Untied States
Environmental Protection
Agency
Municipal Environmental Research
Laboratory
Cincinnati OH 4526a
EPA-600/2-78-123
August 1978
Research and Development
Virus Sensitivity
to Chlorine Disinfection
of Water Supplies
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
• ' .y
This report hag been assigned to the ENVIRONMENTAL PROTECTION TECH-
NOLOC3Y series-' This,.series describes research performed to develop and dem-
onstrate" instrumentation;.'.'equip'ment, and methodology to repair or prevent en-
.viro.nmerttai--degradation from point and non-point sources of pollution. This work
prcVides.iVje new'br improved technology required for the control and treatment
of pollutjon.sources to meet environmental quality standards.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/2-78-123
August 1978
VIRUS SENSITIVITY TO CHLORINE DISINFECTION
OF WATER SUPPLIES
by
Richard S. Engelbrecht
Michael J. Weber
Carl a A. Schmidt
Brenda L. Salter
University of Illinois
Urbana, Illinois 61801
EPA R-803-34fti
Project Officer
John C. Hoff
Water Supply Research Division
Municipal Environmental Research Laboratory
Cincinnati, Ohio 45268
MUNICIPAL ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
This report has been reviewed by the Municipal Environmental Research
Laboratory, U.S. Environmental Protection Agency, and approved for publica-
tion. Approval does not signify that the contents necessarily reflect the
views and policies of the U.S. Environmental Protection Agency, nor does
mention of trade names or commercial products constitute endorsement or
recommendation for use.
ii
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FOREWORD
The Environmental Protection Agency was created because of in-
creasing public and government concern about the dangers of pollution to
the health and welfare of the American people. Noxious air, foul water,
and spoiled land are tragic testimony to the deterioration of our natural
environment. The complexity of that environment and the interplay
between its components require a concentrated and integrated attack on
the problem.
Research and development is that necessary first step in problem
solution and it involves defining the problem, measuring its impact, and
searching for solutions. The Municipal Environmental Research Laboratory
develops new and improved technology and systems for the prevention,
treatment, and management of wastewater and solid and hazardous waste
pollutant discharges from municipal and community sources, for the
preservation and treatment of public drinking water supplies, and to
minimize the adverse economic, social, health, and aesthetic effects of
pollution. This publication is one of the products of that research; a
most vital communications link between the researcher and the user
community.
In this study, the effects of virus type, suspending medium and the
interaction of these, on the kinetics of virus inactivation by chlorine
was examined. The varying effects observed with different viruses
indicate that it would be difficult to select a single virus for use as
a disinfection indicator under all circumstances.
Francis T. Mayo, Director
Municipal Environmental Research
Laboratory
ill
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ABSTRACT
The efficiency of chlorine disinfection of viruses is likely to be
affected both by the type of virus and by the nature of the suspending
medium. The purpose of this study was to examine the effects of virus type,
suspending medium and the interaction of these, on the kinetics of inacti-
vation by chlorine. Six enteric viruses (picornaviruses) as well as SV40
and Kilham rat virus were studied under carefully controlled laboratory con-
ditions. It was found that the different virus types showed a wide range
of sensitivity to chlorine disinfection. The rate of inactivation was
greater at pH 6 than at pH 10; however, the relative sensitivities of the
different viruses were affected differently by changes in pH. This indi-
cates an effect of pH both on the species of chlorine and on the sensitivity
of the virus. The presence of dissolved ions also had an effect on sensi-
tivity to chlorine. The possible effects of virus aggregation and the
appearance of chlorine resistant mutants were investigated as well. The
results indicate that it will be difficult to obtain a single virus type
which will serve as a suitable indicator of disinfection under all circum-
stances.
iv
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CONTENTS
Foreword iii
Abstract iv
Figures vi
Tables vii
Acknowledgment *. . viii
1. Introduction 1
2. Conclusions and Recommendations 5
3. Materials and Methods 7
Preparation and Purification of Stock Virus 7
Virus Assay 8
Preparation of Chlorine Water and Chlorine Demand
Free Buffer 9
Experimental Equipment and Procedures 9
4. Results and Discussion 11
Effect of pH on Chlorine Inactivation of the Six
Picornaviruses 11
Chlorine Inactivation Using Chlorine Demand Free Buffer
Solution 20
Chlorine Inactivation as a Function of Virus Group . . 23
Effect of Temperature and Chlorine Dosage on Virus
Inactivation 24
Effect of Dissolved Ions on Chlorine Inactivation ... 24
Effect of Aggregation on Chlorine Disinfection ..... 29
Genetic Selection for Chlorine-Resistant Virus .... 38
References 42
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FIGURES
Number Page
1 Inactivation kinetics of polio 1 and polio 2 to 0.47-0.51 mg/£
free available chlorine at pH 6.0 and 5.0°C 14
2 Inactivation kinetics of echo 1 and echo 5 to 0.38-0.49 mg/£
free available chlorine at pH 6.0 and 5.0°C 15
3 Inactivation kinetics of coxsackie B5 and coxsackie A9 to
0.46-0.52 mg/£ free available chlorine at pH 6.0 and 5.0°C . . 16
4 Inactivation kinetics of polio 1 and polio 2 to 0.48-0.52 mg/£
free available chlorine at pH 10.0 and 5.0°C 17
5 Inactivation kinetics of echo 1 and echo 5 to 0.49-0.51 mg/£
free available chlorine at pH 10.0 and 5.0°C 18
6 Inactivation kinetics of coxsackie B5 and coxsackie A9 to
0.48-0.51 mg/£ free available chlorine at pH 10.0 and 5.0°C . . 19
7 Inactivation kinetics of polio 1 to 0.46-0.51 mg/t free available
chlorine in phosphate buffer and boric acid-NaOH buffer at
pH 7.8 and 5.0°C 21
8 Inactivation kinetics of echo 1, echo 5, and coxsackie B5 to
0.47-0.52 mg/£ free available chlorine at pH 7.8 and 5.0°C . . 22
9 Inactivation kinetics of polio 1 with three levels of free
available chlorine at pH 6.0 and 2.0°C 25
10 Residual chlorine concentration vs. rate of inactivation of
polio 1 at pH 6.0 and 2.0°C 26
11 Inactivation kinetics of polio 1 by 0.46-0.49 mg/£ free available
chlorine in 0.05 M boric acid-NaOH buffer, with and without
0.05 M KC1 at pH 10.0 and 5.0°C 27
12 Inactivation kinetics of polio 1 by 0.48-0.49 mg/^ free available
chlorine in 0.05 M phosphate buffer, with and without
0.05 M KC1 at pH 6.0 and 5.0°C 28
VI
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Number Page
13 Inactivation kinetics of polio 1 to 0.50-0.55 mg/l free available
chlorine in 0.05 M phosphate buffer, with and without
0.01 M MgCl2 at pH 6.0 and 5.0°C 30
14 Inactivation kinetics of polio 1 to 0.50-0.55 mg/£ free available
chlorine in 0.05 M phosphate buffer, with and without
0.002 M CaCl2 at pH 6.0 and 5.0°C 31
15 Electron micrographs of polio 1 by the phosphotungstate negative
staining technique 32
16 Fraction number vs. density of polio.1 or ribosomes in a 5-30%
sucrose gradient 34
17 Inactivation kinetics of polio 1, used in the gradient studies,
by 0.51 mg/£ free available chlorine at pH 6.0 and 5.0°C ... 35
18 Gradient profile of polio 1, 5-20% sucrose gradient spun for
2 hours at 22,000 rpm at 15°C 36
19 Gradient profile of echo 1, 5-20% sucrose gradient spun for
2 hours at 22,000 rpm at 15°C 37
20 Inactivation of three fractions from the polio 1 gradient profile
by 0.49-0.51 mg/l free available chlorine at pH 6.0 and 5.0°C . 39
21 Inactivation of four fractions from the echo 1 gradient profile
by 0.50-0.51 mg/£ free available chlorine at pH 6.0 and 5.0°C . 40
TABLES
Number Page
1 Time Required for 99 Percent Inactivation by Free Residual
Chlorine at 5.0°C ± 0.2°C 12
2 Comparison of Virus Inactivation by Free Residual Chlorine
at pH 6.0 and 10.0, and 5.0°C ± 0.2°C 13
3 Chlorine Inactivation of Kilham Rat Virus and SV40 Virus by
Free Residual Chlorine at 5.0°C ± 0.2°C 23
vii
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ACKNOWLEDGMENTS
This study was performed primarily in the Environmental Engineering
Laboratories of the Department of Civil Engineering, University of Illinois
at Urbana-Champaign. The laboratory facilities of Dr. Michael J. Weber,
Department of Microbiology, University of Illinois at Urbana-Champaign,
were also available to the study whenever necessary.
