Modification of fluorescence staining method for small-sized microplastic quantification: Focus on the interference exclusion and exposure time optimization.

Microplastics are an emerging pollutant of global concern, and fluorescence staining as an efficient method for small-sized microplastic qualification often undergoes the serious interference from external environments. The key steps affecting the accuracy of fluorescent staining and the corresponding quality assurance measures were rarely known. Therefore, this study took the Nile Red/DAPI co-staining method as an example to explore the key factors affecting its accuracy and effective measures to avoid interference. High background microplastic contamination in typical lab waters (up to 1115 MP/L), glass fiber filter membrane and glassware were identified as dominant factors affecting microplastic quantification. The background microplastics in lab waters mainly originated from the process of water production and storage. A simple filtration process removed 99% of the background microplastic in the lab waters. After burning at 500 °C for 1 h, the microplastic contamination in the filter membrane and glassware was completely eliminated. HO pretreatment and exposure time caused erroneous microplastic size assessment, and were suggested to be set at 48 h and 10 ms, respectively. During the extraction process, the residue in beakers reached ~ 20% and > 50% for 5 μm and 20 μm sized microplastics, respectively, greatly contributing to the microplastic loss. The comprehensive modified measures caused microplastic concentrations in the three typical samples detected by Nile Red/DAPI co-staining method to decrease by 65.7 - 92.2% and to approach the micro-Raman results. This study clarified the reasons for interfering with quantitative microplastics by fluorescent staining and the effective measures to avoid interference, which were conducive to improving the accuracy of quantitative methods of microplastics.