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AbstractPollution by plastic and microplastic impacts the environment globally. Knowledge on the ageing mechanisms of plastics in natural settings is needed to understand their environmental fate and their reactivity in the ecosystems. Accordingly, the study of ageing processes is gaining focus in the context of the environmental sciences. However, laboratory-based experimental research has typically assessed individual ageing processes, limiting environmental applicability. In this study, we propose a multi-tiered approach to study the environmental ageing of polyethylene plastic fragments focusing on the combined assessment of physical and biological processes in sequence. The ageing protocol included ultraviolet irradiation in air and in a range of water solutions, followed by a biofouling test. Changes in surface characteristics were assessed by Fourier transform infrared spectroscopy, scanning electron microscopy, and water contact angle. UV radiation both in air and water caused a significant increase in the density of oxidized groups (i.e., hydroxyl and carbonyl) on the plastic surface, whereby water solution chemistry influenced the process both by modulating surface oxidation and morphology. Biofouling, too, was a strong determinant of surface alterations, regardless of the prior irradiation treatments. All biofouled samples present (i) specific infrared bands of new surface functional groups (e.g., amides and polysaccharides), (ii) a further increase in hydroxyl and carbonyl groups, (iii) the diffuse presence of algal biofilm on the plastic surface, and (iv) a significant decrease in surface hydrophobicity. This suggests that biological-driven alterations are not affected by the level of physicochemical ageing and may represent, in real settings, the main driver of alteration of both weathered and pristine plastics. This work highlights the potentially pivotal role of biofouling as the main process of plastic ageing, providing useful technical insights for future experimental works. These results also confirm that a multi-tiered laboratory approach permits a realistic simulation of plastic environmental ageing in controlled conditions.