EPA-600/2-84-094
May 1984
EFFECT OF PARTICULATES OK DISINFECTION OF
ENTEROVIRUSES IN WATER BY CHLORAMINES
by
Pasqua! e V. Scarp Lno
University of Cincinnati
Cincinnati, Ohio 45221
Grant No. R-806240020
Project Officer
John C. Hoff
Drinking Water Research Division
Municipal Environmental Research Laboratory
Cincinnati, Ohio 4 5268
MUNICIPAL ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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TECHNICAL REPORT DATA
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««-; and primary effluent-solids was
determined. Additionally, comparisons were made between chloramines and other dis-
infectants (i.e. ir/pochlorous acid, H0C1, and hypochlorite ion, OCT) regarding
aggregated vs_. single virus preparations, different temperatures of reactivity, dif
ferent phi's, and different disinfectant combinations. Aggregated poliovirus
inocula were aDout 1.7 times more resistant to NH2C1 than the singles preparation.
Doubling the NH2CI dose from 12 to 22 ppm did not double tne rate of virus disin-
fection. A comparison between virus singles f^Cl disinfection rates at dH 7.0
and 9.0 showed no difference, but NHoCl at pH 7.0 was found to be a more effective
disinfectant for E. coli than at pH 9.0. Forming NHpCl disinfected at a more rapid
rate than performed NHoCl. Repeated exposure of poliovirus to NH2CI apparent in-
creasea resistance to inactivation. Protection of HEp-2 cell-associated poliovirus
when disinfected with NH2C1 occurred, but no differences in the disinfection rates
occurred when B-.M eel 1-dssucrdteti ,'uliuviruv was disinfected by NhiCL-;>
17. <6v WOR'DS ANO OOCwMSNT a,nalv$ S »
J. ' DESCRIPTORS
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RELEASE TO PUBLIC
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UNCLASSIFIED
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i
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DISCLAIMER
The information in this document has been funded wholly or in part by
the United States Environmental Protection Agency under assistance agree-
ment number R-806240020 to University of Cincinnati. It has been subject
to the Agency's peer and administrative review, and it has been approved
for publication as an EPA document. Mention of trade names or commercial
products does not constitute endorsement or recommendation for use.
ii
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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 components require a concentrated and integrated
attack on the problem.
Research and development is that necessary first step in problem solu-
tion and it involves defining the problem, measuring its impact, and search-
ing 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.
This research provides basic and pertinent information concerning the
viral disinfection capabilities of chloramines as compared to free chlorine
forms, and the effect of viral aggregations, and organic turbidities in water
on the effectiveness of chloramines against enteric viruses.
Francis T. Mayo, Director
Municipal Environmental
Research Laboratory
iii
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ABSTRACT
The inactivation kinetics of chloramines (monochloramine and dichlora-
mine) on an enterovirus, poliovirus 1 (Mahoney) and an enteric indicator of
fecal pollution, Escherichia coli 11229, were examined in laboratory bench-
scale studies using the kinetic (stirred beaker) apparatus. The disinfecting
ability of combined chlorine forms of chloramines as affected by viral aggre-
gates and organic particulates was compared to viral inactivation in pure
buffer systems with unassociated viruses and without added particulates.
Additionally, comparisons were made between chloramines, hypochlorous acid
and hypochlorite ion in a variety of different test situations, such as (1)
the type of particulates (enterovirus-associated animal cells, solids-
associated primary effluents and fecal suspensions), (2) aggregated versus
unassociated single viruses, (3) different temperatures of reactivity, (4)
different pH's, and (5) different disinfectant combinations.
Comparison of the relative inactivation rates at pH 9 and 15C at the 99%
inactivation level showed that the aggregated poliovirus preparation was 1.7
times more resistant to monochloramine than the unassociated singles prepara-
tion of poliovirus. Plots of 99% inactivation data for monochloramine disin-
fection showed extended tail ing for the individual survival curves, whereas
the 90% inactivation data provided equally spaced concentration-time rela-
tionships at pH 9 and at temperatures of 5, 15, and 25C. Almost doubling the
monochloramine dose at 5C and pH 9 from 12 mg/L to 22 mg/L did not double the
rate of virus disinfection.
Dichloramine was less effective in the inactivation of poliovirus 1 than
monochloramine. The single viral preparation was inactivated by dichloramine
at the 90% level about eight times as rapidly as the aggregated inoculum.
Using 90% inactivation by dichloramine of poliovirus 1 singles at 5 and 15C,
a 10-degree increase in temperature gave a of 2,5.
Mo no cli lor amine was subsequently formed at pH 9 and then adjusted to pH 7
to give a stable solution of mostly monochl oramines. Monochloramine disin-
fection rates were then examined at the two different pH's. Monochloramine
disinfection rates of single virus particles at pH 7 and 9 showed no differ-
ence, however for E. coli at pH 7 monochloramine was a more effective disin-
fectant than at pH 9. The rate of disinfection of E. coli at pH 7 with 2.0
mg/L monochloramine was about ten times as rapid as that at pH 9 with 2.2
mg/L monochloramine.
Comparison of the disinfection of E. coli using newly formed preformed
and forming monochloramine was made. The forming monochloramine was about
1.2 times more effective than the preformed monochloramine at 5C and pH 9.
Sequential addition of poliovirus 1 after inital exposure of poliovirus 1
iv
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singles to monochloramine resulted in a reappearance of the initial inacti-
vation rate, indicating that the monochloramine Viad not been altered or
destroyed.
Disinfection of HEp-2 cell-associated poliovirus with 2.28 rag/L hypo-
chlorous acid at 5C and pH 6 was 40 times more effective than 12.2 mg/L
monochloramine at 5C and pH 9. Increasing the monochloramine dosage from
12.2 to 21 mg/L at similar turbidities (1.65 NTU and 1.5 NTU, respectively)
reduced the time required for 90% inactivation, from 50 minutes at 12.2 mg/L
to 30 minutes at 21.0 mg/L. At pH 7, increasing turhidities to 20 NTU from
1.5 at almost the same monochloramine levels (i.e. 11.30 and 10.35 mg/L,
respectively) decreased disinfection efficiency.
Comparisons between BGM cell-associated poliovirus disinfection with
hypochlorous acid (1.20 mg/L at 15C, pH 6 and 1.1 NTU), and monochloramine
(1.16 ing/L at 15C, pH 9 and 1.6 NTU) showed that under these conditions
hypochlorous acid is 360 times as effective as monochloramine. When BGM
cell-associated poliovirus 1 and unassociated single virions were disinfected
with dichloramine at 5C and pH 4.5, no differences in disinfection rates were
observed. Changing the pH of monochloramine from 9 to 7 had no apparent
effect on the disinfection rate of unassociated poliovirus 1 singles or
cell-associated viruses.
Disinfection studies of total colifonns in fecal suspensions using 0.71
mg/L hypochlorous acid (final level of 0.27 mg/L) at 5C, pH 6 and 3.2 NTU
showed an inital rapid die-away of more than 99.9% during the first minute,
then the curve became asymptotic for the next 5 minutes. Protection of
naturally-occurring coliforms found in primary effluents after disinfection
with monochloramine (5.1 to 23.2 mg/L) at 5C and pH 7 occurred at various
turbidities (1.8 to 8.0 NTU).
Survivors of poliovirus 1 that had been exposed 8 times at 15C and pH 9
to monochloramine were subsequently disinfected with 8.95 mg/L of monochlora-
mine also under the same conditions. These survivors were now 2.3 times more
resistant than the initially unexposed virus.
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CONTENTS
Foreword iii
Abstract tv
Figures viii
Tables xi
Acknowledgements xii
1. Introduction 1
Objectives 1
Background 2
Chloramines and Trihalomethane Formation 2
Human Enteric Viruses in Water 4
Low Level Transmission of Viruses 6
Infectivity of Particulate-Associated Viruses 6
2. Conclusions 10
3. Recommendations 12
4. Materials and Methods 13
Preparation of Chlorine Solutions 13
Free Available Chlorine 13
Combined Available Chlorine 13
Chlorine Determinations 14
Free and Combined Available Chlorine 14
Neutral izer 14
Preparation of Buffer Solutions 16
Preparation and Purification of Stock Virus 16
Aggregated Poliovirus Preparation 16
Preparation of Virus Singles 16
Selection of Monochloramine-Resistant Poliovirus 17
Preparation of Cell-Associated Viruses 17
Preparation of Solids-Associated Primary
Effluent Colifornis 19
Preparation of Fecal Particulates 19
vi
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Preparation of Bacteria 19
Microbial Assays 19
Viral Assay 19
Bacterial Assay 20
Turbidity Measurement 20
Experimental Procedure 20
5. Results and Discussion 22
Monochloramine Disinfection of Poliovirus 1 Singles
and Aggregates 22
Dichloramine Disinfection of Poliovirus 1 Singles
and Aggregates 26
The Effect of Temperature Upon Inactivation of Polio-
virus 1 Singles by Monochloramine and Dichloramine ... 26
Monochloramine 26
Dichloramine 30
The Effect of pH Upon the Inactivation of Poliovirus 1
Singles and Escherichia coli By Monochloramine 34
Comparison of the Disinfection of Escherichia coll
Using Preformed and Forming Monochloramines 39
Sequential Addition of Poliovirus 1 to Determine the
Extent of Monochloramine Disinfecting Efficiency .... 40
The Effect of Increasing Concentrations of Monochlo-
ramines Upon the Inactivation of Poliovirus 1
Singles 43
The Effect of Chloride Ions Upon Monochloramine
Disinfection of Poliovirus 1 Singles and
Escher ichia col i 43
Selection For Monochloraraine-Resistant Poliovirus 1 .... 47
Disinfection of HEp-2 Cell-Associated Poliovirus 1
With Hypochlorous Acid and Monochloramine 48
Disinfection of BGM Cell-Associated Poliovirus 1
With Hypochlorous Acid, Monochloramine, and
Dichloramine 59
Coliform Disinfection Studies 59
Disinfection With Hypochlorous Acid of Naturally-
Occurring Coliforms Obtained From Human Feces .... 59
Disinfection With Monochloramine of Naturally-Occurring
Coliforms Obtained From Primary Effluent 64
REFERENCES 69
vii
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FIGURES
Number Page
] The formation of trihalomethanes in Ohio River water
after the addition of free and combined chlorine 3
2 Proportions of mono- and dichloramine (NH^Cl and NHCl^)
in water chlorination with equimolar concentrations of
chlorine and amiuonia 15
3 Protocol for preparation of virus inocula used in cyclic
exposure of poliovirus 1 to monochloramine 18
4 Kinetic (stirred beaker) apparatus 21
5 Concentration-time relationship for 99% inactivation
of poliovirus 1 singles and aggregates by mono-
chloramine at pH 9 and 15C 23
6 Individual survival curves of poliovirus 1 singles inac-
tivated by monochloramine at pH 9, and at 7.3 mg/L
at 5C, 7.8 mg/L at 15C, and 7.2 mg/L at 25C 24
7 Individual survival curves comparing poliovirus 1 singles
versus aggregated inocula at similar dichloramine levels
(11.2 mg/L for the singles and 12.4 mg/L for the
aggregates) at 15C and pH 4.5 27
8 Concentration-time relationship for 99% inactivation
of poliovirus 1 singles by monochloramine at pH 9 at
temperatures of 5, 15, and 25C 28
9 Concentration-time relationship for 90% inactivation of
poliovirus 1 singles by monochloramine at 5, 15, and
25C at pH 9 29
10 Individual survival curves of poliovirus 1 singles inac-
tivated by dichloramine at pH 4.5 and 5C, and at con-
centrations of 4.4, 11.2, 19.5, and 25.3 mg/L 31
11 Concentration-time relationship for 90% inactivation of
poliovirus 1 singles at temperatures of 5 and 15C by
dichloramine at pH 4.5 32
viii
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FIGURES
Number Page
12 Comparison of the concentration-time relationships for
90% inactivation of poliovirus 1 singles by mono-
chloramine at 5, 15, and 25C at pH 9, and by
dichloramine at 5 and 15C at pH 4.5 33
13 Inactivation of poliovirus 1 singles by monochloramine
at 5C at pH 9 and pH 7 (preformed at pH 9) by similar
concentrations of the disinfectant 36
14 Disinfection of Escherichia coli by monochloramine at
5C at pH 9 and pH 7 (preformed at pH 9) by similar
concentrations of the disinfectant 37
15 The inactivation of poliovirus 1 singles and
Escherichia coli at 5C by monochloramine at pH 9
and pH 7 (preformed at pH 9) 38
16 Disinfection of Escherichia coli 11229 at 5C and pH 9
by forming and preformed monochl oramiries compared to
a 0.5 mg/L mixture of_hypochlorous acid (HOC!) and
hypochl orite ion (0C1 ) 41
17 Disinfecting efficiency of 10.8 mg/L monochloramine at
5C and pH 9 to inactivate a sequential addition
of poliovirus I singles 42
18 The inactivation kinetics of poliovirus 1 singles with
increasing monochloramine at 5C and pH 9 44
19 Inactivation of poliovirus 1 singles at 5C and pH 9
by monochloramine with and without the addition of
0.02 M chloride ions as the sodium salt 45
20 Disinfection of Escherichia coli 11229 at 5C and pH 7
by 3.2 mg/L monochloramine with and without the addition
of 0.02 M chloride ions as the sodium salt 46
21 Inactivation of Bates et al.-prepared poliovirus 1
at pH 9 and 15C before and after repeated exposure
to monochloramine 51
22 Inactivation of Floyd ct al.-prepared poliovirus 1
at pH 9 and 15C before and after repeated exposure to
monochloramine 52
23 Inactivation of aggregated and HF.p-2 cell-associated
poliovirus 1 with hypochlorous acid at pH 6 and 5C . . . . 54
ix
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FIGURES
Number Page
24 Inactivation of HEp-2 cell-associated pol iovirus 1 at
5C by hypochlorous acid at pH 6 and monoch]oramine
at PH 9 56
25 Concentration-time relationship for 90% inactivation of
poliovirus 1 singles and HEp-2 eel 1-associated polio-
virus 1 at different turbidity levels and concentra-
tions of monochloramine at 5C and pH 7 and 9 57
26 Inactivation of BGM cell-associated poliovirus 1 by
hypochlorous acid at 15C and pH 6 60
27 Inactivation of BGM cell-associated poliovirus 1 by
monochloramine (pH 9) and hypochlorous acid
(pH 6) at 15C 61
28 Concentration-time relationship for 90% inactivation
of BGM cell-associated and unassociated poliovirus 1
by various concentrations of monochloramine at 15C
and p In 9 62
29 Inactivation of BGM cell-associated poliovirus and
poliovirus 1 singles by dichloramine at pH 4.5 and 5C . . 63
30 Inactivation of naturally-occurring coliforms by
hypochlorous acid at 5C and pH 6 66
x
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TABLES
dumber Page
1 Human Enteric Viruses that may be Present in Water
and Their Associated Diseases 5
2 Oral Infective Dose to Man of Enteric Viruses 7
3 Stability of Monochl or amine at pH 7 35
4 Results of Repetitive Exposures to Monochloramincs of
Bates et al.-Prepared Poliovirus 1 at 15C and pH 9 .... 49
5 Results of Repetitive Exposures to Monochloramines of
Floyd et al.-Prepared Poliovirus 1 at 15C and pH 9 . . . . 50
6 Disinfection of IlEp-2 Cell-Associated Poliovirus 1 with
Hypochlorous Acid at 5C and pH 6 55
7 Disinfection of HEp-2 Cell-Associated Poliovirus 1 and
Poliovirus 1 Singles with Honochloramine at 5C and pH 7 ... 58
8 Chlorine Demand of Suspensions of Natural ly-Occurring
Coliforms after a 30 Minute Exposure 65
9 Monochloramine and Turbidity Levels Used for Disinfection at
5C and pH 7 of Naturally-Occurring Coliforras
Associated with Primary Effluent Solids 67
10 The Inactivation by Monochloramine of Naturally-Occurring
Coliforms Associated with Primary Effluent Solids at
5C and pti 7 68
xi
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ACKNOWLEDGEMENTS
The financial sponsorship of this research by the Municipal Environmen-
tal Research Laboratory of the U.S. Environmental Protection Agency is
gratefully acknowledged. The cooperation, continued interest, encouragement,
and patience of the Project Officer, Dr. John C. Hoff, is especially warmly
acknowledged.
