Unraveling health risk of microplastics in bottled water: Evidence of rapid detection and cellular internalization by Nile Red staining

Fluorescence-based detection offers a robust and sensitive strategy for rapid, in situ identification of microplastics (MPs). Herein, we report an improvement to the standardized Nile Red (NR) staining method for rapid identification and establish an intracellular localization method for MPs. This method was applied to quantify the release of MPs in bottled drinking water the five most prevalent brands in the Chinese market. Our results revealed that ingestion of one liter of bottled water may expose consumers to 2860 ± 695-12,080 ± 1419 MPs, with particles in the 1-10 µm size range constituting 60.8-94.4% of total counts. Estimated daily intake (EDI) values ranged from 23.3 to 98.3 particles/kg/day for adults and from 30.7 to 129.4 particles/kg/day for children, indicating a substantially higher exposure burden for pediatric populations. In vitro co-culture assays demonstrated that NR-stained MPs remained stable within cells over 24 h. Confocal laser scanning microscopy (CLSM) revealed distinct red fluorescence signals of MPs interspersed within the green-labeled cytoskeletal network, providing the first direct evidence that MPs originating from commercial bottled water can traverse cellular barriers. These findings establish a robust experimental framework and furnish critical data to inform risk assessments of MPs in bottled water.