Few studies have examined both chlorine and monochloramine disinfection of the same viruses under the same experimental conditions. No studies have examined representatives of all types of CCL2 viruses for disinfection efficacy of chlorine or monochloramine. Because chlorine and monochloramine are the most widely used disinfectants in U.S. drinking water systems, an understanding of the efficacy of each of these disinfectants for a range of viruses is important (2
). The CCL2 and the 2009 CCL3 indicate that adenoviruses, coxsackieviruses, echoviruses, and caliciviruses are potentially important microbiological contaminants for public drinking water systems. Although disinfection data are needed for each of the virus groups, it is also important to understand the range of disinfection resistance for different viral strains within each virus group.
Previous research on chlorine inactivation of multiple enteric viruses in river water identified CVB5 as more resistant than CVB3, E1, and E11 (25
). In the present study, CVB5 also required more time for 2-, 3-, and 4-log10
inactivation than CVB3 and the two echovirus strains. More recently, other investigators have reported inactivation of CVB5 in demand-free water. At pH 7.5 and 5°C, investigators reported CT values of 5.4, 8.4, and 11.5 for 2-, 3-, and 4-log10
inactivations, respectively (7
), which is somewhat consistent with results in the present study for pH 7 and 8 (see Table ). If the CT values are estimated based on reported data, other studies using CVB5 have reported somewhat higher values (~2-fold) for 4-log10
) or ~2-fold lower for 2-log10
) than those found in the present study. In one study, the inactivation rates of CVB3 and CVB5 were the same, and the investigators suggested that this was a result of CVB5 aggregation (18
). No aggregation was found in the virus preparations used in the current study, and other investigators have also found different inactivation rates for CVB3 and CVB5 in river water (25
) (estimated CT for 2 log10
= 8.1 and 19.8, respectively, by another author [15
]). The collective data in the literature indicates that the rate of inactivation of CVB5 with free chlorine is consistently lower than for other enteric viruses, including hepatitis A virus (HAV) (32
). More research is needed to understand mechanisms contributing to the lower rate of inactivation of CVB5 in comparison with other viruses, including other enteroviruses that have been tested.
Investigators have reported similar CT values for chlorine inactivation of HAdV5 and HAdV41 (5
). The results obtained in the current study for HAdV2 are similar to those reported for these two viruses. However, in the current study, HAdV40 and HAdV41 were inactivated more rapidly than in the study by Baxter et al. (5
) or in the study by Thurston-Enriquez et al. (33
). A possible explanation for the different findings of Baxter et al. is the use of borate buffer, in contrast to the phosphate buffer used in the current study. The potential for varying results due to differences in experimental buffers and ionic concentrations has been suggested by others (6
). In addition, both of the previous studies with HAdV's used different types of infectivity assays, either end point analysis of viral antigen production or cytopathic effect endpoint analysis (5
). These approaches could also have produced differences in reported CT values. In the present study, it was not possible to statistically compare the level of inactivation of HAdV2 with HAdV40 and HAdV41, although the CT values for 3-log10
inactivation indicate that HAdV2 required more time for inactivation than HAdV40 and HAdV41.
The second-order inactivation kinetics observed for HAdV2 (Fig. ) could have been due to several factors, including multiple virus populations exhibiting differing resistance to disinfection (15
). Another possibility is that more than one mechanism of inactivation may be in effect for the inactivation of this DNA virus, which has a more complex capsid than the picornaviruses and the norovirus investigated in the present study. The mechanism of inactivation of viruses by chlorine is not known. Conformational changes in capsid structure during inactivation of E1 under certain conditions have been observed and are suggested to play a role in inactivation (44
). More recent studies have suggested that the mechanism of chlorine inactivation of another picornavirus, HAV, was associated with RNA degradation (24
). In the present study, the relatively straight lines for inactivation of the four enteroviruses indicated first-order kinetics, which suggests a single mechanism for inactivation.
In the present study the kinetics of chlorine inactivation of MNV could not be evaluated due to the rapid inactivation of this virus. This is in contrast to previous research reporting the failure of a 3.75-mg/liter concentration of chorine to inactivate Norwalk virus in drinking water (22
). Inactivation was measured by infectivity in human volunteers with an inoculum of a Norwalk virus suspension in broth. The method of evaluation and preparation of the virus could have contributed to the need for a larger dose of chlorine for inactivation. In the present study, use of a partially purified MNV preparation in buffered water could have contributed to the rapid inactivation. Although investigators have used feline calicivirus as a surrogate for studies of human norovirus (11
), more recently investigators have suggested that MNV may be a more relevant surrogate for studying the survival of human noroviruses (3
). Recently, investigators have found that MNV was inactivated more rapidly than poliovirus 1 in treated water from a water treatment plant (23
). Future studies of MNV under different conditions and in drinking water may identify different requirements for chlorine inaction than were found in the present study.
