AbstractThe widespread presence of microplastics in multiple environmental compartments has largely been demonstrated. Assessing the ecological risk that microplastics pose is, at the present stage, hindered due to methodical differences. Moreover, different methods hamper meaningful comparisons between studies and data on microplastics <300 μm is scarce. Therefore, we focused on microplastics >20 μm in freshwater and sampling-related aspects in this concern. Sampling was conducted between 2018 and 2020 in the Tollense catchment in northeastern Germany and was carried out by in situ pump filtration. Two different sampling systems (cutoff sizes 20 μm and 63 μm) were applied to filter water volumes of 0.075–1.836 m3. Retained particles were analyzed by a combination of Nile red staining and micro-Raman spectroscopy. Thereby, we found microplastic concentrations between 123 and 1728 particles m−3 using the 63-μm cut-off size and between 1357 and 2146 particles m−3 using the 20-μm cut-off size. Local hydrodynamics (discharge and flow velocity) and land cover are likely influencing the observed microplastic concentrations and fluxes. The variability between both sampling systems cannot fully be explained by the different mesh sizes used. We argue that differentiation between a theoretical cut-off size (finest mesh) and a factual cut-off size (reliable quantification) can help to understand sampling related differences between studies.