The critical comments, worthwhile suggestions, and encouragement pro-
vided by Dr. John C. Hoff, EPA Project Officer, during the course of this
study were greatly appreciated.
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SECTION 1
INTRODUCTION AND OBJECTIVES OF STUDY
The purpose of disinfecting potable water supplies is the destruction
of pathogenic organisms and thus the elimination and prevention of water-
borne disease. Although disinfection may be accomplished in different ways,
the most widely used disinfectant today is chlorine, primarily because of
its effectiveness, economy, and ease of handling.
Factors affecting the efficacy of chlorine disinfection include:
1) the nature, distribution, and concentration of chlorine and its reaction
products in the water to be disinfected; 2) the nature and condition of the
water to be disinfected; 3) the temperature of the water; 4) the time of
contact between the pathogenic organism and the chlorine; and 5) the nature
of the organisms to be destroyed and their concentration, distribution, etc.
in the water.
Chlorine, when applied to a water supply, may react with various con-
stituents in the water to form a variety of chlorine species with different
disinfecting efficiencies. For example, chloramines, primarily monochlor-
amine and dichloramine, will be formed if the water contains ammonia. These
species of chlorine are referred to in practice as combined available
chlorine. In the absence of ammonia, organic nitrogen compounds, reducing
agents, etc., chlorine may exist in water as either hypochlorous acid (HOC1),
hypochlorite ion (OC1~), or free chlorine, depending upon the pH of the
water. Chlorine existing as HOC! or OC1~ is termed free available chlorine.
Of the two species, HOC1 demonstrates the higher disinfecting efficiency.
Compared with free available chlorine, combined available chlorine is much
less efficient in terms of disinfection. An increased efficiency will also
be achieved with increasing concentrations of the chlorine species.
Further, the nature of the water with respect to other constituents,
such as suspended solids, will affect disinfection efficiency. Another
factor is temperature; disinfection is more rapid with increasing tempera-
ture of the water. Disinfecting efficiency also increases with increased
time of contact or exposure.
The final factor governing disinfection efficiency is the nature of
the pathogenic organisms present. Data exist which indicate that the
resistance of pathogens to free available chlorine varies from one organism
to another. For example, spore forming bacteria, such as 8a&c££u5 anthJuKM,
are more resistant than the non-spore forming bacteria.1 Fortunately, the
most common enteric pathogens do not include spore forming bacteria. The
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bacterial pathogens of intestinal origin which cause typhoid fever,
cholera, and bacterial gastroenteritis can be inactivated by disinfection
with chlorine. Because their resistance is approximately the same or
somewhat less than E&ch
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less effective in inactivating virus than hypochlorous acid (HOC1).
In the case of the studies performed by Kelly and Sanderson,8'9 it
would appear that the viricidal efficiency of HOC1 is more than 50 times
greater than that of the chloramines. However, an investigation reported
by Scarpino e£ ai.10 showed that the hypochlorite ion (OC1~) was more
effective in inactivating poliovirus type 1 than hypochlorous acid (HOC!)*
In fact, OC1" was seven times more effective than HOC!. This observation
is contrary to the findings of others,4'6'9 and to the generally accepted
understanding of chlorine disinfection. It was indicated by the authors
that the borate-KCl-NaOH buffer used may have caused the unusual HOC1-OC1"
effect.
There have been a number of other studies of a similar nature which
have provided additional information on the inactivation of viruses by
chlorine. In many instances, these studies have produced inconsistent
results. The reason for this is not clear but part of the explanation may
be related to the different experimental procedures and conditions that
have been used. Since it is not intended that this be an exhaustive liter-
ature review, these studies will not be discussed. Further, they are of
marginal importance to the following research.
It would seem appropriate, however, to conclude this review by citing
the detailed study performed by Liu
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both adenoviruses and echoviruses are relatively less resistant; and the
polioviruses and coxsackieviruses are the most resistant." However, if a
20 min contact time is arbitrarily assumed, most of the viruses tested
would have been 99.99 percent inactivated at a free residual of 0.5 mg/£,
according to the authors. Although these results are meaningful, they
should not be considered the final answer.
Water supply personnel cannot afford to become complacent regarding
the elimination of enteric viruses in potable water supplies. For example,
although most of the viruses studied by Liu zt aJL. n would be inactivated
with a free chlorine residual of 0.5 mg/l and a contact period of 20 min,
there were still 20 percent of the viruses tested which showed resistance
to this condition of chlorination. It may be pointed out that there have
been instances where viruses have been reported as being detected in finished
water supplies. For example, viruses were detected in samples of water
obtained from the distribution system of two out of three New England com-
munities examined.12
Because of the significance of the results presented by Liu at at.,11
coupled with the importance associated with the question of viruses in
public water supplies, their observations should be independently confirmed.
It was the purpose of the following research to do just this and, at the
same time, to obtain additional information which will provide further
insight and knowledge regarding the inactivation of enteric viruses by
chlorine. Since it has been observed that viruses differ greatly in their
sensitivity to chlorine disinfection, and because the presence of viruses
in water supplies may well prove to be a major public health problem, it is
of critical importance to answer the following questions:
1. What is the full range of resistance of viral pathogens to
chlorine?
2. How much is the effectiveness of chlorination affected by the
quality of the suspending medium, e.g., pH, temperature, ionic
strength?
3. Can genetic selection produce more resistant viral strains?
Answers to these questions should provide knowledge of the effectiveness of
the current practice of chlorination in dealing with waterborne viruses and
a rational basis for improving chlorine disinfection.
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SECTION 2
CONCLUSIONS AND RECOMMENDATIONS
CONCLUSIONS
1. The viruses studied demonstrated a wide range of sensitivity to
free available chlorine. This was true in comparing viruses
which are closely related, i.e., picornaviruses, and even for
different types of the same virus, i.e., poliovirus types 1 and 2.
2. For the six (6) picornaviruses examined, the rate of inactivation
by free available chlorine at pH 10.0 was less than at pH 6.0,
providing the other variables remained the same.
3. The rank ordering of the viruses with respect to their relative
sensitivity to free available chlorine was different at pH 6.0
and at pH 10.0, Indicating that changes in pH have differing
effects on different virus types as well as on the chlorine species.
4. Potassium, calcium and magnesium ions can significantly affect the
rate of chlorine inactivation of poliovirus type 1.
5. At 2°C, the inactivation rate of poliovirus type 1 was observed
not to vary linearly with different free available chlorine
concentrations.
6. Kilham Rat virus and SV40 were found to be more sensitive to
inactivation by free available chlorine than the picornaviruses.
RECOMMENDATIONS
1. Since different viruses vary in their sensitivity to chlorine,
caution should be exercised in using a given virus to determine
the efficacy of disinfection, i.e., chlorination. This observa-
tion may be valid for other disinfectants, so unless the most
resistant virus is known for a given condition, the true efficacy
of a disinfection process in terms of virus inactivation cannot
be determined reliably.
2. The effect of various inorganic ions, alone and in combination, on
the rate of virus inactivation by various disinfectants should be
thoroughly investigated as such information might relate to the
disinfection of natural waters.
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3. The occurrence of disinfectant resistant viruses, resulting from
genetic selection caused by the disinfectant itself, should
receive added attention.
