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

Gabriel Erni-Cassola, Matthew I. Gibson, Richard C. Thompson, Joseph A. Christie-Oleza

Marine plastic debris is a global environmental problem. Surveys have shown that plastic particles <5 mm in size, known as microplastics, are significantly more abundant in surface seawater and on shorelines than larger plastic particles. Nevertheless, quantification of microplastics in the environment is hampered by a lack of adequate high throughput methods to distinguish and quantify 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 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 of small microplastics (i.e. <1 mm) with decreasing particle size. Hence, our data helps to resolve speculation on 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 semi-automated procedure.