&EPA
United States
Environmental Protection
Agency
Municipal Environmental Research EPA-600/2-79-089
Laboratory August 1979
Cincinnati OH 45268
Research and Development
Effect of
Particulates on
Ozone
Disinfection of
Bacteria and
Viruses in Water
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL PROTECTION TECH-
NOLOGY series. This series describes research performed to develop and dem-
onstrate instrumentation, equipment, and methodology to repair or prevent en-
vironmental degradation from point and non-point sources of pollution. This work
provides the new or improved technology required for the control and treatment
of pollution-sources to meet environmental quality standards.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/2-79-089
August 1979
EFFECT OF PARTICULATES ON OZONE DISINFECTION OF
BACTERIA AND VIRUSES IN WATER
by
Otis J. Sproul
The Ohio State University
Department of Civil Engineering
Columbus, Ohio 43210
Charles E. Buck
University of Maine Orono
Department of Microbiology
Maura A. Emerson
Douglas Boyce
Douglas Walsh
Diana Howser
University of Maine Orono
Department of Civil Engineering
Orono, Maine 04401
Grant No. R-804587
Project Officer
John C. Hoff
Drinking Water Research Division
Municipal Environmental Research Laboratory
Cincinnati, Ohio 45268
MUNICIPAL ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
This report has been reviewed by the Municipal Environmental Research
Laboratory, U.S. Environmental Protection Agency, and approved for publica-
tion. Approval does not signify that the contents necessarily reflect the
views and policies of the U.S. Environmental Protection Agency, nor does
mention of trade names or commercial products constitute endorsement or
recommendation for use.
n
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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 enviornment and
the interplay between its components require a concentrated and integrated
attack on the problem.
Research and development is that necessary first step in problem
solution and it involves defining the problem, measuring its impact, and
searching for solutions. The Municipal Environmental Research Laboratory
develops new and improved technology and systems for the prevention,
treatment, and management of wastewater and solid and hazardous waste
pollutant discharges from municipal and community sources, for the preserva-
tion 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 communi-
cations link between the researcher and the user community.
This research provides basic information concerning the disinfection
capabilities of ozone, against coliforms and enteric viruses, when these
microorganisms are associated with or incorporated into particulate mater-
ials. Fecal materials, HEp-2 Cells, .aluminum oxide floe and bentonite clay
were used as the particulate materials at 1 and 5 NTU.
m
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ABSTRACT
This research program was initiated in order to determine the effect of
participates on ozone disinfection of enteric bacteria and viruses adsorbed
to or incorporated into these materials. The participate materials were
fecal material, HEp-2 cells, aluminum oxide floe and bentonite clay. Fecal
coliforms, poliovirus (Sabin Type 1), Coxsackievirus A9, porcine picorna-
virus type 3 (Strain ECPO-6) and f~ bacteriophage were used in this study.
The concentration of particulates to which the bacteria or viruses were
adsorbed to or incorporated into before ozonation was 1 or 5 Nephelometric
Turbidity Units (NTU).
Results of the ozonation study indicate that the encasement or adsorp-
tion of enteric bacteria and viruses in fecal material, both human and
porcine, and HEp-2 cells protects these microorganisms from a concentration
of ozone and contact time that would normally inactivate the bacteria and
viruses in an unadsorbed or free state. The HEp-2 cells gave the greatest
amount of protection for the cell-associated poliovirus and Coxsackievirus
studied. Equipment was developed to maintain a continuous high ozone con-
centration in order to inactivate all of the poliovirus and Coxsackievirus
incorporated into the cell or cellular material. It was necessary to main-
tain a concentration of 5.33 to 4.81 mg/L ozone to inactivate the cell-
associated Coxsackievirus in 5 to 10 minutes. The ozone concentration and
the time required to inactivate the virus was far in excess of the ozone
concentration necessary to inactivate the unadsorbed or free state virus.
Hydrated aluminum oxide floe and bentonite clay afforded little or no
protection to the Escherichia coli, poliovirus and Coxsackievirus adsorbed
to these particles over that of the microorganisms in the free state. The
f? bacteriophage adsorbed to bentonite clay particles was inactivated at a
slower rate than the freely suspended phage.
This report was submitted in fulfillment of Grant No. R-804587 by the
University of Maine Orono under the sponsorship of the U.S. Environmental
Protection Agency. This report covers the period August 20, 1976, to
January 20, 1979.
