Towards quality-assured measurements of microplastics in soil using fluorescence microscopy

Le, Quynh Nhu Phan and Halsall, Crispin and Peneva, Stoyana and Wrigley, Olivia and Braun, Melanie and Amelung, Wulf and Ashton, Lorna and Surridge, Ben W J and Quinton, John (2025) Towards quality-assured measurements of microplastics in soil using fluorescence microscopy. Analytical and Bioanalytical Chemistry. ISSN 1618-2642

Abstract

Fluorescence microscopy is increasingly seen as a fast, user-friendly, and high-throughput method for detecting microplastics (MPs) in soil; however, its effectiveness across diverse MP types and soil properties remains underexplored. This study tested a fluorescence microscopy-Nile red (NR) staining approach on eight MP types, covering both biodegradable and non-biodegradable plastics, in three size ranges (≤ 150 µm, 100-250 µm, 500-1000 µm) across loamy, clayey, and sandy soils. Each sample, processed in triplicate, underwent a relatively quick and straightforward extraction procedure involving density separation, organic digestion, and NR staining, followed by fluorescence and bright-field microscopy. A new digital image analysis pipeline using Image J was developed to expedite and (semi)automate MP quantification. Recoveries ranged from 80% to 90% for MPs with a Feret diameter of 500-1000 µm, regardless of soil type. In contrast, the recovery of smaller MPs (Feret dia. ≤ 250 µm) varied depending on the soils and plastic types: recoveries for low-density polyethylene (LDPE) reached 85% in sandy soil and 90% in loamy soil, whereas those for biodegradable polybutylene adipate terephthalate/polylactic acid (PBAT/PLA) were only 60% and 10%, respectively. The lowest recovery rate was observed in clayey soil and for biodegradable plastics. The method was tested on non-agricultural soil samples, yielding a MP mean number concentration of 20.7 ± 9.0 MPs/g for MPs sized from dia. ≥ 25 µm, comparable to Fourier transform infrared (FPA-µ-FTIR) results of 13.1 ± 7.3 MPs/g (p > 0.05). We conclude that fluorescence microscopy with NR staining and automated particle quantification offers a time-efficient, reproducible, and accurate method for MP detection in light-textured soils, whereas limitations remain for reliable MP analysis in clay-dominated soils.