I am deeply indebted to Dr. Louis Laushey, former Head, now Acting Dean
of the College of Engineering and Dr. James F. KcDonough, Head, of the
Department of Civil and Environmental. Engineering, College of Engineering,
University of Cincinnati, for their active support in the completion of this
study. I am especially appreciative of Dr. Laushey's friendship and
encouragement over the years.
The helpful contributions during the active years of this project of Dr.
Shih Lu Chang, Health Effects Research Laboratory, U.S. Environmental Protec-
tion Agency, and Dr. Gerald Berg, Environmental Monitoring and Support
Laboratory, U.S. Environmental Protection Agency, are particularly appreci-
ated. Although both scientists are now retired, their philosophy of life
directed to research has been instrumental to any success that this research
might have.
The excellent technical assistance throughout this study of Mrs. Sandra
Cronier and Mrs. Constance Wells is acknowledged. I also wish to express my
deep gratitude to all who assisted me in the final review of this manuscript,
expecially to Dr. Jean A. Donnelly. Her presence in the ending days enabled
me to recover more fully from my illness. A warm thanks of appreciation is
given to Ms. Hetty VanKesteren, who assisted in so many ways to complete this
manuscript and to prepare the final typed report.
xii
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SECTION 1
INTRODUCTION
OBJECTIVES
The objectives of this concluded research study were multiple, but
consistent with EPA's goal of providing virus-free water to the consumer that
contain low or no .levels of suspect carcinogens. The main objective investi-
gated was the effects of particulates in water on enterovirus-disinfection
with chloraniines as the disinfectant. The complete study objectives can be
summarized thusly:
a. To determine the effect of water containing particulates on the
disinfection of test microbes (primarily poliovirus 1 and the
reference bacterium Escherichia coli) using combined available
chlorine (the chloramines). These results were then compared to
those of free chlorine (hypochlorous acid and hypochlorite ion).
Particulate material was then examined as to its effect on disin-
fection efficiency, and included human fecal solids, sewage-primary
effluent solids, and animal cell associated-poliovirus 1.
b. The disinfection ability of chloramines (both monochloramine and
dichloramine) was studied at various chloramine levels, tempera-
tures, contact times, and pH values; concentrations and types of
particulates; and single versus aggregated preparations of test
virions. Comparisons were also made of the disinfection efficien-
cies of monochloramine used as a preformed dose to that of forming
doses of monochloramine. The effect of doubling the monochloramine
dose upon virus inactivation, and the addition of multiple doses of
poliovirus 1 during the progress of the experiment were also
studied.
c. The selection of a monochloramine-resistant poliovirus 1 mutant.
All of the disinfection studies were performed using the kinetic appara-
tus of Scarpino et a!. (1,2).
1
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BACKGROUND
Chloramlnes and Trihalomethane Formation
The use of combined chlorine (i.e., chloramines) for disinfection of
water supplies assumes great importance when consideration is given to their
reduced reactivity (compared to chlorine) with precursor organics in water to
form potential carcinogenic trihalomethanes (THMs) (3). Investigations have
shown that chloramines either do not cause trihalomethane (i.e., the most
common types are chloroform, bromodichloromethane, dibromochloromethane, and
bromofo^m) formation, or cause reduced quantities of trihalomethanes to be
formed. Based upon the potential dangers associated with THMs, the U.S.
Environmental Protection Agency added in 1979 a maximum contaminant level
(MCL) for THMs to the Safe Drinking Water Act. This standard included only
the four most common THMs types listed above, and the MCL for them was set at
0.10 mg/L (i.e., 100 \i g/L) .
The 1975 National Organics Reconnaissance Survey (NORS) (5) initially
had noted that the finished water of utilities disinfecting with chloramines
contained total trihalomethane (TTHM) levels that ranged from 1 to 81p g/L
(19 yg/L average), whereas those water utilities using breakpoint chlorina-
tion had TTHM concentrations ranging from 1 to 472 Pg/L in their finished
water, with an average level of 72 >-g/L. The reason why some of the water
utilities in the KORS study using chloramines had high TTHM was probably due
to free chlorine being used prior to the addition of ammonia, with the resul-
tant formation of TTHM in the water (4). Subsequently, Stevens et al. (6)
added both free and combined chlorine for varying contact times to untreated
Ohio River water. Their results are presented in Figure 1.
The TTHM level in the free chlorine dosed sample reached 1601J g/L after
72 hours, whereas that sample dosed with combined chlorine (i.e., chloramines)
formed only 16 yg/I TTHM during the same time period. Thus, the toxicologi-
cal hazard associated with the use of the alternative disinfectant, the
chloramines, is lessened in regard to the THMs. One study (7) indicated,
however, that monochloramine (NH^Cl) was mutagenic in Bacil1 us subtil is,
whereas results from an initial 90-day carcinogenic bioassay study using
Nll^Cl exposed mice showed in their livers increased mitotic figures, bizarre
chromatin patterns, and increased cell size (8). On the other hand, hemo-
lytic anemia has not been found in animals orally consuming levels as high as
100 mg/L of NH^Cl. Obviously, more lifetime animal studies will be required
in order to assess fully the toxicol ogical hazard of chloramines in drinking
water. It is also necessary to conduct further studies regarding chloramine
disinfection capability in regard to turbidities and microbes of pathogenic
significance in water supplies, especially the animal viruses (4,9). This
present study was thus primarily concerned with the virus-kill efficiency of
the chloramines as alternative water disinfectants, singly and in the presence
of natural or simulated turbidities.
2
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180
160
140
100-
X
80-
60
40
^.C_OM^N_E_D- C HIORJ N L ^
48
CONTACT TIME,HOURS
Figure 1. The formation of trihalomethanes in
Ohio River water after the addition
of free and combined chlorine (6)
3
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Human Enteric Viruses In Water
Precise knowledge concerning the inactivation of viruses in water assumes
greater importance as inan turns to an ever increasing degree to the re-use of
his upstream neighbor's wastewater. Since sewage-contaminated water is a
potential health hazard (10), an awareness of the efficiency of applied
disinfectants such as chloramine on human enteric pathogens has increased
significance. This is particularly true with the viruses, which are consid-
erably more resistant to disinfection than the bacteria. Over 100 new human
enteric viruses have been described since the investigations of Enders et al.
(11) on viral propagation techniques using tissue cultures. Enteric viruses
infective for man are the most important viral agents known to be present in
water and wastewater, and more than 100 different types may be expected in
human feces; these are listed in Table 1 along with their associated diseases
(12). Thus, the enteric viruses consist of the enteroviruses (primarily
polioviruses, coxsackieviruses, and echoviruses), hepatitis type A, Norwalk
type agents, rotaviruses, reoviruses, adenoviruses, and parvoviruses. Other
viruses may be swallowed by humans (e.g., influenza, mumps, and cold or fever
sore viruses), and may also be later isolated from our feces. However, these
latter are not believed to be particularly significant in disease transfer
via contaminated water. Clarke _e1^ al. (13) pointed out that since enteric
viruses are found in the feces of infected individuals and are readily iso-
lated from urban sewage, especially in the late summer or early fall, they
may enter water supplies and present health hazards to humans. However, it
was noted that the number of recognized water-borne outbreaks of enteric
virus disease was not large, which indicated that many outbreaks may not be
reported or understood to be viral in origin.
Virologists in a number of countries have now reported the presence of
enteroviruses in drinking water samples obtained from public water supply
systems, including those systems that treat the water by conventionally ac-
cepted methods of filtration followed by disinfection (14-19). More recently,
Sekla et_ al_. (20) and Payment (21) reported the isolation of viruses from
drinking water. Payment's study is particularly important because of his
consistent (i.e., from every sample tested) and high (i.e., most samples
contained 1-10 cytopathogenic units/100 liters of drinking water) recoveries
of viruses from finished drinking water leaving a water treatment plant that
practiced prechlorination, flocculation, sand filtration, and ozonation,
followed by postchlorination. A residual free chlorine level of about 0.2 to
0.3 mg/L at pH 7.5 was maintained throughout this study. This residual,
however, was below the recommended free chlorine residual of 0.5 mg/L (main-
tained for a contact time of 30-60 minutes) as recommended by the World
Health Organization (12). Bacteridogicall y the finished water was safe,
since all the samples were negative for coliforms. However, poliovirus 1 was
a frequent isolate, but many isolates were non-polioviruses. Payment (21)
pointed out that all the studies that have reported the presence of viruses
in finished water share the commonality of being bacteriologically safe and
adequately disinfected with a residual chlorine level considered to be viru-
cidal. It was speculated that the passage of the viruses through the plant's
treatment train could be due to one or more of the following: an enhanced
viral resistance to chlorine, as reported by Bates et_ al.(22); the presence
4
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TABLE 1. Human Enteric Viruses that may be Present
in Water and their Associated Diseases (12)
Virus group
No. of
types
Disease caused
Enteroviruses:
Poliovirus
3
Paralysis, meningitis, fever
Echovirus
34
Meningitis, respiratory disease,
rash, diarrhoea, fever
Coxsackievirus A
24
Herpangina, respiratory disease,
meningitis, fever
Coxsackievirus B
6
Myocarditis, congenital heart
anomalies, rash, fever, meningitis,
respiratory disease, pleurodynia
New enteroviruses
4
Meningitis, encephalitis, respiratory
disease, acute haemorrhagic con-
junctivitis, fever
Hepatitis type A (probably
an enterovirus)
1
Infectious hepatitis
Gastroenteritis virus
(Norwalk type agents)
2
Epidemic vomiting and diarrhoea,
fever
Rotavirus (Reoviridae
family)
1
Epidemic vomiting and diarrhoea,
chiefly of children
Reovirus
3
Not clearly established
Adenovirus
30
Respiratory disease, eye infections
Parvovirus (adeno-
associated virus)
3
Associated with respiratory disease
in children, but etiology not
clearly established
Note: Other viruses which, because of their stability, might contaminate
water are the following:
(1) SV-40 like papovaviruses, which appear in the urine. The JC subtype
is associated with progressive multifocal leukoencephalopathy.
(2) Creutzfeld-Jakob (C-J) disease virus. Like scrapie virus, the C-J
virus resist heat and formaldehyde. It causes a spongiform
encephalopathy, characterized by severe progressive dementia and
ataxia.
5
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of natural particulate matter; the association of the viruses with the alum
used for flocculation; or more probably by virus association with organic
matter (23). In any case these reports on the isolation of viruses from the
treated drinking water appear to be at variance with the statement of the
AWWA Committee on Viruses in Drinking Water (24) that optimal and consist-
ently-applied full water treatment (i.e., coagulation-sedimentation and
filtration steps followed by disinfection, usually with chlorine as in the
United States) will provide reasonable assurance of a virologically safe
finished drinking water. Obviously, enteric viruses are being isolated from
samples of supposedly finished drinking water considered safe for drinking;
concern must therefore be expressed at the possible health risk posed by
these viral isolates. Additionally, if water utilities turn to the use of
chloramines as a water disinfectant because of their lessened effect on the
formation in drinking water of potentially carcinogenic chlorinated organic
compounds, more precise information must be forthcoming as to chloramine
disinfection efficiency alone and in the presence of particulate matter in
drinking water (i.e., high turbidities).
Low Level Transmission of Viruses
Epidemiologically, low level transmission of viruses to man is important
when consideration is given to what constitutes a minimum oral virus dose
capable of producing infection and disease in man. Berg (25) stated that the
ingestion of small quantities of viruses by relatively small numbers of
people daily would result in disseminated illnesses that would produce an
epidemiologic picture consistent with person-to-person transmission. This
situation would more likely produce asymptomatic carriers, making it diffi-
cult to indict the water route. This view has been challenged since no
evidence in support of the low—level transmission hypothesis has yet been
presented (26). However, available data does show that the minimum infective
dose of enteroviruses to man is low, as seen in Table 2. Plotkin and Katz
(27) reviewed the available literature concerned with the minimum virus dose
infective for man by the oral route. Experimentation by these workers with
attenuated poliovirus demonstrated that one tissue culture unit (1 TCID.-^)
could constitute an infectious dose (28). Animal studies by Westwood and
Sattar (29) supported the conclusion of Katz and Plotkin. These studies and
others reported by Westwood and Sattar suggested a near-parity in the cell-
infective doses. The 1981 studies with human volunteers by Stafanovic et al
(34) indicated that 10 plaque-forming units (PFU) of an enterovirus (i.e.,
the ECHO-12 virus) ingested in drinking water resulted in human infections.
Additionally, the infection resulted in a shedding state for as long as 19
days at dosages as low as 10 PFU. An 1AWPRC study group on water virology
concluded that based upon available data, although "the minimal infectious
dose of enteric viruses is generally in considerable excess of 1 PFU, there
is reason to believe that certain highly susceptible individuals may indeed
be infected by a single PFU." (26).
