A MULTI-TECHNIQUE APPROACH BASED ON MICRO-RAMAN SPECTROSCOPY AND ENVIRONMENTAL SCANNING ELECTRON MICROSCOPY FOR THE DETERMINATION OF SMALL MICROPLASTICS IN MILK POWDER: A FOCUS ON ANALYTICAL CHALLENGES AND METROLOGICAL ISSUES

Microplastics (MPs) are ubiquitous anthropogenic pollutants that can be found in the environment and food samples. The European Commission is driving the development of legislation needed to tackle their potential threat to human health and the environment. Currently, interpretation and comparison of analytical data are hampered by the unavailability of standardized and harmonized methods for MP sampling, characterization and counting as well as by the lack of reference materials for metrological traceability.1 In particular, small-microplastics (SMPs, 10-100 μm) are difficult to isolate, enrich and detect in matrices containing high concentration of organic compounds, without compromising the integrity of the particles and altering polymer composition.1 Furthermore, another analytical issue is cross-contamination throughout the analytical workflow: implementing a quality control procedure is essential to assess environmental contamination, define realistic reporting limits (ISO/NP 16094-2) and ensure reliable results. In this context, the present study focuses on the development and validation of an analytical approach for chemical identification, physical characterisation and counting of SMPs in milk powder (infant formula) based on the use of micro-Raman spectroscopy and Environmental Scanning Electron Microscopy.2,3 To maximize the impact of the study, both primary and secondary particles of polystyrene (PS), polyethylene terephthalate (PET) and polyvinyl chloride (PVC) were considered as representative test materials. As part of a collaboration within the European project PlasticTrace, BAM (German Federal Institute for Materials Research and Testing) produced and provided PET tablets to be tested as SMPs reference materials. At first, blank filtration experiments were performed to establish good practices to be implemented into a clean room and develop a multi-step cleaning procedure to minimize cross-contamination. After selecting the appropriate filtration system and filters (Si, 5 μm), the acquisition parameters for micro-Raman measurements were properly selected to obtain high-quality spectra and representative coverage of filter area while avoiding laser-induced SMP degradation and fluorescence phenomena; in addition, the use of ParticleFinderTM and IDFinderTM (Horiba) software for Raman analysis permitted to automate particle analysis. Automatic particle counting on ESEM micrographs was performed using ImageJ-Fiji software. Concerning the isolation of SMPs by selective removal of matrix organic/inorganic matter that inhibits microplastic detection, different approaches for the digestion of organic matrices were tested, obtaining satisfactory results by combining multi-enzymatic digestion with alkaline hydrolysis.4 Excellent recovery values were obtained from SMP fortified milk powder, which were in the 80-120% range for all polymers investigated. Ongoing activities involve the assessment of reporting limits per polymer type within defined size classes. Further activities such as real sample analysis and participation in inter-laboratory testing schemes are planned as a final part of the funded projects. References: [1] T.V. Duncan, S. Afrin Khan, A.K. Patri, S. Wiggins, Anal. Chem. 96 (2024) 4343-4358. [2] M. Mattarozzi, F. Bianchi, M. Maffini, F. Vescovi, D. Catellani, M. Suman, M. Careri, Talanta 196 (2019) 429-435. [3] M. Piergiovanni, M. Mattarozzi, E. Verleysen, L. Siciliani, M. Suman, F. Bianchi, J. Mast, M. Careri, Molecules 29 (2024) 3148. [4] P.A. Da Costa Filho, D. Andrey, B. Eriksen, R.P. Peixoto, B.M. Carreres, M.E. Ambühl, J.B. Descarrega, S. Dubascoux, P. Zbinden, A. Panchaud, E. Poitevin, Sci. Rep. 11 (2021) 24046.