Development of a toolbox for the analysis of microplastic-tissue interactions in two benthic freshwater organisms

Freshwater sediments represent a sink for microplastics (MP) and, hence, especially benthic invertebrates are exposed to these particulate contaminants. Therefore, ingestion of MP by this group of organisms is likely leading to a variety of toxic effects. However, it is still unclear whether these effects of MP are related to the simple passage of the gut for instance at contact or accumulation zones or after translocation of the particles into tissues. To address this issue, histological analyses need to be performed. However, available protocols for these investigations commonly rely on chemicals/solvents that dissolve plastic/polymers and are thus not compatible with MP exposure experiments. Hence, our goal was the adaptation of existing protocols that allow the analysis of MP-tissue interactions in two model systems for benthic freshwater invertebrates Lumbriculus variegatus (Blackworm) and Chironomus riparius (Harlequin fly). We established and optimized protocols for paraffin sectioning and cryosectioning, as well as for SEM (scanning electron microscopy) analysis. In these adapted protocols we did not use plastic damaging reagents. Resulting sections (paraffin and cryo) showed intact tissue and allowed detection of the fluorescence labelled polystyrene (PS) MP inside the digestive system. Furthermore, they represent a basis for classical histological staining, histochemistry and immunohistochemistry. Cryosections additionally allow for a variety of spectroscopic methods such as MALDI-MSI, Raman or FTIR to be applied. The SEM approach provides high magnification and three-dimensional images that enable ultrastructural analysis of the location of MP in the digestive system of both organisms. In conclusion, our adapted protocols can be used as a toolbox for detailed MP-tissue investigations.