Infectivity of Particulate-Associated Viruses
The majority of viruses in the natural environment are associated with
solids and are not in a "free" state (35). Wastewater influent, effluent,
and chlorinated effluent samples were found to have 16. 1 to 100% of their
6
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TABLE 2. Oral Infective Dose to Man of Enteric Viruses
Virus
Human
Subjects
Dose
Method Of
Administration
Percent
Infected
Reference
Poliovirus 1
(Attenuated
SM stain)
Ad ill t s
Infants
0.2 PFU
2.0 PFU
20 PFU
200 PFU
Gelatin capsule
0
67
100
100
Kaprowski, 1955/56 (30,31)
Kaprowski et al . , 1956 (32)
Poliovirus 3
(Attenuated
Fox strain)
Poliovirus 1
(Sabin strain)
Premature
infants
Infants
1.0 TCID
2.5 TCID
10.0 TCID
50
50
50
16
50
90
160
Cavage tube
Aqueous
suspension
30
33
67
0
50
75
100
Katz and Plotkin,
1967 (28)
Minor et al., 1981 (33)
Echovirus 12
(Wild strain)
Young male
adults
10
30
100
Aqueous
suspension
19
29
67
Stephanovic et al . ,
1981 (347
aGiven as plaque-forming units (PFU)
^Civen as the quantity of virus that will infect 50% of the tissue cultures inoculated (TCID^q)
-------
total virus content associated with solids (36). The association of viruses
with solids does not necessarily mean virus inactivation; in fact, clay
solids do not appear to have any deleterious effect on the viruses. Moore et
al . (37) presented data that reaffirmed the findings of others (38-40) that
viruses associated with suspended particulates were infective, by finding
that most of their test enteroviruses were infective by plaque assay in their
particulate-adsorbed form. Thus, monitoring of environmental virus levels
must account tor not only free virus but also for those that are solids-
associated .
The concern that particulates (causing turbidity) in drinking water may
also interfere with the disinfection process is well-founded. Clarke and
Chang (41) believed that turbidity caused by particulates was responsible for
the disinfection failure that resulted in the Delhi, India, infectious hepa-
titis outbreak of 1955. Neefe ert _al. (42) reported that feces from an infec-
tious hepatitis patient after suspension in water and treatment with chlorine
to a final residual of 1.1 mg/L for 30 minutes still caused hepatitis in two
of five human volunteers. Walton (43) detected coliform bacteria in chlori-
nated water from a water-works that usually did not have turbidities greater
than 10 Turbidity Units (TU). Robeck e_t al. (44) demonstrated in a pilot-
plant seeded with viruses that virus attached to floes could penetrate a
granular filter and be recovered from the effluent with as little as a 0.5 TU
increase in turbidity. Sanderson and Kelly (45) recovered coliform organisms
from household taps with water that received no treatment other than chlori-
nation and had turbidities that varied from 4 to 84 TU. Tracy £t al. (46)
recovered coliform bacteria in chlorinated unfiltered water supply with
turbidities of from 5 to 10 TU. Studies on the effects of inorganic turbid-
ity on the disinfection of poliovirus 1 adsorbed onto bentonite clay, and
poliovirus 1 precipitated by aluminum phosphate (AlPO^) were reported by
Symons and Hoff (47). They observed similar disinfection curves for the
poliovirus adsorbed to bentonite or A1P0, at about 5 Nephelometric Turbidity
Units (NTU) and 1.5 mg/L H0C1. No indication of a protective effect was
found when these survival curves were compared to unassociated poliovirus
controls. Stagg et al . (48) reported the inactivation of bentonite-adsorbed
bacteriophage MS-2 with H0C1. At equivalent HOC1 concentrations ranging from
0.02 to 0.6 mg/L, approximately twice the time was required for 99% inacti-
vation of the bentonite-adsorbed bacteriophage MS-2 as for free-associated
virus at turbidities that varied from 2 to 4 Jackson Turbidity Units (JTL).
Boardiuan and Sproul (49) reported that when bacteriophage T^ was adsorbed to
either calcium carbonate, hydrated aluminum oxide, or kaolinite clay and
exposed to chlorine at pH 7 at 22C no protection of adsorbed virus resulted.
(The particulates were measured in mg/L, not as turbidity.) Gerba and Stagg
(50) disputed the results of Boardman and Sproul based on the bacteriophages
ultrastructure and size when compared to that of animal enteric viruses.
They also disagreed with the experimental sampling method employed by the
latter, arguing that short reaction times of less than 1 minute were neces-
sary to detect protective effects. (Stagg et_ _al_. (48) used contact times of
disinfectant and adsorbed-virus of less than 1 minute). Studies by Scarpino
et al^. (51) using Cl 0^ as the disinfectant found a correlation between ben-
tonite protection of poliovirus 1 during disinfection at increasing tempera-
tures and increasing turbidities; i.e., as the temperature and bentonite
8
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turbidity increased, the disinfection efficiency decreased for the bentonite-
adsorbed poliovirus 1.
The effects of organic turbidity on the disinfection process have also
been studied with cell-associated viruses. Cell-associated enteric viruses
are found in domestic sewage, and are produced in cells that line the human
intestinal tract and which eventually slough off, becoming part of the ex-
creta that forms domestic sewage. Cel 1-associated viruses in source waters
may therefore enter potable water treatment plants, and the viruses may then
pass through the disinfection process in a viable state. Hoffa e_t _al. (52)
reporting on the inactivation of cell-associated poliovirus 1 present in
simulated combined sewer overflow water observed that the presence of cellu-
lar material interfered with the effectiveness of disinfection by chlorine
dioxide. Symons and Hoff (47) reported that cell-associated poliovirus 1
with a turbidity of 1.4 Kephelometric Turbidity Units (NTU) was protected
from inactivation by HQC1 at pll 6 at 5 C. The initial H0C1 residual for the
cell-associated poliovirus was about 2.0 mg/L after 5 minutes of contact
time. Free-unassociated poliovirus was reduced by 5 logs in less than I
minute, while cell-associated poliovirus was reduced by about 3 logs in 5
minutes. Scarpino et Ed. (51) determined for ClO^ inactivation of cell-
associated poliovirus 1 versus unassociated poliovirus 1 that no trend was
evident toward protection of the virus at the turbidity levels examined.
This was believed due to the cell-associated poliovirus 1, existing in a
"singles" or non-aggregation state, and that the cellular material was oxi-
dized off the poliovirus by the CIC^-
Waterborne animal viruses may be transported cither inside or on the
surface of cells that are excreted from the intestinal tract of man and ani-
mals. The possibility exists that under natural conditions these cell-
associated viruses may be protected during disinfection. Of particular
interest is the research dealing with minimal infectious dose. For instance,
the turbidity quality of the drinking water may be crucial in altering the
virus minimal infectious dose, since viruses in the water can be protected by
turbidity-causing materials, and thus reach the human gastrointestinal tract
more readily than unprotected virions. This possibility for viral and bac-
terial survival during the water treatment disinfection process led to the
national interim primary drinking water regulations allowing a maximum con-
tamination level of 1 NTU, with up to 5 NTU allowed if it could be demon-
strated that the latter turbidity level did not interfere with disinfection,
prevent maintenance of effective disinfection throughout the distribution
system, or interfere with microbial determinations (53).
9
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SECTION 2
CONCLUSIONS
1. Virus aggregates are, along with organic particulates, a major part of
the mechanism for the survival of virus infectivity in water. In these
studies, aggregated poliovirus 1 (at the 99% inactivation point) at 15C
and pH 9 was about 1.7 times more resistant to disinfection by monochlo-
ramine than unassociated virus singles. The singles virus preparation
disinfected by dichloramine at 15C and pH 4. 5 was inactivated (at the
90% inactivation point) about 8 times as rapidly as the aggregated
virus.
*
2. An average value of 2.75 was obtained in monochloramine-temperature
reactivity studies with poliovirus 1 singles at pH 9 at temperatures of
5, 15, and 25C. For dichloramine, a 10-degree change in temperature
gave a Q of 2.5 for poliovirus 1 singles. Both values are within
the 2 to j factor increase noted by Clarke and Chang (,41).
3. Monochloramine formed at pH 9 and then adjusted to pH 7 was a better
disinfectant for bacteria but not for the test virus. Lowering the pH
from 9 to 7 increased monochloramine disinfection efficiency about 10
times for the bacteria.
4. In the comparison of the disinfection of E^_ coli using preformed and
forming monochloramines, the forming monochloramine was about 1.2 times
more effective than the preformed monochloramine. The faster disinfec-
tion rate could be due to the initial presence of hypochlorous acid
before the monochloramine was completely formed.
5. A gradual progression in the development of virus resistance to mono-
chloramine was found with the Floyd _et_ al. (56) inocula. Survivors of
poliovirus 1 exposed 8 times to monochloramine and then disinfected with
8.95 mg/L monochloramine were 2.3 times more resistant to monochloramine
than either monochloramine-unexposed virus or the virus previously
exposed 7 times to monochloramine.
*
40
ture within defined temperature ranges
is a doubling of the reaction rate per a 10 degree increase in tempera-
10
-------
6. The presence of HEp-2 and BGM cell-associated turbidity interfered with
the disinfection of cell-associated virus by hypochlorous acid and
monochloramine, but not by dichloramine.
7. The solids in human feces and primary effluents offer disinfection
protection to naturally-occurring coliforms.
11
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SECTION 3
RECOMMENDATIONS
Since most finished drinking waters are maintained in the United States
at a pH level below 9 the dramatic increase in monochloramine disinfection
efficiency for coli by lowering the pH of monochloramine from 9 to 7
should be further investigated as to its possible mechanism of action. In
addition, other animal viruses besides poliovirus should be studied to deter-
mine if they also are affected by the pH change.
Additional studies are required with different cell lines to determine
if they have similar viral protective effects during disinfection.
Feasibility studies of the cost effectiveness of reducing the turbidity
levels from 5 to 1 NTU in drinking water treatment should be determined.
Implementation of a reduced turbidity level to 1 NTU is recommended because
of our studies and those of others with coliforms associated with primary
effluent solids, fecal solids and eel 1-associated viruses.
In future turbidity studies, ways of determining the nature of the
particulates (inorganic or organic) must be developed to ascertain their
potential for protection during disinfection.
Methodology guidelines/recommendations should be established as to
disinfection experimental apparatus and the physical state of the test organ-
isms, i.e., viral associated (aggregates or cell-associated) and unassociated
(singles) preparations.
The usefulness of chloramincs, especially the raonochlorainines, in field
situations should be more carefully evaluated. Under certain definite condi-
tions (such as "forming" situations) they may be useful.
Dichloramine1s ability to penetrate organic masses, such as cells,
should be more thoroughly investigated.
The development of resistant strains of viruses in nature should be
thoroughly studied. Laboratory studies can only point out a potential prob-
lem; field studies are required to pin point possible health risks that might
exist in the natural environment.
12
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SECTION 4
MATERIALS AND METHODS
PREPARATION OF CHLORINE SOLUTIONS
Free Available Chlorine
Stock solutions of chlorine were prepared as required by bubbling chlo-
rine gas for 30 minutes into an amber bottle containing deionized distilled
water held at a temperature of 5C and subsequently stored at the same temper-
ature. Chlorine gas reacted with water to form hypochlorous acid (H0C1) and
subsequently hypochlorite ion (0C1 ) according to the following reactions
(Eq. 1 and 2):
Cl2 + li20 ^ ^ H0C1 + H+ + CI" Eq. 1
H0C1 ~ N II+ + OCl" Eq. 2
The dissociation of hypochlorous acid is dependent chiefly upon pH and, to a
much lesser extent, temperature, with almost 100% H0C1 present at pH 5, and
almost 100% OCl present at pH 10. Free available chlorine refers to the
concentration of hypochlorous acid and hypochlorite ion, as well as any
molecular chlorine existing in a chlorinated water. On the day of the tests
using H0C1 or OCl , the desired chlorine concentrations were prepared in
demand-free buffer by the addition of a pre-calculated amount of the stock
chlorine solution.
Combined Available Chlorine
Inorganic chloramines are formed by three successive substitution reac-
tions between aqueous chlorine (i.e., hypochlorous acid) and ammonia (NH^) in
accord with the following equations:
H0C1 + NH3 ^ S NH2C1 + H20 Eq. 3
H0C1 + lffl + h20 Eq. 4
K0C1 + NHC12 ^Z^NC13 + H20 Eq. 5
The proportion of chloramines formed, called monochloramine (Eq. 3), dichlo-
ramine (Eq. 4), and nitrogen trichloride (Eq. 5) depend chiefly upon the
relative amounts of liOCl and NH^, and the pH of the solutions. To a minor
extent, the time of contact and temperature also affects the chloramine
distribution. Dichloramine (NHC12) is the predominant form of chloramine at
13
-------
a 1:1 molar ratio of ammonia to chlorine at pH values of 5 and below, whereas
at pH values of 9 and above, monochloramine (NH^Cl) predominates. Figure 2
shows the proportion of monochloramine and dichloramine formed for pH values
of 4 to 9 and temperatures of OC, IOC, and 25C (54). Combined available chlo-
rine refers to the concentration of inorganic chloramine. Since chlorine
will also react with organic amines, the organic chloramines that are formed
are also Included in the term combined available chlorine.
In these studies, monochloramine was formed by first chlorinating the pH
9 borate buffer to the desired level, and then adding ammonium sulfate
[(NH^)„SO^] to produce an ammonia (NH„) to chlorine ratio of 6:1 (by weight),
as in Eq. 3. This meant 6 mg NH^ to I mg titrable C^, or 23 to 1 on a molar
basis. There was no free chlorine present in the solution by amperometric
titration. At ph 9 and at a 23 to 1 molar ratio of ammonia to chlorine,
practically all titratable chlorine exists as monochloramine. At pH values
greater than 8 only monochloramine is usually noted. Dichloramine was formed
by adding ammonium sulfate to one to two liter quantities of hypochlorous
acid in pll 4.5 demand-free 0.05 M phthalate buffer. The chlorine to nitrogen
weight ratio (C1:N) was 3:1 to insure rapid formation of the dichloramine.
The solution, loosely capped, was allowed to mix on a Magnistir for two hours
at room temperature to allow maximum dichloramine formation. Since
dichloramine was found to be unstable over a 24-hour period, test solutions
were freshly prepared on the day of the experiment.
Nitrogen trichloride has little or no disinfection capability and is of
chief significance in water treatment because of the obnoxious qualities it
imparts to water.
CHLORINE DETERMINATIONS
Free and Combined Available Chlorine
The amperometric titration method employing the Wallace and Tiernan
Amperometric titrator, was used to determine free chlorine and monochloramine
at the time of the experimentation (1,2). Dichloramine was determined by the
FAS:DPD method according to Standard Methods (55) with one exception. Since
the titrations are required to be carried out at a pH of 6.2-6.5, the pH
effect of the phthalate-buffered dichloramine samples had to be overcome. To
accomplish this, pH 6 phosphate buffer was added as necessary to bring the pH
of the titration sample to the proper range. The total recommended titration
volume of 100 ml was held constant, as was the added amount of the DPI) color
indicator. The amperometric titration method was also used for dichloramine
determinat Lons.
NEUTRALIZER
The neutral izer for free and combined chlorine solutions consisted of
sufficient sodium thiosulfate to give a final concentration of 6 mg/L in the
samples removed from the test and control beakers.
