United States
Environ
Agency
pa' Environmental Research
Laboratory
Cincinnati OH 45268
Research and Development
Effect of
Particulates on
Disinfection of
Enteroviruses in
Water by Chlorine
Dioxide
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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)
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8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL PROTECTION TECH-
NOLOGY series. This series describes research performed to develop and dem-
onstrate instrumentation, equipment, and methodology to repair or prevent en-
vironmental degradation from point and non-point sources of pollution. This work
provides the new or improved technology required for the control and treatment
of pollution-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-600/2-79-054
July 1979
EFFECT OF PARTICULATES ON DISINFECTION OF
ENTEROVIRUSES IN WATER BY CHLORINE DIOXIDE
by
Pasquale V. Scarpino
Frank A. O. Brigano
Sandra Cronier
Mary Lee Zink
University of Cincinnati
Cincinnati, Ohio 45221
Grant No. R-804418
Project Officer
John C. Hoff
Drinking Water 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 publication. 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.
11
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FOREWORD
The Environmental Protection Agency was created because of
increasing public and government concern about the dangers of
pollution to the health and welfare of the American people. The
complexity of that environment and the interplay between its com-
ponents 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 Environ-
mental 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 pol-
lution. This publication is one of the products of that research;
a most vital communications link between the researcher and the
user community.
This research provides vital, basic information concerning
the viral disinfection capabilities of chloride dioxide, as well
as the impact of particulate and cell-associated turbidity levels
in water on the effectiveness of this disinfectant against
enteric viruses.
Francis T. Mayo, Director
Municipal Environmental
Research Laboratory
111
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ABSTRACT
The ability of suspended matter and viral aggregation to
affect disinfection efficiency assumes importance in drinking
water treatment. Reduced reactivity of chlorine dioxide
(CIO,,) to form carcinogenic compounds is known, but information
is needed about the disinfecting ability of C102< The in-
activation kinetics of C102 on two enteroviruses, poliovirus 1
(Mahoney) and coxsackievirus A9, and an enteric indicator of
fecal pollution, Escherichia coli, were examined in laboratory
studies. The disinfecting ability of C102 as affected by
particulates and viral aggregates was determined. Comparison
of the relative inactivation rates at the 99% destruction
level (i.e., the Van't Hoff relationship), showed that polio-
virus 1 was 8.9 times and coxsackievirus A9 was 2.3 times more
resistant than E. coli to C102 when compared at 15 C at pH7.0.
Chlorine dioxide at 21 C was found to inactivate poliovirus 1
4.6 times faster at pH9.0 than at pH7.0; and 8.3 times faster
at pH9.0 than at pH4.5. A comparison of the relative in-
activation of poliovirus 1 by C102 and other chlorine species
showed that on a weight basis C102 at 15 C at pH 7.0 was just
as viricidally efficient as HOC1 at pH6.0, while at pH9.0
chlorine dioxide was found to be more efficient than HOCl. In
laboratory studies at 5°C at pH7.0, poliovirus 1 preparations
containing mostly viral aggregates took 2.7 times longer to
inactivate with Cl02 than single state virus preparations.
The latter "singles" contained 93% single and 7% clumped
viruses as determined by electron microscopy. The disinfection
efficiency of C102 at pH7.0 with unassociated poliovirus 1
singles increased as the temperature increased from 5 to 15
to 25 C. However, the disinfection efficiency of C10~ with
bentonite adsorbed-poliovirus 1 singles decreased with in-
creasing temperature compared to the efficiencies obtained
with unassociated poliovirus 1 singles. Poliovirus 1 grown
in association with BGM (Buffalo Green Monkey) tissue culture
cells disinfected with C102, and then reported in the Van't
Hoff relationship at the 99% inactivation levels, showed no
trend towards cellular protection at pH7.0 at either 5 C with
1.10 to 2.00 NTU's, or at 25 C with 1.12 to 3.10 NTU's. Thus,
temperature and the amount of turbidity affected the rate of
inactivation of bentonite-adsorbed poliovirus, while under
the conditions of this study there was no effect seen with
the turbidity levels of the cellular-associated virus ex-
amined.
IV
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This report was submitted in fulfillment of EPA Grant
No. R-804418 from the Municipal Environmental Research Labora-
tory of the U.S. Environmental Protection Agency. This report
covers a period from April 1, 1976 through December 31, 1978,
and work was completed as of August 2, 1978.
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CONTENTS
Foreword
Abstract 1V
Figures lx
Tables Xii
Acknowledgment xiii
1. Introduction l
Objectives of the Study 1
Background of the Study 3
2. Conclusions 9
3. Recommendations 10
4. Materials and Methods 12
Chlorine Dioxide Generation 12
Chlorine Dioxide Analysis 12
Preparation of Virus Stock 12
Preparation of Escherichia Coli (ATCC 11229) . 15
Preparation of Bentonite for Turbidity Studies. 15
Preparation of Poliovirus-Bentonite Suspensions 15
preparation of Cell-Associated Poliovirus . . 16
Electron Microscopy Viral Assay Technique . . 16
Experimental Procedures 16
Microorganism Assay Procedure 18
Tissue Culture Procedures 20
5.
Results and Discussion 21
Chlorine Dioxide Alone 21
The Effect of Bentonite- and Cell-Associated
Turbidity on Virus Inactivation Using the
Disinfectant Chlorine Dioxide 26
Poliovirus 1 Characterization and Quanti-
tation 26
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CONTENTS (continued)
Effect of Viral Aggregation on the Disinfec-
tion Process 26
Temperature Effects of Viral Inactivation
with Chlorine Dioxide 32
The Effects of Inorganic Turbidity on the
Inactivation of Poliovirus 1 by Chlorine
Dioxide 32
The Effects of Cellular Turbidity on the
Inactivation of Poliovirus 1 by Chlorine
Dioxide 44
Other Reports Based on This Research 50
References 51
Vlll
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FIGURES
Number Page
1 Apparatus for the preparation of chlorine dioxide
stock solution 13
2 Kinetic (stirred beaker) apparatus 17
3 Dynamic (flowing stream - rapid mix ) apparatus ... 19
4 Inactivation of poliovirus 1 at 15°C at pH7.0
in the presence of 0.87 mg/1 chlorine dioxide ... 22
5 Destruction of Escherichia coli at 15°C at pH
7.0 in the presence of 0.16 mg/1 chlorine
dioxide 23
6 Concentration-time relationship for 99% destruction
of poliovirus 1, coxsackievirus A9, and Escherichia
coli by chlorine dioxide at 15°C at pH7.0 .... 24
7 Concentration-time relationship for 99% inactiva-
tion of poliovirus 1 at 5°C, 15°C and 25°C by
chlorine dioxide 25
8 The effect of pH on the inactivation of poliovirus
1 at 21°C at pH 4.5, 7, and 9, and at 25°C at
JJil /* • • • • • • • • • • • • • • • • ^* /
9 Comparison of the relative inactivation of polio-
virus 1 by hypochlorous acid, hypochlorite ion,
monochloramine, dichloramine, and chlorine dioxide
at 15°C at different pH values 28
10 Electron micrograph of freeze-thawed "aggregated"
poliovirus 1 preparation depicting single (S)
virions, clumped (C) virions and cellular debris
(D'.e) (63,690X) 29
11 Electron micrograph of freon extracted-density
gradient "singles" poliovirus 1 preparation
depicting viruses of single (S), double (D),
triple (T), and quadruple (Q) aggregation states
(74,500X) 30
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FIGURES (continued)
Number Page
12 Analysis of the fractions obtained in the prep-
aration of freon extracted-density gradient
poliovirus 1 31
13 Concentration-time relationship for 99% inactiva-
tion of poliovirus comparing single virions to
aggregated virions 33
14 Concentration-time relationship for 99% inactiva-
tion of poliovirus 1 singles at 5, 15 and 25°C . . 34
15 Concentration-time relationship for 99% inactiva-
tion of poliovirus 1 singles and bentonite adsorbed
poliovirus 1 singles at 5°C at pH? 35
16 Concentration-time relationship for 99% inactiva-
tion of poliovirus 1 singles and bentonite adsorbed
poliovirus 1 singles at 15°C at pH7 36
17 Concentration-time relationship for 99% inactiva-
tion of poliovirus 1 singles and bentonite adsorbed
poliovirus 1 singles at 25°C at pH7 37
18 Survival curve comparison of the inactivation
kinetics of poliovirus 1 singles (control) to
the polio-bentonite complex at 6.25 NTU at 12.0
mg/1 chlorine dioxide using the dynamic apparatus
at 5°C at pH7 39
19 Survival curve comparison of the inactivation
kinetics of poliovirus 1 singles (control) to
the polio-bentonite complex at 6.7 NTU at 14.3
mg/1 chlorine dioxide using the dynamic apparatus
at 5°C at pH7 40
20 Survival curve comparison of the inactivation
kinetics of poliovirus 1 singles (control) to
the polio-bentonite complex at 16.5 NTU at 11.8
mg/1 chlorine dioxide using the dynamic apparatus
at 5°C at pH7 41
21 Relationship of the product of concentration of C102
times the temperature versus the rate of inactiva-
tion of the unassociated poliovirus, and the <_ 5
and > 5 <_ 17 NTU groups of the poliovirus-bentonite
complexes 42
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FIGURES (continued)
Number
22 Concentration-time relationship for 99% inactivation
of BGM cell-associated poliovirus at various tur-
bidities compared to the 99% inactivation curve
for unassociated poliovirus at 5 and 25°C at
pH7
XI
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TABLES
Number Pag.
