DEVELOPMENT OF A NOVEL PROTOCOL FOR THE EXTRACTION OF SMALL MICROPLASTICS (1-5 µm) FROM BIOLOGICAL TISSUES

Microplastics (MPs) smaller than 5 µm pose a growing concern in marine ecosystems due to their potential for bioaccumulation and toxicity. While there have been increasing publications on exposure to MPs ranging from 100 μm to 5 mm in biological tissues, the analysis of smaller particles is still hindered by several factors, including the lack of harmonised and standardised protocols and technical challenges in the extraction and quantification of MPs. In response to this, a novel protocol is being developed to achieve an efficient and reproducible recovery of small MPs (1-5 µm) from biological tissues, using Mytilus galloprovincialis as model organism. This work addresses the key methodological gaps identified through a comprehensive literature review of existing studies that attempted to develop or optimise the extraction of MPs. The protocol integrates optimised chemical and enzymatic digestion steps that effectively remove the organic matter while minimising polymer degradation. Several digestive agents were tested thus far for the efficient digestion of organic matter and recovery of MPs and were followed by proteases and surfactants, by filtration and resuspension of the filter for subsequent analysis. Analytical validation encompasses techniques such as fluorometry, FTIR/Raman spectroscopy, and SEM-EDS. Preliminary results have shown promising recovery rates. Its outcome will implement the plan outlined in the Marine Strategy Framework Directive 2008/56 by developing innovative solutions, such as enhanced analytical methods and technologies for detecting and measuring MPs in biological tissues and marine environment, and facilitating effective sea monitoring.