IV
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CONTENTS
Foreword i i i
Abstract iv
Tables vii
Figures ix
Acknowledgments x
1. Introduction and Literature Review 1
2. Conclusions 5
3. Materials and Methods 7
Cell Culture 7
LLC-MK2 Cells 7
Minipig Kidney (MPK) Cells 7
HEp-2 Cells 8
Viruses 8
Poliovirus (Sabin Type 1) 8
Coxsackievirus A9 8
Porcine Picornavirus 8
Bacteriophage f? 8
Virus Stock for Cell-Associated Studies 9
Bacteria 9
Escherichia coli 9
Streptococcus fecal is 9
Fecal Escherichia coli Surviving Ozonation 9
Fecal Samples 10
Glassware and Stock Solutions 10
Glassware 10
Solutions 10
Purification of Viruses and Bacteria 11
Poliovirus, Coxsackievirus and Porcine Picornavirus 11
Bacteriophage f2 11
Escherichia colt 12
Titration of Viruses and Bacteria 12
Poliovirus and Coxsackievirus 12
Porcine Picornavirus 13
Bacteriophage f2 13
Escherichia coif 14
Streptococcus fecal is 14
Fecal-Associated Microorganisms 14
Preparation of Particulate Materials 14
Hydrated Aluminum Oxide 14
Bentonite Clay 16
Fecal Samples 17
HEp-2 Cell-Associated Virus 17
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CONTENTS (Continued)
Presonicated HEp-2 Cell-Associated Virus 18
Microscopy 18
Light Microscopy 18
Ozonation and Disinfection Apparatus 18
Ozonation Apparatus 18
Continuous Flow Disinfection Apparatus 20
Declining Residual Batch Reactor 23
Batch Reactor for Continuous Ozonation 25
Ozone Determination 25
4. Results and Discussion 28
The Effect of Fecal Material on Ozone Disinfection
of Coliform Bacteria and Enteric Viruses in Water 28
Results 28
Viruses 28
Bacteria 32
Discussion 36
The Effect of Hydrated Aluminum Oxide Floe on Ozone
Disinfection of £. coli and Viruses in Water 39
Results 39
Poliovirus 39
Coxsackievirus A9 42
Bacteriophage f~ 42
Escherichia coif 46
Discussion 48
The Effect of Human Epithelial Carcinoma on Ozone
Disinfection of Poliovirus (Sabin Type 1) and
Coxsackievirus A9 51
Results 51
HEp-2 Cell-Associated Coxsackievirus 54
Batch Reactor 54
Continuous Ozonation of HEp-2 Cell-Associated
Polio Virus and Coxsackievirus 54
Discussion 58
The Effect of Bentonite Clay on Ozone Disinfection of
£. coli and Virus in Water 60
Results 60
Poliovirus 60
Coxsackievirus A9 60
f~ bacteriophage 60
Escherichia coli 66
Discussion 69
Other Reports Based on this Research 71
References 73
VI
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TABLES
Number Page
1 Concentration of Microorganisms Per Gram of Feces (wet weight) 29
2 Fecal Particle Size Distribution 29
3 Inacti.vatton of Poliovirus (Sabin Type 1) and Fecal-Associated
Poliovirus (Sabin Type 1) 30
4 Inactivation of Fecal-Associated Poliovirus (Sabin Type 1) at
-20°C 31
5 Inactivation of Porcine Picornavirus Type 3 (Strain ECPO-6)
and Fecal-Associated Porcine Picornavirus Type 3
6
7
8
9
10
Strain ECPO-6 j
Inactivation of Eschfirichia coli and Streptococcus fecal is
Inactivation of Escherichia coli and Fecal -Associated
Coli forms
Inactivation of Escherichia coli (ATCC 15766) and Escherichia
coli. Surviving Ozonation When Associated with Fecal Material
Association of Poliovirus (Sabin Type 1) with Hydrated
Aluminum Oxide
Inactivation of Poliovirus (Sabin Type 1) Unadsorbed and
33
34
35
37
40
Adsorbed at 1 and 5Nephelometric Turbidity Units (NTU) of
Hydrated Aluminum Oxtde Floe. 41
11 Association of Coxsackievirus A§ with Hydrated Aluminum Oxide 43
12 rnactivatton of Coxsackievirus A9 Unadsorbed and Adsorbed at
1 and 5 NTU of Hydrated Aluminum Oxide Floe 44
13 Association of f2 Bacteriophage with Hydrated Aluminum Oxide 45
14 Inactivation of f2 Bacteriophage Unadsorbed and Adsorbed at 1
5 NTU of Hydrated Aluminum Oxide Floe 47
15 Association of Escherichia coli with Hydrated Aluminum Oxide 49
VI 1
