Optimization of an Analytical Protocol for the Extraction of Microplastics from Seafood Samples with Different Levels of Fat ^†

The global production of plastics increased from 1.5 million tons in 1950 to 370 million tons in 2020. Microplastics (MPs) are plastic particles smaller than 5 mm, with a concerning ubiquitous occurrence in marine environments, posing risks to wildlife and humans. Seafood is an important source of proteins (including all the essential amino acids), and other essential nutrients with numerous health benefits. Human exposure to MPs through the consumption of contaminated seafood, together with the potential of these particles to cause health risks, is motivating a better understanding of the security of our diets. In recent years, several methodologies have been used to investigate MP occurrence in seafood, although the existence of complex sample matrices, namely, a high level of fats, can pose severe difficulties and compromise the efficiency of MP quantification. To solve this issue, the present study aimed to develop a detailed protocol suitable to process seafood samples with different levels of fats (fish and mollusks, from fresh and canned sources). For the sample digestion, several tests were performed using two solutions (10% KOH, 30% H₂O₂) with different volumes, temperatures (40 °C, 65 °C) and durations (24, 48, 72 h) of incubation. To remove the fat remaining after digestion, three detergents (two laboratory surfactants and a commercial washing-up liquid) and 96% ethanol were tested. Manual recovery of the fat layer was also tested. For filtration, two filter membranes were compared (glass microfiber and nitrate cellulose filters, 0.45 µm pore size). The efficiency of the different experiments was determined through the observation and estimation, in percentage, of the organic matter digestion and post-digestion fat removal. The methodology optimized in this study combined a sample digestion with 30% H₂O₂ incubated at 65 °C, for 24 to 48 h, with a manual separation of the post-digestion fats with immediate observation in a stereomicroscope. After, this methodology was applied to different types of polymers (e.g., polyethylene, polypropylene, polyethylene-terephthalate, polystyrene), to investigate if these procedures altered the integrity of MPs. The results show that this methodology will allow us to efficiently process complex seafood samples with different fat levels, without compromising MPs’ integrity.