1 Human Enteric Viruses and Their Associated
Diseases 4
2 Infective Doses of Viruses for Man
. 6
3 Response of Rabbits to Low Doses of Poxviruses
Administered in 1 ym Aerosols 6
4 Minimal Infective Doses of Virus 7
5 Thermodynamic Values for Unassociated Poliovirus,
<_ 5 NTU Poliovirus-Bentonite Complex, and the
> 5 <_ 17 NTU Poliovirus-Bentonite Complex 43
6 Time for 99% Inactivation at Various C102 Concentra-
tions for Unassociated-Poliovirus 1 as Compared to
BGM Cell-Associated Poliovirus 1 at Various Turbidity
Levels at 5°C 46
7 Time for 99% Inactivation at Various Cl02 Concentra-
tions for Unassociated-Poliovirus 1 as Compared to
BGM Cell-Associated Poliovirus 1 at Various Turbidity
Levels at 25°C 47
8 Comparison of Rates of Inactivation k (log,n/sec),
For Cell-Associated Poliovirus to Unassociated
Singles Poliovirus at Various Chlorine Dioxide
Concentrations Using the Kinetic Apparatus at 5°C ... 48
9 Comparison of Rates of Inactivation k (log., /sec) ,
for Cell-Associated Poliovirus to Unassociated
Single Poliovirus at Various Chlorine Dioxide
Concentrations Using the Kinetic Apparatus at 25°C. . . 49
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ACKNOWLEDGMENTS
The financial sponsorship of this research by the Munici-
pal Environmental Research Laboratory of the U.S. Environmental
Protection Agency is gratefully acknowledged. The cooperation,
continuous interest, encouragement, and patience of the Project
Officer, Dr. John C. Hoff, is especially warmly acknowledged.
We are deeply indebted to Dr. Louis Laushey, Head of the
Department of Civil and Environmental Engineering, College of
Engineering, University of Cincinnati, for his valued criticisms,
active support, and friendship.
The helpful contributions of Dr. Shih Lu Chang, Health
Effects Research Laboratory, U.S. Environmental Protection
Agency, and Dr. Gerald Berg, Environmental Monitoring and
Support Laboratory, U.S. Environmental Protection Agency, are
particularly appreciated.
This research was performed in the Department of Civil and
Environmental Engineering, College of Engineering, University
of Cincinnati.
XI11
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SECTION 1
INTRODUCTION
OBJECTIVES OF THE STUDY
This research study (a) investigated the influence of par-
ticulates in the water on the viral disinfection process using
chlorine dioxide (ClO2) as the disinfectant and bentonite as the
particulate; (b) evaluated the disinfection capabilities of
chlorine dioxide using enteroviruses as the test viruses, along
with Escherichia coli for comparative purposes as the reference
bacterium, at different levels of chlorine dioxide, temperatures,
contact times, pH values, and concentrations of particulate
matter; (c) determined the effect of viral aggregation on survi-
val of the test microbes during the disinfection process through
characterization of the virus inocula by electron microscopic
assay of the viral units; and (d) evaluated the effect of the
disinfectant on enterovirus-associated animal cells which simu-
late naturally occurring cell-associated viruses excreted from
the intestinal tract of man.
The use of chlorine dioxide as a disinfectant in water and
wastewater supplies is proposed for several reasons. For drink-
ing water, one of the advantages is its reduced reactivity with
precursor organics in water to form chlorinated organic com-
pounds which may be carcinogenic. Investigations ' ' have
shown that C102 forms lesser quantities of trihalogenated
methanes (THM) (i.e., chloroform, bromodichloromethane, dibromo-
chloromethane, and bromoform), than would be caused by the use
of chlorine in the same waters. For the disinfection of waste-
water, C102 does not react with ammonia commonly found in waste-
water to form the less effective chloramines.
Symons et al. discussed how levels of trihalomethane could
be reduced using one or more of the following changes in water
treatment:
(a) Treatment to remove the trihalomethanes already
present;
(b) Treatment to remove precursor concentrations prior to
chlorination;
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(c) Modification of the chlorination practices, such as
changing the points of application; or
(d) Replacement of chlorine with an alternative disinfect-
ant such as ozone or chlorine dioxide.
It is this last alternative, the viricidal effectiveness
of replacement of the chlorine disinfectant by chlorine dioxide,
that was evaluated. As pointed out by Symons ejb al. if any
disinfectant or combination of disinfectants is to~~replace
free chlorine as the most commonly used disinfectant, several
criteria must be met. The disinfectant must be easily gen-
erated and in widespread use; it must be a good biocide,
provide an easily measured residual; produce fewer undesirable
by-products than does free chlorine; and must be cost-effective.
In this report we can only focus attention on the biocidal,
predominantly viricidal, effectiveness of chlorine dioxide.
Although chlorine dioxide in the absence of chlorine does
not produce trihalomethanes, chlorine dioxide does introduce
chlorite ion upon partial reduction. Research with animals
(cats) has shown that chlorite has a deleterious effect on
red blood cell survival rate at levels above 10 mg/liter.
Therefore, a chlorine dioxide dosage limit of 1.0 mg/liter has
been proposed by the U.S. Environmental Protection Agency to
prevent potential adverse effects on sensitive individuals
particularly children. Although this proposed limit may be
increased as a result of subsequent studies, one of the more
pertinent objectives of our studies was to ascertain the
levels of chlorine dioxide necessary to inactivate animal
viruses in water.
Another primary objective of our study was to investigate
the effect of particulates in water on the disinfection of
viruses, using chlorine dioxide as the disinfectant and benton-
ite as the particulate. These latter studies were done for
comparative purposes at different levels of chlorine dioxide,
temperatures, contact times, pH values and concentrationsvof
particulate matter. Finally, the effect of viral aggregation
on the survival of the test microbes during the disinfection
process was determined through characterization of the virus
inoculum by electron microscopic assay of the viral units.
Since turbidity may interfere with disinfection efficiency,
the turbidity Maximum Contaminant Level (MCL) was reduced from
5 turbidity units (TU) to one unit in the "Interim Primary
Drinking Water Regulations". Thus, our investigations provide
information as to the actual interference of particulates with
inactivation of viruses by the alternative disinfectant chlorine
dioxide.
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BACKGROUND OF THE STUDY
Information available concerning the inactivation of viruses
and the destruction of bacteria in water by chlorine dioxide were
limited. Information available was equivocal because standard
conditions for testing had not been used, adequate analytical
techniques to differentiate between chlorine dioxide and the var-
ious other chlorine residuals were not available, and the methods
used for chlorine dioxide preparation most probably introduced
interfering substances which would contribute to the decomposi-
tion of chlorine dioxide but would also yield erroneously high
chlorine dioxide values on iodometric analysis. Also, there
was a lack of quantitative data on the aggregate size of the
viruses in the inoculum, and the effect of particulates on viral
and bacterial inactivation mechanisms. Additionally, there had
not been available until now a convenient, accurate technique,q
i.e. the dynamic (flowing stream-rapid mix) apparatus of Sharp ,
for the measurement of short-time inactivation of viruses and
other microbes by disinfectants. The results of Benarde et al.
indicated that although chlorine and chlorine dioxide residuals
were present in their test system after five minutes contact
time, the major bactericidal reductions noted by them occurred
within the first minute of contact time and did not occur appre-
ciably thereafter. Thus, Sharp's dynamic (flowing stream-rapid
mix) apparatus was ideally suited for yielding kinetic data for
short reaction times of less than one minute.
Precise knowledge concerning the inactivation of viruses in
water assumes greater importance as man turns to an ever increas-
ing degree to the re-use of his neighbor's upstream wastewater.
Since sewage-contaminated water is a potential health hazard,
an awareness of the efficiency of applied disinfectants such as
chlorine dioxide on human enteric pathogens has increased signif-
icance. This is particularly true with viruses which are con-
siderably more resistant than the bacteria. Over 100 new human
enteric viruses have been described since the investigations of
Enders et al. on viral propagation techniques using tissue
cultures. Enteric viruses are the most important virus agents
infective for man known to be present in water and wastewater.