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SECTION 3
MATERIALS AND METHODS
PREPARATION AND PURIFICATION OF STOCK VIRUSES
Six of the eight viruses used in the. chlorine disinfection experiments
were enteric picornaviruses. Five of these viruses, poliovirus type 2
(Lansing), coxsackievirus type A9 (Griggs), coxsackievirus type B5 (Faulkner),
echovirus type 1 (Farouk), and echovirus type 5 (Noyce), were obtained from
the National Institute of Health, Bethesda, MD. The sixth picornavirus,
poliovirus type 1 (Mahoney), was obtained from Dr. Gerald Berg, Environmental
Protection Agency, Cincinnati, OH. All six of these viruses were cultured
on and assayed by the plaque technique using Buffalo Green Monkey (BGM)
kidney monolayers.
The BGM cells were grown in medium 199 (Grand Island Biological Co.,
Grand Island, NY) containing 10 percent fetal calf serum (FCS) (Grand
Island Biological Co., Grand Island, NY) and maintained in medium 199 con-
taining 5 percent FCS after reaching confluence on the fifth day. To each
100 m£ of medium, 1.0 ml of an antibiotic solution was added. This anti-
biotic solution contained penicillin, 10,000 y/m£; fungizone, 25 mcg/m£;
and streptomycin, 10,000 mcg/m£; all of these antibiotics were obtained from
Grand Island Biological Co., Grand Island, NY.
Each virus was purified by inoculating a dilute virus suspension onto
a BGM monolayer, growing it up, and isolating a single plaque. The isolated
plaque was serially passaged in BGM cells until stocks with adequate titers
were obtained (10~° to 10~8 PFU/m£). The plaque-purified stocks were sub-
jected to a freeze-thaw procedure three times, and then treated with Freon
113 and sucrose gradient centrifugation to remove any chlorine demand.
Virus stocks were homogenized with Freeon 113 (Trichlorotrifluorethane,
E. I. Dupont de Nemours and Co., Wilmington, DE) (3 parts virus to 2 parts
Freon 113) in a Sorvall Omnimixer (Ivan-Sorvall Inc., Newton, CT) at a
setting of 5 for 2 min.
The homogenized mixture was centrifuged at 2000 rpm in a Sorval centri-
fuge (Ivan-Sorvall Inc., Newton, CT) for 20 min at room temperature. The
supernatant containing virus was removed and an aliquot of 5-6 mt was over-
layed onto a linear 5-20 percent sucrose gradient. The gradients were
centrifuged in a Beckman SW 25.2 rotor at 22,000 rpm for 2 hr (Beckman
Instruments, Inc., Palo Alto, CA). Three ml fractions were collected from
the bottom of the cellulose nitrate-sucrose gradient tube and the highest
titered fractions were pooled to obtain a uniform virus stock. Three mi
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aliquots of the pooled virus were frozen and stored at -70°C until used.
Simian vacuolating virus 40 (SV40), a papovavirus, was obtained from
Dr. Lowell Hager, Department of Biochemistry, University of Illinois. SV40
virus was grown and assayed on CV-1 cells, a derivation of African Green
Monkey Kidney. The cells were grown in medium 199 containing 2 percent
fetal calf serum. The cells reached confluency after one week, at which
time they were split into flasks or plates, SV40 was also purified by the
Freon 113 method followed by sucrose gradient centrifugation as described
above.
Kilham Rat virus, a parvovirus, was obtained along with the Rat Nephroma
cells in which they were grown, from Dr. Lois A. Salzman, National Institute
of Health, Bethesda, MD. Rat Nephroma cells were grown in medium 199 con-
taining 10 percent fetal calf serum and reached confluence in 7 days at
which time they were split and placed into new flasks or plates. Kilham Rat'
virus was also purified using the Freon 113 and sucrose gradient methods.
VIRUS ASSAY
The titers of the six enteric viruses (polio 1, polio 2, coxsackie A9,
coxsackie B5, echo 1, and echo 5) were determined by plaque assay, using
confluent BGM monolayers in 60 x 15 mm tissue culture plates (Falcon Division,
Becton, Dickinson and Co., Oxnard, CA). The virus was diluted in Hank's
balanced salt solution (Grand Island Biological Co., Grand Island, NY) con-
taining 2 percent fetal calf serum, and 0.5 mi of each dilution was inocu-
lated in duplicate onto BGM plates. After incubation at 37°C under 5 per-
cent C02 for 1-2 hr, a 5-6 mi aliquot of overlay medium consisting of medium
199 plus 10 percent fetal calf serum, antibiotics and 0.9 percent agar was
placed on top of the BGM monolayer and incubated at 37°C until the appearance
of plaques. At this time the monolayer was fixed with 70 percent ethanol,
formaldehyde and acetic acid in a volume ratio of 20:2:1. The monolayer was
then stained with a 1 percent solution of crystal violet and the plaques
were counted.
Determination of the titer of SV40 was also done by plaque assay,
except that the assays were performed on CV-1 monolayers. The virus was
diluted in phosphate buffered saline (PBS) and 0.2 m£ of each dilution was
plated in duplicate. After a 2 hr incubation at 37°C, a 5 ml overlay con-
taining 0.9 percent agar, 5 percent fetal calf serum, 0.15 percent NaHCOs
and antibiotics in medium was added. After 5 days of incubation at 37°C,
another 5 mi of the above overlay was added to each plate. Plaques did not
appear until the 13th or 14th day, at which time a 3 m€ staining layer con-
sisting of 0.9 percent agar, 0.005 percent neutral red, 0.15 percent
and antibiotics in medium 199 was added. The final count of plaques was
performed after 16 days.
Use of the plaque assay technique was not possible for the Kilham Rat
virus (KRV) for technical reasons and therefore this virus was assayed by
the hemagglutination technique (HA). Kilham Rat virus will only proliferate
in cells which are actively growing. Therefore, the KRV was grown in cells
which had only recently been sparsely plated, and were therefore actively
8
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dividing. The KRV was diluted in PBS and 0.1 mt of each dilution was added
in quintuplicate onto sparsely plated but attached Rat Nephroma cells
(5 x 105 cells/60 mm culture dish) and allowed to incubate for 2 hr at 37°C
under 5 percent C02. During the 2 hr incubation period, the culture dishes
were gently rocked every 15 rain. After the 2 hr Incubation period, 5 mi of
medium 199 with 10 percent fetal calf serum was added to each plate and they
were incubated for 6 days. By the 6th day, cytopathic effect was apparent
at lower dilutions.
The cells were scraped off the culture dishes with a rubber policeman;
the collected cells were then subjected to a freeze-thaw procedure three
times. An aliquot of 25 yfc of the lysate was placed in the first well of a
microtiter plate in which there was 25 vi of PBS. Serial two-fold dilutions
were then made and 25 v* of a 1 percent solution of guinea pig red blood
cells (diluted in PBS) was added to each well. The microtiter plates were
incubated at room temperature for 1 hr and then the HA titers were recorded
as the highest dilution giving agglutination.
PREPARATION OF CHLORINE WATER AND CHLORINE DEMAND FREE BUFFER
Stock solutions of chlorine were prepared using Clorox (Clorox Corp.,
Oakland, CA), as these solutions proved to be more stable than those pre-
pared from chlorine gas. To compare the two chlorine solutions (Clorox vs.
chlorine gas), an inactivation experiment was performed with polio 1 using
both solutions; no difference was observed 1n the Inactivation rate with
the two chlorine solutions.
Chlorinated water was prepared by adding 5 mg/£ of a hypochlorite
solution, prepared from Clorox, to delon1zed-distilled water and allowing
the chlorinated water to stand several days. This chlorinated water was
then used to prepare chlorine demand-free buffer (CDF buffer). For experi-
ments performed at pH 6.0 and 7.8, a 0*05 M phosphate buffer was prepared
from NaH2P04 and Na2HP04 and the above mentioned chlorinated water. For
those experiments performed at pH 10.0, a 0.05 M borate buffer (HaBOa-NaOH)
without KC1 was used; again the buffer was prepared using chlorinated water.
