Comparison of Raman and fluorescence microscopy for identification of small (< 2 μm) microplastics in soil

The study of microplastics (MP) in soils presents significant challenges due to the absence of standardized extraction techniques and the strong matrix interferences that complicate MP detection, particularly for the smallest particle sizes. As a result, little is known about the behavior and dynamics of these minute MP in soil environments. In this study, we assessed and compared the direct detection of fluorescently labelled small MP (1-2 μm) using Raman and fluorescence microscopy across matrices with varying complexity. We introduced fluorescent polystyrene (PS) microparticles (1.71 ± 0.03 μm in diameter) into different substrates-pure quartz sand and soils with distinct textural properties (sandy loam, silt loam, and clay loam). The soils were analyzed both with and without prior removal of native soil organic matter (SOM). MP were added at concentrations ranging from 0.1 % to 0.001 % (corresponding to a range of natural concentrations), after which their detection was evaluated using μ-Raman and fluorescence microscopy. Raman analysis successfully identified characteristic PS peaks (notably at 1001 cm) in quartz sand at all tested concentrations and, to some extent, in sandy loam and silt loam. However, detection failed in clay loam and in all soil samples still containing the native SOM. In contrast, MP across all concentrations and soil types were consistently visualized by fluorescence microscopy. Furthermore, fluorescent PS particles were directly observed via fluorescence microscopy in field samples collected from an experiment where MP downward transport was monitored, whereas detection was challenging with Raman microscopy. The ability to directly and unequivocally identify fluorescent MP in complex soil matrices eliminates the need for time-intensive extraction methods and offers new opportunities for investigating the movement and behavior of the smallest MP fractions in soil.