Customized digestion protocols for copepods, euphausiids, chaetognaths and fish larvae facilitate the isolation of ingested microplastics

Degradation of oceanic plastic waste leads to the formation of microplastics that are ingested by a wide range of animals. Yet, the amounts that are taken up, especially by small zooplankton, are largely unknown. This is mostly due to the complex methodology that is required for isolating ingested microplastics from organisms. We developed customised, effective and benign digestion protocols for four important zooplankton taxa (copepods, euphausiids, chaetognaths and fish larvae), and assessed their digestion efficacy and their potential to cause particle loss or to alter microplastics using six polymers (HDPE, LDPE, PS, PET, PVC, PMMA). All protocols are based on an incubation of the organic matrix with 10% KOH at 38 °C, which is optionally combined with digestive enzymes (chitinase, proteinase K). This yielded digestion efficacies of > 98.2%, recovery rates of > 91.8%, < 2.4% change in microplastics' size, while no visual alteration of the microplastics and no changes in their spectra were observed when analysing them with a hyperspectral imaging camera. The proposed protocols are inexpensive (< 2.15 € per sample), but require several days when enzymatic digestion is included. They will facilitate research on microplastic ingestion by small marine organisms and thus enable well-founded conclusions about the threat that microplastics pose to these animals as well as about the role of biota in determining the vertical distribution of microplastics in oceanic environments.