Optimizing microplastic extraction from organic-rich and bristle polychaetes (Hermodice carunculata)

Microplastics are ubiquitous and persistent contaminants in marine ecosystems worldwide. Benthic organisms, such as polychaetes, are particularly vulnerable to direct or indirect ingestion of microplastic and have recently been identified as potential microplastic concentrators. Understanding the impacts of microplastics pollution on polychaetes has garnered significant attention given their abundance, species richness, and key ecological role in marine food webs and benthos community structures. However, current extraction protocols are largely adapted from methodologies designed for seafood and fail to account for the morphological diversity of polychaeta species. Thus, harmonization approaches that are broadly applicable across taxa, enabling comparisons between studies, are needed. Herein we proposed an optimized and validated analytical method for the extraction and quantification of microplastics content in the bearded fireworm, Hermodice carunculata , a lipid-rich polychaete characterized by calcareous-chitinaceous structures. Following a comparative assessment in filterability of existing approaches, our optimized method was evaluated for digestion efficiency, microplastic recoveries and alteration on microplastic physio-chemical properties. The protocol included a pre-enzymatic digestion with trypsin, followed by thermo-oxidative treatment using hydrogen peroxide to remove organic matter and a mild acid digestion with acetic acid to dissolve chaetae. To further isolate microplastics, samples are pre-filtered with a stainless-steel mesh, subjected to a flotation step using canola oil, and finally concentrated onto a 1.2 μm filter. This method achieved high efficiency in removing organic and inorganic materials (98.4 ± 1.2% of the initial dry weight and 99.7 ± 0.2% of the initial wet weight of the H. carunculata specimens) while maximizing the recovery of common plastic polymers (ranging from 86.7 ± 5.8% to 94.4 ± 5.0% depending on the polymer type), preserving microplastic integrity. According to our results, the proposed method is a reproducible, sustainable and time-effective approach for microplastics detection in fireworms. Its applicability to other large benthic invertebrates can contribute to standardization of data collection and a better understanding of microplastic ingestion and ecological implications for Mediterranean benthic communities. • Improved green, time-cost effective MP extraction method for marine polychaetes. • Enzymatic-oxidative treatments, weak acid digestion and oil separation are combined. • High recovery rates while preserving MP integrity were achieved. • Applicable to lipid-rich marine invertebrates with chitinous-calcareous bristles.