Chitinase digestion for the analysis of microplastics in chitinaceous organisms using the terrestrial isopod Oniscus asellus L. as a model organism

Chitinaceous organisms have been found to ingest microplastic; however, a standardised, validated, and time- and cost-efficient method for dissolving these organisms without affecting microplastic particles is still required. This study tested four protocols for dissolving organisms with a chitin exoskeleton: 1) potassium hydroxide (KOH) + chitinase, 2) Creon® + chitinase, 3) hydrogen peroxide (H2O2) + chitinase, and, 4) Nitric Acid (HNO3) + hydrogen peroxide (H2O2). The effects on microplastics composed of eight different polymers were also tested. The use of H2O2 followed by chitinase was found to be a highly efficient method. The three other protocols either did not digest the chitin sufficiently or negatively affected the tested polymers. A recovery test using microplastic fibres, beads and tyre particles revealed high recovery rates of 0.85, 0.89 and 1 respectively. This further supported the applicability of the H2O2 and chitinase (protocol 3) for dissolving chitinaceous organisms. Thus, we recommend that future investigations of microplastic (0.05 μm-5000 μm) in chitinaceous organisms (0.3 cm-5 cm) utilise the here presented methodology. This represents an important component of the ongoing validation and harmonization of methodological approaches that are urgently needed for the advancement of microplastic assessments globally.