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Understanding the spatial distribution patterns of microplastics (plastics 5 mm) contributes to the assessment of sources and sinks of pollution thus providing information for the management of biota safety and overall ecosystem functionality. We chose a semi-closed study area, Lake Bracciano (Italy), to assess the environmental variability of contamination, focusing on the water compartment and the exposure of biota, specifically fish, by analysing the ingestion of microplastics. The focus of this study is to evaluate the concentration of microplastics in water (surface and column) across the lake and the ingestion of microplastics by two fish species of economic interest: Atherina boyeri and Coregonus lavaretus, inhabiting demersal and pelagic habitats respectively. Results show a surface contamination of 392,000 ± 417,000 items km