Both the phosphate and borate buffers were boiled for several minutes and
exposed to ultraviolet light for 48-72 hr to achieve dechlorinatlon. All
buffer solutions were analyzed for the absence of chlorine by the ortho-
tolidine test before use.13 When the orthotolldine test was negative, the
buffers were considered to be chlorine demand-free.
EXPERIMENTAL EQUIPMENT AND PROCEDURES
For each experiment, 5 I of the appropriate CDF buffer solution was
prepared and sufficient chlorine added to 4 £ to obtain the desired residual.
The remaining 1 I was set aside to be used 1n the virus control beaker. A
400 mt aliquot of the CDF buffer solution, having the desired chlorine
residual, was placed Into each of three stainless steel beakers labeled
"Test," "Chlorine Control," and "Temperature Control," respectively, and
allowed to stand at room temperature for 1 hr. Another beaker labeled
"Virus Control" was filled with 400 mi of the CDF buffer solution without
any added chlorine. This "soaking" of the beakers eliminated any chlorine
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demand which might have been associated with the stainless steel beakers.
At the end of 1 hr, the contents of each beaker were discarded and replaced
with 400 mi of the appropriate CDF buffer solution. The beakers were then
placed into a circulating water bath and allowed to equilibrate in the
constant temperature water bath for approximately 1 hr or until the desired
temperature was achieved (Masterline Water Bath and Circulating Pump, Forma
Scientific, Marietta, OH),
Before initiation of an inactivation experiment, 200 mt was removed
from the "Chlorine Control" beaker and titrated with an amperometric titrator
(Wallace and Tiernan Titrator, Penwalt Corp., Belleville, NJ) to determine'
the initial free available residual chlorine level. After determining the
chlorine residual, a stirring rod attached to a six-place multiple labora-
tory stirrer (Phipps and Bird Laboratory Stirrer, Phipps and Bird, Richmond,
VA) was placed into each beaker and mixed at maximum speed (approximately
180 rpm); the temperature was allowed to re-equilibrate for approximately
5 min.
Initiation of each inactivation experiment was accomplished by the
addition of 1 mt of a virus preparation to the "Virus Control" and "Test"
beakers. Time-point samples from the "Virus Control" and "Test" beakers
were withdrawn in 5 mt aliquots at pre-determined time intervals and added
to sodium thiosulfate (12 mg/m£) and thoroughly mixed to neutralize the
chlorine. At the end of each experiment, 200 ml of the CDF buffer solution
was removed from the "Chlorine Control" and "Test" beakers to determine the
chlorine residual level. Dilutions were made of the time-point samples and
plated onto B6M cells and allowed to incubate until plaques appeared. The
plaques were counted and the results plotted, i.e., percentage virus sur-
vival vs. time of exposure to chlorine.
10
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SECTION 4
RESULTS AND DISCUSSION
EFFECT OF pH ON CHLORINE INACTIVATION OF THE SIX PICORNAVIRUSES
The hypochlorous acid (HOC!) produced when chlorine is added to water
can be associated (HOC!) or disassociated (OC1~); the distribution of these
two chlorine species is a function of pH. At pH 6.0 and 0°C, about 98.2
percent of the chlorine is in the associated form (HOC!). At pH 10.0 and
0°C, only 0.5 percent of the chlorine is as hypochlorous acid, the remainder
is in the form of hypochlorite ion (OCl').14 Of the two chlorine species,
HOC! demonstrates the greatest disinfecting efficiency.
Experiments were performed with polio 1 and 2, echo 1 and 5, and
coxsackie A9 and B5 at pH 6.0 and 10.0 to evaluate the effect of these two
major species of chlorine, which constitute free available chlorine. The
six viruses were chosen for their wide range of sensitivities to chlorine,
as reported by Liu at oJL. ;n each of the three pairs of viruses represents
a chlorine-sensitive and a chlorine-resistant strain. Chlorine inactivation
studies were also performed at pH 7.8 with echo 1, coxsackie B5, echo 5, and
polio 1 in order to have data comparable to that of Liu and co-workers.
Table 1 summarizes the results, giving the time required for 99 percent (2
logs) inactivation of the six pi coma viruses at pH 6.0 and 7.8 in phosphate
buffer, and at pH 10.0 in borate buffer. These values were obtained by com-
bining the data from at least three separate inactivation experiments for
each virus, and graphically determining the point of 99 percent inactivation.
Experimental error was generally less than 20 percent.
All of the chlorine inactivation experiments were performed at 5°C ±
0.2°C. The pH never varied more than five-tenths of a pH unit during a
given experiment. The range of pH of the separate experiments is shown in
Table 1. The initial virus titer in both the "Virus Control" and "Test"
reactors in each experiment was about 1 x 104 PFU/m£. The dosage of chlorine
employed in each experiment was 0.50-0.53 mg/£. The residual chlorine level,
as measured amperometrically in the "Test" reactor after each experiment,
often decreased but generally not more than 0.03 mg/£. This decrease repre-
sents approximately a 6 percent chlorine-demand by the virus sample. Table 1
shows the range of residual chlorine at the end of each of three separate
experiments for each of the viruses.
The results given in Table 1 indicate that there is a significant dif-
ference in the time of inactivation for the various viruses at pH 6.0 and
10.0. In every case, the rate of inactivation at pH 10.0 was dramatically
11
-------
less than at pH 6.0. For example, echo 1 was 99 percent inactivated in 0.5
min at pH 6.0 and in 96 min at pH 10.0, while polio 1 was 99 percent inacti-
vated in 2.1 min at pH 6.0 and 21 min at pH 10.0.
TABLE 1. TIME REQUIRED FOR 99 PERCENT INACTIVATION
BY FREE RESIDUAL CHLORINE AT 5.0°C ± 0.2°C
Concentration of*
free chlorine
pH mg/£ Virus strain 1
6.00
6.00
6.00-6.02
6.00-6.03
6.00
6.00-6.06
7.81-7.82
7.79-7.83
7.80-7.84
7.81-7.82
10.00-10.01
10.00-10.40
9.89-10.03
9.97-10.02
9.99-10.40
9.93-10.05
0.46-0.49
0.48-0.49
0.48-0.51
0.38-0.49
0.47-0.49
0.51-0.52
0.47-0.49
0.48-0.52
0.46-0.51
0.48-0.50
0.48-0.50
0.49-0.51
0.48-0.50
0.49-0.51
0.50-0.52
0.50-0.51
Coxsackie A9 (Griggs)
Echo 1 (Farouk)
Polio 2 (Lansing)
Echo 5 (Noyce)
Polio 1 (Mahoney)
Coxsackie B5 (Faulkner)
Coxsackie A9 (Griggs)
Echo 1 (Farouk)
Polio 2 (Lansing)
Echo 5 (Noyce)
Polio 1 (Mahoney)
Coxsackie B5 (Faulkner)
Coxsackie A9 (Griggs)
Echo 1 (Farouk)
Polio 2 (Lansing)
Echo 5 (Noyce)
Polio 1 (Mahoney)
Coxsackie B5 (Faulkner)
Minutes
for 99%
inactivation
0.3
0.5
1.2
1.3
2.1
3.4
ND
1.2
ND
1.8
1.3
4.5
1.5
96.0
64.0
27.0
21.0
66.0
Rank
ordering
1
2
3
4
5
6
1
3
2
4
1
6
4
3
2
5
*Range of measured free chlorine residual in the "Test" reactor at the
termination of each of three separate experiments.
ND = not determined
The rank ordering in Table 1 demonstrates the wide range of sensitivi-
ties of related viruses to chlorine disinfection. For example, at pH 10.0,
coxsackie B5 was 40 times more resistant than coxsackie A9. Interestingly,
there are several cases in which the relative sensitivity to chlorine was
altered (rank ordering) between pH 6.0 and 10.0, suggesting important effects
of pH on the virion as well as on the chlorine species. Echo 1 was the
second most sensitive virus at pH 6.0 but was the most resistant at pH 10.0.