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TABLES (Continued)
Number Page
16 Inactivation of Escherichia coli Unadsorbed and Adsorbed
at-1 and 5 NTU of Hydrated Aluminum Oxide Floe. 50
17 Inactivation of HEp-2 Cell-Associated Poliovirus (Sabin
type 1) at 1 and 5 NTU. 52
18 Effect of Presonication on Inactivation of HEp-2 Cell-
Associated Poliovirus (Sabin Type 1) at 1 and 5 NTU. 53
19 Inactivation of HEp-2 Cell-Associated Coxsackievirus A9
at 1 and 5 NTU. 56
20 Batch Inactivation of HEp-2 Cell-Associated Poliovirus
at 5 NTU. 57
21 Batch Inactivation with Continuous Ozonation of HEp-2
Cell-Associated Poliovirus at 5 NTU. 59
22 Association of Poliovirus (Sabin Type 1) with Bentonite Clay 61
23 Inactivation of Poliovirus (Sabin Type 1) Unadsorbed and
Adsorbed at 1 and 5 NTU of Bentonite Clay. 62
24 Association of Coxsackievirus A9 with Bentonite Clay 63
25 Inactivation of Coxsackievirus A9 Unadsorbed and Adsorbed at
1 and 5 NTU Bentonite Clay 64
26 Association of fg Bacteriophage with Bentonite Clay 65
27 Inactivation of f% Bacteriophage Unadsorbed and Adsorbed at
1 and 5 NTU of Bentonite Clay 67
28 Inactivation of Escherichia coli Unadsorbed and in Association
with 5 NTU of Bentonite Clay 68
29 Summary of Inactivation of Unadsorbed Organisms 70
vm
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FIGURES
Number Page
1 Ozonation Apparatus 19
2 Continuous Flow Disinfection Apparatus 21
3 Ozonation and Continuous Flow Apparatus 22
4 Batch Reactor With Declining Residual 24
5 Batch Reactor for Continuous Ozonation 26
6 HEp-2 Cells Before Ozonetion at 5 NTU 55
7 HEP-2 Cells Sonically Treated Before Ozonation at 5 NTU 55
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ACKNOWLEDGMENTS
Following the move of Dr. Sproul, the original Principal Investigator,
from the University of Maine to Ohio State University in August 1977, Dr.
Charles E. Buck of the Department of Microbiology, University of Maine,
assumed responsibility for this research. The efforts of Dr. Buck in assum-
ing this responsibility and seeing the research through to successful
completion are gratefully acknowledged.
We also wish to thank Dr. Dean Cliver and Dr. Donn D'Alessio, University
of Wisconsin, Madison, Wisconsin for supplying the porcine picornovirus, MPK
cell cultures, and porcine feces containing porcine picornovirus (Dr.
Cliver) and infant stool specimens containing vaccine type 1 poliovirus (Dr.
D'Alessio) for use in this study.
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SECTION 1
INTRODUCTION AND BACKGROUND REVIEW
Renovated water may be described as water which has undergone a purifi-
cation process for the removal of contaminants. Although natural biological
and physical processes are capable of renovating water, increased waste loads
have necessitated the use of water and wastewater treatment processes to
supplement natural renovation processes. At the present time, waste loads are
so heavily contaminanted that commonly used technology may not remove the
hazards associated with reused water. In addition, decreasing potable water
sources may soon require widespread utilization of renovated waste waters for
agricultural and domestic purposes, whereas in the past most renovated water
was used solely for industrial purposes. Thus, extensive evaluation of the
capabilities of purification processes both natural and otherwise are needed
to ensure the safety of consumers (1).
The potential hazards associated with direct potable reuse of waste-
water have been summarized by Long and Bell (2). One of the primary health
threats posed by direct use of renovated water is the transmission of
bacterial and viral pathogens. The large number of waterborne disease out-
breaks which have been reported in the literature are reasons for concern.
