APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2010, p. 1028–1033
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 76, No. 4
Inactivation of Adenoviruses, Enteroviruses, and Murine Norovirus in
Water by Free Chlorine and Monochloramine?
Theresa L. Cromeans,1,2Amy M. Kahler,1,2and Vincent R. Hill1*
Atlanta Research and Education Foundation1and Centers for Disease Control and Prevention, National Center for Zoonotic,
Vector-Borne, and Enteric Diseases, Division of Parasitic Diseases,2Atlanta, Georgia 30341
Received 9 June 2009/Accepted 7 December 2009
Inactivation of infectious viruses during drinking water treatment is usually achieved with free chlorine. Many
drinking water utilities in the United States now use monochloramine as a secondary disinfectant to minimize
disinfectant by-product formation and biofilm growth. The inactivation of human adenoviruses 2, 40, and 41
(HAdV2, HAdV40, and HAdV41), coxsackieviruses B3 and B5 (CVB3 and CVB5), echoviruses 1 and 11 (E1 and
E11), and murine norovirus (MNV) are compared in this study. Experiments were performed with 0.2 mg of free
chlorine or 1 mg of monochloramine/liter at pH 7 and 8 in buffered reagent-grade water at 5°C. CT values
the efficiency factor Hom model. The enteroviruses required the longest times for chlorine inactivation and MNV
the least time. CVB5 required the longest exposure time, with CT values of 7.4 and 10 mg ? min/liter (pH 7 and 8)
for 4-log10inactivation. Monochloramine disinfection was most effective for E1 (CT values ranged from 8 to 18
mg ? min/liter for 2- and 3-log10reductions, respectively). E11 and HAdV2 were the least susceptible to monochlo-
ramine disinfection (CT values of 1,300 and 1,600 mg-min/liter for 3-log10reductions, respectively). Monochlora-
mine inactivation was most successful for the adenoviruses, CVB5, and E1 at pH 7. A greater variation in
inactivation rates between viruses was observed during monochloramine disinfection than during chlorine disin-
fection. These data will be useful in drinking water risk assessment studies and disinfection system planning.
Disinfection is a critical step in the drinking water treatment
process to inactivate infectious viruses because primary treat-
ment is less effective for the removal of viruses. Chlorine and
monochloramine are the most widely used disinfectants in the
United States (2). Free chlorine is widely used as a primary
disinfectant following filtration and also as a secondary disin-
fectant in distribution systems. Under the Long Term 2 En-
hanced Surface Water Treatment Rule (38), monochloramine
can also be used as a primary disinfectant, but because it
requires longer contact times to achieve the same level of
disinfection as free chlorine it is primarily used as a secondary
disinfectant to maintain a stable disinfectant residual in the
distribution system and minimize disinfection by-product for-
mation and biofilm growth.
The efficacy of chlorine disinfection for viruses has been
evaluated in numerous studies over the years. Many early stud-
ies focused on the disinfection of polioviruses by chlorine (14,
17, 26, 28, 30, 39, 40, 43). Early investigators suggested a
number of variables that must be controlled in the disinfection
of viruses: contact time, temperature, ionic strength, pH, chlo-
rine concentration, and virus aggregation (29, 30). These re-
searchers concluded that comparisons and general trends of
disinfection efficacy can only be discerned for viruses when the
same disinfection parameters are applied.