14
-------
1.0
0.8
0.6 -
04 -
,r 02 _
0.2
04
0-6
0.8
1.0
pH
Figure 2. Proportions of mono- and dichloramine
(NH„C1 and NHCl^) in water chlorination
witn equimolar concentrations of chlorine
and ammonia (54)
15
-------
PREPARATION OF BUFFER SOLUTIONS
Phosphate (KH^PO^-K^HPO,), borate (H^BOyNaOH), and phthalate (KHCgH^O^-
NaOH) buffer systems at 0.05 M were used at various pH values to determine
the effect of pH on the antiviral activity of free and combined available
chlorine. The buffer system for the hypochlorous acid and hypochlorite ion
studies consisted of 0.05 M, pH 6 phosphate buffer (KH„P0,-K^HPO,), and
0.05 M, pH 10 borate buffer (H„B0_-Na0H), respectively (l,2j. For the rnono-
chloramine work, the test buffer was 0.05M borate (H^BO^-NaOH) at pH 9, or
the above phosphate buffer at pH 7. For the dichloramine work, the test
buffer was 0.05M potassium phthalate at pH 4.5. The buffers were made demand-
free by the addition of the free available chlorine stock solution to provide
a residual of 3 to 5 mg/L. After standing for several days, the water was
dechlorinated at room temperature for at least 24 hours under ultraviolet
light (chlorine is highly photoreactive at ultraviolet wavelengths of about
2600 X and decomposes rapidly) until the DPD spot plate test method for
chlorine was negative. The buffer was then considered to be chlorine demand-
free .
PREPARATION AND PURIFICATION OF STOCK VIRUS
The poliovirus 1 (Mahoney strain) stocks in these studies were prepared
in two different ways. In both methods the polioviruses were grown in mono-
layers of Buffalo Green Monkey (BGM) kidney continuous cell line obtained
from Cercoplthecus aethiops, the African Green monkey.
Aggregated Poliovirus Preparation
Poliovirus 1 was prepared from BGM cells which were infected approxi-
mately 24 hours earlier at a multiplicity of infection of 10 PFU per cell,
and then incubated for one hour at 37C. MEM's maintenance media with 2%
fetal calf serum was added to the infected tissue cultures and incubation was
continued for 11 hours. When cytopathologieal effect (CPE) was apparent, the
cells and fluids were collected and subjected to freezing (-70C) and thawing
three times to release the virus particles contained in the cells.
The cell debris was spun out at 1,300 x g for 30 minutes, and the super-
natant was spun at 128,000 x g for 3 hours. The pellet was suspended in
chlorine demand-free water and spun to pellet the virus. This pellet was
resuspended in chlorine demand—free water and tested for chlorine-demand. If
none was present, the virus was diluted to 10 PFU/ml, placed into demand-
free vials in 1 ml aliquots, and these vials were frozen at -70C. These
virus preparations were referred to in this work as aggregated poliovirus
preparations. If demand was present, the virus was resuspended in demand-
free buffer and the precedure was repeated.
Preparation of Virus Singles
Purified poliovirus 1 inocula containing mostly single virions was
prepared using the method of Floyd e_t _al (56). BGM monolayers were inoculated
with poliovirus 1 at a multiplicity of 100 PFU per cell. The virus was
allowed to adsorb for 1 hour at 37C, after which the MEM's maintenance medium
16
-------
was added and the tissue, culture cells were further incubated at 37C for 11
hours. The cells were then removed from incubation and chilled to 4C. 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 tissue culture containers were then washed
twice with phosphate buffered saline. The BGM monolayers were then 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 horaogeni-
zation (2 minutes) in a Waring blender, with separation of the virus contain-
ing aqueous phase from the freon phase by centrifugation (800 x g for 10
minutes). This freon extraction was carried out three times, each time the
Freon phase was re-extracted with another 6 ml of phosphate buffered saline;
and the phases were again separated. The upper virus-containing aqueous
phases were collected, held in an ice bath, and made up to a final volume of
20 ml. The virus was now further purified and concentrated by density gra-
dient centrifugation. The aqueous-virus phase was layered onto a 10% to 30%
(wt/wt) sucrose gradient made with 0.05 M phosphate buffer at pH 7.2. The
gradient was centrifuged at 80,000 x g in a Beckman L2-65B ultracentrifuge
for 2.25 hours at 4C. Fractions of 2 ml each were collected from the centri-
fuge tubes and examined by sucrose refraction. Singles viruses appeared in
the fractions containing 18-2 2% sucrose. All relevant fractions were pooled
and stored at 4C without any attempt to remove the sucrose. The greatest
concentration of purified virus was found at a concentration of 22% sucrose.
The resultant virus preparation contained no cell debris, or debris of any
kind, and consisted of greater than 93% single virus particles (51). This
preparation was called poliovirus singles.
SELECTION OF MONOC H LOR AM 1N E- R KSI. S TAN T POLIOVIRUS
Both the Bates jet al_. (22) and Floyd e^t al. (56) procedures for prepar-
ing the inocula used in their studies on selection of disinfectant—resistant
poliovirus was utilized in our experimentation. The procedures used for
preparation of the virus inocula are outlined in Figure 3.
PREPARATION OF CELL-ASSOCIATED VIRUSES
Enterovirus-associated animal cells were prepared to simulate naturally
found cell-associated viruses which can be excreted from the intestinal tract
of man. Two cell lines were used, i.e., HEp-2 (Human Epidermoid Carcinoma)
and BGM cells. The HEp—2 cell-associated viruses were prepared by Dr. J.
Hoff of MERL, U.S. EPA (57). Monolayer cultures were infected with polio-
virus 1 at a multiplicity of infection of 5 PFU/cell. Infected cells were
harvested, washed 6 times with chlorine demand-free 0.05M phosphate buffer to
remove chlorine-demand, and titered. The total quantity of viruses associat-
ed only with the cells and cell debris was determined by first centrifuging
the eel 1-associated virus preparation, and determining viral presence in the
supernatant. The total amount of virus present which was associated only
with the cell and cell debris was then determined by lysing the pelleted
infected cells and cell debris with chloroform, thus releasing the associated
viruses, and determining the total released viral titer.
17
-------
BGM cell sheets were inoculated with
poliovirus 1 (Mahoney) at a multiplicity
of infection of 100 PFU/cell. The virus
was adsorbed for one hour at 37C. MEM's
(containing 20% fetal calf scrum) was
added, and the cultures were incubated for
11 hours at 37C until CPE was observed.
1
Follow Protocol For
Bates et al. (22)
Floyd ct al. (56)
Freeze and Thaw Fluids and Cells 3 Times
Centrifuge at 10,000 x g for 20 Minutes
to Remove Cell Debris
Centrifuge Supernatant Fluids at
135,000 x g for 3 Hours
Resuspend Pelleted Virus in 1.8 ml
of Phosphate-Buffered Saline
Disperse Virus by Sonic Oscillation
Pass Virus through a 0.45 m
Cellulose Nitrate Membrane Pretreated
with 5 ml of a 1:5 Dilution of Fetal
Calf Serum in Phosphate-Buffered
Saline
After Filtration, 0.2 ml of Fetal
Calf Serum was Added to the Virus Sample
to Give a Final Volume of 2 ml
Determine Virus Titer by Plaque Assa
- Wash Cells with Phosphate-
Buffered Saline and Scrap off
Cells
- Extract Cells with Freon 113
in Blender for 2 Minutes
- Centrifuge to Separate Layers
- Extract 3 Times, Removing
Aqueous Virus Layer Each Time
- Layer Aqueous Virus Layer onto
10 to 30% (wt/wt) Sucrose
Gradien
- Centrifuge 80,000 x g for 2.25
Hours.
- Collect 2 ml Fractions
- Determine Sucrose % of Frac-
tions By Refraction
- Pool 18-22% Fractions Con-
taining Singles of Polio-
virus 1
- Determine Virus Titer by
Plaque Assay
Figure 3. Protocol for preparation of virus
inocula used in cyclic exposure
of poliovirus 1 to monochloramine
18
-------
BGM cell-associated poliovirus 1 inocula were prepared by inoculating
cell monolayers at a multiplicity of 100 PFU/ml. The virus was allowed to
adsorb one hour at 37C prior to addition of MEM maintenance medium, and the
cells were further incubated at 37C for 11 hours. The infected cells were
then chilled to 4C. The monolayers were then scraped from the bottles, and
they and their fluids were spun at 1,300 x g. The BGM cell-associated polio-
virus 1 complex was then washed and spun 6 times with chlorine-demand-free
0.05 M phosphate buffer to remove chlorine-demand due to the animal cell
presence.
PREPARATION OF SOLIDS-ASSOCIATED PRIMARY EFFLUENT COLIFORMS (58)
Five gallons of the City of Cincinnati's Little Miami Sewage Treatment
Plant's unchlorinated primary effluent was passed through 90, 45 and 38 ym
sieves. The collected solids were washed from the 38|_ m sieve with 250 ml of
pH 7, 0.05 M phosphate buffer. The solids were then washed, centrifuged for
15-20 minutes at 1,700 x g, and the pelleted solids were suspended in the
buffer (100 ml volume/wash). This procedure was repeated 3 times to remove
soluble chlorinc-demand substances.
PREPARATION OF FECAL PARTICULATES
One gram of human fecal material was suspended in 100 ml chlorine demand-
free pH 7, 0.05 M phosphate buffer by homogenizing the fecal material in a
Sorval Orani-Mixer for 0.5 minutes at 11,500 RPM. The suspension was allowed
to settle overnight, then the supernatant was centrifuged at 2300 x g the
pellet was washed 3 times in the pH 7, 0.05M phosphate buffer, the pellet was
resuspended in a small quantity of the demand-free phosphate buffer and was
now ready for use.
PREPARATION OF BACTERIA
Escherichla col i (ATCC 11229) was grown in trypticase soy broth for
16-18 hours at 35C, centrifuged for 20 minutes at 2300 x g, and then the
pellet was resuspended in chlorine-demand-free pH 7, 0.05 M phosphate buffer.
The buffer washings of the cells were repeated 3 times. The final suspension
was adjusted to an optical density (using a Klgtt-Summerson photoelectric
colorimeter) equal to a cell concentration of 10 bacteria/ml. The suspen-
sion was usually found to be chlorine-demand-free. When necessary the cells
were rewashed with demand-free phosphate buffer until the demand-free state
was obtained.
MICROBIAL ASSAYS
Viral Assay:
Animal viruses were titered by the plaque technique of Dulbecco and Vogt
(59), as modified by Hsiung and Melnick (60), in a continuous cell line, BGM,
derived from primary African Creen Monkey kidney cells. The BCM cell line has
been found to be more sensitive to many enteroviruses (61). Most tissue
cultures were prepared jointly by us and the Virology Section of the Biologi-
cal Methods Branch, EMSL, U.S. Environmental Protection Agengy, Cincinnati,
19
-------
but BGM cell-associated poliovirus 1 inocula were prepared by us. HEp-2
(Human Epidermoid Carcinoma) cell cultures for the poliovirus 1 cell-associ-
ated studies were supplied by Dr. John Hoff of the U.S. Environmental Protec-
tion Agency, MERL.
Bacterial Assay
E. col i survivors in these studies were recovered and enumerated using
surface-inoculated trypticase soy agar plates, or, for primary effluent work,
using the Most Probable Number multiple-tube fermentation technique through
the confirmed test (55). The plates were incubated for 24 hours at 37C prior
to be ing counted.
TURBIDITY MEASUREMENT
Turbidity was measured in Nephelometric Turbidity Units (NI'U) using a
Hach 2100A Turbidimeter. The instrument was always standardized before use
using turbidity standards obtained from the liach Co., Loveland, Colorado
80539.
EXPERIMENTAL PROCEDURE
On the day of the experimentation, the desired free or combined chlorine
concentrations were prepared in chlorine demand-free buffer by the addition
of a calculated amount of the stock chlorine solution. The procedures out-
lined in (1) Preparation of Chlorine Solution and (2) Chlorine Determination
above were used to prepare and analyze for the free and combined chlorine
used in these studies.
Four hundred ml volumes of the disinfectant-treated buffer were then
placed in 600 ml test beakers according to the test scheme used by Scarpino
et al. (1,2), (Figure 4). These beakers were covered by a loose metal lid
through the center of which passed a glass stirring rod. One buffer control
beaker contained 400 ml of the untreated (no disinfectant was added) demand-
free buffer, while a second neutralizer control beaker contained a treated
neutralized buffer (containing the highest free chlorine, monochloramine or
dichloramine concentrations being used that day). The chlorine species in
the second beaker was neutralized with sodium thiosulfate just before the
start of the experiment. Disinfectant control beakers were prepared that
were similar in every aspect to the test beakers except that the test virus
or bacterium was not added. The glass stirring rods were connected to an
overhead variable-speed device that was adjusted to 81 RPM, and all beakers
were allowed to equilibrate to the test temperature in a carefully regulated
water bath. After temperature equilibrium was obtained, the disinfectant
control beakers were titrated at the start (and end) of each experiment,
whereas the test beakers were titrated at the end of each study. One ml
standardized amounts of the test virus or bacteria were added to the buffer
control, neutralizer control, and the test beakers; 5 ml samples were removed
at intervals and rapidly added into 5 ml of the neutralizer. The neutralized
samples were assayed immediately following the conclusion of the test. The pH
of the test and control solutions were also determined at this time.
20
-------
WATER INLET
I - ~*m1i
WATER OUTLET
glass proMllor
38*
M
K3
8*'
WATER BATH
Figure 4. Kinetic (stirred beaker) apparatus (1,2)
-------
SECTION 5
RESULTS AND DISCUSSION
MONOCHLORAMINE DISINFECTION OF POLIOVIRUS 1 SINGLES AND AGGREGATES
A number of viral aggregation studies have implicated aggregates in the
viral inocula as the cause of aberrations in survival curves when viruses are
exposed to destructive chemical and physical agents, such as disinfectants
(62,63). Scarpino et al. (64) concluded that their observed variations in
chlorine dioxide disinfection survival rates occurred due to viral aggrega-
tion. In a series of publications, Sharp and his colleagues (56,63,65-67),
at the University of North Carolina investigated this phenomena and pointed
out that the time and concentration of the disinfectant necessary to inacti-
vate the virus will be dictated by the aggregates present. They concluded
that aggregates were doubtless a major part of the mechanism for the survival
of poliovirus infectivity in treated water (65).
In order to determine the effect of aggregation on poliovirus 1 survival
during monochloramine, disinfection tests were done to compare the survival of
poliovirus singles and aggregates. Figure 5 is a concentration-time plot
showing the relationship of monochloramine concentration and time (in minutes)
for 99% inactivation of poliovirus 1 at pH 9 and 15C. This plot is based on
individual survival curves with different concentrations of Nt^Cl in the
borate buffer system at the same pH (9) and temperature (15C) . At pH 9,
monochloramine is the predominant species of chlorine present (see Figure 2).
