Optimisation of enzymatic digestion and validation of specimen preservation methods for the analysis of ingested microplastics

Microplastics are considered to be a widespread environmental contaminant. Due to their small size microplastics have the potential to be ingested by a range of aquatic organisms which mistake them for a food source and can suffer adverse impacts as a result. Development of standardised methods is imperative to provide reliable and meaningful data when analysing microplastic ingestion by marine fauna. A range of proteolytic digestive enzymes (trypsin, papain and collagenase) were tested to establish optimum digestion efficacy of biological samples and assess the effects of enzymes on microplastics; additionally the applicability of freezing and formaldehyde followed by ethanol as specimen preservation techniques for microplastic research was investigated. Of the enzymes investigated, trypsin yielded the greatest digestive efficacy based on weight reduction (88% ± 2.52 S.D.) at the lowest concentration (0.3125%) with no observed impacts on microplastics. Enumeration of microplastics from wild collected Mytilus edulis revealed mean numbers of 1.05 ± 0.66 S.D. (minimum) to 4.44 ± 3.03 S.D. (maximum) microplastic particles per g wet weight mussel tissue depending on location. There was no significant difference based on preservation method on the quantification of ingested microplastics and no detrimental impacts were observed on the microplastics directly. Enzymatic digestion using trypsin therefore provides a suitable, time and cost effective method to extract microplastics from M. edulis. Furthermore the preservation methods did not have detrimental effects on microplastics, serving to highlight the suitability of biological samples preserved either way for future inquiries into ingested microplastics.