A Fluorescence-Based Protocol for Quantifying Microplastics in Soil: Protocol Optimization and Field Investigation

The increasing accumulation of microplastics (MPs) in terrestrial environments has intensified the need for reliable methods to detect and quantify MPs in complex soil matrices. This study aimed to establish and optimize a protocol for quantifying MPs in soil. Important steps include soil organic matter (SOM) digestion, MPs extraction, Nile red staining, fluorescence microscopy detection, and MPs counting after image processing, in which the efficiencies of SOM digestion and MPs extraction play the crucial roles and were systematically optimized. Digestion reagents including H₂O₂, Fenton's reagent, and HNO₃ were evaluated for their abilities to remove SOM that can induce background fluorescence interfering with MPs quantification. Among the tested reagents, HNO₃ achieved the highest background fluorescence removal efficiency (89% in loam and 82% in clay) without causing significant damage to polypropylene (PP) and polyvinyl chloride (PVC) MPs. CaCl₂ (1.4 g/cm³) and ZnCl₂ (1.6 g/cm³) showed similar efficiencies for MPs extraction but CaCl₂, which yielded recoveries of 78-80% for PP and 61-70% for PVC in tested soils, was selected over ZnCl₂ due to its lower environmental toxicity. The established protocol was applied for soil samples collected from three sites designated for different land uses. The highest MPs concentration was found in samples collected from a farmland (1.04×10⁵ particles/kg), followed by a landfill site (7.10×10⁴ particles/kg) and a park (4.00×10⁴ particles/kg), with MPs < 100 μm being the dominant size in all samples. Overall, the established protocol shows potential for application in assessing MPs contamination in terrestrial environments.