The 99% inactivation (i.e., the 1% survival) points used in the construction
of the concentration-time plot were obtained from 5 individual survival
studies for poliovirus 1 singles and 8 individual survival studies for polio-
virus 1 aggregates. Examples of the individual survival curves obtained with
poliovirus singles from which 99% inactivation points were obtained are shown
in Figure 6 for temperatures of 5, 15, and 25C.
As pointed out by Fair e_t aL. (68), the principal factors that affect
the efficiency or rate of destruction or inactivation of a particular species
of organisms are: time of contact, concentration of organisms, concentration
of disinfectant, temperature, and nature of the disinfectant. The rate of
destruction or inactivation of microbes has been usually expressed by a
first-order relationship referred to as Chick's Law (69,70),
dN = k . N Eq. 6
_ __ c
where, _ dN is the rate of destruction or inactivation expressed
dt as the change in the number of viable microorganisms,
22
-------
a>
Poliovirus I'Singles'
Poliovirus 1 Aggregates
US
o>10
£
I I I I
100
Minutes
J I L
Figure 5. Concentration-time relationship for 99 percent inactivation of
poliovirus 1 singles and aggregates by monochloramine at pH 9 and 15C
-------
1001
10
7.8
7.2
.10
.01
60
240
180
300
120
Minutes
Figure 6. Individual survival curves of poliovirus 1
singles inactivated by monochloramine at pH 9,
and at 7.3 mg/L at 5C, 7.8 mg/L at 15C, and
7.2 mg/L at 25C
24
-------
N, with time t, and k is a proportionality rate
factor which varies with the disinfectant concen-
tration, temperature, or other conditions, but is
independent of the organism number or time.
Integration gives,
e —jt— = - kt fcq. 7
ft
O
where, N and N are the number of microorganisms living initially
and at time, t, respectively; and k is the rate of
the reaction, a constant.
Thus, a plot of log N/N against t for various contact times should give a
linear relationship, i.e., follows first-order kinetics. As illustrated in
Figure 6, the logarithm of the percentage of surviving poliovirus 1 singles
is plotted as the ordinate, whereas the time in minutes is the abscissa.
These studies were done at similar monochloramine levels, but at temperatures
of 5, 15, and 25C. Although the process of inactivation was kinetically of
the first-order, it should be more properly considered as pseudo-first order
(69). As the temperature increased from 5 to 25C at almost the same level of
monochloramine, the inactivation rate of the viruses increased, i.e., their
inactivation was faster as shown by the time in minutes it took to reduce the
poliovirus 1 singles population by 99%. Also, some "tailings" of survivors
were observed at temperatures of 5 and 15C.
The concentration-time relationships were plotted on log-log paper, as
in Figure 5, using in this case parameters of the level of monochloramine
versus the contact time in minutes of the disinfectant. Concentration-time
relationships have been expressed in this manner since J.H. van't hoff (71)
demonstrated in 1896 that the disinfectant concentration coefficient, n,
indicated the order of the reaction. Chick (69,70) found n to be an exponen-
tial function between concentration of the disinfectant and time, i.e.
C t = constant or k
where, t is the time required to destroy or inactivate a given
percentage of organisms (i.e., 99% inactivation of polio-
virus 1 singles or aggregates based on individual survival
curves), C is the concentration of the disinfectant
reactant, n is a constant which is characteristic of a
particular disinfectant, and may also be called the
concentration exponent, while k is the reaction constant.
Figure 5 shows the plotting of the 99% inactivation points obtained from
individual survival curves. The data in all cases formed a straight line
that had slopes close to 1, indicating similar first-order inactivation
mechanisms. The aggregated poliovirus 1 inoculum was found to be about 1.7
25
-------
times more resistant to monochloramine than the singles inoculum. This
difference was quite significant, since it showed a change in disinfection
kinetics of the same virus type due to the viral, aggregation.
DICHLORAMINE DISINFECTION OF POLIOVIRUS 1 SINGLES AND AGGREGRATES
A similar difference in disinfection kinetics of the same virus type
(i.e. poliovirus 1, Mahoney strain) due to viral aggregration was observed in
dichloramine disinfection, when singles viral preparations were compared to
aggregated ones. Figure 7 shows the differences in survival characteristics
of individual survival curves at similar dichloramine levels (11.22 mg/L for
the singles and 12.4 mg/L for the aggregates) at 15C. The singles viral
preparation was inactivated at the 90% level approximately eight times as
rapidly as the aggregated inoculum. The retardant, stepladder inactivation
of the aggregated inoculum in Figure 7 exposed to dichloramine demonstrates
the clumped nature of the inoculum.
THE EFFECT OF TEMPERATURE UPON INACTIVATION OF POLIOVIRUS 1 SINGLES BY
M0N0CHL0RAMINE AND DICHLORAMINE
Monochloramine
Since chemical disinfection is a rate process there is an increase in
the chemical reaction rate with increasing temperatures. The empirical "rule
of thumb" used is that the rate of the reaction increases by a factor of 2 to
3 for each 10-degree rise in temperature. After an extensive review of the
literature, Clarke and Chang (41) concluded that the temperature coefficient
for a 10-degree change (Qjq) i-11 thc destruction of virus by free chlorine
increased the rate of virus inactivation by a factor of 2 to 3 (200 to 300
times). In these present studies, Figure 8 shows the concentration-time
relationship for 99% inactivation of poliovirus 1 singles at temperatures of
5, 15, and 25C by monochloramine at pH 9. Although all three curves illus-
trated linear reaction rates, they were not equally spaced. A 10—degree
temperature change from 5 to 15C showed a Qm of 1.5, whereas that from 15 to
25C was 4, giving an average value of 2.75. Poliovirus 1 singles were
inactivated faster at the higher temperature of 25C when 99% inactivation
points were used to construct the concentration-time figure. However, when
the NH^Cl data were regraphed using 90% inactivation points (see Figure 9),
all three curves were equally spaced. The Q.q from a temperature of 5 to 15C
was npw 2, while that from 15 to 25C was 1.9, giving an average Q.„ value of
1.95. The lower parts (the "tails") of the monochloramine survival curves at
temperatures especially of 5 and 15C (see Figure 6) were in part non-linear
on sem.il ogarithmic plots, whereas the upper portions (the 90% inactivation
points) reflected first-order kinetics. Although poliovirus 1 singles were
used in these studies, the observed "tailings" were similar to those seen
with aggregated preparations of poliovirus. The non-linearity (i.e.,
"tailings") of the survival curves appeared more frequently at lower tempera-
tures (i.e., 5 and 15C) rather than at the higher temperature of 25C.
26
-------
Singles
1 2.4
Ag g regate
100
IS
>
>
~
3
1.0
.08
500
400
300
200
100
Minutes
Figure 7. Individual survival curves comparing poliovirus 1
singles versus aggregated inocula at similar dichloramine
levels (11.2 mg/L for the singles and 12.4 mg/L for the
aggregates) at 15C and pH 4.5
27
-------
MINUTES
ure 8. Concentration-time relationship for 99% inactivation of poliovirus 1
singles by monoch lor amine at pH 9 at temperatures of 5, 15, and 25C
-------
to
vc
k.
0
\
0)
J
0
M
1
Z
10
1.0
\ \ (ffi\
\ ® \ \
E
~ \ \. N.
\. ¦ N.
\ \ \ 0
X \ 5 c
\ > 0
\ 15 C
25°C
1 I I 1 1 III 1 1 1 1 1 II 1 ll 1 1
I I I
1.0 10 100
1000
MI ri utea
Figure 9. Concentration-time relationship for 90% inactivation of poliovirus I
singles by monochloramine at 5, 15, and 25C at pH 9
-------
Dichloramine
Individual dichloramine (NHCI^) survival curves of poliovirus 1 singles
at both 5 and 15C showed more extended kinetic rate patterns than those
encountered with monochloramine (Figure 10). The rate of inactivation ini-
tially followed first-order kinetics for at least part of the survival curve.
In several cases there was evidence of a retardant rate process, where the
survival curve demonstrated "tailing" of the survivor fraction of the original
inoculum. This might have been due to the presence of poliovirus clumps or
aggregates still in the inoculum. Clumping would restrict the penetration of
the disinfectant into the aggregates and thus enable internal viral survival
to occur in the tissue culture recovery system.
Although so-called "singles" preparation of poliovirus 1 were used in
this work, clumps of virions were still evident. The freon extracted-density
gradient method of preparing poliovirus singles was found in a previous study
(65) to yield no detectable cellular debris upon examination by electron
microscopy, and to be composed of 93.1% single virus particles and 6.9%
aggregated virions. The aggregated viral fraction was further characterized
and found to have 3.9% of the virions in pairs and 3.0% existing in a state
of + 5 virus particles. The aggregated poliovirus inoculum contained, on the
other hand, considerable cellular debris, a virus content of 90.7% single
virions, and 9.3% aggregated forms. The aggregated virus fraction consisted
of 4.4% pairs, 0.1% triplets, and 4.8% of + 5 virus particles. Thus, the
difference (except for the cellular debris) was not great; but was signifi-
cant when aggregates and singles inocula were compared (Figure 5). The
dichloramine (and monochloramine) survival curves were similar in shape; as
the percentage of recovered virus particles declined, there occurred a gradual
slowing of the inactivation rate followed by a leveling off of the curve.
Survival curves of this type are usually indicative of interference in the
die-away kinetics of the process (Chick's Law) and were due to the presence
of clumps or aggregates of virions in the inoculum. Although the fractional
rate of inactivation of the virus for a given set of conditions should be a
constant according to Chick's Law, deviations from Chick's Law occur, as
documented here with aberrant survival curves. Chang's (72,73) multi-Poisson
distribution model for treating disinfection data illustrates the inactiva-
tion of increasing virus clump sizes; i.e., the smallest clump size or single
virions were inactivated first since more surface area per virion was exposed
to the disinfectant, followed by the inactivation of increasing clump sizes.
Thus, the inactivation rate appeared to slow down as larger and larger clumps
were encountered until the survival curve "tailed off".
Figure 11 is a log-log plot of the concentration-time relationship for
90% inactivation by dichloramine of poliovirus 1 singles at 5 and 15C. A
10-degree increase in temperature gave a Q1 . of 2.5 for poliovirus 1, which
was within the 2 to 3 factor increase notea by Clarke and Chang (41). When
the data from Figures 9 and 11 were combined in Figure 12, the dichloramine
90% concentration-time relationships did not parallel those encountered for
monochloramine. The inactivation kinetics may thus be different.
30
-------
100
10
(0 uo
N H Cl2 Level
0.1
4.4
25.3
.01
240
60
120
180
Minutes
Figure 10. Individual survival curves of poliovirus 1 singles
inactivated by dichloramine at pH 4.5 and 5C, and
at concentrations of 4.4, 11.2, 19.5, and 25.3 mg/L
31
-------
I I I I I I Hi I I i i i n ill I i i i i ml i I
1.0
10
100
n utes
1000
Figure 11. Concentration-time relationship for 90% inactivation of poliovirus 1
singles at temperatures of 5 and 15C by dichloramine at pH 4.5
-------
-X-
5c NHCI2
15°c N HCI2
5°c
o
15c
N HCI
Nhysi
25 c NH2CI
I I I II 111 I I I
1jO
10
10U
M i nutes
1000
ure 12. Comparison of the concentration-time relationships for 90% inactivation
of poliovirus I singles by monochloraraine at 5, 15, and 25C at pH 9,
and by dichloramine at 5 and 15C at pH 4.5
-------
THE EFFECT OF pH UPON THE INACT1VATION OF POLIOVIRUS i SINGLES AND
ESCHERICHIA COLI BY MONOCHLORAMINE
The pH range of most mineral-bearing waters are generally within the
narrow range of 6 to 9 (74). The Classes AA and A used by the State of New
York for fresh surface waters have a recommended pH range between 6.5 and
8.5, and after appropriate treatment are considered safe and satisfactory for
drinking water purposes (75). Most finished drinking waters in the United
States are maintained at a pH level below 9, usually between 7 and 9 (76).
At pH values of 9 and above, the chloramine that is formed when hypochlorous
acid reacts with ammonia is predominately monochloramine. Thus, many research
studies are done at pH 9 or above. However, pH values lower than 9 can be
encountered in drinking water treatment. In order to evaluate whether a
still predominately monochloramine system at a pR lower than 9 is a better
disinfectant, the following procedure was followed.
Levels of monochloramine were first preformed at pH 9 in 0.05 M borate
buffer. The pH of this preformed monochloramine was then dropped immediately
to 7 with 0.2 N HC1. The monochloramine was found to be stable at this pH
for greater than four hours (see Table 3). This confirms previous studies by
Snead and Olivieri (77). If the chloramines had been initially formed at pH
7, the chloramines present would have been a mixture of both monochloramine
and dichloramine. By forming the chloramines at pH 9, and then dropping the
ph to 7, it was now possible to determine the effect of a lower pH along with
mostly monochloramine upon the. inactivation of poliovirus 1. As a comparison,
the study was also done using the test bacterium Escherichia col i. The
elucidation of the effect of solely a pH change to 7 was now possible.
Figure 13 therefore compares the disinfection of poliovirus 1 singles at 5C
by 11 mg/L of preformed monochloramine adjusted to pH 7 compared to 10.8 mg/L
of monochloramine formed and held at pH 9. The change in pH had no apparent
effect on the disinfection of the poliovirus 1 singles. This study was
repeated using coli as the test organism. In this study (shown in Figure
14), monochloramine levels of 2.0 mg/L at pH 7, and 2.2 mg/L at pH 9 were
tested. Contrary to what occurred with poliovirus, the lowering of the pH
from 9 to 7 increased the rate of monochloramine disinfection efficiency (at
the 99% inactivation level) about ten times. Figure 15 is another comparison
study of the disinfection at 5C of poliovirus 1 singles with monochloramine
at pH 9 and 7. The E. coli results shown here are from Figure 14. Again, as
seen in Figure 13, there was a distinctly different pattern of inactivation
for the virus than for the bacterium. At similar concentrations of mono-
chloramine the virus was inactivated at the same rate. On the other hand,
approximately the same concentration of monochloramine inactivated the test
bacterium more rapidly at pll 7 than pH 9. Thus, monochloramine was shown to
be a better disinfectant for bacteria at pH 7 than at 9, but this was not so
in the case of poliovirus 1.
Viruses are generally considered to be stable in the ptl range of 5 to 9
encountered in natural waters, whereas the growth of bacteria is usually
characterized by a similar range but have a pH optima near neutrality. The
effect of pH upon transport mechanisms across the bacterial cell membrane
may have influenced the greater monochloramine destructive effect at pR 7
compared to pH 9. For example, Chang (78,79), while studying the inactiva-
34
-------
TABLE 3. Stability of Monoehloramine at pH 7
Time after formation NH„CL content
(hr) (mg/L)
0 11
1.0 11
2.5 11
3.0 11
3.5 11
4.0 11
The pH of preformed N^Cl was dropped to pH 7.0 immediately after
NH^Cl formation.
