Zoological biobanks as a resource for microplastic analysis: a Py-GC-MS/MS case study on sea cucumbers.

Microplastics have spread through every ecosystem. Yet, documenting their spatiotemporal distribution remains challenging, particularly in marine environments where sampling is costly and difficult. Archived specimens in zoological biobanks offer an underused resource for investigating microplastic contamination. However, false-positive signals can arise from storage conditions or procedural contamination. This case study addresses these methodological challenges showing that careful experimental design can reliably account for such artefacts, using sea cucumbers as a model organism. As deposit feeders, they are directly exposed to microplastics accumulating on the seafloor. We adapted state-of-the-art sample preparation protocols to quantify five common plastic polymers from sea cucumber intestines using pyrolysis-gas chromatography-tandem mass spectrometry after chemical digestion of the intestines of five specimens of Holothuria forskali from the Atlantic and five specimens of Holothuria tubulosa from the Mediterranean. Samples of the biobank's preservation fluids were also analyzed, revealing low polymer concentrations compared to intestinal tissues. Procedural contamination was negligible. High purification rates confirmed effective elimination of tissue matrix before polymer analysis. Our analytical approach achieves low detection limits. It also uses a conservative multi-marker strategy to distinguish polyethylene from confounding organic compounds such as lipids. Analyzed specimens contained total polymer concentrations ranging from 9.4 to 169.2 μg per gram of dry weight, consistent with levels found in other marine organisms, and predominantly polyethylene terephthalate and polystyrene. These results confirm the method's robustness and support the wider use of zoological biobanks for microplastic pollution monitoring across time and space.