This group includes all viruses known to be excreted in quantity
in the feces of man; they are listed in Table 1 along with their
associated diseases. Thus, the enteric viruses consist of the
enteroviruses (poliovirus, coxsackievirus, and echovirus), infec-
tious hepatitis, adenoviruses, and reoviruses. Other viruses
may be ingested by man (e.g. influenza, mumps, and cold or fever
sore viruses), and may also be later isolated from his feces.
However, these latter are not believed to be particularly signify
icant in disease transfer via contaminated water. Clark et al.
pointed out that since enteric viruses are found in the feces
of infected individuals and are readily isolated from urban sew-
age, especially in the late summer or early fall, they may enter
water supplies and present health hazards to humans. However,
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TABLE 1. HUMAN ENTERIC VIRUSES AND THEIR ASSOCIATED DISEASES5
Major subgroup
Number
of
types
Associated disease
Poliovirus
Coxsackievirus 23
Group A
Group B 6
Echovirus 31
Infectious
hepatitis
Adenovirus 31
Reovirus 3
Paralytic poliomyelitis, aseptic
meningitis
Herpangina, aseptic meningitis,
paralytic disease
Pleurodynia, aseptic meningitis,
and infantile myocarditis
Aseptic meningitis, fever and
rash, diarrheal disease,
respiratory infections
1(?) Infectious hepatitis
Respiratory and eye infections
Fever, respiratory infections,
and diarrhea
aFrom References 12, 13, 14, 15
DType 23 was found to be Identical with echovirus tyje 9,
and A23 has been dropped and the number is unused.
"Echovirus serotypes 10 and 28 have-now been reclassified,
and these numbers are now unused.
Isolation uncertain.
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it was noted that the number of recognized water-borne outbreaks
of enteric virus disease was not large, which indicated that
many may not be reported or understood to be viral in origin.
The enteric virus density of domestic sewage has been esti-
mated atfi700 virus units per 100 ml of sewage. Northington
j_t>
e_t al. while studying the health aspects of wastewater re-use,
noted that if such sewage underwent activated sludge treatment
with a subsequent virus removal efficiency of 80 to 90 per-
cent, i^'lb'-L/'lbthe secondary effluent would contain 70 virus
units per 100 ml. Further flocculation processes would effect
a 90 to 99 percent virus reduction so that the tertiary efflu-
ent would contain about 1 to 7 virus units per 100 ml. A figure
of 5 units per 100 ml was then assumed to be in the renovated
water prior to chlorination. If a 99.99 percent reduction of
virus units occurred after chlorination, the virusfidensity would
be reduced to 1 unit per 50 gallons. Thus, 1 x 10 virus units
could be present in a 50 million gallon per day water supply.
If 0.2 percent of this water is consumed as drinking water, about
2000 virus units could be ingested daily by consumers, say
in a metropolitan area of 250,000 to 500,000 persons.
The importance of such low level transmission to man is
evident when consideration is given to what constitutes a mini-
mal virus dose capable of producing infection and disease in man.
Plotkin and Katz reviewed the available literature concerned
with the minimal dose of viruses that would be infective for
man via the oral route, to infect a human if it comes in contact
with susceptible cells. Subsequent experimentation by these
workers with the attenuated poliovirus demonstrated that one
tissue culture unit (1 TCID,-0) constituted an infectious dose
(see Table 2). More-recent animal studies (see Table 3) by
Westwood and Sattar support the conclusion of Plotkin arid
Katz, and coupled with other evidence in the literature (see
Table 4) suggests a near-parity in the cell-infective doses of
a wide array of viruses and their infective doses for various
hosts. In addition, recent research has focused attention upon
the infectivity of particulate-associated viruses. For e^fm§5e26
Moore e_t a!L. have presented data that reaffirm findings ' '
that certain viruses associated with suspended particulates are
infective, by finding that most of their test viruses were in-
fective by plaque assay in their particulate-absorbed form. Thus,
monitoring of environmental virus levels must account for not
only free virus but also for those that are solids-associated.
The studies reported herein extend these cited particulate virus
studies by viewing the survival characteristics of enteroviruses
associated with particulates after dosing with chlorine dioxide.
The use of enterovirus-associated animal cells will focus atten-
tion on the survival characteristics of such cell-associated
viruses after application of varying levels of chlorine dioxide.
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TABLE 2. INFECTIVE DOSES OF VIRUSES FOR MAN*
Virus
Poliovirus 1
(SM strain)
Poliovirus 3
(Fox strain)
Virus Route of
Dose** Inoculation
2 p.f.u. Oral
(gelatin
capsule)
1 TCDC_ Gavage
50
, c Percent of
Number of Persons
Persons Infected
Inoculated
3 67
10 30
*From References 20 and 21
**Given as plaque-forming units (p.f.u.) or as the quantity of
virus that will infect 50% of the tissue cultures inoculated
(TCD5Q) -
TABLE 3. RESPONSE OF RABBITS TO LOW DOSES OF POXVIRUSES
ADMINISTERED IN ONE MICROMETER AEROSOLS22
Virus
Vaccinia
Rabbit pox
Rabbit pox
Rabbit pox
Dose: p.f.u.
of Virus
4
9
1
0.4
No. of
Rabbits
Exposed
3
4
3
3
No.
Infected
3
4
1
1
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TABLE 4. MINIMAL INFECTIVE DOSES OF VIRUS
22
Virus
Poliovirus 1(SM)
Poliovirus 3(Foxl3)
Coxsackievirus A21
Coxsackievirus B4
Influenza A(PR8)
Influenza A (Asian)
Parainf luenza 1,2,3
Parainf luenza 3
Rhinovirus
Rabbit pox
Vaccinia
Yellow fever
Foot & mouth
Newcastle disease virus
Infectious bronchitis
Host
Man
Man
Man
Man
Mouse
Mouse
Hamster
Hamster
Man
Rabbit
Rabbit
Man
Cattle
Chicken
Chicken
Dose
2.0
10.0
18.0
1.3
0.02
0.2
1.0
0.3
1.0
0.4
2.0
5.0
1.0
1.0
1.0
Unit
p. f .u.
TCD5()
TCD5()
Mouse LD
MP.EID50
MP.EID5Q
TCD5()
TCD5()
EID5Q
p.f .u.
p. f .u.
Mouse LDj-0
TCD5Q
EID50
EIDCA
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The use of chlorine dioxide as a disinfectant in water
supplies assumes great importance when consideration is given to
its reduced reactivity with precursor organics in water to form
chlorinated organic compounds which may be later identified as
carcinogenic. ' Investigations have shown that chlorine diox-
ide when used as a water disinfectant does not produce measurable
quantities of trihalogenated methanes (i.e. chloroform, bromo-
dichloromethane, dibromochloromethane, and bromoform) and that
when chlorine dioxide is used in combination with chlorine, chlo-
rine dioxide also appears to^have a retarding action on trihalo-
genated methane formation. ' Additional information, however,
is essential concerning not only the usefulness of chlorine diox-
ide in minimizing chlorinated organic formation, the possible
toxicity of the organic by-products resulting from the reaction
of chlorine dioxide with organic matter in water, the toxicity
of chlorite and chlorate (possible products o| |he reactions of
chlorine dioxide when added to natural water) ' but also con-
cerning its disinfecting capability in regard to animal viruses
of pathogenic significance in water supplies. As chlorine diox-
ide becomes a more used disinfectant because of its lessened
capability to form chlorinated organics, more information is
required concerning its disinfecting capability and factors that
influence such ability.
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SECTION 2
CONCLUSIONS
In summary, CIO2 has been found to be an excellent disin-
fectant even when compared to chlorine, especially at the pH of
most drinking waters. The test viruses were found to be signi-
ficantly more resistant to disinfectants than the bacterial
fecal indicator organism, E. coli. Therefore, present micro-
biological standards for water quality need to be reevaluated to
include a disinfectant standard for the destruction of animal
viruses of enteric origin which are more resistant to inactiva-
tion by chlorine compounds and chlorine dioxide than the coliform
bacteria.
Variations in disinfection rates occur due to viral aggre-
gation even with the same virus type. This affect was clearly
seen when the data for aggregated virus, which would be similar
to the virus state in the natural environment, is compared to
the inactivation rates of the unassociated single poliovirus
preparation. Thus, this indicates a need for closer examination
of the aggregated state of the virus when consideration is given
to the time of exposure of the virus to the disinfectant residual
to insure proper viral destruction.
Chlorine dioxide inactivation of cell-associated poliovirus
versus unassociated poliovirus showed no trend towards protec-
tion at the turbidity levels examined. This is believed due to
the cell-associated poliovirus 1 existing in a "singles" or non-
aggregation state, and that the cellular material is readily
oxidized by the chlorine dioxide.
Finally, a correlation exists between bentonite protection
of poliovirus 1 during disinfection at increasing temperatures
and increasing turbidities, i.e. as the temperature and benton-
ite turbidity increases, the disinfection efficiency decreases
for the bentonite adsorbed poliovirus.