35
-------
10 OCT
N H jC I
Level
9.0
10
10
t/i
,10
240
60
180
120
Mi nutes
ure 13. Inactivation of poliovirus 1 singles by monochloramine at 5C
pH 9 and pH 7 (preformed at pH 9) by similar concentrations
of the disinfectant
36
-------
1000C
2.2 mg/L NHp pH9
2.0 mg/ L NhyCI pH7
100
10
pH 9
1.0
pH 7
10 30
Minutes
40
50
Figure 14. Disinfection of Escherichia coli by monochloramine at 5C
at pH 9 and 7 (preformed at pH 9) by similar concentrations
of the disinfectant
37
-------
pH
100
• Poliov I r us 1
~ Poliovirus 1
8.5
8.7
2.0
2.2
70
9.0
7.0
9.0
10
.01
120
300
60
180
240
Minutes
Figure 15. The inactivation of poliovirus 1 singles and Escherichia
coll at 5C by monochloramine at pH 9 and pH 7 (preformed
at pH 9)
38
-------
tion of Entamoeba histolytica cysts by chlorine, noted greater uptake of
chlorine and less survival at low pH than at high pH. Dennis (80), in a more
recent study, found that the inactivation rate of the bacterial virus f2
increased with decreasing pH. The incorporation of chlorine into the f2
bacterial virus was dependent on pH, and the higher rates of incorporation
occurred at lower pH values. The fact that in my study poliovirus 1, an
animal virus, did not demonstrate a similar effect as that noted by Dennis
with the bacterial virus points out once again the caution that should be
displayed when using a surrogate animal virus, such as the f2 bacteriophage.
These studies should be continued using other animal viruses, to determine if
there is greater inactivation by monochloramine at pH 7 compared to pH 9.
COMPARISON OF THE DISINFECTION OF ESCHERICHIA COLI USING PREFORMED AND
FORMING MONOCHLORAMIKES
Chloramine research studies usually utilize preformed monochloramines as
the disinfectant. The combination of ammonia (NH^) with chlorine (C^) to
form chloramines for the treatment of drinking water was practiced for many
years. Ammonia is still deliberately added to some chlorinated public water
supplies to provide a combined available chlorine residual (i.e. chlora-
mines) . Monochloramine is the principal chloramine that is encountered in
drinking water treatment. Monochloramine formation is very rapid at the
concentrations and conditions of water treatment, the reaction (shown in Eq.
3) being usually 90% complete in about 1 minute (81,82). As shown in Figure
2, the reaction rate is maximum at the pH range 8.5 to 9.0 (81,82). Chlora-
mines are less effective oxidizing and disinfecting agents than free chlorine
residuals (i.e., hypochlorous acid and hypochlorite ion), but they are more
stable and can retain a residual in water for a longer period of time.
Chloramines can, however, be successfully used, and even preferred, under
certain use conditions. Since many waste waters contain large amounts of
nitrogenous substances, chloramines are usually the only form of chlorine
present unless breakpoint chlorination is practiced. In recent years chlora-
mines have not been recommended as a primary disinfectant because of low
germicidal efficiency. Criticism has been directed towards this decision of
the U.S. Environmental Protection Agency (83). It was pointed out that under
field conditions, disinfectants with high lethality coefficients may not be
in the final analysis as good as those, such as chloramines, with lower
lethality coefficients (83). Thus, factors such as raw water quality, the
presence of oxidLzable compounds, the contact times, the construction of the
chlorine contact chamber to maximize chlorine feed solution mixing rates, and
the ammonia to chlorine application ratios (for chloramines) must all be
evaluated and specific guidelines developed to maximize disinfection effi-
ciency (83,84) .
Another concern that has been expressed by some is that forming mono-
chloramines were better disinfectants than the application of preformed
monochloramines. Thus, this study attempted to cast more light upon the "real
world" situation where monochloramines are formed during the process of
disinfection and are not added in the preformed state. The disinfecting
efficiencies of preformed monochloramine (NH2CI) and forming monochloramine
(free chlorine and NH^) against the test bacterium E^_ coli were compared in
the same study, along with reference to the disinfecting ability of free
39
-------
chlorine alone. In the forming monochloramine study, the pH 9 buffer was
first dosed with sufficient free chlorine to give a 1.86 mg/L concentration,
then sufficient ammonium sulfate was added to produce an ammonia to chlorine
ratio of 6:1 (by weight). The ammonia solution and the E. coli inoculum were
added at the same time. A previous study showed that the E. coli was not
adversely affected by the ammonia alone. The results of these studies are
shown in Figure 16. The tests were done at 5C and pH 9. The forming mono-
chloramine was found to be more effective (about 1.2 times as effective) than
the preformed monochloramine. Split-second exposure of the E. coli inoculum
to the hypochlorite-hypochlorous acid mixture which existed at pH 9 in the
forming monochloramine study may have been responsible for the initial faster
kill of the test bacteria. This conclusion is made because when a mixture of
hypochlorite ion and hypochlorous acid at pH 9 (0C1 + HOC! curve in Figure
16) was used as a disinfectant in a separate test in this study, the latter
disinfectants destroyed 99% of the inoculum within a contact time of 3 min-
utes. In the first 3 minutes in the forming monochloramine curve in Figure
16, 30% of the original bacterial inoculum was destroyed, leaving fewer
bacteria to be disinfected by the forming monochloramines. The preformed
monochloramine curve showed a "hump" in the initial part of the curve, but
then after 15 minutes the bacteria were destroyed in a linear first order
fashion. No "hump" was observed in the forming monochloramine study, and
after 15 minutes this latter curve paralleled the preformed monochloramine
destruction curve. These results showed that the disinfection rates for the
forming and preformed monochloramines were the same after the first 15 min-
utes of the study; the observed differences in the positioning of the lines
can be attributable to the differences in bacterial numbers after the first 3
minutes of the study. Although the original bacterial inocula were similar,
there was a greater initial kill of the bacteria in the forming monochlora-
mine study than in the case of the preformed monochloramine. After the first
3 minutes of the forming monochloramine experiment, the ability of the newly
formed monochloramine to kill the remaining bacteria was the same as that
encountered in the preformed study, but there were more bacteria to disinfect
in the preformed study. It appears that the brief initial exposure of the
bacteria in the inoculum to the free chlorine present before the monochlora-
mine was completely formed accounts for the differences between the two mono-
chloramine survival curves.
SEQUENTIAL ADDITION OF POLIOVIRUS 1 TO DETERMINE THE EXTENT OF MONOCHLORAMINE
DISINFECTING EFFICIENCY
Survival curves seen in these studies often show retardant die-away/
inactivation patterns. Although the disinfectant level was never depleted
during the course of the experimentation, the question arose whether changes
had nevertheless occurred in the disinfectant's efficiency which would
account for retardant curves. Thus, a second inoculum of poliovirus 1 was
added two hours after the first virus administration (see Figure 17) to
determine if the inactivation rate of this subsequent virus inoculum would
mimic the first portion of the curve. As seen in Figure 17, there was a
reappearance of the rapid initial inactivation rate, indicating that the
disinfecting efficiency of the original monochloramine present had not been
effected or altered, and that this monochloramine was still capable of inact-
ivating the additional inoculum.
40
-------
100
Preformed NH2CI
10:
OCL" + HOCL
M I x t u r e
1.0
g/ L(HOCI + OCI-)
A 0.5
.01
TO
40
M I nutes
SO
BO
Figure 16. Disinfection of Escherichia col i 11229 at 5C and
pH 9 by forming and preformed monochloraraines
compared to a 0.5 tr.g/L mixture of hypoehlorous acid
(110C1) and hypochlorite ion (0C1 )
41
-------
1000
100
^^Pol iovi rus
Add i t ion
1X>
Pol lovl rus
v Control
.10
240
60
120
180
MInutes
Figure 17. Disinfecting efficiency of 10.8 mg/L monochloramine
at 5C and pll 9 to inactivate a sequential addition
of poliovirus 1 singles
42
-------
THE EFFECT OF INCREASING CONCENTRATIONS OF MONOCHLORAMINES UPON THE INACTIVA-
TION OF POLIOVIRUS 1 SINGLES
In the process of this research, it was noted that increasing the mono-
chloramine concentration did not proportionally increase the rate of inacti-
vation of poliovirus 1 singles. Figure 18 shows the effect of the almost
doubling of the monochloramine level from 12 to 22 mg/L at 5C and pH 9.
Although 12 mg/L of monochloranine was about 4 times more effective at the
99% inactivation point than the lower concentration of 5.4 mg/L monochlo-
ramine, 22 mg/L was found to be as effective as 12 mg/L. This was contrary
to the work of Butterfield and Wattie (85) who believed that "without excep-
tion" an increase in the amount of chloramine present increased the rate of
kill of their test organism, E. coli. However, what they termed "marked"
increases in the extent of kill was not observed in 60 minutes of exposure at
2 to 6C, with less than 1.2 mg/L residual at pH 8.5, and 1.5 mg/L residual at
pH 9.5. At the higher temperature range of 20 to 25C that was used they
found that monochloramine residuals of about 0.3 and 0.6 mg/L were required
to obtain about the same kill rate at pH 8.5 and 9.5, respectively. On the
other hand, Snead and Olivieri (77), while studying the inactivation of f2
bacterial viruses by monochloramine at 25C and pH 7, found that above a
monochloramine concentration of 4.0 mg/L the extent of f2 inactivation was
relatively independent of the monochloramine concentration. Below 4.0 mg/L
the degree of inactivation was found to be dependent on the monochloramine
concentration. My work described in this report is more consistent with the
results of Snead and Olivieri (77) than Butterfield and Wattie (85), but
differences might be due to the use of viruses versus bacteria as the test
organisms.
THE EFFECT OF CHLORIDE IONS UPON MONOCHLORAMINE DISINFECTION OF POLIOVIRUS 1
SINGLES AND ESCHERICHIA COLI
Studies by Scarpino et al . (1), and confirmed by Engelbrecht e_t al. (86)
and Jensen et al. (87), found that poliovirus 1 (Mahoney) was inactivated
more rapidly by chlorine in the form of hypochlorite ion (0C1 ) at pH 10 than
by hypochlorous acid (H0C1) at pH 6. Scarpino et al. (1) suggested that the
borate buffer system (containing KC1) had an influence on the hypochlorite
ion and hypochlorous acid virucidal relationships. Since 0. 2 N 11C1 had been
used to prepare preformed monochloramie at pH 9 (see The Effect of pH upon
Inactivation .... in Section 5)), it was decided to investigate the effect of
the chloride ion on the disinfection process. The addition of 0.2 N HC1 made
the 0.05 M borate buffer system about 0.02 M with respect to chloride ions.
A study was thus done testing the presence and absence of 0.02 M chloride
ions, added as the sodium salt (0.02 M NaCl), on the inactivation by monochlo-
ramine of poliovirus 1 singles at 5C and pll 9 using borate buffer prepared
without KCl. The results in Figure 19 (characteristic not only of this study
but one other) showed that the chloride ions had little or no effect on the
monochloramine disinfecting process, since the survival curves closely paral-
leled each other. If the chloride ions (as 0.02 M NaCl) had influenced
disinfection, a difference in inactivation kinetics would have been apparent.
A similar study with E. col i was also performed at 5C (see Figure 20) where
3.2 mg/L monochloramine was formed at pH 7, 0.02 K chloride ions were added
as the sodium salt, and disinfecting comparison was made to the same level of
43
-------
100
N H2CI Level
¦ 22. 0 mg/L
~ 12. 0 mg/L
10
IQ
>
>
k.
1.0
a
.10
.01
300
240
180
120
60
Minutes
Figure 18. The inactivation kinetics of poliovirus 1 singles
with increasing monochloramine at 5C and pH 9
44
-------
O 8.9
10
CD
>
>
.10
.01
50
150
250
Minutes
Figure 19. InactivatLon of poliovirus 1 singles at 5C and pH 9 by
monochloramine with and without the addition of 0.02 M
chloride ions as the sodium salt
45
-------
100
o no cr
• 0.02 M CI
10
<0
>
>
L.
3
(0
<*
1.0
.10
10
20
M Inutes
Figure 20. Disinfection of Escherichia coli 11229 at 5C and pli 7 by
3.2 mg/L monochloramine with and without the addition of
0.02 K chloride ions as the sodium salt
46
-------
monochloramine at pH 7 but without the added chloride ions. No effect of the
added chloride ions was observed. Therefore, the observed (Figure 14) dif-
ference in disinfection at pH 9 and pH 7 for E. coli was due to the pH change
to 7 and not due to the addition of chloride ions when the 0.2 N HC1 was
added to the buffer system.
SELECTION FOR MONOCHLORAMINE-RESISTANT POLIOVIRUS 1
Bates e^t a1_. (22) reported that a laboratory strain of poliovirus 1
(LSc) could be made resistant to free chlorine at pH 7 after repeated expo-
sures to initial free chlorine levels of 0.8 mg/L for up to 30 minutes of
inactivation. They exposed the virions to free chlorine, grew the survivors,
and then reexposed than to the free chlorine. After ten such exposures,
their data suggested increased resistance to free chlorine. There was a
gradual enhancement of resistance to the free chlorine doses over several
cycles of exposure to free chlorine, rather than a single-step process of
resistance. They believed that this gradual development of resistance sug-
gested an evolutionary or adaptive alteration in the virus population after
repeated sublethal exposures to free chlorine. Subsequently, Bates et al.
(88) exposed the same poliovirus strain previously used to 10 cycles of
inactivation by chlorine at pH 5 and pll 9 and compared their results to the
data from their earlier research (22). Virus exposed to chlorine at pH 9 (as
mostly hypochlorite ion) demonstrated more progressive development of resis-
tance than to the chlorine at pH 5 (as mostly hypochlorous acid). They
reported that polioviruses with resistance developed at pll 7 showed rates of
inactivation when exposed to chlorine at pH 9 similar to those of the virus
which was repetitively exposed to chlorine at pH 9. This suggested to them
that the mechanism of resistance development was the same at pH 7 and pll 9.
The development of poliovirus resistance at pH 5 to chlorine was not as
apparent as at pH 7 and 9. This was explained by them on the basis of the
faster inactivation rate in all the cycles at pH 5, which was due to the
predominate chlorine species, hypochlorous acid. They concluded that in-
creased amounts of hypochlorous acid or environmental conditions on the acid
side of neutrality would not favor selection of resistant viruses but would
favor virus inactivation. Also, at pli 7 and 9 where hypochlorite ion domi-
nated as the chlorine species, virus inactivation occurred more slowly and
facilitated the detection of virus plaques containing the most resistant
virus survivors. If the latter conclusion is correct, monochloramine (a
slower combined chlorine disinfectant than hypochlorite ion) could also be
successfully used as the chlorine species at pH 9 to develop resistant polio-
virus. However, increased resistance reported by Bates j^t al_. (22) might
have been partly due to the formation of aggregates in the virus suspension.
