Effects of routine laboratory parameter variations on diatom preservation: Implications for the standardization of forensic diatom testing.

Forensic diatom testing remains controversial in drowning diagnosis and site inference, highlighting the need for standardized protocols. However, despite being involved in nearly all diatom testing methods, routine laboratory parameters have often been overlooked, and their effects on diatom preservation remain poorly investigated. This study systematically evaluated the effects of digestion parameters and washing procedure on diatom recovery and assemblage preservation, including digestion temperature and duration, centrifugation force and duration, washing frequency, and the residual pellet volume retained after each centrifugation. Controlled spiking experiments were performed using pooled natural water samples and human lung tissue. Procedural performance was assessed based on the diatom recovery rate and multiple assemblage-based indicators, including Bray-Curtis dissimilarity, Jaccard similarity, Spearman's rank correlation, and replicate dispersion. Both diatom recovery and assemblage integrity were better maintained under the selected combination of heating at ≤ 80 °C for ≤ 60 min, centrifugation at 3,000 rpm (1,313×g) for 5 min, and one washing cycle with 500 µL retained after centrifugation. Based on these parameters, we established an optimized extraction workflow that increased diatom recovery rates from 16.45% to 41.00% for acid digestion and from 80.87% to 84.42% for enzymatic digestion, while maintaining assemblage composition comparable to that of the reference water samples. Our findings suggest that routine laboratory parameters should be incorporated into the standardization of forensic diatom testing, as their optimization can substantially improve diatom preservation.