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SECTION 3
RECOMMENDATIONS
1. Methodology guidelines should be established as to disin-
fection apparatus (i.e. kinetic, dynamic or other), and
viral (and other raicrobial) preparations (i.e. singles
or aggregates) used in disinfection studies.
2. A complete literature survey should be conducted to compare
thermodynamic parameters to the site or sites of inacti-
vation for viruses and bacteria by various inactivation
agents.
3. Feasibility studies on the cost effectiveness of reducing
the turbidity levels from 5 to 1 NTU in drinking water
treatment should be determined. Implementation of a re-
duced turbidity (to 1 NTU) level is recommended in light
of our studies with poliovirus-adsorbed bentonite.
4. The effectiveness of chlorine dioxide as a suitable drink-
ing water disinfectant should be determined at the treat-
ment plant scale. Cost of conversion of present day
"chlorine" water treatment plants to chlorine dioxide use
should be evaluated. Different types of water should be
used, such as surface versus ground.
5. Analyses of the chemical species formed after the use of
chlorine dioxide in drinking water treatment must be fully
assessed, along with the formed chemical species' concen-
trations, properties, and mutagenicity/carcinogenicity.
6. Toxicological and epidemiological studies should be con-
ducted on finished drinking water disinfected with chlor-
ine dioxide.
7. Inactivation kinetics of various enteric microorganisms
using chlorine dioxide in conjunction with other drinking
water disinfectants (e.g. chlorine) should be determined.
8. Analysis of the mechanism of inactivation of chlorine
dioxide, as has been done for chlorine, iodine, and other
disinfectants, should be determined with whole and iso-
lated viral components (i.e. naked nucleic acids).
Nucleic acids may be viable inside of disinfectant de-
10
-------
stroyed viral protein, or may be released from disinfec-
tant damaged or incompletely formed viral particles.
Therefore, the rate of destruction of the naked or
partially protected nucleic acid should be investigated
along with that of whole, intact virions.
11
-------
SECTION 4
MATERIALS AND METHODS
Chlorine Dioxide Generation
A stock solution of chlorine dioxide (ClO2) was prepared by
the generation of C102 gas by the following reaction:
The C1O? stock solution was generated in the apparatus depicted
in Figure 1. The Cl02 gas was the result of the reaction of a
sodium chlorite (NaC102) solution (4.0 g/50 ml deionized dis-
tilled water) and a potassium persulfate (K2S2Ofi) solution
(2.0 g/100 ml deionized distilled water). The evolved gas was
swept from solution (vessel 1) by purified nitrogen gas and passed
through a column of sodium chlorite (vessel 2) to absorb any
chlorite gas or volatilized hypochlorous acid that might also be
present. Any sodium chlorite dust was retained in an empty
vessel (vessel 3) prior to collection of the gas in deionized
distilled water (vessel 4) held at 5°C. The stock solution was
prepared prior to experimentation, and had a concentration of
500 to 1,000 mg/1 Cl02after 15 to 20 minutes of generation time.
Chlorine Dioxide Analysis
The concentration of C102 was determined by the DPD (diethyl-
p-phenylene diamine) Method or Palin ' ' and as also set forth
in the 14th Edition of . Standard Methods for the Examination of
Water and Wastewater. This is a titrimetric and colorimetric
procedure, with DPD (DPD powder #1) as the colorimetric indicator
and ferrous ammonium sulfate (FAS) as the titrant. The volume
of titrant used to neutralize the pink color produced by the indi-
cator in a 100 ml sample is multiplied by 1.9 and expressed as
mg/1 Cl02 . The DPD Method was used to calibrate a Gary 14
Spectropfiotometer for determination of the C102 concentration in
the bentonite turbidity studies.
Preparation of Virus Stocks
The animal viruses used in these studies were poliovirus 1,
Mahoney strain, and coxsackievirus A9. The poliovirus 1 was
prepared by two different methods. In both methods the polio-
viruses were grown in monolayers of Buffalo Green Monkey (BGM)
12
-------
1. Potassium persulfate & sodium chlorite solution
2. Dry sodium chlorite
3. Empty trap
4. 5°C double distilled water
U)
Figure 1. Apparatus for the preparation of chlorine dioxide stock solution.
-------
kidney continuous cell line obtained from Cercopithecus aethiops,
the African Green monkey. The coxsackievirus A9 was grown in a
primary cell line also obtained from the kidney of Cercopithecus
aethiops.
Poliovirus 1 was prepared from BGM cells which were infected
approximately 24 hours earlier at a high multiplicity per cell
and exhibited a definite cytopathological effect (CPE). The
infected cells and fluids were collected and subjected to freez-
ing and thawing twice to -70 C to release the virus particles
from the cells. The virions were partially purified by differ-
ential centrifugation (i.e. 30 minutes at 20,000 rpm); dialysis
of the supernatant against polyethylene glycol; and finally by
ultra-centrifugation (i.e. 2 hours at 50,000 rpm). All centrif-
ugation was done using a Beckman L2-65B Ultra-Centrifuge with a
60 Ti rotor. The resultant virus pellet was repeatedly resus-
pended and re-pelleted with demand-free water until the virus
suspension was made C102 demand-free. The coxsackievirus A9
was prepared as just stated for the poliovirus 1, except it
was grown in a primary cell line (as previously stated) and the
dialysis step was eliminated.
A poliovirus 1 preparation of high purity and containing
mostly single virions, as determined bv_electron microscopy, was
prepared by the method of Floyd et. al. BGM monolayers were
inoculated with poliovirus 1 at a multiplicity of 100 plaque
forming units (PFU) per cell. The virus was allowed to adsorb
for 1 hour at 37 C, after which a maintenance medium of Minimum
Essential Medium (Eagle) was added and the cells were further
incubated for 11 hours. The infected cells were then removed
from incubation and chilled to 4°C. The maintenance medium
was now separately collected and centrifuged at 250 x g for 10
minutes to harvest the cells remaining in the medium itself.
The chilled cells remaining in the containers were then washed
twice with phosphate buffered saline. The BGM monolayers were
scraped from the bottles, harvested by centrifugation (250 x g
for 10 minutes) and pooled with the cells collected from the
maintenance medium. The combined BGM cells were resuspended
with phosphate buffered saline (6 ml) and the virus extracted
by the addition of Freon 113 (4 ml) followed by homogenization
(2 minutes) in a Waring blender, with separation of the aqueous
phase from the freon phase by centrifugation (800 x g for 10
minutes). This freon extraction was carried out 3 times with
addition of phosphate buffered saline each time. The virus-
containing aqueous phase was collected and held in an ice bath.
The aqueous extractions weire pooled and brought to a final volume
of 20 ml. The virus was now further purified and concentrated
by density gradient centrifugation. The aqueous-virus phase
was layered onto a 10 to 30% (wt/wt) sucrose gradient made with
0.05 M phosphate buffer (ClO2 demand-free) at pH 7.2. The
gradient is centrifuged at 25,000 rpm in a Beckman L2-65B
14
-------
Ultracentrifuge for 2.25 hours at 4°c. Fractions of 2 ml each
were collected from the centrifuge tubes and examined by elec-
tron microscopy for the presence of virus. All relevant frac-
tions were pooled and stored at refrigerator temperature with-
out any attempt to remove the sucrose.
Preparation of Escherichia coli (ATCC 11229)
The Escherichia coli used in these studies were grown on
trypticase soy agar slants for 16 to 18 hours at 35 C. The
bacterial cells were then removed from the slants by washing with
0.05 M phosphate buffer (C102 demand-free). The E. coli was
then pelleted and repeatedly washed with phosphate buffer at
2500 rpm for 10 minutes in an International Centrifuge (size 2)
using an International 250 rotor until the suspension was made
demand-free. The final E. coli suspension was adjusted to a
concentration of 10 bacteria/ml by optical density using a
Klett-Summerson photoelectric colorimeter.
Preparation of Bentonite for Turbidity Studies
A bentonite suspension used in the turbidity studies was pre-
pared by the method of Stagg e_t al. The procedure consisted
of adding 5 grams of bentonite~~to 2000 ml sterile deionized
distilled water. The bentonite suspension was mixed for 2 hours
and then left undisturbed for 24 hours to precipitate out the
larger bentonite particles. The top liter of the suspension was
carefully removed and then washed 3 times with sterile demand-
free water. The bentonite was harvested by centrifugation,at
800 x g for 15 minutes after each washing. The washed bentonite
was resuspended to a volume of 2000 ml with sterile demand-free
water and then passed through a sterile, prewashed 90 mm, 0.45
ym porosity, type HA membrane filter (Millipore). The filter-
trapped bentonite was removed from the filter and then resus-
pended in sterile demand-free water. This suspension was 'then
centrifuged at 2100 x g for 20 minutes with the resultant pellet
resuspended to a final desired volume with sterile demand-free
water. This procedure yielded bentonite particles of approxi-
mately 2 ym or less. This bentonite stock was stored at 4°C.
