C01 | Validation of an analytical protocol for the isolation of microplastics and microfibers in milk: preliminary study of contamination in raw milk samples

Purpose. Microplastics (MP) can contaminate numerous food matrices and, consequently, be ingested through the diet. Among the various routes of human exposure to these contaminants, food consumption is one of the most significant. To estimate the presence of MP in food, it is essential to have accurate and reliable analytical methods that allow both their isolation and quantification. In this study, a method was validated for the isolation of natural and synthetic MP and microfibers (MF) in samples of raw bulk milk and heat-treated milk. The method was then applied to samples of raw milk collected by mechanical milking. Methods. To validate the method, a known number of MF in polyethylene terephthalate (PET; n=4), MF in cellulose (n=4), and MP in low-density polyethylene (LDPE; n=4) were added to each sample (n=3 raw milk; n=3 heat-treated). The heat-treated milk samples were digested by adding 30% hydrogen peroxide (H₂O₂), incubated at 45°C for 48 hours, and finally filtered. For the raw milk samples, incubation was carried out at 50°C in order to improve the subsequent filtration phase. Each sample was analyzed in triplicate. The method was then applied to the analysis of raw milk samples collected from a dairy farm. MP and MF were counted by microscopic observation and characterized by FTIR microscopy. Results. The MP and MF, added in known quantities to the samples, showed no signs of deterioration or color change after digestion. The average recovery rates were between 91.6 and 116.6% for PET MF, 91.6% for cellulose MF, and between 91.6 and 100% for LDPE MP. In raw milk samples obtained by mechanical milking, synthetic MP and MF ranged from 1.3 ± 0.5 to 5.5 ± 5 particles/100 mL. In addition, natural MF was detected, with an average of 5.5 ± 5.7 MF/100 mL, in positive samples. The average lengths of the contaminants detected were: 1090.6 ± 810.1 µm for natural MF, 1168.5 ± 1260.1 µm for synthetic MF, and 321.5 ± 226.3 µm for fragments. The most common MF were black (48%) and blue (18%), while transparent fragments prevailed among the fragments (47%). FTIR analysis confirmed the synthetic nature (41% MP in polypropylene, 12% MF in polyester and polyvinyl chloride) or natural/artificial nature (47% cellulose MF) of the isolated particles. Conclusions. The validated method allows for effective recovery of natural/artificial MF and MP present in milk. An important advantage of the method is its applicability to both raw and heat-treated milk samples, requiring only optimization of the incubation temperature. This flexibility makes the protocol suitable for use in supply chain studies, allowing for rapid and effective analysis of MP and MF contamination throughout the various stages of production. Preliminary results highlight the presence of PM and FM in raw milk, suggesting possible contamination attributable to mechanical milking equipment and/or the clothing of operators involved in the initial stages of production.