A validated Nile Red fluorescence method following KOH/H₂O₂ digestion for microplastic detection in marine molluscs

Microplastic (MP) in marine ecosystems poses an increasing risk to the environment and human health. However standardized rapid methods for monitoring large numbers of samples are still lacking. This study validates a semi-automated Nile Red dye (NRD) fluorescence method combined with a two-step KOH/H₂O₂ digestion protocol for MP detection in marine molluscs. Twelve reference materials, including six synthetic polymer types (PE [two size fractions], PP, PA-66, PET, PMMA) and six natural materials (cotton, hemp, cellulose, wool, viscose, olive stone powder), were evaluated before and after digestion. Treatment resulted in 83.2 (± 4.8%) digestion of the natural reference, and reduced fluorescence intensity by 83.9 (± 14.9%). In comparison, the synthetic polymers lost only 13.0 ± 11.6% of their initial mass and 42.7 ± 18.4% of their fluorescence intensity, thus increasing the fluorescence discrimination ratio between synthetic and natural materials from 3-fold to 16.9-fold. The protocol retains the chemical integrity of the synthetic polymers (FTIR Hit Quality Index: 89.7-99.3%), which allows further reliable identification of the particles by FTIR. The protocol was applied to samples of Black Sea invertebrate organisms (M. galloprovincialis and R. venosa, 67 specimens) and achieved near-complete organic matter removal (>99.8%). The MP load was higher in M. galloprovincialis (median: 1.3 MPs g^-1 WW; Q1-Q3: 0-3.2; n = 27) than in Rapana venosa (median: 0 MPs g^-1 WW; Q1-Q3: 0-0.2; n = 40). This validated workflow provides a reproducible and cost-effective approach for large-scale MP monitoring in marine bioindicator species.