HNO3-PCF: A new lossless light microscopy method for forensic diatom drowning analysis

Five simple methods for diatom drowning analysis, including three newly developed ones, were compared for diatom yield and species composition consistency. Saponification of fats using sodium hydroxide solution significantly improved the yield of the extraction step, as well as the use of filtration instead of centrifugation. The combination of nitric acid digestion, vacuum filtration, and polycarbonate filter melting led to the development of the lossless light microscopy diatom forensic method (HNO3-PCF), with no detectable alteration in diatom community composition. When applied to 4-week submerged pig cadavers and six confirmed drowned human cases, the method exhibited unprecedented sensitivity. Among eight tissue types, lung diatom concentration was found to be the only reliable indicator for distinguishing between non-drowned and drowned cases. To minimize false positives from systematic contamination of the thoracic cavity, subtraction of heart diatom concentration from lung values was recommended. Provisional diatom concentration thresholds for drowning determination were set. The method's low equipment requirements (simple vacuum filtration, light microscope, hot plate) and high efficiency make it a promising tool for widespread use in forensic practice.