Quantitative image analysis of microplastics in bottled water following Nile Red staining and fluorescence microscopy

The ubiquitous occurrence of microplastics (MPs) in the environment and the use of plastics in packaging materials result in the presence of MPs in the food chain and the exposure of consumers. Yet, no fully validated analytical method is available for MP quantification, which prevents the reliable estimation of the level of exposure and, ultimately, the assessment of the food safety risk associated with MP contamination. In this study, an integrated method for the analysis of MPs in bottled water based on Nile Red staining, fluorescent microscopy, and automated image analysis was developed and validated, featuring a partial interrogation of the filter, thereby boosting the analysis time. The image analysis provided the number of particles in the region of interest of the analyzed filter and the size of their major and minor axis. From these data, a rough estimation of the mass of the individual MPs, and consequently of the mass concentration in the sample, could be obtained as well. The applicability of the method for accurate MP size measurements and mass quantification was critically evaluated: the method showed to be highly sensitive in sizing MPs down to 10 µm with a limit of detection and quantification of 1.1 ppb and 3.4 ppb, respectively. Linearity and linearity range were studied between 10 ppb and 1.5 ppm resulting in a regression coefficient of (R2) of 0.99. Method precision was demonstrated by a repeatability of 11% - 12% RSD (n=7) and within-laboratory reproducibility of 16 - 29% RSD (n=21). Accuracy based on recovery was 92.6 ± 23.3 % - 97.3 ± 14.5 %. Finally, the method was successfully applied to the analysis of 15 commercial samples of bottled water with and without gas and additives.