Inactivation of coxsackievirus B5 by free chlorine under conditions relevant to drinking water treatment

Microbial control of drinking water experienced a significant paradigm shift after several outbreaks caused by Cryptosporidium in the 1990s due to its high persistence to chlorine (Mac Kenzie et al. 1994). For example, the Interim Guideline for Cryptosporidium Treatment in the Water Supply was established in Japan in 1996 and reformed in 2007 (MHLW 2007). The guideline mandated the strict control of turbidity by optimizing coagulation and filtration conditions at drinking water treatment plants and required it not to exceed 0.1° (approximately 0.14 NTU) after the stage of filtration. Since the implementation of the guideline, no Cryptosporidiosis outbreaks via the water supply have been reported in Japan. However, the reduction efficiency of another important enteric pathogen, viruses, has been shown to be limited (Kato et al. 2018; Canh et al. 2019). As such, the disinfection process is essential to reduce enteric viruses to a sufficient level in drinking water.

Coxsackievirus B5 (CVB5) is a genotype of the Enterovirus genus that is included in drinking water contaminant draft candidate list five (CCL5) of the U.S. Environment Protection Agency (U.S. EPA). CVB5 was reported to be one of the most frequently detected viruses among the Enterovirus genus in clinical and wastewater surveillance (Cromeans et al. 2010; Kahler et al. 2011; Brinkman et al. 2017; Bisseux et al. 2020). Also, CVB5 was found to exhibit higher persistence to disinfection (e.g., free chlorine) compared to other waterborne viruses (Sobsey et al. 1988; Black et al. 2009; Cromeans et al. 2010; Kahler et al. 2011; Torii et al. 2022a). In fact, infectious CVB5 was detected in disinfected drinking water (Payment et al. 1985; Lee & Kim 2002). Therefore, CVB5 is often considered a representative pathogen for worst-case scenarios in drinking water and wastewater reclamation treatment.

Free chlorine is one of the most commonly used disinfectants for drinking water treatment. For example, more than 70% of water treatment plants in the United States use it as a disinfectant (AWWA 2017). In Japan, residual chlorine at the tap is mandatory, so free chlorine disinfection is applied to drinking water treatment. It is widely known that the virucidal efficacy of free chlorine differs depending on the pH and thus the recommended disinfectant concentration × contact time (CT) values in U.S. EPA Guidance Manual are provided depending on these two parameters (U.S. EPA 1991). The influence of temperature and pH on the kinetics of virus inactivation by free chlorine was also thoroughly investigated in previous studies. In addition, recent studies found that the source water quality except for pH and temperature (i.e., chloride concentration, turbidity and mater matrices) can also affect the efficiency of virus inactivation (Kahler et al. 2011; Wati et al. 2019; Szczuka et al. 2022). For example, Wati et al. (2019) reported that the disinfection efficiency of CVB5 inactivation is lower at a turbidity of 20 NTU compared with that at a turbidity of less than 5 NTU. However, such effects have merely been tested under conditions relevant to drinking water treatment such as a turbidity level of less than 0.1° (approximately <0.14 NTU) and a lower total organic carbon (TOC) level in the Japanese case.

To fill this gap, we investigated the CVB5 inactivation by free chlorine using a total of 61 pH-adjusted water samples collected at a stage between filtration and disinfection processes.

A laboratory strain of CVB5 (CVB5.Faulkner) was used in the current study. CVB5 was propagated by buffalo green monkey kidney (BGM) cells and purified by density gradient centrifugation, as described elsewhere (Torii et al. 2021a). The purified virus stock was stored at −20 °C until conducting experiments.

The infectious CVB5 was enumerated by the most probable number (MPN) assay using BGM cells on 96-well plates (Meister et al. 2018; Torii et al. 2022b). The samples were serially diluted 10-fold with five replicates per dilution. The number of positive wells was counted with microscopy and analyzed with an R package {MPN} (Ferguson & Ihrie 2019).