Extensive reviews of waterborne outbreaks have been published by Long and
Bell (2), Craun et al. (3) and the Committee on Environmental Quality Manage-
ment (4). Low level transmission of viruses must be considered a source of
endemic disease and hence a cause for concern (2).
The majority of waterborne disease outbreaks of known etiology are
bacterial in origin. The most commonly identified pathogen during the period
between 1971-1974 was Shi gel!a (3). Other genera of pathogenic bacteria
found in water as a result of fecal pollution were Salmonella, Vibrio,
Mycobacterium, yersinia and Leptospira (5).
Unlike sporadic outbreaks of waterborne disease caused by bacterial
pathogens, illnesses caused by viral transmission in water are difficult to
detect. The only viral agent which has definitely been linked to trans-
mission in water is infectious hepatitis virus. However, "any virus excreted
in the feces and capable of producing infection when ingested should theore-
tically be transmissible in water". Therefore, viruses of concern include the
enteroviruses (poliovirus, coxsackievirus and echovirus), the adenoviruses
and the reoviruses, as well as infectious hepatitis virus (4). In all, more
than 100 types of viruses fall into the enteric group and, with the exception
of hepatitis virus have been isolated from wastewater and other contaminanted
sources of water (6) (7).
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The failure of epidetniological studies to implicate water as a route of
viral dissemination may result from the fact that small quantities of virus
only produce infection rather than disease when ingested (8). Infected
individuals, however, excrete viruses in concentrations as high as 10^
viruses/gm feces (9). Thus, secondary infections may occur and these may
result in overt disease (8). Since epidemiological studies normally use
disease as the index of pathogen occurrence rather than infection, the role of
water in viral transmission may not be apparent.
Viral densities in raw sewage were estimated by Clarke (10) to be about
7000 plaque forming units (PFU)/1. Direct examination of wastewater suggests
that densities may actually be somewhat higher than these estimates. As
many as 463,500 viruses/liter of raw sewage have been detected in some parts
of the world (9), while virus densities in the United States appear to be
considerably lower (11).
Despite the high levels of viruses in wastewater, a minimal amount of
viruses are present in receiving waters. Commonly used water and wastewater
treatment processes will not completely remove viruses (12). Once effluents
are discharged to receiving waters, virus dilution, dieoff and other factors
combine to reduce virus levels significantly. Reduction of viruses to levels
as low as 1 to 2 infectious doses/100 gal may occur (11). These levels of
viruses, though difficult to detect, may constitute a health hazard. It
has been demonstrated that 1 TCID (tissue culture infectious dose for 50
percent response) or 2 PFU are capable of producing infection when ingested
by gavage (13). Due to the manner in which viruses, particularly entero-
viruses, are produced, excreted and transported (intestinal tract+feces-»water)
and the number and variety of particles in natural waters, it is likely that
the majority of viruses in the natural environment are associated with sus-
pended and particulate solids (14). Virus association with solids does not
necessarily imply inactivation. Virions may remain infective while
associated with solids or they may be eluted from the solids and still retain
infectivity (15).
The possibility that bacterial and viral pathogens associated with
solids may be protected from disinfectants during treatment and hence remain
viable has been a question of considerable importance in recent years. In
reviewing the 1962 U.S. Public Health Service Drinking Water Standards, con-
sideration was given to changing the standard for turibidity. The National
Interim Primary Drinking Water Regulations established a turbidity Maximum
Contaminant Level (MCL) of 1 Nephelometric Turbidity Unit (NTU) with up to 5
units allowed provided the turbidity does not interfere with disinfection,
prevent maintenance of effective disinfection throughout the distribution
system or interfere with microbiological determinations (16).
The extent of protection given to microorganisms by encasement,
adsorption or competitive oxidation of turbidity causing materials, depends on
a number of factors. These include the size of the microorganism in question
relative to particulate size, the relative innate disinfection resistance of
the microorganisms and the type of particle(s) causing turbidity. The
importance of these factors are discussed by Hoff (17) in a comprehensive
review of the relationship of turbidity to disinfection of potable water.
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Hoff concluded that the interference of turbidity with disinfection depends
primarily on the type of turbidity present rather than the amount of turbid-
ity. The protection given to microorganisms by inorganic particulate matter
is generally negligible or relatively minor. In comparison, organic turbid-
ity offers an appreciable amount of protection to associated microorganisms.
