Cryptosporidium is an extremely chlorine-resistant pathogen. To effectively inactivate Cryptosporidium, the U.S. Centers for Disease Control and Prevention recommends that aquatic venue operators achieve free chlorine concentrations of 20 mg/L for 12.75 hours. Inactivation times might need to be at least 8 times longer when the chlorine stabilizer cyanuric acid (as a stand-alone additive or in the form of “dichlor” or “trichlor”) is present in the water. These longer contact times are not feasible for most aquatics venues. Recent laboratory research indicates that chlorine dioxide (ClO2) is highly effective against C. parvum and that the presence of free chlorine might shorten inactivation times. However, it is unclear what effect CYA has on ClO2 inactivation rates. The aim of this study was to determine if ClO2 can serve as an effective alternative to hyperchlorination of aquatic venues that utilize stabilized chlorine. The technical objective was to determine the time required to achieve a 3- log10 inactivation of C. parvum oocysts at 5 mg/L ClO 2 ; 2 mg/L free chlorine; and 50, 100, or 150 mg/L CYA.
Laboratory studies were conducted under ideal conditions (oxidant-demand- free water [ODF] at 2 mg/L free chlorine, pH 7.5, 25 °C) with CYA added to achieve a target concentration of 50, 100, or 150 mg/L. A concentrated solution of ClO2 was prepared and added to triplicate experimental flasks to achieve a final ClO2 concentration of 5 mg/L. A fourth flask was used to measure ClO2 decay over experimental time periods. Control experiments included flasks containing: 1) 2 mg/L free chlorine and 5 mg/L ClO2 ; 2) 20 mg/L free chlorine; and 3) ODF water to measure natural C. parvum decay.
Flasks were continuously stirred and samples were removed at select time points for C. parvum infectivity testing. Samples were quenched using sodium thiosulfate, concentrated by centrifugation, inoculated onto MDCK mammalian cells and incubated for 48–60 hours at 37 °C under 5% CO2. A Cryptosporidium-specific monoclonal antibody was used to fluorescently label C. parvum living stages before microscopic counting. Images of fluorescing living stages were collected using a digital camera attached to a Zeiss Axiovert microscope at 100X magnification. Zeiss AxioVision and ImageJ software were used to quantify the number of living stages associated with sample and back-calculation provided an estimate of the log inactivation of oocysts over contact time.
A total of six individual experimental flasks were tested for each CYA concentration. At an average of 53 mg/L CYA and 2.4 mg/L free chlorine, dosing to 5 mg/L ClO2 resulted in a 3- log10 inactivation of oocysts in < 3 hours. At an average of 119 mg/L CYA and 2.3 mg/L free chlorine, a 3-log10 reduction was achieved in 3 hours. At 186 mg/L CYA and 2.1 mg/L free chlorine, a 2.4-log10 reduction was achieved within 5 hours. All control assays indicated that oocysts did not exhibit differences in inactivation or die-off rates that were substantially different than those reported in previously published research reports.
These data indicate that ClO2 might serve as an effective alternative to hyperchlorination in aquatic venues that utilize stabilized chlorine, even at CYA concentrations that exceed the maximum of 90 mg/L recommended by the U.S. Model Aquatic Health Code. Our previous work indicates that at CYA concentrations of 50–100 mg/L, utilizing hyperchlorination to achieve 3-log10 inactivation of C. parvum requires >102 hours at 20 mg/L free chlorine concentration. Alternatively, 3-log10 C. parvum inactivation in the presence of 50–100 mg/L CYA can be achieved within 3 hours at 5 mg/L ClO2. Use of ClO2 as an alternative to hyperchlorination, might allow aquatic venues to avoid or reduce the time for disruptive, potentially costly, closures. However, more research is needed before ClO2 -based C. parvum remediation procedures that incorporate occupational and swimmer health and safety considerations are implemented.