Microplastic is ubiquitous in the aquatic environment, however little is still known whether microplastic has a negative impact on freshwater macroinvertebrates and the potential risk of this for freshwater ecosystems. In this thesis, sources of microplastic to the freshwater environment are described, followed by a discussion of current methodological challenges and the need for a focus on method optimization and harmonization – which form the foundations of trustworthy and comparable data; a prerequisite for the management and risk assessment of microplastics. This is followed by a review of in situ studies of microplastic in freshwater macroinvertebrates, which is compared to results from exposure experiments. The current knowledge and its limitations are discussed. In general, it is evident from literature that observed effects of microplastic are reduced at environmental relevant concentrations or when studies are conducted in a natural environment. Finally, microplastic as a stressor is compared to other primary and documented stressors for freshwater macroinvertebrates. Based on this, which also includes my own findings as reported in papers I to IV, I conclude, that microplastic >50 μm at the current environmental concentrations are not likely to pose any risk to freshwater benthic macroinvertebrates, alone or in combination with other stressors. This is in agreement with a risk assessment carried out by Adam, Yang, and Nowack (2019) on freshwater concentrations of microplastics and exposure studies on freshwater biota, including macroinvertebrates, who conclude that the present concentrations do not pose a risk to the freshwater environment. Paper I describes, tests, and assesses a new method which was developed to digest chitinaceous organisms. Chitin is a recalcitrant organic compound found in many freshwater macroinvertebrates and has been documented to hinder analysis of microplastic content in this central group of organisms in the freshwater food web. The method developed combined the use of hydrogen peroxide (H2O2) followed by chitinase digestion. The results showed that the method was highly efficient in removing organic matter, including chitin. It is therefore suggested that future microplastic studies of chitinaceous organisms apply the presented method. Paper II examines the effect of urbanisation on microplastic concentration in the freshwater macroinvertebrate Rhyacophila sampled at two sites in each of five streams, representing a gradient in urbanisation and pollution pressure. In general, microplastic was found in very low concentrations in organisms, all below the limit of quantification and no effect of urbanisation on the microplastic content in the macroinvertebrates could be found. Based on this, it is discussed what constitute the minimum sample size required for accepting a null-result; i.e. that microplastics are not present. In relation to this discussion, we describe methods to assess this minimum value and advocate for the necessity of incorporating such calculations prior to conducting microplastic studies on biota in the future. Paper III addresses the pertinent issue of microplastic in freshwater sediment and biota and how this might be linked to the land use in catchments. Macroinvertebrates and sediment from a Norwegian stream receiving microplastic from a plastic recycling plant were investigated. High concentrations of polyethylene films in the sediment downstream of the plastic recycling plant were found. A relatively stable concentration of polypropylene film at all sites, both upstream and downstream the recycling plant was observed. Despite the high sediment concentrations, microplastic was not found in the >500 individuals of macroinvertebrates analyzed. Thereby, this paper illustrates how microplastic contamination is intimately tied to plastic waste management and demonstrates how a plastic recycling plant can result in microplastic contamination of an upland stream with a catchment dominated by forested and natural areas. Lastly, the factors that potentially mediate the uptake of microplastics by macroinvertebrates in streams are discussed. This gives important context for how to address microplastic risk in different environmental settings. Paper IV unites several components of environmental microplastic research. This paper sheds light on the interplay between terrestrial sources of microplastic, freshwater surface sediments and a component of freshwater biota. This was performed by analyzing zebra mussels (Dreissena polymorpha) and sediment from five Danish lakes covering a potential gradient in overall environmental quality and microplastic loading. Low concentrations in the sediments and just a single particle from a total of 150 mussels were recorded. No compelling links or relationships could be ascertained between microplastic concentrations and in-lake environmental data, discharges from point sources or agricultural practices in the catchment. It is therefore argued that actual sources and not just potential sources of microplastics in the catchment must be determined.