Polio 1 was one of the most resistant viruses at pH 6.0; however, it was
relatively sensitive at pH 10.0 when compared to the sensitivity of the
12
-------
other viruses at this pH value. This can be seen most clearly by inspection
of Table 2 in which the time required for 99 percent inactivation of the
viruses and the ratios of inactivation rates at pH 6.0 and pH 10.0 are com-
pared. Even at pH 7.8, differences in relative sensitivity appeared when
rank ordered and compared to results at pH 6.0 (Table 1). Polio 1 and
echo 1 were inactivated at approximately the same rate at pH 7.8, while
their rates were quite different at pH 6.0. Of particular interest was
the observation that polio 1 was inactivated more rapidly at pH 7.8 than at
pH 6.0. This suggests that the effect of pH in this range (pH 6.0-7.8) may
be greater on the structure and reactivity of the virus than on the species
of chlorine.
TABLE 2. COMPARISON OF VIRUS INACTIVATION BY FREE RESIDUAL
CHLORINE AT pH 6.0 AND 10.0, AND 5.0°C ± 0.2°C
Virus strain
Coxsackie A9 (Griggs)
Echo 1 (Farouk)
Polio 2 (Lansing)
Echo 5 (Noyce)
Polio 1 (Mahoney)
Coxsackie B5 (Faulkner)
Minutes
pH 6.0
0.3
0.5
1.2
1.3
2.1
3.4
for 99%
pH 10
1.5
96.0
64.0
27.0
21.0
66.0
Inactivation
.0 Ratio*
5
192
53
21
10
19
*Time required at pH 10.0
Time required at pH 6.0
Figures 1 through 6 show the differences in the kinetics of inactiva-
tion of the two types of polio, coxsackie and echo viruses at pH 6.0 and
10.0, with 0.5 mg/£ of free available chlorine and a temperature of 5°C.
Each pair of viruses from the same subgroup is shown on one figure for pur-
poses of comparison and each curve represents the average of three or more
separate experiments. It is significant to note that some virus types
closely resemble each other in their kinetic response to chlorine while
others do not. For example, in Figure 3, coxsackie A9 at pH 6.0 shows a
rapid decrease in titer followed by a somewhat slower rate of inactivation,
whereas coxsackie B5 appears to be dramatically more resistant to chlorine.
The half-life of coxsackie A9 is about 2 sec, whereas the half-life of
coxsackie B5 is approximately 0.5 min, a 15-fold difference.
13
-------
100
10
1 —
en
c.
s-
3
C/5
5-9
0.1 —
0.01 —
0.001
Polio 1 (Mahoney)
A Polio 2 (Lansing)
2345
Contact Time, Min.
Figure 1. Inactivation kinetics of polio 1 and polio 2 to 0.47-0.51 mg/£
free available chlorine at pH 6.0 and 5.0°C.
14
-------
TOO
10 _'
10
3
s-
c
>
t
0.1
0.01
0.001
1
Echo 1 (Farouk)
Echo 5 (Noyce)
I
I
I
I
0.5 1.0 1.5 2.0 2.5 3.0 3.5
Contact Time, Min.
Figure 2. Inactivation kinetics of echo 1 and echo 5 to 0.38-0.49 mg/l
free available chlorine at pH 6.0 and 5.0°C.
15
-------
TOO
I
CO
10
0.1
0.01
0.001
Coxsackie B5 (Faulkner)
Coxsacki'e A9 (Griggs)
I I I
I I
0 0.5 1.0 1.5 2.0 2.5
Contact Time, Min.
3.0 3.5
Figure 3. Inactivation kinetics of coxsackie B5 and coxsackie A9 to 0.46-
0.52 mg/£ free available chlorine at pH 6.0 and 5.0°C.
16
-------
TOO
10
1 —
3
S-
CT)
C
s_
3
CO
0.1 —
0.01 _
0.001
Polio 1 (Mahoney)
A Polio 2 (Lansing)
I
1
0 20 40 60 80 100
Contact Time, Min.
120
140
Figure 4. Inactivation kinetics of polio 1 and polio 2 to 0.48-0.52 mg/£
free available chlorine at pH 10.0 and 5.0°C.
17
-------
100
in
=3
S-
O)
c
S-
Echo 1 (Farouk)
Echo 5 (Noyce)
0.01
0.001
60 80 100 120
Contact Time, Min.
Figure 5. Inactivation kinetics of echo 1 and echo 5 to 0.49-0.51 mg/£
free available chlorine at pH 10.0 and 5.0°C.
18
-------
100
to
3
S-
c
>
•r-
S-
C/0
10 - -
0.1
0.01
0.001
Coxsackie B5 (Faulkner)
Coxsackie A9 (Griggs)
1
I
1
10 20 30 40 50
Contact Time, Min.
60
70
Figure 6. Inactivation kinetics of coxsackie B5 and coxsackie A9 to 0.48-
0.51 mg/i free available chlorine at pH 10.0 and 5.0°C.
19
-------
Figure 1 shows the kinetics of inactivation of polio 1 and 2, at pH 6.0
and 5°C with 0.5 mg/£ free available chlorine residual. These viruses dis-
play inactivation kinetics which are similar to each other; this is contrary
to the results for the two coxsackie viruses (Figure 3). Initially, both
polio viruses have a half-life of approximately 8-10 sec. However, a sig-
nificant difference in the rate of inactivation for polio 1 and polio 2
appeared following approximately 30 sec of exposure to chlorine. Two logs
of polio 1 were inactivated after 2 min, whereas polio 2 was inactivated
to the same extent in half the time. This is a consequence of the non-
linearity of the inactivation curves.
This non-linearity is most likely due to a difference in degree of
aggregation between the two viruses, although differences in the rate at
which chlorine and the neutralizing thiosulfate solution penetrate the
capsid could also contribute to this non-linearity. Limited results using
sucrose gradients suggest that virus aggregation amounted to 0.5-8 percent
of the particles in the virus stock preparations. This is consistent with
the deviation from single-hit kinetics of polio, between 1 and 2 logs
inactivation (see Figure 1). Whether strain-specific variations in the
degree of aggregation consistently generate differences in inactivation
kinetics remains to be determined. The results with polio 1 quantitatively
agree with those reported by Weidenkopf,6 including the biphasic nature of
the curve.
Since phosphate buffer was used in preparing the CDF buffer solution
at pH 6.0 and borate at pH 10.0, it was important to determine whether the
buffers themselves affected the rates of inactivation. Figure 7 compares
the kinetics of chlorine inactivation of polio 1 at pH 7.8, using both the
borate and phosphate CDF buffer solutions. The curves are indistinguishable
indicating that the buffer ions are unlikely to be responsible for the pre-
vious results.
CHLORINE INACTIVATION USING CHLORINE DEMAND FREE BUFFER SOLUTION
Chlorine inactivation experiments were performed at pH 7.8 with echo 1,
coxsackie B5, and echo 5 in order to have data comparable with Liu e£ at.11
Figure 8 compares the chlorine inactivation of these three viruses at pH 7.8
with 0.5 mg/£ of free available chlorine and at 5°C ± 0.2°C. The times for
99.99 percent inactivation of these three viruses were less than the times
reported by Liu at cUt. n in their Potomac River water study. Liu
-------
TOO
10
CTl
J_
0.1 —
0.01
A Boric Acid - NaOH Buffer
• Phosphate Buffer
Polio 1 (Mahoney)
2 3
Contact Time, Min.
Figure 7.
Inactivation kinetics of polio 1 to 0.46-0.51 mg/l free available
chlorine in phosphate buffer and boric acid-NaOH
buffer at pH 7.8 and 5.0°C.
21
-------
100
in
i.
•r~
>
U>
c
•I—
>
•r-
ZJ
00
10 r5
0.1
0.01
0.001
0
Echo 1 (Farouk)
Echo 5 (Noyce)
Coxsackie B5 (Faulkner)
I
I
1
345
Contact Time, Min.
Figure 8. Inactivation kinetics of echo 1, echo 5, and coxsackie B5 to
0.47-0.52 mg/£ free available chlorine at pH 7.8 and 5.0°C.
