Ozonized Water in Microbial Control: Analysis of the Stability, In Vitro Biocidal Potential, and Cytotoxicity

O₃ dissolved in water (or ozonized water) has been considered a potent antimicrobial agent, and this study aimed to test this through microbiological and in vitro assays. The stability of O₃ was accessed following modifications of the physicochemical parameters of water, such as the temperature and pH, with or without buffering. Three concentrations of O₃ (0.4, 0.6, and 0.8 ppm) dissolved in water were tested against different microorganisms, and an analysis of the cytotoxic effects was also conducted using the human ear fibroblast cell line (Hfib). Under the physicochemical conditions of 4 °C and pH 5, O₃ remained the most stable and concentrated compared to pH 7 and water at 25 °C. Exposure to ozonized water resulted in high mortality rates for Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, and Candida albicans. Scanning electron micrograph images indicate that the effects on osmotic stability due to cell wall lysis might be one of the killing mechanisms of ozonized water. The biocidal agent was biocompatible and presented no cytotoxic effect against Hfib cells. Therefore, due to its cytocompatibility and biocidal action, ozonized water can be considered a viable alternative for microbial control, being possible, for example, its use in disinfection processes.