Virus stock solution (50 μL) was spiked into testing water (20 mL), which was gently stirred in glass flasks. The testing water was maintained at temperatures of 19.6 ± 1.4 °C and pH 7.0 buffered by 10 mM phosphate buffer (1 mL) (Wako, Japan). Then, the chlorine stock solution was added to testing water to obtain an initial concentration of 0.6 mg/L. A 0.5 mL aliquot was collected every 25 s (0, 25, 50 and 75 s) and mixed with sodium thiosulfate (5 μL, 5,000 mg/L) to quench the residual free chlorine. The collected sample was used for virus enumeration. All experiments were performed in duplicate. Glassware was soaked with 50 mg/L of sodium hypochlorite overnight and rinsed with Milli-Q water to quench chlorine demand before the experiments. Free chlorine concentration was measured using the N,N-diethyl-p-phenylenediamine colorimetric method in a DR890 colorimeter (HACH, USA).

Inactivation rate constants (k) (mg⁻¹·min⁻¹·L) were estimated by fitting a Chick-Watson model (N_T/N₀ = e^−kCT) (Gyürék & Finch 1998), where N₀ and N_T are the virus concentration (MPN/mL) at time 0 and T of the disinfection experiment, and the CT value (mg·min/L) is the free chlorine concentration (C, mg/L) multiplied by the contact time (T, min).

Microsoft Excel 2023 was used for all statistical analyses. The one-way analysis of variance (ANOVA) was employed to evaluate the difference in the inactivation rate constants among the drinking water samples collected at plants A, B, C and D. Pearson's correlation coefficient (R) was used to evaluate the correlation between water quality parameters (TOC, turbidity and EC) and the efficiency of chlorine disinfection. Statistical significance was determined with a threshold of p-values less than 0.05.

TOC was determined using the TOC-V CSH instrument (Shimadzu Corporation, Tokyo, Japan). Turbidity was measured using the integrating-sphere photoelectric photometry method with the PT-200 device (Mitsubishi Chemical Corporation, Tokyo, Japan) for samples collected from plants A and D. For samples collected from plants B and C, turbidity was measured using the particle counting method with the MILPAzero3t-P instrument (Mikunikikai, Tokyo, Japan). Electrical conductivity (EC) was measured by conductivity meters: the MM-60R model (TOA DKK, Tokyo, Japan) for plant A, the CM-30G model (TOA DKK, Tokyo, Japan) for plant B, the DS-71 T model (HORIBA, Kyoto, Japan) for plant C and the CM-41X model (TOA DKK, Tokyo, Japan) for plant D.

It was found that there was no clear correlation between the levels of TOC, turbidity and EC in tested drinking water and the efficiency of chlorine disinfection (Pearson's correlation coefficient, R_TOC = 0.05, R_Turbidity = 0.13, R_EC = 0.19). In addition, no significant difference was observed in the inactivation rate constants for CVB5 among the tested drinking water collected from plant A (K_A = 8.2 ± 3.0), plant B (K_B = 9.4 ± 2.2), plant C (K_C = 7.1 ± 1.9) and plant D (K_D = 8.1 ± 2.8) (ANOVA, p > 0.05). These results indicate that the efficiency of chlorination was not influenced by the tested drinking water samples collected from plants A, B, C and D. The CT values for 4-log₁₀ inactivation in the current study ranged from 1.01 to 1.27 mg·min/L (Table 1), which was consistent with previous studies, reporting 1.02 mg·min/L (Torii et al. 2021b) and 1.15–1.19 mg·min/L (Shirasaki et al. 2020). These previous studies were performed at a similar condition (CVB5 Faulkner strain, pH = 7, water temperature = 20 °C) as the current study, but PBS/PB buffer was used instead of actual drinking water. Thus, it was likely that the efficiency of chlorination for CVB5 was similar between the tested drinking water and PBS/PB buffer. This might also imply that the quality of tested drinking water in the current study did not interfere with the efficiency of chlorination.

