Enzymatic digestion method development for long-term stored chitinaceous planktonic samples

Different extraction methods have been proposed to study the ingestion of microplastics by marine organisms, including enzymatic digestion. While mussels have been the focus of research, crustaceans' enzymatic digestion has received little attention. An overlooked source of information for microplastic research is analysis of long-term time-series biotic samples. These collections are invaluable for the detection and monitoring of changes in ecosystems, especially those caused by anthropogenic factors. Here, crustacean larvae collected in two periods, 1985 and 2020, in the central North Sea were used to develop and optimise an effective and gentle enzymatic digestion method suitable for microplastic research. Sequential breakdown of these chitinaceous samples via a mechanical and surfactant (Sodium Dodecyl Sulphate 1% v/v) pre-treatment, followed by proteinase K (100 mU/mL) and chitinase (50 mU/mL) digestion, efficiently removed >96% of biomass of 1985 and 2020 samples. The optimised method was effective without interfering with the identification of naturally weathered microplastics via FTIR Spectroscopy.