Turbidity was measured in Nephelometric Turbidity Units (NTU)
using a Hach 2100A Turbidimeter.
Preparation of Poliovirus-Bentonite Suspensions
Virus-bentonite suspensions were prepared by allowing the
poliovirus 1 of high purity and mostly single virions to asso-
ciate with the bentonite for 1 hour with constant mixing in
0.05 M phosphate buffer (ClO2 demand-free). This contact time
allows the virus to adsorb to the bentonite. The virus-benton-
ite complex is harvested by centrifugation at 4,600 x g for 20
minutes. The supernatant was discarded and the resulting pellet
was resuspended with phosphate buffer to the desired volume.
15
-------
The virus associated with bentonite was then ready for use in
experimentation.
Preparation of Cell Associated Poliovirus
Cell associated-poliovirus 1 was prepared from virus infected
BGM cells. BGM monolayers were inoculated with poliovirus 1 at
a multiplicity of 100 PFU/ml. The virus was allowed a 1 hour
attachment period to the BGM cells at 37 °C prior to addition of
maintenance medium and further incubationQ(37 C) for 11 hours.
The infected cells were then chilled to 4°C. The monolayers
were then physically removed from the containing vessel surface.
The resultant medium and cells were centrifuged at 2000 rpm in
an International Centrifuge (size 2) employing an International 240
rotor. The BGM cells containing poliovirus 1 were washed 6 times
prior to experimentation with C102 demand-free 0.05 M phosphate
buffer. The virus-cell complex is then resuspended in this
buffer to the desired volume. Despite these repeated washings
this preparation still maintained a considerable C10_ demand due
to the BGM cells.
Electron Microscopy Viral Assay Technique
The virus inocula was quantitated and characterized-by elec-
tron microscopy using the kinetic attachment procedure. This
technique consisted of placing a drop of virus suspension, 1 mm
thick, on an aluminum-coated collodian film covering a standard
electron microscope grid. Precautions were taken to prevent
drying by keeping the area around the grid moist. Virus parti-
cles, by diffusion and Brownian motion, came into contact with
the grid surface; and due to the difference in the charges be-
tween the aluminum (+) and the virus (-), they attached to the
grid. The drop was washed away after a 30 minute contact time.
Washing was continued for 30 minutes to remove all unattached
virus before drying was allowed. The grids were then shadow
cast with chromium for examination and counting purposes. The
grids were examined at a low magnification (5000 X) in an elec-
tron microscope (JEM 100 B, JEOL Co.) with the resulting random-
ly-taken micrographs being projected onto a gridded screen for
virus counting and characterization.
Experimental Procedures
The C10~ disinfection studies-were performed using the ki-
netic (stirfed beaker) apparatus ' and the dynamic (flowing
stream-rapid mix apparatus).
The kinetic apparatus (Figure 2) consisted of CIO- test and
control solutions in stainless steel beakers held at the desired
temperature in a water bath, and stirred throughout by glass
stirring rods connected to an overhead stirring device. Each
experiment consisted of five experimental solutions. Two of
16
-------
WATER INLET
X
glan propel kx
38"
Q
WATER BATH
*3 C O C
Figure 2. Kinetic (stirred beaker) apparatus '
-------
these solutions were controls to assure that the test organisms
were not adversely affected by the pH of the buffer system, the
chemicals themselves or the experimental temperature. The re-
maining three experimental solutions contained test levels of
C1C>2 - The buffering system used in these studies was 0.05 M
phosphate buffer. Each stainless steel beaker (capacity 600 ml)
contained 400 ml of solution. These solutions were constantly
stirred by the glass stirring rods throughout the experiment at
100 rpm. Prior to and during experimentation the beakers and
their contained solutions were equilibrated and maintained at
the desired test temperature by a carefully regulated waterbath.
The actual, timed-experiment, began individually at the inocula-
tion of the test organism into the rapidly stirring experimental
solution. Samples of 5 ml each were withdrawn at specified con-
tact time intervals from the experimental solution and placed
into 5 ml of CiCU-neutralizing thiosulfate solution with subse-
quent serial dilutions made in 0.05 M phosphate buffer.
The dynamic apparatus (Figure 3) is ideally suited for yield-
ing kinetic data of short reaction times of less than one minute.
The apparatus provides rapid injection and mixing of the inocu-
lum in the flowing stream of buffer-containing ClO2. The ClO--
buffered water (i.e. 0.05 M phosphate buffer) is contained with-
in a 5 gallon Nalgene carboy. The buffered water turbulently
flows through a 1 cm (I.D.) polyethylene tubing by maintaining
a Reynolds number greater than 3000. A 5 ml inoculum contained
within a syringe is injected into the turbulent flowing stream
by a constant drive motor. Rapid mixing of the inoculum is
assured by the presence of a mixing disc within the stream at
the point of inoculation and also by the 3000 Reynolds number.
Samples of 1 ml were rapidly withdrawn from the turbulent-flow-
ing stream at the syringe sampling ports at appropriate time
intervals by spring-loaded 5 ml syringes. The sample was imme-
diately mixed with 1 ml of sodium thiosulfate which was contained
within each syringe in order to stop the reaction. The time of
transit of the moving stream of water from the injection point
to a sampling point was determined by the flow rate and the
distance involved.
Microorganism Assay Procedure
The surviving E._ coli from these studies were recovered on
surface inoculated plates of tergitol-7 agar supplemented with
triphenyl tetrazolium chloride. The plates were incubated for
24 hours at 37°C prior to them being counted. The use of this
medium was previously shown not to interfere with recovery of
the test bacterium. 40
The surviving poliovirus 1 and coxsackievirus A9 from these
studies were assayed by the plaque forming system in BGM
cells. ' The association of bentonite with poliovirus has
18
-------
vo
CI02
&
BUFFER
CONTINUOUS FLOW APPARATUS
for
VIRUS DISINFECTION STUDIES
VIRUS
INJECTION
MOTO
m**
SAMPLING PORTS
Figure 3. Dynamic (flowing stream-rapid mix) apparatus.
-------
previously been shown not to affect the plaque forming ability
of the virus.
Tissue Culture Procedures
BGM cultures for viral assay were prepared in 6 oz. rubber-
lined, screw-cap, prescription bottles (Brockway Glass Co., Inc.),
The cultures were maintained in equal concentrations of Minimum
Essential Medium (Eagle) or MEM's from Grand Island Biological
Company and L-15 (Leibovitz) from Kansas City Biological contain-
ing 0.22% NaHC03. Then 2-5% heat-inactivated fetal calf serum
(from Flow Laboratories) was added, and the solution was adjusted
to pK 7.6. Each milliliter of medium contained 100 units peni-
cillin, 100 yg streptomycin, 0.0125 mg tetracycline, and 1.0 ug
amphotericin B. Stock cultures of BGM cells were maintained in
a similar manner except for a 10% fetal calf serum concentration
and the size of the vessels in which the cells were contained.
The cultures were grown and maintained at 37°C.
The BGM cells were prepared for inoculation by washing the
cell monolayer once with Earle's Lactalbumin Hydrolyzate with
Earle's salts (ELH) (Grand Island Biological Co.) without any
fetal calf serum, but containing 0.22% NaHCO_., 100 units peni-
cillin, 100 Mg streptomycin, 0.0125 mg tetracyline and 1 yg
fungizone per milliliter. The Earle's-washing medium was re-
moved prior to inoculation of 0.5 ml per bottle of inoculum. The
virus was allowed to attach to the BGM cells for 2 hours at ambi-
ent temperature or 1 hour at 37°C. The infected cells were then
"overlayed" with MEM's containing 2% fetal calf serum (Flow Lab-
oratories) , 1.5% Difco Bacto-agar, 1% milk (California "Real-
Fresh"), 0.1 mg/1 MgCl2, 15 yg/ml neutral red (Baltimore Bio-
logical Lab.), along with the constituents as described for the
Earle's-washing medium. The overlayed inoculated BGM cultures
were inverted and incubated at 37°C. Plaques were first enumer-
ated at 48 hours and counted every 24 hours thereafter for the
next 3 days.
20
-------
SECTION 5
RESULTS AND DISCUSSION
CHLORINE DIOXIDE ALONE
Numbers derived from the control beakers were used to
establish 100% survival times. Survival curves were obtained
by plotting the log of the percent survival against the time
of exposure to C10-, as shown in Figure 4. This figure shows
a typical disinfection curve of poliovirus 1 in contact with
0.87 mg/1 C102 at 15°C at pH 7.0. On the horizontal axis is
plotted time in minutes of exposure of the virus to CIO-/
whereas on the vertical axis is plotted the percent survival
of the virus.