22
-------
appear that the inactivation of virus in natural water, i.e., Potomac River
water, may differ significantly from that which occurs in a chlorine demand
free (deionized-distilled water) buffer solution.
CHLORINE INACTIVATION AS A FUNCTION OF VIRUS GROUP
The chlorine sensitivity of different viruses was also considered,
particularly with respect to those virus groups which pose a threat to
health because of their possible occurrence in water supplies. The picorna-
viruses, having RNA as their genetic material, appear to demonstrate a wide
variation in their sensitivity to chlorine (Table 1). Kilham Rat virus, a
parvovirus containing single stranded DNA, was also studied as to its
chlorine sensitivity, because it has been speculated that the virus of
infectious hepatitis may also belong to this same group of viruses. Table 3
gives the kinetics of inactivation of the'Kilham Rat virus at pH 6.0 with
0.5 mg/£ free available chlorine and at a temperature of 5°C.
TABLE 3. CHLORINE INACTIVATION OF KILHAM RAT VIRUS AND SV40 VIRUS BY
FREE RESIDUAL CHLORINE AT 5.0°C + 0.2°C
Kilham Rat Virus
Cone, of free
Exp. chlorine in test
No. pH reactor, mq/£
1 6.01 0.48
2 6.04 0.49
H.A. Titer
% Survival
H.A. Titer
% Survival
Hemaqglutination Titer (H.A. Titer)
Control
1:1638
100
1:563
100
10 sec
1:1792
109
1:243
43
20 sec
1:3.2
0.19
1:3.2
0.56
30 sec 60 sec
1:1.6
0.09
0
0
0
0
0
0
SV40 Virus
Exp.
No.
1
2
3
6.
6.
6.
pH
01
02
02
Cone, of
chlorine
reactor,
0.49
0.48
0.48
free
in test
mg/£
%
Control
100
100
100
; vi
10
1
1
0
rus
sec
.13
.67
.69
Surviving
20
0.
sec
0
02
0
30 sec
0
0
0
Kilham Rat virus was no more resistant to chlorine than any of the
picornaviruses, being 99 percent inactivated in less than 20 seconds. The
chlorine sensitivity of SV40 virus, a papovavirus containing double-stranded
DNA, was also studied. Table 3 shows the kinetics of inactivation of SV40
virus at pH 6.0 with 0.5 mg/t free available chlorine and at a temperature
23
-------
of 5°C. The SV40 virus was even more sensitive to chlorine than Kilham Rat
virus, being 99 percent inactivated in 10 sec. Even though the SV40 virus
has a reputation among tumor virologists as being very hardy under adverse
conditions, it appears to be extremely sensitive to chlorine.
EFFECT OF TEMPERATURE AND CHLORINE DOSAGE ON VIRUS INACTIVATION
Chlorine inactivation experiments were performed at 2°C ± 0.5°C and a
pH of 6.0 with polio 1 and different levels of free available chlorine
residuals, ly M (0.07 mg/£), 7y M (0.5 mq/l) and 28y M (2.0 mg/£), for com-
parison with the data reported by Sharp.5-5 Sharp observed that at 2°C,
pH 6.0, with HOC!, the rate of inactivation of polio 1 increased from about
0.48 log/min with ly M of HOC! to twice this rate at 20y M. However, there
was no further increase in the inactivation rate with 40y M HOC1.15
Figure 9 shows the results of inactivating polio 1 with the three
chlorine residuals given above; as the level of free available chlorine
increases the rate of inactivation increases as well. In Figure 10, the
initial rate of inactivation for polio 1 is plotted as a function of
residual chlorine. It would appear from Figure 10 that the rate of inacti-
vation does not decrease linearly with the residual chlorine concentration.
Below 7y M of residual chlorine, the rate appears to decrease rapidly in
order to intersect the vertical axis at zero residual chlorine concentration.
These data are similar to those of Sharp,15 and are also in agreement with
the work of Weidenkopf in his studies at 0°C, as presented by Floyd at at.lB
This phenomenon seems to appear only at low temperatures.
EFFECTS OF DISSOLVED IONS ON CHLORINE INACTIVATION
Experiments were performed at 5°C with 0.5 mg/£ of free available
chlorine, using 0.05 M phosphate buffer (pH 6.0) and boric acid-NaOH buffer
(pH 10.0) with and without 0.05 M KC1 added, to further investigate the
reversal effect of KC1, as observed by Scarpino
-------
100
in
Z3
S_
cn
c
3
t/)
• 28 yM (2.0 mg/£)
A 7 yM (0.5 mg/t)-
O 1 MM (0.07 mg/t)
0.001
I
I
0
12 16 20
Contact Time, Min.
24
28
Figure 9. Inactivation kinetics of polio 1 with three levels of free
available chlorine at pH 6.0 and 2.0°C.
25
-------
ro
10 15 20
Residual Chlorine Concentration (yM)
25
30
Figure 10. Residual chlorine concentration vs. rate of inactivation of polio 1 at pH 6.0 and 2.0°C.
-------
TOO
10
00
3
s-
>
s-
oo
0.1
0.01
0.001
Boric Acid-NaOH Buffer with
* 0.05 M KC1
A Boric Acid-NaOH Buffer
1
I
10 15 20 25
Contact Time, Min.
30
35
Figure 11. Inactivation kinetics of polio 1 by 0.46-0.49 mg/£ free available
chlorine in 0.05 M boric acid-NaOH buffer, with and without
0.05 M KC1 at pH 10.0 and 5.0°C.
27
-------
100
10
a*
0.1 _
0.01 —
0.001
• Phosphate Buffer with 0.05 M KC1
A Phosphate Buffer
I
I
345
Contact Time, Min.
Figure 12. Inactivation kinetics of polio 1 by 0.48-0.49 mg/t free available
chlorine in 0.05 M phosphate buffer, with and without
0.05 M KC1 at pH 6.0 and 5.0°C.
28
-------
inactivation of polio 1 by free available chlorine.
Polio 1 was also used to study the effect of calcium and magnesium on
chlorine inactivation. These experiments were performed at 5°C, and with
0.05 mg/£ free available chlorine in pH 6.0 phosphate buffer using different
concentrations of magnesium and calcium. Floyd and Sharp17 showed by
electron microscopy that polio 1, suspended in 0.05 M phosphate buffer at
pH 6.0, was markedly aggregated. However, this aggregation was found to be
sensitive to the ionic strength of the solution, and could be prevented by
appropriate concentrations of MgCl£» i.e., at least 0.01 M Mgd2 was required
to inhibit aggregation at pH 6.0. Figure 13 shows the inactivation of
polio 1 by 0.5 mgfl of free available chlorine at pH 6.0 in 0.05 M phosphate
buffer, with and without 0.01 M MgC^. Chlorine inactivation with only
phosphate buffer shows a biphasic curve, while inactivation performed in
phosphate buffer plus 0.01 M MgCl? demonstrates first-order kinetics. It
appears likely that MgCl2 does inhibit the formation of aggregates in phos-
phate buffer at pH 6.0 and this, in turn, alters the inactivation kinetics.
Using an electron microscope, Floyd and Sharp17 observed that polio 1
(7 x 10lO particles/ni^) in a phosphate buffer solution with 0.001 M CaClg
did not produce aggregation, but in 0.01 M CaCl2 aggregation occurred. Using
phosphate buffer at pH 6.0, the highest concentration of CaClg that could be
studied without the formation of insoluble calcium phosphate was 0.002 M.
Figure 14 compares the inactivation of polio 1 in pH 6.0 phosphate buffer,
with and without 0.002 M CaCl2. There appears to be no significant differ-
ence between the two systems; both appear biphasic in nature.
It is possible that the presence of calcium ions causes increased
aggregation. This is evidenced by the rate change in inactivation when
polio 1 is suspended in phosphate buffer with CaCl2 (Figure 14). In the
system with CaCl2» the rate change occurs at 30 percent survival; while in
the absence of CaCl2, this does not happen until survival is 18 percent.
