Developing an Efficient Protocol for Quantifying Microfibres in Wastewater Treatment Sludge

Microfibres (MFs) are among the most frequently detected microplastics in wastewater treatment plants. This study aims to evaluate the methodologies commonly used to characterise MFs in sludge samples and to identify their main critical points, with special attention to organic matter removal, density separation, cross-contamination, and MF quantification. Three oxidation methods (peroxidation, ozonation, and peroxone) were evaluated for organic matter removal. In addition, density separation techniques using NaCl and NaI solutions were tested to assess MF recovery from the sludge matrix. The influence of cross-contamination, particularly from conditioning reagents, was also evaluated. Ultrasound was applied before oxidation and density separation to determine whether it improved MF release from the solid fraction. Peroxidation was the most effective oxidation treatment, achieving 98.9% organic matter removal without altering the integrity of the MF colour. Density separation showed limitations, since more than 55% of MFs remained in the solid fraction. In addition, segregation may occur by fibre type, as less-dense MFs separated more easily, while denser fibres tended to remain with the solid residue. The application of ultrasound before oxidation and density separation did not produce a significant improvement in MF recovery. Based on these results, a protocol for MF quantification in sludge samples is proposed, which consists of conditioning the sample to have a maximum of total solids of 14 g/L, performing a peroxidation, followed by distribution of the oxidised sample across three filters before stereomicroscope quantification. This method reduces cross-contamination, reagent consumption, and experimental time, while ensuring reliable and comprehensive MF characterisation.