Figure 5 shows a typical disinfection curve for E coli
using 0.16 mg/1 C102 at 15°C at pH 7.0. The 99% inactivation
or destruction points (which are the 1% survival points) were
then extrapolated from the survival curves, as shown in Figure
4, to give the time necessary for 99% inactivation of viruses
or destruction of bacteria. These 1% survival points for each
of the CIO- levels used were then replotted on log-log paper
to show CIO- concentration vs the previously determined 99%
inactivation or destruction times. These new concentration-
time plots, as shown in Figure 6, were used to compare the
rates of disinfection of E_ coli, poliovirus 1 and coxsackie-
virus A9 by C10-. The closer a concentration vs time curve
lies to the lower lefthand corner of the qraph, the faster
the reaction, i.e., the quicker the inactivation or destruc-
tion of the microbes. From the relative positions of these
curves, it was found that poliovirus 1 was 8.9 times and
coxsackievirus A9 was 2.3 times more resistant than E coli
to C102 when compared at 15°C at pH 7.0. The data in all
cases formed a straight line that had a slope very close to
1.0. Therefore, it appeared that the reactions involved were
of the first order.
In addition to the 15°C data, tests were performed on
poliovirus 1 at 5°C and 25°C, as shown in Figure 7. This
was done to obtain the Q, Q or relative effect of a 10°C
change in temperature on the rate of inactivation of the
virus. As was expected, poliovirus 1 was inactivated faster
at higher temperatures, although not at an equal rate at
each 10°C increment.
21
-------
100
10
3
Cfl
« .1
.01
.001
.0001
1
Figure 4
Minutes
Inactivation of poliovirus 1 at 15°C at pH 7.0
in the presence of 0.87 mg/1 chlorine dioxide.
22
-------
2
C/5
H
Z
OS
tt
0U
SECONDS
Figure 5.
Destruction of Escherichia coli a't 15°C at pH 7.0
in the presence of 0.16 mg/1 chlorine dioxide.
23
-------
10
1.0
c*-Poliovirusl
0.1
^•Coxsackie-
virus A9
_ E.coli —*
i i i i ri i
i i i i i i
10
100
1000
SECONDS
Figure 6. Concentration-time relationship for 99% destruction
of poliovirus 1, coxsackievirus A9, and Escherichia
coli by chlorine dioxide at 15 C at pH 7.0.
24
-------
K>
U>
0.1
10
5°C
I i i i i i I I
i I I i I I i
100
1000
SECONDS
Figure 7. Concentration-time relationship for 99% inactivation
of poliovirus 1 at 5°C, 15 C and 25 C by
chlorine dioxide.
25
-------
The studies in Figure 8 were conducted to investigate the
effect of pH on inactivation of poliovirus 1 by CIO-. Chlorine
dioxide was found at 21°C at pH 9.0 to inactivate poliovirus 1
4.6 times faster than at pi! 7.0, and 8.3 times faster than at
pH 4.5.
Finally, the comparison in Figure 9 of the relative inac-
tivation of poliovirus by CIO- and other chlorine species shows
that on a weight basis. C10~was an efficient viricidal agent,
even when compared to HOC1. 35' 36'40' 41 The pH of the
chlorine-containing buffer solutions varied in order to produce
the desired chlorine species. For example, more than 95% of
the chlorine present was in the HOCl form at pH 6, while at
pH 10, 99.7% of the free chlorine existed as OC1.
THE EFFECT OF BENTONITE- AND CELL-ASSOCIATED TURBIDITY ON VIRUS
INACTIVATION USING THE DISINFECTANT CHLORINE DIOXIDE.
Poliovirus 1 Characterization and Quantitation
The electron microscopic kinetic attachment technique.
was used to characterize and quantitate the poliovirus 1 used
in these disinfection studies. The freeze-thawed poliovirus
preparation (Figure 10) on examination by electron microscopy
was found to have considerable cellular debris associated with
the virus particles. This made accurate non-biased viral
quantitation impossible. Non-random electron micrographs
gave-a biased estimate of viral quantitation to be 90.7% sin-
gle and 9.3% aggregated poliovirus particulates. The agareaa-
ted virus fraction consisted of 4.4% pairs; 0.1% triplets and
4.8% of > 5 virus particles. The freon extracted-density
gradient~poliovirus preparation (Figure 11) yielded no detect-
able debris on examination by electron microscopy. Analysis
of the individual density gradient fractions collected after
ultra-centrifugation yielded the relationship seen in Figure 12.
The greatest number of virus particles were found to occur in
fraction 13 or at a sucrose percentage of 22. The relevant
fractions were pooled. Electron micrographs taken in a random
fashion gave a non-biased estimate of the virus particle state
of the pooled gradient fractions to be 93.1% sinale and 6.9%
agareaated virions. The aggregated viral fraction was further
characterized and found to have 3.9% of the virions in pairs
and 3.0% of the virions existing in a state of >_ 5 virus particles,
Effect of Viral Aggregation on the Disinfection Process
The two poliovirus preparations differ primarily in their
aggregation states with the freon extracted-density gradient
preparation consisting of a greater percentage of single and
paired virus particles and a lesser percentage of >_ 5 viral
clumps as compared to the freeze.-thawed viral preparation.
When these two preparations were subject to disinfection with
26
-------
Ktr
1.0
0.1
15"
pH9
J 1 1 I I I I I
J I I ' ' ' ' '
SECONDS
Figure 8. The effect of pH on the inactivation of poliovirus 1
at 21 C at pH 4.5, 7 and 9, and at 25°C at pH7.
27
-------
1000
KX>
10 __
^v —
E 1.0 _
0.1
NHCI.
4.5)
NH2CI
(pH9)
HOC!
(pH6)
•Oil 1 I I Illlll I III Mill iii. mil i . . i mil t i i i u
.01 0.1 1.0 10 100 1000
Time in Minutes for 99% Inocfivation
Figure 9. Comparison of the relative inactivation of polio-
virus 1 by hypochlorous acid, hypochlorite ion,
monochloramine, dichloramine, and chlorine dioxide
at 15 C at different pH values.
28
-------
•
:
Figure 10. Electron micrograph of freeze-thawed "aggregated" poliovirus 1 preparation
depicting single (S) virions, clumped (C) virions, and cellular debris (De)
(63,690X).
-------
U)
G
Figure 11.
Electron micrograph of freon extracted-density gradient "singles"
poliovirus 1 preparation depicting viruses of single (S), double (D)
triple (T), and quadruple (Q) aggregation states (74,500X)
-------
Figure 12.
. o
6 8 10 12 14 )6 18 20
f'ac t i on Number
Analysis of the fractions obtained in the prepara-
tion of freon extracted—density gradient polio-
virus 1.
LEGEND
= number of virus particles
= percentage of sucrose
31
-------
C10_ at 5 C at pH 7 using the kinetic apparatus differences in
their inactivation kinetics were evident. A log-loa, concentra-
tion-time plot (Figure 13) for the 99% inactivation of the polio-
virus showed that the freon extracted-density gradient "singles"
virus preparation reached 99% inactivation at a rate 2.7 times
faster than the freeze-thawed "aggregated" virus preparation.
This difference was significant since it indicated a variation
in disinfection kinetics of the same virus type (i.e., polio-
virus 1, Mahoney strain) due to viral aggregation. Caution
must be taken then when comparing and interpreting differences
of disinfectant efficiencies without knowledge of the viral
aggregation state.
Temperature Effects on Viral Inactivation with Chlorine Dioxide
The effect of temperature on the viral inactivation kinetics
of C102 was determined at pH 7 at 5, 15, and 25°C using the freon
extracted-density gradient "singles" poliovirus and the dynamic
apparatus. The results for 99% inactivation of the virus are
represented on a log-loa, concentration-time plot (Figure 14),
with the curves closer to the left hand corner of the graph rep-
resenting the faster reaction rates. The 99% inactivation rates
were found to increase with increasing temperature. That is,
the inactivation of the virus at 15°C was 2.26 times faster than
at 5°C, while inactivation at 25°C was 4.25 times faster than at
5°C or 1.99 times faster than at 15°C. The mean Q,0 value for
these inactivation kinetics with C10_ was 2.13.
The Effects of Inorganic Turbidity on the Inactivation of
Poliovirus 1 by Chlorine Dioxide
Studies on the effect of inorganic turbidity on the dis-
infection of bentonite adsorbed-poliovirus 1 singles were done
at pH 7.0 and at 5, 15, and 25°C usinn the dynamic apparatus.