Sproul (12) has indicated that current water and wastewater treatment
practices do not effect complete removal of viruses. Berg (8) (14) also
maintained that treatment processes constitute adjunctive removal of viruses.
In addition, Berg concluded that by removing those substances that interfere
with terminal disinfection, treatment processes facilitate the final achieve-
ment of total removal or destruction of viruses. In the final analysis,
however, reliance for complete destruction of pathogens must be placed on
terminal disinfection.
Chlorine traditionally has been the most widely used disinfectant in the
United States. Recently, however, its ability to form carcinogenic or
potentially carcinogenic compounds such as chloroform and other trihalo-
methanes has become widely documented (18) (19) (20). As a result of these
findings, a review of possible alternate disinfectants has been initiated
by the Environmental Protection Agency (18).
Ozone is a possible alternative to chlorine. Although widely used out-
side the United States, ozone has failed to gain widespread acceptance with-
in the United States. One of the primary reasons for this is the inability
of ozone to persist in a distribution system for any length of time. Ozone
is a very unstable gas with an effective half life ranging from 20 to 30
minutes. Maintenance of a residual in the distribution system is, therefore,
not possible. This problem may be overcome by adding small quantities of a
disinfectant, such as chlorine, just before the water passes into the distri-
bution system.
The advantages of ozone far outweigh its disadvantages as a disinfecting
agent. Ozone is a potent biocide which does not impart a taste or odor to
treated water. To date no obvious effects on the health of consumers from
ozonated byproducts have been documented (18). Several extensive reviews of
the bactericidal and viricidal capabilities of ozone in water (18) (21) (22)
(23) and wastewater (21) (22) treatment have been published. Despite the
apparent superiority of ozone, the lack of standardization in disinfection
research precludes comparison of most of the data which have been generated.
The principal objective of this investigation was to develop information
on the disinfection protential of ozone for viruses and bacteria which have
been attached to or incorporated within various solids. Disinfection
studies were conducted over a range of 0, concentrations, using viruses and
bacteria in both unadsorbed and adsorbed state, in order to determine whether
or not a protective effect was afforded the adsorbed organisms. Experiments
were carried out with the solids at 1 and 5 NTU so that a comparison of
disinfection data could be made at both turbidity levels. The viruses used
for this study were poliovirus (Sabin type 1), coxsackievirus A9 and
bacteriophage f?. In addition, information on the inactivation by ozone of
Escherichia coif Hfr K-13 (ATTC#15766) and Streptococcus fecal is (ATTC 19433)
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was obtained.
The solids used included fecal materials, aluminum oxide floe, clay and
cells.
1. Fecal materials were used to simulate the disinfection condition
of wastewater treatment and water treatment plants using a secondary
wastewater plant effluent to produce potable renovated water. The
naturally occurring fecal associated coliforms, poliovirus (Sabin
Type 1) and porcine picornavirus Type 3 (Strain EPCO-6) were used
for this study.
2. Hydrated aluminum oxide floe was used to simulate the floe particles
formed during the coagulation process used in conventional water and
wastewater treatment plants when A12 (^K is used as a coagulant.
The viruses used were poliovirus (Sabin type l)j coxsackievirus A9,
and f? bacteriophage. Inactivation studies were also obtained for
Escherichia coli with the aluminum oxide.
3. Bentonite clay suspensions were used because they are natural com-
ponents of some soils and are known to be an adsorbant of many
bacteria and viruses. The viruses used were poliovirus (Sabin type
1), coxsackievirus A9, and f? bacteriophage. Escherichia coli was
also used in this study.
4. Human epithelial carcinoma cells were used in order to simulate
infected and dying cells of the intestinal tract of an individual
infected with enteric viruses. These cells may be a source of fecal
pollution in a potential drinking water source. Poliovirus (Sabin
type 1) and coxsackievirus A9 were used in this study.