These results, although limited, suggest that magnesium might inhibit
aggregation of polio 1, while calcium might tend to maintain or increase
the aggregation of the virus at pH 6.0. However, to substantiate this
observation, further investigation's required.
EFFECT OF AGGREGATION ON CHLORINE DISINFECTION
Electron microscope studies to determine viral aggregation were initi-
ated. Unfortunately, the studies could not be completed because of the
inaccessibility of the electron microscope. Before this situation developed,
however, a few electron micrographs were taken of the polio 1 virus particles
on a grid prepared by the phosphotungstate negative staining technique. The
majority of the virus particles appeared to be singles as shown by electron
micrograph A 1n Figure 15. Electron micrograph B, Figure 15, shows two
virus particles that, at first, appear to be aggregated, but upon closer
examination appear not to be touching each other. After looking at this
grid and others, it was concluded that only a small percentage (approximately
1 percent) of the viruses 1n this particular preparation were aggregated.
29
-------
TOO
10 _
l-
CD
S-
<**
1 —
0.1 _
0.01 -
0.001
9 Phosphate Buffer with 0.01 M MgCl2
A Phosphate Buffer
I
345
Contact Time, Min.
Figure 13. Inactivation kinetics of polio 1 to 0.50-0.55 mg/l free available
chlorine in 0.05 M phosphate buffer, with and without
0.01 M MgClg at pH 6.0 and 5.0°C.
30
-------
i.
•r-
01
•r~
>
00
• Phosphate Buffer with 0.002 M CaCl,
i
A Phosphate Buffer
0.001
I
I
0
• 345
Contact Time, Min.
Figure 14. Inactivation kinetics of polio 1 to 0.50-0.55 mg/l free available
chlorine in 0.05 M phosphate buffer, with and without
0.002 M CaCl2 at pH 6.0 and 5.0°C.
31
-------
V
•
*
Micrograph A
'
Micrograph B
Figure 15. Electron micrographs of polio 1 by the phosphotungstate negative
staining technique
32
-------
Sucrose density gradients were also used in an attempt to determine
the aggregation state of the polio 1 stock preparation employed in the
inactivation experiments. The experiment consisted of three separate 5-30
percent sucrose gradients. The first gradient was layered with a ribosome
marker (70 S), the second gradient was layered with a sample of polio 1
(150 S) used in the chlorine inactivation experiments, and the third gradi-
ent contained the same polio 1 preparation but treated with 0.001 M EDTA.
The EDTA was added to the sample of polio 1 to break up aggregates by
chelating divalent cations. After centrifugation at 40,000 rpm for 30 min,
the gradients were collected into 0.33 mi fractions and each fraction was
titered by plaque assay. The results are shown in Figure 16. In plotting
the density of virus in each fraction against the fraction number (40 frac-
tions), aggregated virus should appear as either a separate peak from the
fraction having single virus particles, or as a shoulder on the heavy side
of this single virus peak. In Figure 16, the single virus peak for untreated
polio 1 (150 S) should be approximately fraction number 26, based upon that
for the known ribosome preparation (70 S).
The untreated polio 1 gradient (Figure 16) shows a peak at fraction 22,
presumably consisting of single particles; this is further down the gradient
than expected. Likewise, polio 1 treated with 0.001 M EDTA shows a peak at
fraction 14. It would appear that treatment with 0.001 M EDTA did not break
up the aggregates as anticipated. Further studies would be necessary to
ascertain the significance of these preliminary results. Assuming that the
major peak of the untreated polio 1 is composed of single virus particles,
then possibly the shoulder on the side close to the bottom of the gradient
may be due to aggregates. The shoulder accounts for about 8 percent of the
total area under the peak in the gradient.
Inactivation of the polio 1 virus sample used in the above gradient
studies with 0.5 mg/l free available chlorine at 5°C is shown in Figure 17.
The results indicate the possibility of multi-hit kinetics of inactivation;
this is consistent with the presence of viral aggregates, i.e., about 8 per-
cent of the surviving virus.
The chlorine sensitivity of various fractions from a sucrose gradient
fractional on of polio 1 and echo 1 was determined. Each virus was extracted
with Freon 113 and centrifugation through a 5-20 percent sucrose gradient
was performed simultaneously on each virus. The gradients were fractionated
into 3 mi fractions and each was titered by plaque assay. A gradient profile
with a virus peak was obtained from each gradient as shown 1n Figures 18
and 19.
Representative fractions were selected from different areas along the
virus peak: the left (bottom) shoulder, the peak, and the right (top)
shoulder. In the case of echo 1 (Figure 19), a distinct shoulder was evi-
dent on the side of the peak closer to the bottom of the gradient. Believ-
ing this to represent aggregates, a fraction (#5) In this region was also
selected to be tested for chlorine sensitivity. Chlorine Inactivation
experiments were performed on each of the three fractions for polio 1 and
on the four fractions for echo 1.
33
-------
u>
n.
3-
o
X
«l
•?—
1ft
c
OJ
O
to
13
S-
60
50
40
30
20
10
0
30% Sucrose
• Ribosomes
A Polio 1 Untreated
O Polio 1 Treated with
0.001 M EDTA
04 8 12 16
20 24 28 32 36 40
Fraction Number
5.0
4.0
3.0 |
2.0 -j
1.0
0
re
o
Q.
O
5% Sucrose
Figure 16. Fraction number vs. density of polio 1 or ribosomes in a 5-30% sucrose gradient
-------
100
10 —
10
>
S-
0.1
0.01
0.001
0
1
I
I
345
Contact Time, Min.
Figure 17. Inactivation kinetics of polio 1, used in the gradient studies,
by 0.51 mg/e free available chlorine at pH 6.0 and 5.0°C.
35
-------
Q-
£>
O
X
to
c
20
18
16
14
12
10
8
6
0
Fraction from
Bottom
Shoulder
Peak Fraction
Fraction from
Top
Shoulder
0 2
Bottom
6 8 10 12
Fraction Number
14
16
Top
Figure 18. Gradient profile of polio 1, 5-20% sucrose gradient spun for
2 hours at 22,000 rpm at 15°C.
36
-------
u_
0.
iO
o
I
44
40
36
32
28
24
20
16
12
8
Fraction from
Bottom
Shoulder
Fraction
#5
J I 1
Peak Fraction
Fraction from
Top
Shoulder
0
Bottom
6 8 10
Fraction Number
12 14
16
Top
Figure 19. Gradient profile of echo 1, 5-20% sucrose gradient spun for
2 hours at 22,000 rpm at 15°C.
37
-------
The results obtained with polio 1 (Figure 20) conformed to the antici-
pated degree of viral aggregation in each fraction, i.e., the fraction on
the left or bottom shoulder, most likely to contain aggregated virus, showed
the slowest inactivation (2.8 min for 4 logs inactivation), the peak fraction
was more rapid (2.34 min), while the top or right fraction gave the most
rapid inactivation (2.1 min).
Also of interest is the fact that the fractions from the peak and the
top shoulder showed single-hit kinetics, while the fraction from the bottom
shoulder of the virus peak showed a biphasic inactivation pattern. This
latter fraction had possibly less than 1 percent aggregates as determined
from the break in the curve.
The results with the different fractions of echo 1 showed much the
same pattern as with polio 1, i.e., the fractions closest to the bottom
required the greatest length of time to inactivate, and the top fraction
the least (Figure 21). Fraction number 5, selected from the distinct
shoulder on the left of the virus peak, gave the slowest inactivation, i.e.,
4 logs inactivation in 2.90 min. This fraction also demonstrated biphasic
kinetics, with the break in the curve occurring when about 20 percent of the
virus still remained. A larger amount of aggregated virus was probably
present in fraction number 5 than in the other fractions of echo 1. The
fraction on the bottom side of the peak showed a break in the inactivation
curve at about 1 percent survival and the peak fraction at about 0.4 percent
survival, while the top fraction showed single-hit kinetics.