Only "singles" virus that had been adsorbed to the bentonite
particles were used in these studies. The results for 99% in-
activation of the poliovirus at these temperatures and turbid-
ities are graphically represented in log-log, concentration-
time plots (Figures 15, 16, 17). The left portion of each graph
represents the unassociated "singles" poliovirus-control, with
data points and curve for the 99% inactivation of the unassociat-
ed "singles" poliovirus. The right portion represents the polio-
virus-bentonite complex at various turbidity levels as depicted
by the data points shown for 99% viral inactivation. The solid
line on the poliovirus-bentonite complex side of the graph rep-
resented the 99% inactivation curve for the unassociated "singles"
poliovirus-control (without data points) as shown in the left
portion of each graph. At 5°C (Figure 15), there was no evident
trend towards protection from C102 inactivation offered by the
bentonite to the attached poliovirus with turbidities ranging
from 1.14 to 16.5 NTU's. A slight trend towards protection
from inactivation by the bentonite develops at 15°C (Figure 16),
32
-------
10
1.0
0.1
5'C
pH7
Poliovirus 1
A Aggregates
•Singles
25
100
Seconds
1000
Figure 13.
Concentration-time relationship for 99% inactivation of poliovirus
comparing single virions to aggregated virions.
-------
OJ
1001—
o
u
o>
10
pH 7
25C
I
10
Seconds
100
Figure 14. Concentration-time relationship for 99% inactivation of poliovirus 1
singles at 5, 15, and 25 C.
-------
100
10
u>
Ul
o>
Free Poliovirus
5C pH7
10
Polio-Bentonite
NIL
1.14
2.20
3.09
4.15
6.25
6.70
12.38
16.50
100 1
Seconds
10
100
Figure 15. Concentration-time relationship for 99% inactivation of poliovirus 1
singles and bentonite-adsorbed poliovirus 1 singles at 5 C at pH 7.
-------
100
cs
o
U)
(Ti
\
o>
10
15 C pH7
Free Poliovirus
I
Polio- Bentonite
NTH
* 0.64
* 1.65
o 2.03
o 2.48
• 3.93
• 5.18
^ 12.25
10
100 1
Seconds
10
100
Figure 16. Concentration-time relationship for 99% inactivation of poliovirus 1
singles and bentonite-adsorbed poliovirus 1 singles at 15°C at pH 7.
-------
100
o
u
_l
\
O)
10
25'C
Free Poliovirus
U)
pH7
Polio-Bentonite
o
o
0
K)
100 1
Seconds
10
o
NTU
1.35
1.67
2.29
3.22
6.30
7.94
10.20
14.10
1 1
1 L.
100
Figure 17. Concentration-time relationship for 99% inactivation of poliovirus ;
singles and bentonite-adsorbed poliovirus 1 singles at 25 C at pH 7
-------
with turbidity levels that vary from 0.64 to 12.24 NTU's. At
25°C (Figure 17), a definite trend toward protection by the
bentonite was evident. The 99% inactivation points of adsorbed
poliovirus at turbidities of from 1.35 to 2.29 NTU's at 25°C
were found clustered around the free poliovirus inactivation
curve. As the turbidity increased from 3.22 to 14.10 NTU's,
the 99% inactivation points were found further from the free
poliovirus inactivation curve, thus showing a definite protective
effect at higher levels of turbidity at 25°C. This data indicat-
ed- that the amount of protection from inactivation with the C10-,
by the bentonite to its adsorbed poliovirus increased with in-
creasing temperature and turbidity.
.Comparison of survival curves at 5°C and at pH 7 of the
poliovirus-bentonite complex to unassociated "singles" polio-
virus showed in each case examined that the poliovirus-bentonite
complex was inactivated at a rate faster than the unassociated
"singles" poliovirus. At 6.25 NTU and 12.1 mg/1 C102 (Figure 18),
99% inactivation of the poliovirus-bentonite complex was achieved
1.5 seconds faster than the unassociated virus. The same was
true for 6.7 NTU poliovirus-bentonite at 14.3 mg/1 C102 (Figure
19) and for 16.5 NTU poliovirus-bentonite at 11.8 mg/1 C102
(Figure 20), as the time for 99% inactivation was reached 7.6
and 5.75 seconds, respectively, faster than the unassociated
virus controls. These comparative survival curves verify the
results seen on the Van't Hoff plots, i.e., at 5°C there was
no apparent protection offered to the surface adsorbed polio-
virus due to the hentonite.
When the data was placed into turbiditv groupings of <^ 5
NTU' s and > 5 <^ 17 NTU' s and graphed along with the unassociated
"singles" poliovirus data onto a plot of rate of inactivation
versus the product of concentration times the temperature, a
linear relationship resulted(Figure 21). From this relationship
it was found that bentonite-adsorbed virus of the <_ 5 NTU aroup
was protected to an extent of 11.4% (or 88.6% unprotected) when
comparison was made to the unassociated poliovirus. The >5 <_ 17
NTU group was protected to 24.8% (or 72.2% unprotected).
Thermodynamic analysis of 'the data (Table 5) yielded mean
values for Qln/ Energy of Activation (E ), Enthalpy of Activation
(AH) and Entropy of Activation (AS) forathe unassociated polio-
virus, the <_ 5 NTU group, and > 5 <_ 17 NTU group. This informa-
tion gave us values which were consistent with those obtained in
protein denaturation reactions. ' ' ' b' b' ' This in-
dicated that the mechanism of inactivation for the poliovirus
by the CIO- seemed to be due to protein denaturation. The
values for the > 5 <_ 17 NTU group indicated that the bentonite
was interacting with the CIO-, thus, inhibiting the chlorine
dioxide's ability to react with the virus and cause its in-
activation.
38
-------
100
* Poliovirus
" Bentonite assoc.
Poliovirus(6.25NTU)
Figure 18.
SECONDS
Survival curve comparison of the inactivation kinet-
ics of poliovirus 1 singles (control) to the polio-
bentonite complex at 6.25 NTU at 12.0 nig/I chlorine
dioxide using the dynamic apparatus at 5 C at pH7.
39
-------
100
Poliovirus
Bentonite assoc.
Poliovirus (6.7NTU)
10
15
20
SECONDS
Figure 19. Survival curve comparison of the inactivation kinet-
ics of poliovirus 1 singles (control) to the polio-
bentonite complex at 6.7 NTU at 14.3 mg/1 chlorine
dioxide using the dynamic apparatus at 5°C at pH7.
40
-------
• Poliovirus
" Bentonite assoc
Poliovirus
(16.5 NTU)
SECONDS
Figure 20. Survival curve comparison of the inactivation kinet-
ics of poliovirus 1 singles (control) to the polio-
bentonite complex at 16.5 NTU at 11.8 nig/1 chlorine
dioxide using the dynamic apparatus at 5 C at pH7.
41
-------
0.50
0.40
0 0.30
c
o
u
O)
o
0.20
0.10 •
— Unassociated Poliovirus
•"• <5 NIL) Polio-Bentonite
— >5<17NTU Polio-Bentonite
10 20 30 40
10-4 °C moles/I
50
Figure 21.
Relationship of the product of concentration of Cl09
times the temperature versus the rate of inactivation
of the unassociated poliovirus, and the <_5 and >5
<17 NTU groups of the poliovirus-bentonite complexes.
42
-------
TABLE 5. T'HERMODYNAMIC VALUES FOR UNASSOCIATED POLIOVIRUS, <_5NTU POLIOVIRUS- BENTON-
ITE COMPLEX, AND THE>5<17 NTU POLIOVIRUS-BENTONITE COMPLEX.
Ul
GROUP
Unassociated
Poliovirus
< 5 NTU
Polio- Bentonite
> 5<17NTU
Polio- Bentonite
°10
2.13
2.35
1.56
a
cal/mole
12353
14020
7289
AH
cal/mole
11781
13448
6717
A S
cal/mole-deg
98
104
80
AE = Energy of Activation
AH = Enthalpy of Activation
AS ~ Entropy of Activation
-------
The Effects of Cellular Turbidity on the Inactivation of Polio-
virus~l by Chlorine Dioxide"~""
The disinfection of BGM cellassociated poliovirus by CIO
was done at pH 7 using the kinetic apparatus. The results ob-
tained were compared to "singles" poliovirus subjected to the
same disinfection methodology. The amount of cellular material
present was measured as turbidity in NTU's. The data generated
from these experiments were examined by various means to deter-
mine what role, if any, the cellular material plays in the dis-
infection reaction. When the data for the time necessary to
inactivate 99% of the cell-associated virus was graphed with
similar data for unassociated poliovirus (i.e., the control) on
a log-log, concentration-time plot and compared, no apparent
trend towards protection offered to the poliovirus by the
cellular material was evident at the turbidity levels examined.
These experiments were conducted at 5 and 25 C. These results
(Figure 22) are depicted at these temperatures with the data
points only for the cell-associated virus. The solid line at
both temperatures represents the 99% inactivation curve without
data points for the unassociated "singles" poliovirus. These
same data are numerically presented in Tables 6 and 7. Further
studies are needed to confirm these results, and the actual
state of the cell-associated virus preparation.