Fewer studies have investigated the disinfection efficacy of
monochloramine, but monochloramine disinfection has been
found to be less effective than free chlorine for viruses. In
comparative studies of chlorine and monochloramine disinfec-
tion, coxsackievirus B5, adenovirus 2, and adenovirus 41 were
found to be inactivated far more readily by chlorine than
monochloramine (4, 5, 32). For drinking water treatment sys-
tems where monochloramine is used as a secondary disinfec-
tant, it is important to know its efficacy for a wide range of
viruses, as infectious viruses may be introduced in the distri-
bution system where only monochloramine is present. In ad-
dition, relatively few studies have investigated the efficacy of
monochloramine as systematically as free chlorine; frequently
only one concentration, pH, or temperature has been investi-
gated. Two notable exceptions were investigations that exam-
ined monochloramine disinfection of human adenovirus 2
(HAdV2) and coxsackievirus B5 (CVB5) at multiple pH levels
In 2005, the U.S. Environmental Protection Agency
(USEPA) published its second candidate contaminant list
(CCL2). The CCL2 is comprised of unregulated microbial and
chemical contaminants of potential public health concern that
are known or anticipated to occur in drinking water systems
and includes: echovirus, coxsackievirus, adenovirus, and calici-
virus (36). A number of researchers have reported the disin-
fection efficacy of free chlorine for representatives of the CCL2
viruses (4, 5, 7, 11, 13, 18, 20, 22, 27, 33, 34, 35), but fewer
studies have investigated the disinfection efficacy of monochlo-
ramine on these viruses (4, 5, 21, 31). In addition, comparison
between existing studies of chlorine or monochloramine disin-
fection is difficult because of differences in the viruses exam-
ined, experimental parameters investigated, and analytical
The present study compared the inactivation kinetics for
* Corresponding author. Mailing address: Centers for Disease Con-
trol and Prevention, National Center for Zoonotic, Vector-Borne, and
Enteric Diseases, Division of Parasitic Diseases, 4770 Buford Highway,
Mail Stop F-36, Atlanta, GA 30341-3724. Phone: (770) 488-4432. Fax:
(770) 488-4253. E-mail: email@example.com.
?Published ahead of print on 18 December 2009.
representative CCL2 viruses with levels of free chlorine and
monochloramine recommended for drinking water disinfec-
tion. Duplicate experiments with both disinfectants were car-
ried out in pH 7 and 8 buffered chlorine-demand-free (CDF)
water at 5°C, with eight viruses chosen to represent the CCL2
virus types. Coxsackieviruses B5 and B3 (CVB5 and CVB3)
and echoviruses 1 and 11 (E1 and E11) were chosen based on
existing data suggesting resistance to free chlorine, disease
implications, and likelihood of presence in higher numbers in
natural water. Three representative human adenoviruses were
studied, including both serotypes of species F HAdV (40 and
41) that cause gastroenteritis and HAdV2, a representative of
respiratory HAdV that may be found in water because they are
present in fecal excretions (9). Murine norovirus (MNV), phy-
logenetically similar to human norovirus and the only norovi-
rus that can be propagated in cell culture, was used as a
surrogate for human norovirus. Kinetic inactivation curves are
presented, and CT values (disinfectant concentration ? time,
reported in mg ? min/liter) were calculated by using the effi-
ciency factor Hom (EFH) model (16).
MATERIALS AND METHODS
Virus propagation and infectivity assays. CVB5 (Faulkner strain), CVB3
(Nancy strain), E1 (Farouk strain), and E11 (Gregory strain) were obtained from
the American Type Culture Collection (ATCC; Manassas, VA) and propagated
in Buffalo green monkey (BGM) cells (Scientific Resources Program, Centers for
Disease Control and Prevention (CDC; Atlanta, GA). Clones of CVB5 and E1
selected from larger plaques were propagated and used in experiments, in order
to shorten assays to 2 days. HAdV2 (strain 6), HAdV40 (Dugan strain), and
HAdV41 (a clinical isolate) were obtained from the CDC (Respiratory Virology
Diagnostics, GRVLB, DVD, CDC, Atlanta, GA) and propagated in A549 (hu-
man epithelial lung carcinoma) cells obtained from the CDC (Scientific Re-
sources Program, CDC, Atlanta, GA). HAdV40 and HAdV41 were selected and
propagated as described previously (10). MNV was obtained from H. W. Virgin
and C. E. Wobus (19) and propagated in RAW 264.7 (murine monocyte/mac-
rophage) cells obtained from the ATCC.
