Particle size-dependent quantitative and qualitative differences of common microplastic detection procedures: Nile Red-assisted fluorescence microscopy and confocal micro-Raman spectroscopy

Microplastics (MPs) are pervasive and widespread pollutants penetrating ecosystems worldwide, including aquatic environments and sediments. The lack of standardised evaluation procedures and limited sample throughput hampers accurate assessment of global MP pollution. High-throughput analytical methods are crucial for advancing our understanding of MP cycling in the environment. This study compares MP observations by confocal micro-Raman spectroscopy and Nile Red-assisted fluorescence microscopy to evaluate their effectiveness for high-throughput MP analysis using the percentage differences (%DIF) between the results of the two methods. The results show the influence of particle size on the detected percentage differences and demonstrate that both methods deliver better matching results at smaller particle sizes. The overall percentage difference in the number of detected MP counts between the two methods is 421%, with variations ranging over three orders of magnitude depending on morphological characteristics (particles and fibres) and particle size, whereas the analysis of the distribution of the detected polymers across the particle size fractions does not indicate that specific polymer types influence the observed %DIF between the two methods in this study. The combination of the Fenton reagent's limited organic matter removal and the resulting increased risk of false-positive MP detection, along with Raman spectroscopy's ability to reliably distinguish MPs from organic components, offers opportunities for data validation and correction to enhance accuracy and reliability of the results. This study contributes to the development of robust methods for high-throughput MP analysis, enabling improved spatial and temporal monitoring of its fate and transport in natural fluxes.