Effect of Direct Pre-Ozonation on the Antimicrobial Activity, pH and Available Chlorine Content of Endodontic Irrigants: An In Vitro Study

This study examined whether direct pre-ozonation changes inhibition-zone diameters and selected chemical properties of endodontic irrigants. Sodium hypochlorite (NaOCl; 5.25%), chlorhexidine digluconate (CHX; 2%), ethylenediaminetetraacetic acid (EDTA; 17%), and saline underwent a fixed procedure (gas-phase ozone, 80 µg/mL; 30 L/h; 5 min; 200 mL). Disk diffusion against Candida albicans, Staphylococcus aureus, and Enterococcus faecalis was evaluated in eight independent paired runs. pH was measured, and available chlorine in NaOCl was determined in three independent paired batches. Mean inhibition-zone diameters were smaller after ozonation in all nine comparisons, and eight comparisons were statistically significant after Holm correction; the EDTA comparison against C. albicans was not statistically significant. CHX showed the largest pH change (9.05 to 6.34; −2.71 units). Available chlorine decreased from 5.10 ± 0.06% to 2.84 ± 0.04%, a paired reduction of 2.26 percentage points (95% CI, 2.02–2.50). Under this protocol, pre-ozonation did not increase inhibition-zone diameters. These findings cannot distinguish changes in intrinsic antimicrobial activity from altered agar diffusion and do not establish a clinical effect. Dissolved ozone was not measured, and chemical transformation products were not characterized.