Besides, when compared to other viruses tested in previous studies, such as murine norovirus (MNV) and adenovirus type 40 (AdV-40) at a similar temperature (20–25 °C) and pH (about 7) conditions to the current study, the CT value for 4-log10 inactivation of CV.B5 (1.01–1.27 mg.min/L) was obviously higher than that of MNV (0.25 mg.min/L) and AdV-40 (< 0.04 mg.min/L) (Kitajima et al. 2010; Shirasaki et al. 2020). A similar result was also observed when comparing the CT value for 4-log₁₀ inactivation of CVB5 (7.4 mg·min/L) with AdV-2 (0.15 mg·min/L), AdV-40 (<0.04 mg·min/L), echovirus 1 (1.3 mg·min/L) and echovirus 11 (0.97 mg·min/L) in water at pH 7 and 5 °C (Cromeans et al. 2010). These results suggest that CVB5 exhibited greater resistance to free chlorine compared to the other tested enteric viruses. The higher resistance of CVB5 can be attributed to its capsid protein, which contains a low number of sulfur-containing amino acids, making it less reactive to oxidants (Torii et al. 2021b).

It has been theorized that water quality characteristics (e.g., total organic carbon, alkalinity, hardness, ionic strength and turbidity) might affect the efficiency of chlorination. However, the results of this current study appear that these parameters of drinking water, particularly in Japanese drinking water with controlled turbidity levels (<0.1°), may not be a key factor influencing the efficiency of disinfection by free chlorine. In fact, several previous studies reported that virus inactivation rates for AdV2, CVB5, E1 and MNV were higher in drinking water with a higher ionic strength level (Kahler et al. 2010). Additionally, calcium hardness as CaCO₃ (0–150 mg/L) was not found to affect AdV inactivation by free chlorine and no effects on chlorination efficiency for AdV2 were reported as alkalinity increased from 150, 200 to 300 mg/L as CaCO₃ (Page et al. 2009). In a previous study, no association between increased TOC and decreased inactivation rates was observed when comparing drinking waters with different TOC concentrations (1.9, 2.2 and 18 mg/L) (Kahler et al. 2010).

Notably, the temperature and pH of tested drinking water were controlled in this current study, and so, the effects of these factors on the efficiency of chlorination were unknown. Generally, the rate of virus inactivation was decreased with a decreasing temperature or increasing pH. In fact, a previous study reported that the CT value required for 4-log₁₀ reduction of CVB5 in buffered reagent-grade water at 5 °C was 7.4 mg·min/L at pH 7, but this value was 10 mg·min/L at pH 8 (Cromeans et al. 2010). When investigating the free chlorine inactivation of CBV5 in drinking water at 15 °C, the CT value required for 3-log₁₀ virus reduction was also found to increase from 1.0 to 1.6 mg·min/L as pH increased from 7 to 8. These findings suggest that water temperature and pH may more strongly affect the chlorination inactivation rate of viruses in drinking water.

Japanese turbidity control may play an indirect role in reducing the uncertainty of free chlorine disinfection, thus contributing to reduce the risk of waterborne viruses. U.S. EPA's Guidance Manual for disinfection requirements for public systems recommends a CT value of 3 to achieve 4 virus log₁₀ inactivation in water at 20 °C, pH 6–9 (U.S. EPA 1991). In the current study, the CT value required for 4-log₁₀ reduction (1.01–1.27 mg·min/L) was found to be lower than the recommended value. Therefore, chlorination treatment at the Japanese drinking water treatment plant is considered efficient and achieves a safe level. However, as the current study controlled water temperature and pH, it was not possible to investigate the efficiency of chlorine disinfection under the worst-case scenario, which was characterized by low temperature and high pH. Therefore, further studies should be conducted to investigate the kinetics of virus inactivation in actual drinking water, considering the variations in pH and temperature.

The results of this current study provided evidence that water quality might not play a significant role in affecting the inactivation rates of viruses by chlorination in drinking water, while water temperature and pH were more likely to influence the efficiency of chlorination.

This work was supported by JSPS KAKENHI Grant 20H00259, by the Ministry of Health, Labour, and Welfare, Japan through a Health and Labour Sciences Research Grant (22LA1007) and by the Bureau of Waterworks, Tokyo Metropolitan Government.

V.D.C. conceptualized the work, performed methodology and formal analysis, investigated the work, wrote the original draft, and visualized the work. S.T. investigated the work, did methodology, and wrote, reviewed and edited the manuscript. T.S. investigated the work and wrote, reviewed and edited the manuscript. H.K. conceptualized the work, analyzed the resources, wrote, reviewed and edited the manuscript, supervised the work, and did funding acquisition.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.