In an attempt to prepare a monochloramine resistant poliovirus we guarded
against the possible effect of aggregates influencing the appearance of
"resistant" variants by using the procedure of Floyd et al. (56) for polio-
virus singles preparation as well as the Bates et_ _al. (22) procedure. Both
procedures used for preparation of the virus inocula are outlined in Figure
3, page 19, of this report. The poliovirus prepared by both procedures were
exposed separately under the same test conditions for similar time periods.
After exposure to monochloramine, the more resistant plaques were isolated
and regrown and then re-exposed to monochloramine. Eight repetitive mono-
47
-------
chloramine exposure cycles were performed for viruses prepared by both proce-
dures .
Tables 4 and 5 summarized the results of these studies. The studies of
Table 4 with the Bates et^ _al1.-prepared viruses (22) did not show the devel-
opment of resistance to monochloramine. For instance, Figure 21 shows the
plotted % survival, data of study 10 (in Table 4) where viruses exposed 7 and
8 times to monochloramine were combined as the inoculum. The latter had to
be combined because of the low numbers grown-out of poliovirus survivors in
study 9. Comparison was made in this Figure to that obtained with the Bates
e_t _al_.-prepared virus (22) which had never been exposed to monochloramine. No
differences between the survival curves were apparent. However, the Table 5
survivor data showed development of virus resistance using the Floyd et
al_,-prepared virus (56) exposed to monochloramine. Although the viral resis-
tance patterns were at time irregular, such fluctuations might be due to the
possible heterogeneity of resistance of virus in separate, surviving plaques,
as noted by Bates e_t al. (22). Using this base of reasoning, this would
explain the demonstration of resistance to monochloramine as plotted in
Figure 22 from the percent survival data in studies 8 and 10. The virus in
study 8 had been exposed 7 times to monochloramine, whereas that in study 10
was exposed 8 times. When comparisons were made to Floyd et al.-prepared
poliovirus (56) which had not been previously exposed to monochloramine, a
development of resistance of the 8 times-exposed poliovirus was quite evident.
The 8 times-exposed poliovirus was exposed to 8.95 mg/L of monochloramine,
and was 2.3 times more resistant to monochloramine than either the unexposed
Floyd et ^1_.-prepared virus (56) or the virus exposed 7 times to monochlora-
mine in study 8. A gradual progression in the development of virus resis-
tance was evident in the research with the Floyd _et al. (56) inocula. Per-
haps if we had carried out the studies further and exposed the regrown virus
several more times to monochloramine more consistant resistance patterns
could have emerged.
DISINFECTION OF HEp-2 CELL-ASSOCIATED POLIOVIRUS 1 WITH HYPOCHLOROUS ACID AND
MONOCHLORAMINE
Disinfection studies with animal cell-associated poliovirus 1 were per-
formed using two continuous cell lines, Human Epidermoid Carcinoma (HEp-2)
and Buffalo Green Monkey (BGM) kidney cells. The cell-associated virus
system approximates the state of viruses as they are excreted from the body
into domestic sewage. Wastewater however contains many organic substances
which consume free chlorine. On the other hand, chlorine which has combined
with ammonia to form chloramines can be an important factor in disinfection.
Thus, these studies were performed to ascertain the effects on disinfection
rates of HEp-2 and BGM eel 1-associated poliovirus 1, which also provided the
turbidity to the systems. The first study described below was with HEp-2
cell-associated poliovirus.
Disinfection of HEp-2 eel 1-associated poliovirus 1 with 2.2 mg/L and
2.28 mg/L hypochlorous acid at 5C and pH 6 is shown in Figure 23. This HEp-2
cell-associated poliovirus 1 preparation showed protection of virions when
compared to the aggregated preparation of poliovirus 1 (See Section 4, Mate-
rials and Methods for preparation of cell-associated viruses and virus aggre-
48
-------
TABLE 4. Results of Repetitive Exposures to Monochloraaines of
Bates et al.-Prepared Poliovirus I (22) at 15C and pH 9
Exposures to NK^Cl
0
0
1
2
3
4
5
6
7
8
7.8®
Study Number
10
1
2
3
4
5
6
7
8
9
10
Date of Study
Initial Titer
8/29
1.6xl05
4/1
2.5xl05
4/18
4.IxlO4
5/2
3.2x10
5/9
'' 1.4xi03
7/8
9.4xl04
7/18
6.1x10^
7/25
5. 2xl03
8/8
3.5xl03
8/15
2.7xl03
8/29
8.9xl02
(PFU/nl)
Time*5
Percent Survival
t
162
80.0
62.5
50.0
25.7
54.2
TNTC0
TNTC
42.9
72.7
135
10
_d
-
-
-
-
-
31.1
-
21.7
-
-
1 5
106
-
-
-
-
10.4
-
18.0
-
24.7
82.0
20
-
-
-
-
-
-
14.9
-
28
-
-
30
27.5
TNTC
1.6
0.41
Nl)e
2.5
2.5
2.1
7.7
7. 1
28.1
45
35.0
-
-
-
-
0.49
1.5
0.15
0.17
9.1
22.5
60
17.5
TNTC
0.05
0.028
ND
0.21
0.05
0.05
0.037
2.5
15.7
75
6.9
-
-
-
-
-
KD
0.08
ND
1.1
6.3
90
TNTC
-
-
-
ND
0.04
0.07
ND
ND
0.29
5.5
105
-
-
-
-
-
-
0.01
ND
m
0.24
-
] 20
TM"C
TNTC
0.014
ND
ND
0.004
0.003
-
ND
0.19
3.9
180
TNTC
1.2
0.003
ND
ND
ND
ND
KD
ND
ND
0.45
240
0.36
-
ND
ND
ND
ND
ND
ND
ND
0.3
Final fcH^CLCmg/L)
9.6
12.U
11.2
12.2
11.1
11.3
13.1
11.6
10.15
10.2
9.3
a Viruses exposed 7 and 8
b In minutes after initial
tines were
exposure.
cotabined
as the
inoculum.
c Too numerous to
d Not sampled.
count.
e Not detected, no
recovery.
-------
TABLL 5. Kesults of Repetitive lixposurts to Monochloramine of
Floyd et al.-Prepared I'o 1 iovlrus 1 (56) ac 15C and pH 9
Exposures to L
0
0
1
2
3
4
5
6
7
8
8
Study I Amber
10
1
2
3
4
5
6
7
•8
9
10
UiLt; of Study
8/21
4/1
4/18
5/2
5/9
7/8
7/18
7/25
8/8
8/15
*8/21
Initial Titer 9
.9xl03
l.QxlO5
5. <*103
7.3x10
1 4.5xl02
2.4xl05
1.2xl06
7. lxlO4
4.6xl03
1.6cl04
1.2cl04
(PFU/nil)
tine*
Percent Survival
t
94.9
26
112
61.6
4.7
TNTCT
TNTC
46.5
89.1
76
104
10
_c
-
-
-
-
-
TNTC
-
11.5
-
-
15
23.2
-
-
-
0.22
TNTC
-
8.6
-
25
45.8
20
-
-
-
-
-
-
TVTC
-
3.9
-
-
30
2.2
0.75
0.3
0.41
NDd
15
TNTC
2.0
2.4
5.7
18.3
45
0.66
-
-
-
-
5
TKIC
0.04
0.43
6
4.9
l>0
0.52
0.24
ND
0.14
ND
2.1
0.11
0.04
0.46
4.3
2.3
75
0.2
-
-
-
-
-
ND
-
0.22
1.4
0.89
90
0.1
-
-
-
ND
0. 74
ND
0.002
0.115
0.37
-
105
-
-
-
-
-
-
ND
-
0.029
0.36
-
120
0.05
0.003
0.18
ND
ND
0.12
ND
ND
0.059
0.14
0.28
180
NO
SO
ND
ill
ND
0.008
ND
ND
ND
N>
0.011
240
ND
-
ND
ND
-
0.002
0.003
0.002
ND
~
ND
Final NHX1 fag/L)
9.15
12. i
11.4
12.3
11.1
11.75
12.8
12.1
10. IS
10.4
B.9S
a In minutes after initial exposure,
b 'ioo numerous to count.
c Not Bamplud.
d Not detected, no recovery.
-------
1000
A Initial Bates' prepared Poliovirus 1 exposed to
9.6 mg/L NH2CI
• Bates' prepared Poliovirus 1 survivors of 7
and 8 exposures and finally disinfected
with 9.3 mg/L NH2CI
100
w.
3
<0
1.0
0.1
3
2
1
4
Hours
Figure 21. Inactivatlon of Bates et a.1 .-prepared poliovirus 1 (22)
at pll 9 and L5C before and after repeated exposure to
raonochloramine.
51
-------
200
«
>
3
l/l
*
Initial Singles Pollovirus
exposed to 9.15 mg /L NH2CI
Pollovlrusi Singles
survivors of 7 exposures
now exposed tolO. 5 mg/L
H2CI
llovirus1 Singles
rvivors ofSexposures
w exposed to 8.95 mg/L
H CI
Hours
Figure 22. Inactlvation of Floyd e^t -prepared poliovirus 1 (56)
at pK 9 and 15C before and after repeated exposure to
monochloramine
52
-------
100
O 2.2 mg 'L ; Cell-Assoc. (2.45NTU)
A 2.28 mg/L ; Cell-Assoc- (1.25NTU)
• 2.98 mg/L ; Aggregated
~ 3.04 mg/L : Aggregated
10
- O
1.0
HEp-2
Cell •associated
Pol io v i r u s 1
.10
Aggregated
Po llovlrus 1
.01
Minutes
Figure 23. InaetLvation of aggregated and HEp-2 eel 1-associated
poliovirus 1 with hypochlorous acid at pH 6 and 5C
53
-------
gates). This effect is most dramatically demonstrated at the 99.9% through
99.99% inactivation portion of the curves. Percent survival data including
final turbidity levels in nephelometric turbidity units (NTU), final hypo-
chlorous acid levels and test dates are contained in Table 6. An earlier
study by Hoff (23) on the relationship of turbidity to disinfection of pota-
ble water using HEp-2 cell-associated poliovirus showed a similar interfer-
ence with hypochlorous acid inactivation of the cell-associated viruses.
Comparison of the disinfection at 50 of HEp-2 cell-associated poliovirus
1 with monochloramine at pH 9 and hypochlorous acid at pH 6 is shown in
Figure 24. As can be seen from the graph, 3.OA mg/L hypochlorous acid inac-
tivated 99% of the HEp-2 cell-associated virions in 3 minutes, while 12.2
mg/L monochloramine inactivated 99% in 120 minutes, making the hypochlorous
acid 40 times more effective as a disinfectant than the monochloramine under
these test conditions.
Studies on the effect of organic turbidity on the disinfection of HEp-2
cell-associated poliovirus with monochloramine concentrations ranging from
4.15 to 21.0 mg/L at 5C and pH 7 and 9 are shown in Figure 25. This is a
concentration-time plot at the 90% death point which also shows the inactiva-
tion of poliovirus 1 singles with monochloramine at 5C and pH 9. Increasing
the monochloramine dosage almost twice from 12.2 to 21.0 mg/L at pH 9 in the
presence of almost the same turbidity reduced the time required for 90% virus
inactivation, i.e., from 50 minutes at 12.2 mg/L to 30 minutes at 21.0 mg/L
monochloramine. Increasing the turbidity in Figure 25 from 0.8 NTU to 2.0
NTU at almost the same monochloramine levels in two cases (10.35 and 11.3
mg/L, respectively) at pH 7 significantly decreased disinfection efficiency.
Turbidity caused by the presence of animal cells interfered with the disin-
fection process. The pK change from 9 to 7 had no apparent effect on the rate
of inactivation of poliovirus 1, whether or not the virus was associated with
animal cells or not.
Some tests on HEp-2 cell-associated poliovirus 1 were run concurrently
with Mr. Donald Berman of the U.S. Environmental Protection Agency, using
0.05 M phosphate buffer at 5C and pli 7. Table 7 gives for these studies the
% survival data, final turbidity levels in Nephelometric Turbidity Units
(NTU), initial and final monochloramine levels, and test dates. The same
amount of HEp-2 cell-associated poliovirus 1 inoculum per unit volume of
buffer was added for each test. As seen in Table 7, poliovirus 1 singles
were used as positive virus controls on test dates 10/31/80 and 11/14/80. By
inspection of the table, it can be seen that the poliovirus 1 singles inacti-
vation rates are more rapid at the end of the exposure time period. Some
protection of poliovirus was occurring due to the association with HEp-2
tissue culture cells.
HEp-2 cell-associated viruses have been also used by Foster e_t al. (89)
and Emerson et _al. (90) in their studies with ozone inactivation of cell-
associated viruses (both poliovirus and coxsackievirus). Although the disin-
fectant was different, their results indicated again the protective effect of
virus-association with HEp-2 cells. Their inactivation data on poliovirus
and coxsackievirus indicated that cell-associated viruses required higher
ozone residuals for inactivation than unassociated viruses. They emphasized
54
-------
TABLfc 6. Distrit ection of HEp-2 Cell-Associated Pol. lov true 1 with Hypochlorous Acid at 5C and pH 6
HEp-2 Cell-Associated Follovlrus 1
Date of lest:
7/12/79
7/26/79
Pollovlrus 1 Control (A^gre^ated)
7/12/79 7/26/79
Concentration
of U0C1 in mg/L:
Initial :
l- ina!:
NTUa:
Control liter:
3.08
2.28
1.25
1.05xl05
3.04
2.20
2.45
7.55x10*
3.08
3.04
0.51
6.05x10*
3.04
2.98
0.45
4.85x10
Tine
10 Sec.
15 "
20 "
25 "
30 "
40 "
50 "
60 "
75 "
90 "
2 Min.
3 "
4 "
5 "
10 "
20 "
30 "
40 "
50 "
60 "
PFU/ml
2.8x10"
1.7x107
1.4x10*
1.3x10*
7.5x10:?
5.3x10,
5.4x10;:
2. 1x10^
1.7x10;
6.6x10,
1.9x10:
2.9x10,
l-OxlO*.
3.0x10
NlJ
ND
ND
ND
ND
ND
X Survival
26.7
16.1
13.2
12.4
7.1
5.1
5. 1
2.0
1.6
0.63
0.35
0.055
0.020
0.006
ND
ND
ND
ND
ND
ND
PFU/nl
2.2x10*
5.4xlof
1.5x10?
1.1x10*
5.8x10^
2.5x10^
2. Ox 10
1.1
4.3x10!