In these sucrose gradient studies, there is a definite relationship
between the position of the fraction on the virus peak (or on the gradient),
and its inactivation response to free available chlorine. By pooling the
different fractions (fraction numbers 9 through 11), more consistent inac-
tivation results can be obtained but the virus particles in the pooled sample
are probably in various states of aggregation and, as a result, tend to
demonstrate multiphasic inactivation.
GENETIC SELECTION FOR CHLORINE-RESISTANT VIRUS
In an attempt to obtain chlorine-resistant virus variants, ten plaques
of polio 1 were picked after three successive inactivation experiments. The
virus from each plaque was grown and stocks prepared. Each stock was then
examined for resistance to chlorine. The contact times for 99 percent
inactivation of the virus from each of the ten plaques were in the range
of 0.2 to 1.4 min longer than the parent culture. This difference is not
believed to be significant. Following completion of these experiments,
Bates ojt o£.18 reported the progressive increase in resistance of polio 1
(LSc) to free available chlorine as a result of repeated exposure of the
virus to a sublethal concentration of chlorine.
Polio 1 was also exposed to ultraviolet (UV) light in hopes of causing
a mutation which, in turn, would produce a chlorine-resistant variant.
Using a virus stock suspended in phosphate buffer, aliquots were withdrawn
following various times of exposure to UV and the UV exposed samples were
plaque assayed. A sample showing about two logs inactivation, i.e., after
38
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100
10 _
CO
c
•r™
>
•r—
$-
3
to
1 —
0.1 __
0.01 —
o.ooi L
0
9 Fraction from Bottom
Shoulder
A Fraction from Peak
O Fraction from Top Shoulder
I
3 4
Time, Min.
Figure 20. Inactivation of three fractions from the polio 1 gradient profile
by 0.49-0.51 mg/£ free available chlorine at pH 6.0 and 5.0°C.
39
-------
s_
01
c
>
s-
Echo 1 (Farouk)
• Fraction #5
• Fraction from Bottom Shoulder
A Fraction from Peak
0 Fraction from Top Shoulder
0.001
I
3 4
Time, Min.
Figure 21. Inactivation of four fractions from the echo 1 gradient profile
by 0.50-0.51 mg/l free available chlorine at pH 6.0 and 5.0°C.
40
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1.5 to 2.0 m1n exposure to UV light, was grown up in BGM cells and subse-
quently purified by Freon 113 extraction and sucrose gradient. A chlorine
inactivation experiment was performed on the purified virus stock. A
sample showing two to three logs Inactivation (2.75 to 3.50 min) was
selected and again grown on BGM cells, purified, resuspended in phosphate
buffer, and re-exposed to UV light. Two exposures to UV light and chlorine
were performed. The contact times for 99 percent Inactivation were in the
range of 1.6 min, which is somewhat more rapid than the parent culture.
This difference, however, is not believed to be significant.
41
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REFERENCES
1. Brazis, R. A., e£ al. Special Report to Department of the Navy, Bureau
of Yards and Docks: Sporicidal Action of Free Chlorine. USPHS Report,
R. A. Taft Sanitary Engineering Center, Cincinnati, OH, 1957.
2. Trask, J. D., zt al. Chlorination of Human, Monkey Adapted and Mouse
Strains of Poliomyelitis Virus. Amer. Jour. Hyg., 41:30, 1945.
3. Ridenour, G. M., and Inglos, R. S. Inactivation of Poliomyelitis Virus
by Free Chlorine. Amer. Jour. Pub. Health, 36:369, 1946.
4. Clarke, N. A., and Kabler, R. W. The Inactivation of Purified Coxsackie
Virus in Water by Chlorine. Amer. Jour. Hyg., 59:119, 1954.
5. Clarke, N. A., tf. al. The Inactivation of Purified Type 3 Adenovirus
in Water by Chlorine. Amer. Jour. Hyg., 64:314, 1956.
6. Weidenkopf, S. Inactivation of Type 1 Poliomyelitis Virus with
Chlorine. Virology, 5:56, 1958.
7. Clarke, N. A., e£ al. Human Enteric Viruses in Water: Source, Survival
and Removability. In: Proceedings of the First International Confer-
ence on Water Pollution Research, Pergamon Press, London, 1962. p. 523.
8. Kelly, S. M., and Sanderson, W. W. The Effect of Chlorine in Water on
Enteric Viruses. Amer. Jour. Pub. Health, 48:1323, 1958.
9. Kelly, S. M., and Sanderson, W. W. The Effect of Chlorine in Water on
Enteric Viruses * II. The Effect of Combined Chlorine on Poliomyelitis
and Coxsackie Virus. Amer. Jour. Pub. Health, 50:1, 1960.
10. Scarpino, P. V.,
-------
14. Gulp, R. L. Breakpoint Chlorination for Virus Inactivation. Jour.
Amer. Waterworks Assoc., 12:699, 1974.
15. Sharp, D. G. Virus Particle Aggregation and Halogen Disinfection of
Water Supplies. EPA-600/2-76-287, U.S. Environmental Protection
Agency, 1976.
16. Floyd, R., Johnson, J. D., and Sharp, D. G. Inactivation by Bromine
of Single Poliovirus Particles in Water. Appl. and Environ. Micro.,
31:298, 1976.
17. Floyd, R., and Sharp, D. G. Aggregation of Poliovirus and Reovirus
by Dilution in Water. Appl. and Environ. Micro., 33(1):159, 1977.
18. Bates, R. C., vt al. Development of. Poliovirus Having Increased
Resistance to Chlorine Inactivation. Appl. and Environ. Micro.,
34:849, 1977.
43
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
i. REPORT NO.
EPA-600/2-78-123
3. RECIPIENT'S ACCESSION NO,
4. TITLE AND SUBTITLE
VIRUS SENSITIVITY TO CHLORINE DISINFECTION
OF WATER SUPPLIES
5. REPORT DATE
August 1978 (Lssuing J)ate)
0. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Richard S. Engelbrecht, Michael J. Weber,
Carla A. Schmidt, Brenda L. Salter
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Civil Engineering
University of Illinois-Champaign
Urbana, Illinois 61801
10. PROGRAM ELEMENT NO.
ICC 614
11. CONTRACT/GRANT NO.
R 803346
12. SPONSORING AGENCY NAME AND ADDRESS
Municipal Environmental Research Laboratory--Cin.,OH.
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
13. TYPE OF REPORT AND PERIOD COVERED
Final 8/74-4/78
14. SPONSORING AGENCY CODE
EPA/600/14
15. SUPPLEMENTARY NOTES
Project Officer: Dr. John C. Hoff (513) 684-7331
16. ABSTRACT
The efficiency of chlorine disinfection of viruses is likely to be
affected both by the type of virus and by the nature of the suspending
medium. The purpose of this study was to examine the effects of virus
type, suspending medium and the interaction of these, on the kinetics of
inactivation by chlorine. Six enteric viruses (picomaviruses) as well
as SV40 and Kilham rat virus were studied under carefully controlled
laboratory conditions. It was found that the different virus types
showed a wide range of sensitivity to chlorine disinfection. The rate
of inactivation was greater at pH 6 than at pH 10; however, the relative
sensitivities of the different viruses were affected differently by
changes in pH. This indicates an effect of pH both on the species of
chlorine and on the sensitivity of the virus. The presence of dissolved
ions also had an effect on sensitivity to chlorine. The possible effects
of virus aggregation and the appearance of chlorine resistant mutants
were investigated as well. The results indicate that it will be difficult
to obtain a single virus type which will serve as a suitable indicator
of disinfection under all circumstances.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Potable water, Water Treatment,
Chlorination, Disinfection,
Enteroviruses, Polioviruses,
Coxsackie viruses, ECHO viruses,
Inorganic salts, Ph
Kilham rat virus
SV40 virus
Virus Inactivation
13 B
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
10. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
52
20. SECURITY CLASS (Thispagej
UNCLASSIFIED
22. PRICE
EPA Form 2220.1 (R.v. 4-77)
44
TNMTIMOmCC, 1OT- 657-040/ U»2
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