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

25 November 2022, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

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.

Keywords

microplastics
method validation
bottled water
nile red

Supplementary materials

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Supplementary materials
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The supplementary material contains (i) sample details of commercial samples of bottled water analysed in the manuscript; (ii) data related to microplastics quantification in bottled water and related bootstrap estimate; (iii) particle size and particle size distribution of microplastics isolated from bottled water; (iv) schematic diagram for the fluorescence microscopy image acquisition; (v) image of the Nile Red stained polyethylene MP used for image processing
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