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SECTION 2
CONCLUSIONS
The results of this study indicate that the encasement or adsorption of
enteric bacteria and viruses in fecal material and HEp-2 cells protects these
microorganisms from a concentration of ozone that would normally inactivate
the bacteria and viruses in an unadsorbed or free state. Protection of fecal-
associated coliform bacteria, poliovirus (Sabin Type 1) and porcine picorna-
virus Type 3 (strain ECPO-6) at 5 Nephelometric Turbidity Unit (NTU) was
demonstrated at initial ozone concentrations of 0.096, 0.013 and 0.024 mg/L
ozone, respectively. The cell-associated poliovirus and Coxsackievirus at 1
and 5 NTU was not inactivated by ozone concentrations up to 4.68 mg/L ozone
for 30 seconds exposure in the Sharp continuous flow disinfection apparatus.
Even when the cells were sonicated to break up the clumps of cells and
particles, total inactivation was not obtained. However, there was an in-
crease of about one log reduction in the sonicated sample. A batch reactor
with decreasing residual and an initial ozone concentration of 3.93 mg/L
ozone and an initial titer 1.2 x 10^ PFU/ml ozonated stream was also ineffec-
tive in inactivating the cell-associated poliovirus at 5 NTU after 75 minutes
contact. In the batch reactor with continuous ozonation and initial ozone
concentrations of 6.82 and 6.50 mg/L ozone and initial poliovirus concen-
trations of 2.3xl05 and 3.6 x 104 PFU/ml.respectively the cell-associated
poliovirus was inactivated in two minutes. The Coxsackievirus was inactivated
in five to fifteen minutes when the initial ozone concentrations were 5.33 to
4.81 mg/L ozone and the initial virus concentrations were 1.9 x 10^ to 1.9 x
105 PFU/ml. The initial ozone concentration and residual was somewhat less
than used for the poliovirus.
The hydrated aluminum oxide floe offered no protection at either 1 or 5
NTU for poliovirus, Coxsackievirus or £. coli. The inactivation data for
adsorbed f^ bacteriophage were inconclusive since most of the absorbed virus
was unrecoverable after being adsorbed to the aluminum oxide floe.
Poliovirus, Coxsackievirus, and Escherichia coli associated with bento-
nite clay were inactivated by ozone at rates similar to the freely suspended
organisms. The inactivation of adsorbed f2 bacteriophage was slower than the
unadsorbed phage. The bentonite clay absorbed virus complex was prepared in
concentrations of 1 and 5 NTU for disinfection studies with poliovirus,
Coxsackievirus and f? bacteriophage. Excherichia coli was tested at only 5
NTU.
The particle size of the fecal material, hydrated aluminum oxide floe
and bentonite clay was comparable to particulates that may escape water
filtration plant. The particle size of the HEp-2 cell-associated virus,
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10 to 15 ym, was at the upper limit of size causing turbidity after filtra-
tion. Therefore, most of the turbidity particles of HEp-2 cell-associated
viruses would have been filtered out before the start of the disinfection
process if the water had been filtered.
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SECTION 3
MATERIALS AND METHODS
CELL CULTURE
LLC-MK2 Cells
LLC-MK2 cells (Hull, Monkey Kidney, Rhesus type) were obtained from
(Microbiological Associates, Walkersville, Maryland) and maintained at 37 C
in growth medium consisting of Eagle's Minimal Essential Medium (MEM) with
Earle's balanced salt solution and L-glutamine (Grand Island Biological
Company, Grand Island, New York) containing 2 percent newborn calf serum
(Grand Island Biological Company, Grand Island, New York). Antibiotics
were routinely incorporated in all media. Sodium penicillin G (The Upjohn
Company, Kalamazoo, Michigan), streptomycin sulfate (Eli Lilly and Company,
Indianapolis, Indiana) and amphotericin B (E. R. Squibb and Sons, Inc.,
New York, New York) were employed in the growth medium at levels of 100 units/
ml, 100 yg/ml and 5 yg/ml, respectively.
Cells were grown in 32 oz. Brockway prescription bottles (Microbiological
Associates, Walkersville, Maryland) until a confluent monolayer was obtained.
The monolayer was then dispersed using 0.2 percent trypsin. Approximately
100 ml of fresh medium were added and the suspension was equally divided into
3 bottles.
Minipig Kidney (MPK) Cells
The minipig kidney (MPK) cell line (ATCC CCL166) was propagated at 37°C
in growth medium consisting of MEM containing 20 percent fetal calf serum
(Grand Island Biological Company, Grand Island, New York), 1 percent essential
vitamin mixture (Microbiological Associates, Walkersville, Maryland), 2 per-
cent essential amino acid mixture (Microbiological Associates, Walkersville,
Maryland), 300 units/mg penicillin G, 300 ug/ml streptomycin sulfate and 5
yg/ml amphotericin B.