All virus titers were determined by plaque assay. Appropriate cell monolayers
were infected nearly or completely confluent at 2 days after seeding or at 1 day
for RAW 264.7 cells. Tenfold dilutions of virus or experimental samples (0.25- or
0.7-ml MNV samples) were added to each 60-mm2cell monolayer; two or more
dilutions of each sample were assayed in duplicate. After 1 h adsorption at 37°C,
the infected cells were overlaid with 5 ml of the cell-appropriate medium con-
taining 0.5% SeaKem ME agarose. Plaque assays for adenoviruses and entero-
viruses also contained an additional 30 mM MgCl2. After a 2-day incubation of
MNV and enterovirus assays, a second agarose overlay containing 2% neutral
red was added to visualize plaques within 4 to 6 h. HAdV2 was incubated 5 days;
HAdV40 and HAdV41 were incubated 12 and 9 days, respectively, as previously
described (10). Adenovirus plaque assays were stained by the addition of a 1/10
volume of 0.5% thiazolyl blue tetrazolium bromine (Sigma-Aldrich) in phos-
phate-buffered saline (PBS) for 2 h.
CAV preparations. Cell monolayers were infected at a multiplicity of infection
of ca. 0.5 to 1.0 and cultured in serum free medium. At predetermined times
postinfection for maximum virus titer (based on kinetic studies of each virus), the
supernatant was removed, and 10 ml of CDF Dulbecco’s PBS (DPBS) was added
to the flask. These cell-associated virus (CAV) preparations were frozen at
?70°C and stored for days or up to 2 months before use. After thawing, the CAV
preparation was incubated overnight at 4°C with 8% polyethylene glycol and 0.3
M NaCl. After centrifugation at 10,000 ? g, the pellet was suspended in CDF
DPBS and extracted by vigorous shaking with an equal volume of chloroform for
2 min. After centrifugation at 10,000 ? g, the upper aqueous layer (purified CAV
[pCAV]) was taken for use in disinfection experiments on the same day. Electron
microscopy indicated that all pCAV preparations did not contain virus aggre-
gates, and aggregates were not formed upon dilution in CDF water. Only single
particles were observed, with rare to occasional doublets.
Reagents and glassware. CDF DPBS and CDF reagent-grade water were
prepared according to standard method 4500-Cl C (1). CDF water was buffered
to 0.01 M and brought to pH 7 and 8 by dissolving 0.83 g of Na2HPO4(anhy-
drous) and 0.58 g of NaH2PO4(monohydrate) per liter and 1.3 g of Na2HPO4
and 0.1 g of NaH2PO4per liter, respectively. A free chlorine stock solution was
prepared by diluting 5.65 to 6% sodium hypochlorite (Fisher Scientific, Fair
Lawn, NJ) in CDF water. Prior to each experiment, this stock was added to
the experimental waters to achieve the desired free chlorine concentration. The
starting concentration of the water was adjusted so that the addition of the
inoculum did not cause a ?0.05 mg/liter decrease in the free chlorine concen-
tration in order to achieve a final starting value of 0.2 mg/liter. Free and total
chlorine were measured by the DPD method using colorimetric methodology
with a DR/850 colorimeter (Hach Company, Loveland, CO). A monochloramine
stock solution was made by mixing equal volumes of 200 mg of free chlorine/liter
and 800 mg of ammonium chloride/liter in pH 8 CDF water and stored at 4°C for
2 weeks. Before each experiment, a 1-mg/liter monochloramine solution was
prepared by diluting the stock solution in pH 7 or 8 CDF water. Monochloramine
was measured by using a Hach DR/850 colorimeter. CDF glassware was pre-
pared by soaking in ?5 mg of free chlorine/liter overnight. The glassware was
rinsed five times with CDF water, covered with clean foil, and baked at 200°C for
2 h. All glassware and water were prechilled at 5°C before each experiment.
Experimental protocol. All experiments were conducted at 5 ? 0.2°C in a
recirculating water bath inside a biological safety cabinet. A multiplace stir plate
placed under the water bath allowed for continual mixing during an experiment.
For monochloramine experiments lasting longer than several hours, samples
were moved to a refrigerator maintained at 5 ? 0.5°C. For each pH, four 50-ml
Erlenmeyer flasks were used, each containing 40 ml of CDF water with 1 mg of
monochloramine/liter or, for free chlorine experiments, 20 to 40 ml of CDF
water with ?0.2 mg of free chlorine/liter. Two flasks were used for the experi-
mental replicates; one flask was used to monitor free and total chlorine residual
or monochloramine residual, and one flask was used to monitor virus titer at
selected points during the experiment. At time zero, 0.2 to 1 ml of a pCAV stock
was inoculated into each flask. At selected time points, a 5-ml sample was
removed and added to a tube containing sodium thiosulfate to achieve a final
concentration of 50 mg/liter.
