Small microplastic particles in Lake Superior: A preliminary study coupling Nile red staining, flow cytometry and pyrolysis gas chromatography-mass spectrometry

Microplastic particles (< 5 mm) are now found throughout earth's ecosystems, with smaller microplastics often showing greater impacts on organismal health than larger ones. Unfortunately, there are no readily available analytical approaches that can couple microplastics enumeration and polymer determination for smaller microplastics (< 10 mu m), and 1-20 mu m particles are difficult to quantify with existing techniques. This study presents a method using Nile red (NR) staining and flow cytometry (FCM) to quantify and isolate small microplastic particles for subsequent identification by pyrolysis gas chromatography-mass spectrometry (pyGCMS). Results using standard plastic particles showed that FCM sorting can provide sufficient material for pyGCMS analyses; the polymer composition remains identifiable after the processing steps. The post-sorting concentration step yielded recovery of 58%-83% of the original plastic polymer mass. Analysis of a mixed plastic standard solution showed no significant difference in plastic counts obtained by microscopy and FCM, although blank correction reduces the FCM counts to 62% of the microscopy counts. The applicability of NR staining and FCM was demonstrated through analysis of small microplastic particles (5-45 mu m) from Lake Superior surface water samples, which showed particle abundances two to three orders of magnitude higher than particles > 100 mu m that were counted using FTIR microscopy. PyGCMS analysis of a test lake sample showed the presence of polyethylene in this small size fraction. Careful attention to blanks and longer FCM sorting times (> 2 h) are recommended for successful analysis of natural aquatic samples processed by this approach.