Subcultivation of MPK cells was conducted according to the procedure
supplied by Sandy Wolens at the University of Wisconsin, Madison, Wisconsin
(Personal Communication). After a monolayer had been obtained in 32 oz.
prescription bottles it was washed 3 times with 25 ml aliquots of calcium
and magnesium-free phosphate buffered saline (PBS). Ten ml of an enzyme
preparation consisting of PBS with 0.01 M sodium ethylenediamine tetraacetate
(EDTA) (Fisher Scientific Company, Fair Lawn, New Jersey) and 0.01 percent
pancreatin (Grand Island Biological Company, Grant Island, New York) was
then added to the flask. After incubating the flask at 37°C for 5 to 10
minutes, the cells were dispersed by aspirating them several times against
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side of the bottle. Approximately 60 ml of fresh medium were then added and
the cells were equally divided into 2 bottles.
HEp-2 Cells
The HEp-2 cell line was propagated in growth medium consisting of (MEM)
with Earle's balanced salt solution, supplemented with 5% mycoplasma tested
and virus screened newborn calf serum (Grand Island Biological Company, Grand
Island, New York). Penicillin sodium, N.F. (Upjohn Company, Kalamazoo,
Michigan) 200 units/ml, streptomycin sulfate, U.S.P. (Eli Lilly and Company,
Indianapolis, Indiana) 200 meg/ml and fungizone (E.R. Squibb and Sons, New
York, New York) 5 meg/ml were routinely added to growth and maintenance media.
Cultures were grown in 32 oz. prescription bottles (Microbiological Asso-
ciates, Walkersville, Maryland) at 37°C.
VIRUSES
Poliovirus (Sabin-Type 1)
Poliovirus (Sabin-Type 1) which had been maintained in the Sanitary
Engineering research laboratory at the University of Maine at Orono for
several years was used for virus stock development. Virus stock was devel-
oped by infecting monolayers of LLC-Mk2 cells with poliovirus. The growth
medium was removed from the monolayer and 5 ml of poliovirus suspension were
placed on the cells and allowed to adsorb for 1/2 hours. After adsorption,
40 ml of fresh medium were added and the cells were incubated for approxi-
mately 24 hours. When the majority of cells showed evidence of cytopathic
effect (CPE), the bottle was shaken to remove the cells. The crude stock
was then frozen at -2QQC. This procedure was repeated using the crude stock
to infect additional cells until a sufficient amount of stock was accumulated.
Coxsackievirus A9
The Coxsackievirus A9 stocks were prepared in the same manner as polio-
virus (Sabin type 1). These stocks had also been maintained in the Sanitary
Engineering research laboratory at the University of Maine for several years.
Porcine Picornavirus
Porcine picornavirus Type 3 (Strain ECPO-6) was supplied by Dr. Dean
Cliver at the University of Wisconsin, Madison, Wisconsin. Monolayers of
MPK cells were infected with 5 ml porcine picornavirus and allowed to adsorb
for 2 hours. The virus was swirled over the cell sheet every 15 minutes
during the adsorption period. Fresh medium was then added and the cells were
incubated at 37°C until extensive CPE was noted (usually 2 to 4 days);
crude stock was frozen at -20°C.
Bacteriophage f?
The bacteriophage fo (ATCC #15766 B) was obtained from the American Type
Culture Collection, Rockville, Maryland. The virus stock was prepared by a
-------
procedure described by Watson and Drewry (24) and Cooper and Zinder (25), and
modified by Gentile (26). An aliquot of 100 ml of an 18 hour broth culture
of Escherichia coli Hfr K-13 (ATTC #15766) was inoculated into 5 liters of
sterile broth. The broth was incubated for 3 hours at 37°C with continuous
agitation and aeration. After 3 hours, enough virus was added to yield a
5:1 ratio of virus to E_. coli. Incubation for 6 hours at 37°C with agitation
and aeration followed. Lysozyme (0.25 mg/ml) was added and incubation
continued for 30 minutes. One ml/L of chloroform was introduced, agitated
for 30 minutes, and then aerated to remove the residual chloroform. (Both
lysozyme and chloroform served to aid lysis).
