Combination of microscopic and spectroscopic techniques to study the presence and the effects of microplastics in mussels

The growing concern due to the presence of plastics, especially micro and nanoplastics, in environmental aquatic media requires the development of new methodologies to study the distribution of these particles and the effects that might cause in many organisms. In this work we have performed experiments using synthetic polystyrene microplastics (6-90 µm diameter) and mussels (Mytilus galloprovincialis) and we have studied the distribution of these particles by different techniques including FTIR and Raman spectroscopy, light and polarized light microscopy after being exposed for different periods of time (1-72 h). As a result of this work we were able to fine tune the preparation of the samples, from conservation to image and spectra analysis, and it was concluded that it was better to freeze the samples and to prepare the cryosections instead of embedding in paraffin. Regarding the light microscopy darkfield illumination offered less background signals than polarized one and therefore it was more suitable for small size particles. Finally, Raman spectroscopy allowed the characterization of the polystyrene particles better than FTIR allowing the development of image analysis techniques.