Development of a method for the detection of polystyrene microplastics in paraffin-embedded histological sections

The concerns about the presence of microplastics (MPs) in marine ecosystems have widely increased in the past years. This is reflected in a growing number of studies addressing the effects of exposure to these materials in indigenous, farmed and even laboratory marine animals subjected to toxicity-oriented bioassays. There have been, however, many constraints in the detection of MPs in biological tissues, as routine histological techniques tend to degrade these materials, which are especially sensitive to organic solvents. This issue hinders the application of standard histopathological procedures based on convenient paraffin wax-embedding protocols, with consequences for biomonitoring and bioassay procedures. The method described here was developed and validated for the detection of polystyrene microplastics in biological tissue processed for paraffin-based histology. The strategy was developed and tested from whole-soft body sections of marine mussels that internalised the MPs following dedicated bioassays. The protocol is based on the replacement of xylenes with isopropanol for the purpose of intermediate infiltration and deparaffinization. Special modifications for staining, mounting and archiving are needed and are detailed as well. The protocol is shown to be a highly cost- and time-effective procedure compatible with formalin-based fixatives plus standard sectioning and staining, yielding complete preservation of MPs and optimal tissue conditioning. The method also produced excellent results with pre-stained MPs, with fluorochromes included, altogether providing excellent localisation of polystyrene MPs in paraffin-processed biological tissue.