Enzymatic digestion combined with μFT-IR imaging for recovery and characterisation of polymer particles from Mytilus galloprovincialis tissue

Abstract

Microplastic contamination in marine organisms requires analytical approaches capable of efficiently removing biological matrices while preserving polymer integrity. Building upon previous work identifying enzymatic digestion as a suitable treatment method, the present study applies a pancreatic enzyme protocol (KreonR25000) to recover polymer particles from Mytilus galloprovincialis tissue and to evaluate particle characteristics by μFT-IR imaging. Frozen mussel tissue (2 g) was artificially spiked with reference particles of PVC, HDPE, PA, and PET and subjected to enzymatic digestion under controlled conditions. Digestion removed 99.8% of the biological matrix, allowing subsequent filtration, microscopic inspection, and spectroscopic identification. A total of 2334 particles were detected. Recovery varied among polymers, with HDPE showing the highest numerical recovery and PET the lowest. Particle size distributions differed markedly: HDPE, PA, and PVC were dominated by particles <50 μm, whereas PET particles were predominantly larger. Morphological analysis revealed irregular fragment-like shapes across all polymer types. μFT-IR imaging enabled polymer identification and spatial mapping, revealing heterogeneous particle distribution and localized clustering patterns on filter surfaces. Comparison of ATR-FTIR reference spectra with μFT-IR spectra obtained after digestion confirmed preservation of diagnostic polymer bands, indicating that enzymatic treatment did not alter polymer chemical structure. The results demonstrate that enzymatic digestion combined with μFT-IR imaging provides a reliable and polymer-preserving workflow for microplastic analysis in marine biological matrices. The findings highlight the influence of particle size and spatial distribution on recovery and detection, underscoring the importance of standardized imaging strategies for accurate quantification.