Microplastics (MPs) are recognized as emerging pollutants in aquatic environments, where they are rapidly colonized by microbial communities that form biofilms. These biofilms can alter the environmental behaviour, transport characteristics, and ecological impact of MPs. Although many studies have simulated biofilm formation under laboratory conditions, fewer have examined natural biofilm development on MPs in freshwater systems. This study investigates biofilm formation on polystyrene (PS) MPs (shape: fragments) in different flow conditions of a natural riverine setting. The goal is to develop a protocol for producing environmentally relevant biofilm-coated MPs. PS MPs of two size classes, namely 20–75 \(\upmu\) m and 600–1000 \(\upmu\) m, were incubated for four weeks in the Rhine River using a perforated box (low flow environment) and a tube setup (high flow environment). Immersion microscopic observations revealed widespread microbial colonization across all MPs, with higher flow conditions supporting visibly more surface coverage and diverse biofilms. Scanning electron microscopy revealed the presence of various microorganisms-including diatoms, bacteria, ciliates, and choanoflagellates-in the high-flow tube setup, whereas they were largely absent in the low-flow box setup. Spectrophotometric analysis using crystal violet staining confirmed significantly higher biofilm biomass (higher absorbance values) in the tube incubation setup (0.2374 ± 0.0865) compared to the box setup (0.0764 ± 0.0225). The results demonstrated that flow velocity plays a critical role in shaping biofilm density and microbial composition. Higher flow conditions likely promoted greater nutrient exchange and surface contact, facilitating enhanced colonization. These findings underscore the importance of mimicking realistic hydrodynamic conditions when preparing biofilm-coated MPs for environmental studies. The methodology developed in this study is a step towards a standardized approach to generating environmentally relevant MPs, which can improve the accuracy of future research on MP behaviour, transport, and ecological interactions.