1.2x10'
1.3x10,
1.0x107
3.2x10:
1.6x10;:
2.0x10
ND
ND
ND
ND
2.0x10
5xlCj
X Survival
29.1
7.2
19.9
14.6
7.7
3.3
2.6
1.5
0.57
0.16
0.167
0.135
0.04
0.02
0.003
ND
ND
ND
ND
0.003
PFU/ml
7.2x10*
5. 7x1of
4. 4xlof
4.0x10^
1. 5x 1 of
l.OxlO,
1.1x10,
2.0x10,
1.0x107
1.0x10
ND
ND
ND
ND
% Survival
11.9
9.4
7.3
6.6
2.5
1.7
1.8
0.33
0.165
0.0165
ND
ND
ND
ND
PFU/ml
1.9x10*
1.2x10*
6.OxlO~
4.1x10,
3.8x10,
1.9x10::
1.8x10,
6.2xl0r
2.7x10,
9.4x10!,
2.0x10
ND
ND
ND
Z Survival
39.2
24.7
12.4
8.45
7.8
3.9
3.7
1.28
0.56
0.19
0.004
ND
ND
ND
a Nephelometric Turbidity Units (NIU)
b Not detected.
-------
00
10
i £. £ in y/ l. n n-v ¦
pH 9.0,1.65 NTU
1.0
U1
3.04 mg/L HOC i
pH 6.0, 1.25 NTU
01
60
180
120
240
Minutes
Figure 24. Inactivation of HEp-2 ce]1-associated poliovirus 1 at 5C
by hypochlorous acid at pH 6 and monochloramine at pH 9
56
-------
Ln
* 20
D>
E
"cm10
I
Z
—
*
NH£ILevel
(m g / l)
4.1 5
Turbidity
(ntu)
0.8
pH
7
-
c
10.35
1.5
7
-
•
1 1.3
2.0
7
¦
1 22
1.6 5
9
~
21.0
1.5
9
O Poliovirusl Singles
\—i—i—H-H
9KT
I I I I I I 1
TOO
Minutes
Figure 25.
Concentration-time relationship for 90% inactivation of poliovirus 1 singles
and HEp-2 cell-associated poliovirus 1 at different turbidity levels and
concentrations of monochloramine at 5C and pH 7 and 9
-------
1ABLE 7. Ulstniectlon ui Htjj-2 Cei l-At>4>ociatt:d Pulioviruti 1 and Poltoviruu 1 Singles
with Koncr.hlornmine at 5C ar.d pH 7
Dat«= ui Test;
10/24/BU
HEp-2 Cell-Associated
10/3i/80
11/14/80
Pellovlrus 1 Stories
10/31/80 11/14/80
Cuncontrat ion
of NH CI in n\fjL
Initial:
Final:
N'l'Ua;
Control Titer:
4.30
4.5
O.fc .
4.6x10^
11.5
11.3
2.0
4.6x10
10.6
10.35
1 5
9.8x10
11.5
11.
0.2
1.85x10
10.6
10.55
b
2.3xl0H
Cn
oo
1 tltn.
2
5
10
15
20
3C
40
50
60
75
90
120
130
15C
180
PFU/tt.I
4.55x10S
3.2.110.
1.8x10'
7.1x10^
1.2x10'
1.2x10?
4. 6x 10,
4.4x107
5.9x1c"!
5.3x10,
4.0x10^
4.0x10
4.Ox 1C
h
b
4.0x10
,3
% Survival
98.9
69.6
39.1
15.4
26.0
26.0
10.0
9.6
12.8
11.5
8.7C
0.87C
0.87C
h
b
0.87°
PFU/rnl
6.9x10"
4.0x10^
1.2x10*
6.0x10*
1.5x10*
8.6x10?
8.7x10^
7. 3x 107
1.9xln;
2.1x10*
1.6x10*
1.9x10
h
b
8.4x10
% Survival
150.0
8/.0
26.0
13
32.6
18.7
18.9
7.4
15.9
4. 1
4.6
3.5
4.1
b
b
1.8
5
PFli/ml
i, 3x10;!
5.0x10*
6.0x10,*
1.6x10*
1.9x10,
1.6x10
9.3x10*
6.5x10*
3.1x10*
4.7x10*
4.0x10*
2.0x10
1.7x10*
7.8x10^
1.9x10
X Survival
13.3
51
6.1
16.3
19.4
16.3
9.5
6.6
3.2
4.8
4.1
2.0
b
1.7
0.79
0.27
PFU/ml
9. Ox i 0^
1.3x1^
2.1x0*
1.3x10*
1.3x10*
9. 5x10;:
3.6x 10
1.65x10
9.0x10
b
2.3x10:
I. /xiO'
4.7x10
b
b
2.5x10
2
% Survival
48.6
70.3
113.5
70.3
70.3
51.4
19.5
8.9
4.9
b
1.2
0.92
0.25
b
b
0. 14
PFU/skI
1.65x10*
9.4x10":
1.1x10*
6.0x10.
5.Ox 10,
1 .Ox 10^
7.2x10^
3.9x10
b „
1.7x10'
9.7x10
b
2.7x10
b
2.5x10
Survival
71. ;
40.9
47.8
26. 1
21.7
12. 2
4.8
3.1
1. 7
b
0. /6
0.42
b
0.13
b
0. 11
a Nephelometric turbidity unite
b Not done
c Estimated value?
-------
that since the HEp-2 cells were approximately 10 to 15 y m in size, such sized
particles could be easLly removed from finished water by filtration (89).
Similar sized particles are also found in wastewater effluent after activated
sludge treatment.
DISINFECTION OF BGM CELL-ASSOCIATED P0LI0V1RUS 1 WITH HYPOCHLOROUS ACID,
MONOCHLORAMINE, AND DICHLORAMINE
The inactivation of Buffalo Green Monkey (BGM) cell-associated polio-
virus 1 was studied using three disinfectants, i.e. hypochlorous acid (H0C1)
at 15C and pH 6.0, monochloramine (NH^CI) at 15C and pH 9, and dichl oramine
at 5C and pU 4.5. Figure 26 is a comparison of the disinfection at 15C and
pH 6 of BGM cell-associated poliovirus 1 by 0.03, 0.42, and 1.20 mg/L hypo-
chlorous acid at turbidity levels of 1.75, 1.05, and 1.10 NTU, respectively.
All the survival curves showed extended tailings caused by the association of
the poliovirus 1 to the cells and to themselves (aggregation) during the
disinfection process. A comparison between the disinfection of BGM cell-
associated pol iovirUvS 1 by monochloramine and hypochlorous acid at similar
turbidities and concentrations is shown in Figure 27. Whereas 90% of the
cell-associated viruses were inactivated in 15 seconds by the hypochlorous
acid, 95 minutes was required to reach the 90% inactivation point with the
monochloramine. Even under conditions as difficult as this to disinfect
viruses, hypochlorous acid was about 380 times as effective as the monochlo-
ramine. Figure 28 is a summary monochloramine concentration-time plot for
the 90% inactivation of BGM cell-associated and unassociated viruses. The
BGM cell-associated poliovirus 1 points are represented by bold symbols.
Most of these "associated" points were above the poliovirus 1 unassociated
singles curve, indicating that the cell-associated viruses were being pro-
tected from inactivation. In another study by us of the disinfection of BGM
cell-associated poliovirus 1 by chlorine dioxide we were unable to demon-
strate such protection (64).
A final study was done (see Figure 29) comparing the inactivation of BGM
cell-associated polioviruses to the survival of unassociated poliovirus 1
singles, using diclilorainine as the disinfectant at 5C and pH 4.5. No differ-
ences were observed in the rates of disinfection between the two poliovirus
preparations, although the dichloramine concentrations were similar (17.0
mg/L for the unassociated versus 17.35 mg/L for the associated poliovirus).
The lack of protection could be due to rapid penetration of the cell mass by
the dichloramine.
CO L IF 0 KM DISINFECTION STUDIES
Disinfection studies using coliforms were divided into two groups: (a)
disinfection of naturally-occurring coliforms from fecal suspensions, and (b)
disinfection of fecal coliforms associated with primary effluent solids.
Disinfection with Hypochlorous Acid of Naturally-Occurring Coliforms Obtained
From Human Feces
Fecal suspensions were prepared as outlined in Preparation of Fecal
Particulates in Section 4 of this report. These fecal suspensions provided
59
-------
NTU
100
NTU
NTU
.4 2 mg
M i nutes
Figure 26. Inactivation of BGM cell-associated poliovirus 1 by
hypochlorous acid at 15C and pH 6
60
-------
100
.10
0 1.16 mg't N H2CI ; 1.6 NTU
~ 1.20 mg/L HOCI ; 1.1 NTU
60
120 180
Minutes
240
Figure 27. Inactivation of BGM eel 1-associated po1iovirus 1 by
raonoch lor amine (pti 9) and hypochlorous acid (pH 6) at 15C
61
-------
Turbidi ty
(ntuI
NHjCI Level
(mg/Lj
tv>
- ~ 0,7
~ 1.0
t 20
- n 1.9 8
_i
O 2.0
"v.
a
¦ .6 5
E
+ .90
10
O 1.6
5
- 9 Pol
X
—
z
—
iovirus 1
3.3 2
rigies
M inutes
Figure 28. Concentration-time relationship for 90% inactivation of BGK cell-
associated and unassoeiated poliovirus 1 by various concentrations
of monochloramine at 15C and pH 9
-------
• Singles 17.0 mg/L NHCIj.l NTU
a B6M eell-issocljted 17.3 5
mg/L NHClj.2.1 NTU
100
10
m
>
>
10
w
3
(0
#
0.1
.01
60
120
180
240
Minutes
Figure 29. Inactivation of BGM cell-associated poliovirus and
poliovirus 1 singles by dichlorainine at pH 4.5 and 5C
63
-------
an uncontested source of naturally-occurring coliforms which had not been
nurtured by laboratory media. Difficulties soon arose due to the chlorine
demand of the feca! suspensions. Repeated washings (10 times) of the fecal
suspension did not remove the chlorine demand or result in a suspension
containing a consistant chlorine demand. Table 8 shows the shifting levels
of chlorine demand encountered, although the chlorine demand was found to be
sometimes lower at 5C than 20C. Therefore, it was decided to do further
studies at 5C. Figure 30 shows a typical hypochlorous acid disinfection
study at 5C and pll 6 with naturally occurring coliforms at a turbidity of 3.2
NTU. In similar studies done by Foster _et _al. (89), no protection of fecal-
associated coliforms was evident at 1.0 NTU, although some protection was
shown with a turbidity of 5.0 NTU and an inital ozone level of 0.10 mg/L and
below.
Disinfection With Monochloramine of Naturally-Occurring Coliforms Obtained
From Primary Effluent
Laboratory studies were performed on naturally-occurring total coliforms
obtained from primary effluent. These coliforms were associated with parti-
cles which, when suspended in 0.05 M phosphate buffer, gave a turbidity level
of 5.0 NTU. Table 9 is a summary of the test levels of monochloramine used
and their corresponding turbidity levels. Survival data for the naturally-
occurring coliforms after their exposure to different monochloramine levels
at different turbidities (Table 9) are recorded in Table 10. Coliforms were
present after 60 minutes contact time in tests 2 and 3, at initial mono-
chloramine levels of 12.2 and 5.1 mg/L, respectively. The turbidity levels
were similar in these two tests (5.8 NTU in test 2, 5.5 NTU in test 3). Tests
1, 4, and 5 at initial monochloramine levels of 23.2, 10.3, and 10.3, respec-
tively, showed a more rapid coliform decline after 1 minute in test 1, and
after 10 minutes in tests 4 and 5. In tests 1, 4, and 5 coliforms were
detected erratically. For instance, coliforms were found after 105 minutes
in tests 1 and 5, although substantial kill had occurred after one minute and
10 minutes, respectively. Coliforms were present after 120 minutes contact
time with monochloramine during test 3, which contained the lowest level of
monochloramine (4.95 mg/L N^Cl) used. These results with naturally-
occurring coliforms obtained from primary effluent were substantially consis-
tent to those of Hoff (23), who used 0.5 mg/L hypochlorous acid and turbid-
ities of 1 and 5 NTU. His survival curves extended over a 60 minute period
at both 1 and 5 NTU's. The results reported here were similar in that the
organic material from the primary effluent associated with the coliforms and
particles demonstrated a protective effect in tests 2 and 3, and to a more
limited extent in tests 4 and 5.
64
-------
TABLE 8. Chlorine Demand of Suspensions of Naturally-Occurring
Coliforms after a 30 Minute Exposure
Chlorine
Initial
Final
Demand of
Temperature
NTUa
H0C1
R0C1
1.0 ml of
(C)
Level
Level
Suspension
(mg/L)
(mg/L)
(mg/L)
0.91
0.34
0.57
20
_b
0.66
0.27
0.39
5
3.0
0.71
0.27
0.43
5
3.2
0.74
0. 16
0.58
20
1.75
0.50
0.11
0.40
5
1.75
0.74
0.32
0.42
5
1.75
1.05
0.33
0.72
20
2.2
0.75
0.25
0.50
5
2.2
0.85
0.30
0.55
5
2.0
1.05
0.51
0.54
20
-
1.05
0.55
0.50
20
-
1.05
0.66
0.39
5
-
1.05
0.61
0.44
5
-
0.66
0.42
0.24
5
2.0
0.74
0.64
0.10
5
2.0
2.65
0.14
2. 51
5
16
3.35
0.10
3.25
5
18
^Nephelometric Turbidity Units.
Not done.
65
-------
100
HOCI Turbidity
(mg/L)
-------
TABLE 9. Monochloramine and Turbidity Levels Used For Disinfection
at 5C and pH 7 of Naturally-Occurring Coliforras Associated
with Primary Effluent Solids
Test number
1 2 3 4 5
Monochloramine (NH^Cl) Levels
Initial NH CI 23.2 12.2 5.1 10.3 10.3
(mg/L)
Final NH CI 22.6 11.95 4.95 9.9 9.95
(mg/L)
Turbidity (NTU) Concentrations
NIL Test 1.8a 5.8 5.5 8.0 to 1.75b 5.5
aTurbidity level run at 5C, the remaining turbidity measurements were done at 25C.
occulation in test beaker made turbidity reading fluctuate.
-------
TABLE 10. The Inactivation by Monochloramine of Naturally-Occurring Coliforms
Associated with Primary Effluent Solids at 5C and pH 7
Test number
1
2
3
4
5
Most
Probable Number/100 ml
Initial
5.4x10"*
1.8xl05
5.4x10^
9.2x 105
3.5xl05
Final
3.6xl04
6.3x104
5.4xl05
7.9xl05
3.5xl05
Average
2.9xl05
1.2xl05
5.4xl05
8.5xl05
3.5xl05
Test times
(minutes)
C
£
1
1.4x10
1. 8x10
5.4x10
5.4x10
1.6x10
10
NDb
1.3xl02
1.6xl05
1.6xl03
2.8xl03
20
ND
13
2.4xl04
ND
2
30
ND
1.3xl02
5.4xl03
ND
5
45
ND
23
1.6xl03
2
ND
60
2
23
17
ND
ND
75
ND
ND
27
2
2
90
ND
ND
2
ND
ND
105
2
ND
2
ND
2
120
ND
ND
2
ND
ND
180
c
-
ND
—
—
a Analysis of coliforms through confirmed most probable number test procedure,
b Not detected, 110 recovery,
c Not sampled.
-------
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