Free and total chlorine residuals were measured immediately before an ex-
periment, immediately after virus inoculation, at the midpoint when possible,
and at the end of an experiment. Monochloramine residual was measured im-
mediately before an experiment, immediately after virus inoculation, at the
midpoint, and at the end of an experiment, at a minimum. These values were
incorporated into the kinetic modeling and CT calculations. Prior to virus inoc-
ulation into the virus control flask, 50 mg of sodium thiosulfate/liter was added
to quench the free chlorine or monochloramine residual. This flask was sampled
immediately after virus inoculation and at the end of the experiment to ensure
that virus infectivity was stable without disinfectant present. Only HAdV40 and
HAdV41 were found to lose infectivity during the time frame of the monochlo-
ramine experiments; therefore, control samples were taken at every sampling
point. After sampling at the indicated time points, 10? PBS containing 10% fetal
bovine serum was added to each sample in order to have isotonic samples for
assay. Samples were assayed on the same day as the experiment when possible or,
in the case of lengthy monochloramine experiments, held at 4°C until the final
samples were taken.
Kinetic modeling and CT calculations. Viral inactivation was determined by
calculating the survival ratio (N/N0; infectious viruses at time t divided by infectious
viruses at time zero) for each experimental sample. The EFH model was used to
calculate predicted survival ratios based on experimental conditions, including dis-
infectant decay over time using a first-order kinetic equation (16). Samples were
included in the EFH modeling and CT calculations only if the plaque assay counts
averaged ?10 PFU/plate. Inactivation curves were created by using Microsoft Excel
to compare observed versus predicted inactivation values. CT values were calculated
for 2-, 3-, and 4-log10inactivation for each virus and condition through application
of the EFH model. Linear regression using a quadratic response function was used
to compare viral inactivation between different viruses and pH levels using SAS
version 9.0. Statistical significance was set at ? ? 0.05.
A summary of CT values (mg ? min/liter) obtained for free
chlorine and monochloramine disinfection of all study viruses
at pH 7 and 8 are shown in Tables 1 and 2, respectively. Each
CT value is an average of replicate experiments with the vari-
ation between replicates less than 25%. CT values were ob-
tained directly from experimental data by using the EFH
model. Calculation of extrapolated 4-log10CT values using the
EFH model was possible for many of the viruses that did not
VOL. 76, 2010CHLORINE AND MONOCHLORAMINE DISINFECTION OF VIRUSES1029
achieve 4-log10inactivation experimentally. However, extrap-
olated CT values for monochloramine disinfection of HAdV40,
HAdV41, and E1 could not be calculated due to limitations in
the EFH model at predicting inactivation reaching asymptotic
levels (see Fig. 3 and 4, note the scales).
HAdV40, HAdV41, and MNV were the most readily inac-
tivated study viruses by free chlorine, with at least 3-log10
inactivation within 5 s. The rapid rate of inactivation did not
allow for statistical comparisons to be performed. CVB5 re-
quired the longest time for inactivation, significantly more than
CVB3 (P ? 0.0001) at pH 7 and at pH 8. CVB5, E1, and
HAdV2 were each inactivated more rapidly at pH 7 than at pH
8 (P ? 0.0001). In contrast, E11 was inactivated more rapidly
at pH 8 (P ? 0.0001), and CVB3 showed no difference in
inactivation rates at the different pH values.
Chlorine inactivation curves for the four enteroviruses (Fig. 1
and 2, note scales) indicate that these viruses were inactivated
according to first-order reaction kinetics. Inactivation curves for
HAdV2 (Fig. 2) exhibited a second-order tailing effect.
All of the viruses were significantly different from each other
in their relative resistance to monochloramine disinfection,
including viruses of the same type. Inactivation of CVB3 was
two to three times faster than CVB5, depending on pH (P ?
0.0001). At both pH levels, inactivation of E1 was more than
100 times faster than E11 (P ? 0.0001). Inactivation of
HAdV41 was slightly faster than HAdV40 (P ? 0.0002 at pH
7, P ? 0.0006 at pH 8); however, inactivation of HAdV40 was
five to seven times faster than HAdV2, depending on pH (P ?
