The increasing contamination of the environment with microplastic requires efficient methods for the separation and detection of these plastic particles. In this work, we present a protocol that uses Fenton oxidation to remove biological material, centrifugation to separate microplastics from soil, and Nile Red staining, fluorescence microscopy, and image processing to detect and quantify of microplastic. The main component of this work was the separation process using centrifugation. All the main polymers used in this work, polyethylene, polypropylene, polystyrene, poly (vinyl chloride), and poly (ethylene terephthalate), were efficiently recovered at more than 94 wt% from heat-altered soil using CaCl2 solution with a density of 1.4 g ml-1. The hydrophilicity of the polymer had a greater effect on the recovery than density. The protocol was then tested on agricultural soil sampled near a contaminated site. The number of microplastic particles was quantified, and the weight of microplastic in the soil was estimated. The highest contamination was observed near the hotspot at a distance of 1 m with 75✕103 particles kg-1, corresponding to a weight between 20 and 6 mg kg-1. The number of particles decreased logarithmically to 30✕103 particles kg-1 or 5 to 2 mg kg-1.
Keywords: Image processing; Microplastic weight; Nile red staining; Poly(ethylene terephthalate); Polyethylene.
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