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There are many issues in the evaluation protocols based on CT (mg min/L) values, which have been used to assess the germicidal effect of highly oxidative and unstable ozonated water. The major problems include the carryover of culture medium components in virus inactivation assays and the reaction volume ratio between the virus suspension and ozonated water. Furthermore, it is essential to correct the CT value with the decay curve of dissolved ozone under the same conditions as the inactivation assay. In this study, these concerns were reexamined to obtain quantitative CT values. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) inactivation test using ozonated water prepared from pure water was assessed by determining the corrected concentration time (CCT) values. Moreover, a possible inactivation mechanism of SARS-CoV-2 was discussed with the aid of findings from this study and previous reports. The findings revealed that the CCT value required for 99.95% inactivation of SARS-CoV-2 with ozonated water was 0.97 mg·min/L. To quantitatively evaluate the SARS-CoV-2 inactivation test, the virus purification procedure during the pretreatment and the CT value correction using a dissolved ozone decay curve obtained under the same condition as the inactivation test were demonstrated to be essential. We proposed a possible mechanism of SARS-CoV-2 inactivation with ozonated water. Amino acids such as tyrosine, tryptophan, methionine, cysteine, and histidine in the SARS-CoV-2 spike protein are susceptible to oxidative attack by the ozone dissolved in water. This attack may induce structural destruction of the spike protein and inhibit its binding to the angiotensin converting enzyme 2 (ACE2) receptor, an essential host receptor for viral infection, resulting in viral inactivation and contributing to infection suppression.

Quantitative evaluation of the inactivation effect of ozonated water on SARS-CoV-2 based on corrected CT values