Fewer published data are available for comparing the disinfection efficacy of monochloramine for viruses. The enteroviruses examined in the present study each exhibited markedly different responses to monochloramine disinfection. E11, CVB3, and CVB5 exhibited first-order inactivation curves (Fig. ), while the inactivation curves of E1 exhibited a second-order tailing effect (Fig. ). Inactivation of CVB5 and E1 was less effective at pH 8, but inactivation of E11 and CVB3 was similar at pH 7 and 8. The most notable difference between the enteroviruses was the 100-fold difference between CT values for E1 and E11. The differences in the responses of the enteroviruses to monochloramine are not readily explained, but they could suggest that the mechanism of monochloramine disinfection is not the same for all enteroviruses. There has been no prior research to compare monochloramine disinfection on multiple enteroviruses under the same experimental conditions. However, previous research found that enteroviruses responded differently to free chlorine disinfection, both in the time required for disinfection and in the shape of the inactivation curves (13
Comparison of monochloramine data from the present study with previous findings is limited due to differences in experimental conditions, such as buffering capacity of the water, temperature, and pH. However, a few studies have investigated monochloramine disinfection of CVB5 under conditions similar to those used here. Using 10 mg of monochloramine/liter at pH 8, Sobsey et al. reported a 4-log10
inactivation by 104 min, which, if translated into a CT value (1,040) is similar to the 4-log10
CT value reported here (1,100) (32
). In addition, the inactivation kinetics and pH trend (monochloramine less effective at pH 8) reported by Kelly et al. for CVB5 are similar to the results of the present study (21
Like the enteroviruses, the adenoviruses were notably different in their responses to monochloramine disinfection. HAdV40 and HAdV41 produced similar inactivation curves that exhibited tailing, but the inactivation curves for HAdV2 exhibited first-order kinetics. In addition, the magnitude of the differences between CT values for HAdV2 and those for HAdV40 and HAdV41 were substantial. However, one similarity for the three adenoviruses was a decreased effectiveness of monochloramine at pH 8 versus pH 7.
The differences in CT values and monochloramine inactivation kinetics between HAdV2 and both HAdV40 and HAdV41 might be explained by the fact that HAdV2 is a species C adenovirus and HAdV40 and HAdV41 are species F adenoviruses. However, Baxter et al. examined monochloramine disinfection of HAdV41 and HAdV5 (a species C HAdV) and found similar CT values for both viruses (5
). They also reported a CT value for 2.5-log10
inactivation of HAdV41 (300) that was similar to the 2.5-log10
CT value from the present study (320; data not shown). Inactivation kinetics and pH data for HAdV2 from the present study were also consistent with previous research (31
For each of the study viruses, the CT values were consistently higher using monochloramine than free chlorine, although the magnitude of this difference varied by virus type. Monochloramine disinfection yielded a 2-log10 CT value of 8 for E1 at pH 7, whereas chlorine disinfection yielded a 2-log10 CT of 0.96. The greatest difference between chlorine and monochloramine efficacy reported in the present study was for HAdV2, for which monochloramine was over 37,000 times less effective than chlorine in achieving a 2-log10 reduction at pH 7. In addition, the relative inactivation rates of the study viruses to disinfection were dramatically different for chlorine and monochloramine. Whereas HAdV2 was one of the least resistant viruses to chlorine (2-log10 CT = 0.02, pH 7), it was one of the most resistant viruses to monochloramine. The 2-log10 free chlorine CT values for E1 and E11 were similar (E1 = 0.96 and E11 = 0.82, pH 7), but the monochloramine CT values for these viruses were different by >100-fold.
Both free chlorine and monochloramine were highly effective for inactivation of MNV. Although there are no reports demonstrating that human noroviruses are less susceptible to chlorine or monochloramine inactivation than reported in the present study for MNV, human noroviruses have been identified as the etiologic agents in numerous waterborne disease outbreaks, including outbreaks in which free chlorine residuals were reported (12
The susceptibility of the study viruses to monochloramine varied greatly, both between and within virus types. Monochloramine was least effective for inactivating HAdV2 (at pH 8) and E11 (at pH 7), whereas monochloramine disinfection was most effective for E1 (at both pH values). The HAdV2 results from the present study indicate that a CT value of 2,300 may be needed to achieve a 4-log10
inactivation of HAdV2 with monochloramine at 5°C and pH 8, which is above the CT value of 1,988 recommended in the USEPA Guidance Manual for Compliance with the Filtration and Disinfection Requirements for Public Water Systems Using Surface Water Sources
to achieve a 4-log10
inactivation with chloramines at pH 8 (37
). Within virus types, differences in monochloramine 2-log10
CT values were 2- to 3-fold between CVB5 and CVB3, 5- to 10-fold between HAdV2 and HAdV41, and 110- to 130-fold between E11 and E1. These data indicate that monochloramine inactivation modeling and system design should incorporate monochloramine efficacy data for multiple viruses of concern.
Because of the increasing level of treated wastewater entering natural water sources and use of reclaimed water, a complete understanding of the disinfection of different types of viruses in drinking water sources is needed. These data are also needed as input for the USEPA's ongoing CCL process to evaluate pathogens for potential rulemaking. The comparative effectiveness of free chlorine and monochloramine for disinfection of eight different CCL viruses in water has been demonstrated in the present study. Similar comparative studies at different temperatures and pH values in typical sources of drinking water are needed to better understand the level of disinfectant needed for these types of water.