concern with far-reaching implications for ecosystems, environmental sustainability, and human/wildlife health. Among plastic-derived contaminants, micro- and nanoplastics (MNPs) are of particular concern due to their small size, persistence, and surface reactivity. Although commercial pristine MNPs have been widely used in early toxicological studies, they are increasingly considered inadequate to reflect real-world scenarios. Once into the environment MNPs undergo substantial physicochemical changes due to aging processes, which may alter their interactions with other pollutants and influence their biological effects. In light of this, growing attention is being directed toward more environmentally relevant forms of MNPs. In this context, we investigated the impact of various polystyrene-based MNPs – pristine, COOH-functionalized, and laboratory-weathered – using both in vitro and in vivo biological models. Given their dual role as pollutants and vectors, interactions between MNPs and two widespread environmental contaminants, bisphenol A (BPA) and cadmium (Cd), were also examined, along with their combined biological effects. The biological impact of 5 µm commercial microplastics (MPs) – both pristine and COOH-functionalized (COOH-MPs) – was investigated across multiple cell models, including 3T3-L1 preadipocytes, HepG2 hepatocytes, GT1-7 hypothalamic neurons, and BAE-1 endothelial cells. COOH-MPs, bearing surface carboxyl groups representative of oxidized plastics, induced mild cytotoxicity in GT1-7 and BAE-1 cells, and showed significantly enhanced BPA adsorption and desorption compared to pristine MPs, as revealed by HPLC-MS/MS analysis. Moreover, exposure to COOH-MPs, either alone or pre-adsorbed with BPA, affected triglyceride accumulation in 3T3-L1 adipocytes. Further experiments in HepG2 cells using 500 nm nanoplastics (NPs) revealed oxidative stress induction without noticeable cytotoxic effects. Interestingly, co-exposure with Cd attenuated Cd-induced toxicity and, during in vitro steatosis induction, led to a reduction in lipid accumulation. This effect was likely driven by complex interactions among fatty acids, Cd ions, and NPs, as supported by electron microscopy and dynamic light scattering analyses. To expand our investigations, we focused on the biological effects of NPs with greater environmental relevance at a whole-organism level. Specifically, the effects of the exposure to ⁓100 nm simulated environmental particles (SEPs), derived from the degradation and laboratory-weathering of plastic cutlery, were compared to those of pristine NPs in zebrafish embryos and larvae. SEPs elicited stronger toxic responses than pristine beads, primarily causing developmental delays. Furthermore, analyses at 6 days post-fertilization revealed that exposure to SEPs at environmentally relevant concentrations (0.1 mg/L) caused elevated cortisol levels, activation of stress and hypoxia pathways, and altered locomotor behavior. Overall, these findings confirm that surface chemistry and environmental weathering critically influence MNPs toxicity and their interactions with pollutants. Moreover, they underscore the necessity of incorporating realistic exposure scenarios in environmental toxicology research.