Lost, but Found with Nile Red: A Novel Method for Detecting and Quantifying Small Microplastics (1 mm to 20 μm) in Environmental Samples

Marine plastic debris is a global environmental problem. Surveys have shown that <5 mm plastic particles, known as microplastics, are significantly more abundant in surface seawater and on shorelines than larger plastic particles are. Nevertheless, quantification of microplastics in the environment is hampered by a lack of adequate high-throughput methods for distinguishing and quantifying smaller size fractions (<1 mm), and this has probably resulted in an underestimation of actual microplastic concentrations. Here we present a protocol that allows high-throughput detection and automated quantification of small microplastic particles (20-1000 μm) using the dye Nile red, fluorescence microscopy, and image analysis software. This protocol has proven to be highly effective in the quantification of small polyethylene, polypropylene, polystyrene, and nylon-6 particles, which frequently occur in the water column. Our preliminary results from sea surface tows show a power-law increase in small microplastics (i.e., <1 mm) with a decreasing particle size. Hence, our data help to resolve speculation about the "apparent" loss of this fraction from surface waters. We consider that this method presents a step change in the ability to detect small microplastics by substituting the subjectivity of human visual sorting with a sensitive and semiautomated procedure.