Dyeing to Know: Harmonising Nile Red Staining Protocols for Microplastic Identification

With the escalation of microplastic (MPs) pollution and the laborious nature of existing MPs identification methods, new approaches for large-scale sampling of MPs in the environment are necessary. A promising solution lies in the fluorescence staining of Nile Red (NR), whose fluorescence is polarity-dependent, offering the potential for classification based on fluorescence. However, the choice of carrier solvents to dissolve NR remains unstandardised, and methods to represent and differentiate the fluorescent behaviour of MPs are lacking. This study addresses these gaps by testing eight NR-carrier solvents (n-hexane, chloroform, acetone, methanol, ethanol, acetone/hexane, acetone/ethanol, and acetone/water) applied to ten most common polymer-types of MPs (HDPE, LDPE, PP, EPS, PS, PC, ABS, PVC, PET, and PA). The fluorescence behaviour, including fluorescence intensity and Stokes shift, was compared across solvents, and their effects on polymer degradation were evaluated. Additionally, the effectiveness of various polarity measures with the proposed HSV colour spaces in reflecting Stokes shift for MPs identification was assessed. To differentiate natural organic materials (e.g., eggshells, fingernails, wood, and cotton) from polymers, Fenton oxidation was found to quench the fluorescence of natural organic matter with minimal changes observed in NR-stained MPs. The findings identified acetone/water [25% (v/v)] as the best compromise, effectively mitigating the adverse effects of acetone while maintaining strong fluorescence behaviour suitable for identification.