Laboratory guidelines for the detection and quantification of plastics particles from freshwater environmental samples

The present document develops the laboratory protocols to analyse plastic pollution in freshwater environmental samples, although the methods followed can be extrapolated to other ecosystems. These protocols are developed in the framework of the PLASTICØPYR project, which aims to tackle plastic pollution associated with mountain tourism activities by developing transversal actions involving several stakeholders. These guidelines are developed in order to quantify plastic pollution in different river habitats, from large items (macro- and mesoplastics) to tiny plastic particles (microplastics). The protocols are developed for the analysis of microplastics (MicP) up to 63 µm, from samples from the water column, the fluvial sediments, the fluvial biota (i.e., brown trout and biofilm), as well as plastic particles present in the atmosphere (either wet or dry deposition). The MicP analysis is done following the Nile Red method, a fluorescent dye that facilitates the observation of small particles. Although Nile Red cannot provide chemical characterisation, it can be compatible with MicP identification techniques, such as FTIR (Fourier-transform infrared spectroscopy). The samples procedure includes: the extraction of MicP from the inorganic matrix by density separation (using ZnCl2), a digestion step with hydrogen peroxide and catalysed with Fenton's reagent to eliminate the organic remains and finally, the staining of the sample with Nile Red.