0.0001). Overall, monochloramine disinfection was most effec-
tive for E1 (P ? 0.0001) and least effective for E11 and HAdV2
(P ? 0.0001).
Monochloramine disinfection was more effective at pH 7
than pH 8 for HAdV2 (P ? 0.006), HAdV40 (P ? 0.0001),
HAdV41 (P ? 0.001), CVB5 (P ? 0.0001), and E1 (P ? 0.029).
There was no significant difference between the efficacy of
disinfection at pH 7 and pH 8 for CVB3 (P ? 0.18), E11 (P ?
0.37), and MNV (P ? 0.07).
For each virus, monochloramine inactivation kinetic curves
were similar at pH 7 and pH 8 (Fig. 3 and 4). HAdV2, CVB3,
TABLE 1. CT values for free chlorine inactivation of study viruses
with 0.2 mg of free chlorine/liter at 5°C
CT value (mg ? min/liter)
pH 7pH 8 pH 7 pH 8pH 7 pH 8
aCT value for this level of inactivation extrapolated using the EFH model.
bOnly one replicate achieved the desired inactivation (the CT value for the
second replicate was extrapolated).
cND, no data (the CT value could not be extrapolated due to asymptotic
TABLE 2. CT values for 2-, 3-, and 4-log10inactivation of study
viruses with 1 mg of monochloramine/liter at 5°C
CT value (mg ? min/liter)
pH 7 pH 8pH 7 pH 8pH 7pH 8
aCT value for this level of inactivation extrapolated using the EFH model.
bOnly one replicate achieved the desired inactivation (the CT value for the
second replicate was extrapolated).
cData from one replicate only.
dND, no data. The CT value could not be extrapolated due to asymptotic
FIG. 1. Free chlorine inactivation of CVB5 at pH 7 (f) and pH 8
(?) and E1 at pH 7 (F) and pH 8 (E).
FIG. 2. Free chlorine inactivation of HAdV2 at pH 7 (f) and pH
8 (?), CVB3 at pH 7 (F) and pH 8 (E), and E11 at pH 7 (Œ) and pH
1030CROMEANS ET AL.APPL. ENVIRON. MICROBIOL.
CVB5, and E11 exhibited first-order inactivation curves,
whereas inactivation curves for HAdV40, HAdV41, E1, and
MNV exhibited a second-order tailing effect.
Few studies have examined both chlorine and monochlora-
mine disinfection of the same viruses under the same experi-
mental 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, coxsackievi-
ruses, echoviruses, and caliciviruses are potentially important
microbiological contaminants for public drinking water sys-
tems. 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
Previous research on chlorine inactivation of multiple en-
teric 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-log10inactivation 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 val-
ues of 5.4, 8.4, and 11.5 for 2-, 3-, and 4-log10inactivations,
respectively (7), which is somewhat consistent with results in
the present study for pH 7 and 8 (see Table 1). If the CT values
are estimated based on reported data, other studies using
CVB5 have reported somewhat higher values (?2-fold) for
4-log10inactivation (32) or ?2-fold lower for 2-log10inactiva-
tion (13) 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 prep-
arations 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 ). The collective data in the
literature indicates that the rate of inactivation of CVB5 with
free chlorine is consistently lower than for other enteric vi-
ruses, 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, 30). In addi-
tion, 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,
33). 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 in-
activation than HAdV40 and HAdV41.
The second-order inactivation kinetics observed for HAdV2
(Fig. 2) could have been due to several factors, including mul-
tiple virus populations exhibiting differing resistance to disin-
fection (15). Another possibility is that more than one mech-
anism 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
FIG. 4. Monochloramine inactivation of CVB5 at pH 7 (f) and pH
8 (?), CVB3 at pH 7 (F) and pH 8 (E), E11 at pH 7 (Œ) and pH 8 (‚),
and HAdV2 at pH 7 (?) and pH 8 (?).
FIG. 3. Monochloramine inactivation of HAdV40 at pH 7 (f) and
pH 8 (?), HAdV41 at pH 7 (F) and pH 8 (E), E1 at pH 7 (Œ) and pH
8 (‚), and MNV at pH 7 (?) and pH 8 (?).
