Comparative Assessment of Protocols for Microplastic Quantification in Wastewater

Microplastics are an increasing concern due to their widespread occurrence in aquatic environments worldwide. The lack of a harmonised protocol for their reliable quantification remains a major challenge in current scientific efforts. This study presents a comparative evaluation of three protocols for the detection and quantification of microplastics in aqueous samples. The protocols were assessed based on quantification efficiency, risk of particle degradation, staining performance, operational complexity, and cost per sample. Protocol A combined Rhodamine B and ethanol staining with NaCl-based density separation, demonstrating strong isolation performance while maintaining minimal chemical hazards and moderate cost (2.45€ per sample) that could be further reduced to 0.45€ per sample by substituting reagent-grade NaCl with table salt. Protocol B offered moderate isolation capacity and presented the highest risk of particle fragmentation, likely due to the use of acetone and high-temperature digestion. Protocol C, based on the combined use of Nile Red and ZnCl2, also presented a risk of particle fragmentation, resulting in the highest MP count for small and hydrophobic particles. In addition, its high cost (15.23€ per sample) limits its suitability for routine application.