Analysis of the initial reaction rates, k(log,Q/sec)
(Tables 8 and 9) from survival curves for the two virus prep-
arations showed little variation between the obtained k values
at respective CIO.-., concentrations at 5 and 25 C. At 5 C and a
C107 level of 0.61 mg/1, the inactivation rate of the un-
associated poliovirus was equal to or slower than rates
obtained with lesser C102 concentrations used in cell-associated
studies. Similar results were seen at 25 C where 0.17 mg/1
C102 used with unassociated poliovirus produced a k of 0.12
log,Q/sec, which was slower than the cell-associated poliovirus
inactivation rates obtained at 0.14 and 0.16 mg/1 C109 with
different turbidities.
44
-------
5°C
NTU
• 1.10
-1.47
• 1.48
o2.00
25°C
NTU
•1.13
'133
• 3.10
1000
100
1000
Seconds
Figure 22
Concentration-time relationship for 99% inactivation of RGM cell-associated
poliovirus at various turbidities compared to the 99% inactivation
curve for unassociated poliovirus at 5 and 25 C at pH7.
-------
TABLE 6. TIME FOR 99% INACTIVATION AT VARIOUS C102 CONCENTRA-
TIONS FOR UNASSOCIATED POLIOVIRUS 1 AS COMPARED TO
BGM CELL-ASSOCIATED POLIOVIRUS 1 AT VARIOUS TURBIDITY
LEVELS AT 5°C.
Virus NTU mg/1 of
Preparation C10~
BGM Cell-Associated 1.10 0.54
Poliovirus 1
1.47 0.48
1.48 0.51
2.00 0.47
Unassociated Poliovirus 1 0.39
(Control)
0.45
0.49
0.52
0.57
0.61
0.78
0.98
99% Inactivation
Time (Seconds)
435
382
370
570
425
425
540
265
305
375
245
185
46
-------
TABLE 7. TIME FOR 99% INACTIVATION AT VARIOUS Cl02 CONCENTRA-
TIONS FOR UNASSOCIATED POLIOVIRUS 1 AS COMPARED TO
BGM CELL-ASSOCIATED POLIOVIRUS 1 AT VARIOUS TURBIDITY
LEVELS AT 25°C.
Virus NTU
Preparation
BGM Cell- Associated 1.13
Poliovirus 1
1.33
3.10
Unassociated Poliovirus 1
(Control)
mg/1 of
cio2
0.16
0.14
0.14
0.17
0.24
99'i Inact i vat ion
Time (Seconds)
342
310
389
282
175
47
-------
TABLE 8. COMPARISON OF RATES OF INACTIVATION k(log 1Q/sec),
FOR CELL-ASSOCIATED POLIOVIRUS TO UNASSOCIATED
SINGLES POLIOVIRUS AT VARIOUS CHLORINE DIOXIDE
CONCENTRATIONS USING THE KINETIC APPARATUS AT 5°C.
Virus Preparation
NTU mg/1
of CIO
(Final) "
Cell- Associated
Poliovirus 1
Unassociated Singles
Poliovirus 1
(Control)
1.10 0
1.47 0
1.48 0
2.00 0
0
0
0
0
0
.54
.48
.51
.47
.39
.52
.57
.61
.78
k(log , n/sec
0.009
0.011
0.010
0.009
0.005
0.013
0.015
0.009
0.025
48
-------
TABLE 9. COMPARISON OF RATES OF INACTIVATION k(log 1Q/sec),
FOR CELL-ASSOCIATED POLIOVIRUS TO UNASSOCIATED
SINGLES POLIOVIRUS AT VARIOUS CHLORINE DIOXIDE
CONCENTRATIONS USING THE KINETIC APPARATUS AT 25°C.
Virus Preparation
Cell -Associated
Poliovirus 1
NTU
1.13
1.33
3.1
mg/1 of CIO.,
(Final)
0.16
0.14
0.14
k (log1Q/sec
0.016
0.021
0.020
Unassociated Singles — 0.24 0.019
of Poliovirus 1
(Control) -- 0.17 0.120
49
-------
OTHER REPORTS BASED ON THIS RESEARCH
Additional published material, based on research conducted
under this grant includes the following:
Cronier, S., P. V. Scarpino, M. L. Zink, and J. C. Hoff.
Destruction by Chlorine Dioxide of Viruses and Bacteria
in Water. Abstracts of the Annual Meeting—1977,
American Society for Microbiology, N58 (1977).
Scarpino, P. V., S. Cronier, M. L. Zink, F. A. 0. Brigano,
and J. C. Hoff. Effect of Particulates on Disinfection
of Enteroviruses and Coliform Bacteria in Water by
Chlorine Dioxide. In: Proceedings AWWA Water Quality
Technology Conference, "Water Quality in the Distri-
bution System," Kansas City, Missouri, December 4-7,
1977, 2B-3:1-11 (1978).
Brigano, F. A. 0., P. V. Scarpino, S. Cronier, M. L. Zink,
and J. C. Hoff. Effect of Particulates and Viral
Aggregation on Inactivation of Enteroviruses in Water
by Chlorine Dioxide. Abstracts of the Annual Meeting—
1978, American Society for Microbiology, Q30 (1978).
Cronier, S., P. V. Scarpino, and M. L. Zink. Chlorine
Dioxide Destruction of Viruses and Bacteria in Water.
Chapter 51 In: Water Chlorination, Environmental Impact
and Health Effects, Volume 2 (R. L. Jolley, H. Gorchev,
and D. H. Hamilton, Jr., Editors). Ann Arbor Science
Publishers, Ann Arbor, Michigan. (1978) p. 651-658.
Scarpino, P. V. Viricidal Effectiveness of Disinfection
Process-Chlorine Dioxide. In: Proceedings AWWA Water
Quality Technology Conference, Atlantic City, New Jersey,
June 25-30, 1978.
Brigano, F. A. 0., P. V. Scarpino, S. Cronier, and M. L. Zink.
Effect of Particulates on Inactivation of Enteroviruses
in Water by Chlorine Dioxide. In: Proceedings of the
USEPA National Symposium on Wastewater Disinfection,
September 18-20, 1978, Cincinnati, Ohio. In Prepara-
tion (1979).
50
-------
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55
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TECHNICAL REPORT DATA
(Please read fnitnictions on the reverse before completing]
1. REPORT NO.
EPA-600/2-79-054
3. RECIPIENT'S ACCESSI ON- NO.
4. TITLE AND SUBTITLE
EFFECT OF PARTICULATES ON DISINFECTION OF ENTERO-
VIRUSES IN WATER BY CHLORINE DIOXIDE
5. REPORT DATE
July 1979
(Issuing Date)
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Pasquale V. Scarpino, Frank A.O. Brigano,
Sandra Cronier, Wary Lee Zink
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Civil Engineering
University of Cincinnati
Cincinnati, OH 45221
10. PROGRAM ELEMENT NO.
1CC824 SOS #2 Task 9
11. CONTRACT/GRANT NO.
R-804418
12. SPONSORING AGENCY NAME AND ADDRESS
Municipal Environmental Research Laboratory- Cin, OH
Office of Research & Development
U. S. Environmental Protection Agency
Cincinnati, OH 45268
13. TYPE OF REPORT AND PERIOD COVERED
Final 3/76 - 12/78
14. SPONSORING AGENCY CODE
EPA/600/14
15. SUPPLEMENTARY NOTES
Project Officer: Dr. John C. Hoff (513!) 684-7331
16. ABSTRACT
The inactivation kinetics of C102 on two enteroviruses, poliovirus 1 (Mahoney)
and coxsackie virus A9, and an enteric indicator of fecal pollution, (Escherichia
coli, were examined in laboratory studies. In addition, the disinfecting ability
of C102 as affected by particulates (both inorganic (bentonite) and cell-associated
virus preparations), and viral aggregates, was determined. C102 was found to be an
excellent disinfectant even when compared to chlorine, especially at the pH of most
drinking waters. The test viruses were found to be significantly more resistant to
disinfectants than the bacterial fecal indicator organism, _E. coli. Variations in
disinfection rates occurred due to viral aggregation. Chlorine dioxide inactivation
of cell-associated poliovirus versus unassoclated poliovirus showed no protection
at the turbidity levels examined. This is believed due to the cell-associated
poliovirus 1 existing in a "singles" or non-aggregation state, and that the
cellular material is readily oxidized by the chlorine dioxide. Finally, a cor-
relation exists between bentonite protection of poliovirus 1 during disinfection at
increasing temperatures and increasing turbidities, i.e. as the temperature and
bentonite turbidity increases, the disinfection efficiency decreases for the
, b'entonite-adsorbed poliovirus.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Enteroviruses, Polioviruses, Coxsackie
viruses, Escherichia coli, Particles,
Disinfection, Microorganism Control,
Chlorine Oxides, Water Treatment Chemical;
Potable Water, Water Supply, Turbidity,
Protection, Coliform Bacteria
Chlorine dioxide
13B
13. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF PAGES
70
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
56
*U,S. GOVEBNMENI PRI»IIWOFFICE; 1979 -6 57-060 /54Z8
------- |