A new approach for routine quantification of microplastics using Nile Red and automated software (MP-VAT)

Microplastics are widespread contaminants in the environment. However, most identification protocols rely on long and subjective visual counting, which could be improved using staining dyes. Thus, the objective of this work is to identify the best staining dye protocol and create an objective and quick automated counting software for microplastics. Tests were conducted to identify the most appropriate of eight staining dye solutions and of six wavelengths for virgin and weathered synthetic polymers, textile fibers, natural organic matter and filters. Nile Red produced the best results (without interfering in infrared spectra) rendering microplastics fluorescent at 254 nm, but with limited number of fluorescent polymers, and at 470 nm (with orange filter), with fluorescence of plastics as well as natural organic matter (requiring a digestion step). Next, a script was developed in ImageJ for the automatic quantification and characterization in shape (fiber, fragment, particle) and size of fluorescent microplastics, the Microplastics Visual Analysis Tool (MP-VAT). MP-VAT was evaluated, producing recovery rates in the range of 89.0-111.1% in spiked filters under 470 nm. Furthermore, this package is accompanied by a script that sets a scale from a known filter diameter, MP-SCALE, and a script that allows user threshold setting, MP-ACT.