Protocol optimization for microplastic analysis in sea turtles: Including the first blood digestion method

Sea turtles are particularly vulnerable to bioaccumulation of microplastics due to their long life cycles, trophic diversity, and migratory behavior. However, standardized methodologies for processing complex biological samples remain limited. This study introduces a standardized, reproducible two-step digestion protocol for the extraction of microplastics from sea turtle tissues, including blood, yolk sac, liver, kidney, muscle, and gonadal tissues. Samples were obtained from two distinct biological contexts: (i) blood from reproductive females and yolk sacs from their respective eggs to evaluate potential maternal transfer, and (ii) liver, kidney, muscle, and gonadal tissues from stranded individuals along the Brazilian Northeast coast to assess environmental bioaccumulation. The digestion efficiency was initially tested in reproductive matrices, resulting in an optimized two-step protocol comprising 10% KOH digestion followed by 30% H₂O₂ oxidation, which was subsequently applied to tissues from stranded turtles. All post-digestion membranes exhibited minimal organic residues, allowing reliable visualization of microplastic particles. The optimized method achieved recovery rates of 83.5% for blood, 73.1% for yolk sac, 75.4% for liver, 70.9% for kidney, and 82.0% for muscle. In addition, a preliminary, illustrative physical characterization of microplastics recovered from real samples was conducted to demonstrate the applicability of the protocol. To our knowledge, this study represents the first standardized chemical digestion workflow systematically applied across multiple sea turtle tissues, including a validated protocol for blood. Overall, this methodological framework establishes a baseline for future studies investigating microplastic contamination and potential maternal transfer pathways in marine reptiles and other vertebrates.