VOL. 76, 2010 CHLORINE AND MONOCHLORAMINE DISINFECTION OF VIRUSES 1031
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 indi-
cated first-order kinetics, which suggests a single mechanism
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 mea-
sured 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 inactiva-
tion. Although investigators have used feline calicivirus as a
surrogate for studies of human norovirus (11, 33–35), more
recently investigators have suggested that MNV may be a more
relevant surrogate for studying the survival of human norovi-
ruses (3, 8, 41). 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 disin-
fection efficacy of monochloramine for viruses. The enterovi-
ruses examined in the present study each exhibited markedly
different responses to monochloramine disinfection. E11,
CVB3, and CVB5 exhibited first-order inactivation curves (Fig.
4), while the inactivation curves of E1 exhibited a second-order
tailing effect (Fig. 3). 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 en-
teroviruses was the 100-fold difference between CT values for
E1 and E11. The differences in the responses of the enterovi-
ruses to monochloramine are not readily explained, but they
could suggest that the mechanism of monochloramine disin-
fection is not the same for all enteroviruses. There has been no
prior research to compare monochloramine disinfection on
multiple enteroviruses under the same experimental condi-
tions. 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 inacti-
vation curves (13, 25).
Comparison of monochloramine data from the present study
with previous findings is limited due to differences in experi-
mental conditions, such as buffering capacity of the water,
temperature, and pH. However, a few studies have investigated
monochloramine disinfection of CVB5 under conditions sim-
ilar to those used here. Using 10 mg of monochloramine/liter
at pH 8, Sobsey et al. reported a 4-log10inactivation by 104
min, which, if translated into a CT value (1,040) is similar to
the 4-log10CT 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 dif-
ferent 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 simi-
larity for the three adenoviruses was a decreased effectiveness
of monochloramine at pH 8 versus pH 7.
The differences in CT values and monochloramine inactiva-
tion 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 adenovi-
ruses. However, Baxter et al. examined monochloramine dis-
infection of HAdV41 and HAdV5 (a species C HAdV) and
found similar CT values for both viruses (5). They also re-
ported a CT value for 2.5-log10inactivation of HAdV41 (300)
that was similar to the 2.5-log10CT 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 consis-
tently higher using monochloramine than free chlorine, al-
though the magnitude of this difference varied by virus type.
Monochloramine disinfection yielded a 2-log10CT 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-log10reduction 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 resis-
tant viruses to chlorine (2-log10CT ? 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 effec-
tive 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 identi-
fied as the etiologic agents in numerous waterborne disease
outbreaks, including outbreaks in which free chlorine residuals
were reported (12, 42).
The susceptibility of the study viruses to monochloramine
varied greatly, both between and within virus types. Monochlo-
ramine 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-log10inactivation of HAdV2 with mono-
chloramine at 5°C and pH 8, which is above the CT value of
1,988 recommended in the USEPA Guidance Manual for Com-
pliance with the Filtration and Disinfection Requirements for
Public Water Systems Using Surface Water Sources to achieve a
4-log10inactivation with chloramines at pH 8 (37). Within virus
types, differences in monochloramine 2-log10CT values were
2- to 3-fold between CVB5 and CVB3, 5- to 10-fold between
1032CROMEANS ET AL.APPL. ENVIRON. MICROBIOL.
HAdV2 and HAdV41, and 110- to 130-fold between E11 and Download full-text
E1. These data indicate that monochloramine inactivation
modeling and system design should incorporate monochlora-
mine efficacy data for multiple viruses of concern.
Because of the increasing level of treated wastewater enter-
ing natural water sources and use of reclaimed water, a com-
plete 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 disin-
fection of eight different CCL viruses in water has been dem-
onstrated 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.
We thank Charles Humphrey (CDC) for electron microscopy analysis
Funding for this project was provided by the Water Research Founda-
tion (Project #3134, Contaminant Candidate List Viruses: Evaluation of
The use of trade names and names of commercial sources is for iden-
tification only and does not imply endorsement by the CDC or the U.S.
Department of Health and Human Services. The findings and conclusions
in this presentation are those of the authors and do not necessarily rep-
resent those of the Centers for Disease Control and Prevention.
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