Suitability of Nile Red Dye for In-Situ Microplastic Detection

Plastic pollution is a widespread and significant environmental problem. Of the 359 million tonnes of plastic produced in 2018, 5.5-14.5 million tonnes entered the ocean; it is unknown how long plastic remains in the environment (Wayman & Niemann, 2021). Understanding the transport and fate of plastic particles in the environment is critical for assessing impacts and mitigation approaches. However, the definition of a "microplastic" is nebulous; the most common definition is <5mm (Arthur et al. 2009). Microparticles observed in this paper are less than 1 mm, in accordance with the definition and categorization framework from Hartmann et al. (2019). Environmental plastic particles may be further divided into "primary" and "secondary". "Primary" refers to plastic particles that are purposefully manufactured in the form of small particles (e.g. microbeads from cosmetics) and "secondary" being produced from the breakdown of larger plastics. It is estimated 70-80% of microplastics released into the environment are secondary while 15-31% are primary microplastics (Mariano et al., 2021). Common plastic polymers include polyethylene (PE), polypropylene (PP), polyurethane, polyethylene terephthalate (PET), polystyrene (PS), and polyvinyl chloride (PVC).Identification of MPs consists of two goals: physical characterization of observed particles and chemical characterization of the observed particles (Mariano et al., 2021). Physical characterization is typically achieved with microscopal techniques (e.g., stereo-, fluorescence, atomic force, transmission electron, scanning electron), which can rapidly identify the shape, size, and color of the particles. Some microscope techniques (e.g., SEM) have analytical potential by identifying both the physical and chemical properties of plastic polymers (Mariano et al., 2021). Most of these techniques involve complex equipment that is unsuitable for use in the field. Portable microscopes can visually identify MPs in situ but lack the ability to chemically identify the observed particles. Chemical characterization can be achieved with spectroscopy techniques (e.g., Fourier-Transform Infrared, Raman, Thermal Analysis); however, traditional spectrometers are expensive and complex machines and are unsuitable for in-situ use. Portable spectrometers are available, but these devices are costly and must be paired with in-situ microscopy techniques to detect MPs. An efficient in-situ MP detection method ought to be portable, economical, able to determine MPs visually and chemically, and ideally, able to classify found MPs.A novel solution for in-situ MP detection, Nile Red (9-diethylamino-5-benzo[a]phenoxazinone; NR) offers a rapid and low-cost approach to identify MPs both visually and chemically. Nile Red is a lipophilic stain that preferentially bonds to plastic polymers and renders them fluorescent when illuminated with blue light. Organic detritus such as seaweed, wood, feathers, and various mollusk shells stain weakly or not at all (Maes et al., 2017). In-situ detection of stained MPs can be accomplished with photography and an appropriate light filter setup. Microplastic detection to date has involved sample preparation, typically including filtering and drying the sample. To detect MPs in situ (i.e., an aqueous sample), Nile Red is applied to environmental seawater samples, and fluorescent particles are identified and extracted for closer analysis. NR solutions of acetone, acetone:n-hexane, and methanol were tested for their interactions with water. Nile Red is a practical solution for the in-situ detection of microplastics for its relative ease of use and ability to identify plastic polymers visually and chemically. Nile Red staining can be a valuable tool for rapid screening to complement other identification/characterization methods.