Optimization of a protocol for the extraction and chemical characterization of microplastics in Chamelea gallina by Raman microspectroscopy

Microplastics (MPs) are now present in all environments, from marine to terrestrial, representing a problem for food safety as well as for public health. Bivalve molluscs are often used as bioindicators of the healthiness of seawater: given their nature of filter feeding, they process a large quantity of water and act as concentrators of pollutants, including MPs. Many methods based on spectroscopic techniques have been employed for the analysis of MPs, in particular Raman microspectroscopy (µRaman) which allows to reach dimensions up to 1 µm and preserves the morphological characteristics of the microparticles. A method for the extraction of MPs from the clam Chamelea gallina was developed and adapted for chemical characterization using µRaman. The clams are collected off the Central Adriatic coast (Abruzzo, Marche and Molise regions), according to a sampling plan started in June 2023 that includes a total of 108 samples. The extraction method was validated using MPs of different dimensions (6 µm, 160 µm, 3 mm) as reference materials and processing environmental and reagent blanks to take under control the contamination. The procedure involves an alkaline digestion with KOH at 60 °C for 24 hours, 2 gravimetric separations without and with KI to ensure also the recovery of polymers with higher density. The filtration is carried out on Polycarbonate filters for counting and sorting the microparticles with the stereomicroscope. A variety of filters were evaluated to apply the protocol for the chemical characterization using Raman spectroscopy, taking into consideration parameters including time required for the filtration step, recovery rate of standard materials, cost and readability of the filters. This study was financially supported by Ministry of Health, PLASTICVONG research project (2022).