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 might be exposed to high concentrations of those particles. Therefore, ingestion of MP by this group of organisms is highly likely which is concerning as it can lead to a variety of toxic effects. However, it is still unclear whether the effects of MP are related to the simple passage of the gut, if they only occur at MP 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 rely on plastic damaging chemicals that are therefore not compatible with MP experiments. Hence, our goal was the adaption 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 without plastic damaging reagents for paraffin sectioning, cryo sectioning, as well as for SEM (scanning electron microscopy) analysis in MP research. Resulting sections (paraffin and cryo) showed intact tissue and allowed detection of, for instance, the fluorescence labelled PS-MP inside the digestive system. Further, they represent a basis for classical histological staining, histochemistry and immunohistochemistry. Cryo sections additionally allow a variety of spectroscopic methods such as MALDI-MSI, Raman or FTIR to be applied. The SEM approach provides a high magnification, three dimensional images, enabling 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. Also see: https://micro2024.sciencesconf.org/559546/document