Virus Stock For Cell Associated Studies
A crude virus stock was prepared by removing the growth medium from con-
fluent monolayers of HEp-2 cell cultures and adding 5 ml of purified Sabin
Type 1 poliovirus containing 10° plaque forming units (PFU) per ml. After
adsorption for 30 minutes, growth medium was replaced. Infected cell cultures
were incubated 12 to 18 hours until (CPE) of the monolayer was apparent. The
remaining cells were removed by gentle snaking, dispensed to test tubes, and
stored at -20°C until use.
Crude virus stocks of Coxsackievirus A9 were obtained in a identical
manner. These stocks were stored in test tubes at -20°C until use.
BACTERIA
Escherichia coli
Escherichia coli Hfr K-13 (ATCC 15766) were obtained from the American
Type Culture Collection, Rockville, Maryland. The bacteria were grown on
nutrient agar (Difco Laboratories, Detroit, Michigan) slants for 24 hours at
37°C. Slants were stored at 4 C and maintained throughout the duration of
the project by periodic transferring.
In preparation for an experiment an initial culture of E_. col i was
prepared by inoculating 100 ml of nutrient broth (BBL, Cockeysville, Maryland)
with _E. coli from nutrient agar slant. The culture was then incubated 12 to
18 hr at 37°C. Subsequent cultures were prepared by inoculating 100 ml of
nutrient broth with 1 ml of a previous broth culture which had been main-
tained at 37°C.
Streptococcus fecal is
Streptococcus fecal is (ATCC 19433) was processed in a manner similar to
that described for E_. col i. Trypticase soy agar (BBL, Cockeysville, Maryland)
was used in lieu of nutrient agar and trypticase soy broth (BBL, Cockeys-
ville, Maryland) was used in preparations of broth cultures of £. fecal is.
Fecal E. coli Surviving Ozonation
L- coli which survived ozonation were isolated from an EMB agar (Difco
-------
Laboratories, Detroit, Michigan) plate. The identity of the £. col i was con-
firmed by an IMViC test conducted according to Standard Methods (27).
Fecal Samples
Adult stool specimens were obtained from volunteers at the University of
Maine at Orono, Orono, Maine. One gram samples were stored at -20°C in 4 oz.
ozone-demand free specimen bottles. These samples were used as a source of
fecal-associated coliform bacteria.
Porcine feces containing porcine picornavirus were furnished by Dr. Dean
Oliver at the University of Wisconsin, Madison, Wisconsin; samples were
stored at -70°C.
Infant stool specimens containing poliovirus (Sabin-Type 1) were sup-
plied by Dr. Donn D'Alessio at the University of Wisconsin, Madison,
Wisconsin. Sample titers were determined in the following manner. One-half
gram portions of fecal samples were homogenized in 10 ml PBS containing 1500
units/ml penicillin G, 1500 yg/ml streptomycin sulfate and 3 yg/ml amphoter-
icin B by mixing 30 seconds with a Vortex-Genie (Scientific Industries Inc.,
Bohemia, New York). Samples were then centrifuged 30 minutes at 14,000 x g,
5 C in a fixed angle rotor (Head 870) in a Model B20a 1EC refrigerated
centrifuge (Damon/lEC Division, Needham Heights, Massachusetts). Supernatant
titers were determined. Those samples with the highest titers were pooled
for subsequent use; all samples were stored at -20°C.
GLASSWARE AND SOLUTIONS
Glassware
Ozone was used exclusively as the disinfectant in these experiments and
glassware associated with sample preparation, adsorption, and ozone
determination was treated to make it demand free. Cleaning with a chromic
acid solution was followed by 9 rinses with tapwater and 5 rinses with dis-
tilled water. The glassware was soaked in distilled water which had been
ozonated at a high rate, then dried in a 180°C oven to ensure the elimina-
tion of ozone. A cabinet was maintained for the dust free storage of the
glassware.
Solutions
Solutions used in these experiments were prepared so as to make them
ozone demand free. Distilled water was glass-distilled twice and stored in
20 L carboys. This triple distilled water (TDW) was ozonated at a high rate
and solutions were immediately prepared. Excess ozone was allowed to dis-
sipate or was removed by autoclaving the solutions. The pH was maintained
at a constant value for all experiments by using TDW which was 0.01 M in
NaCl and buffered to pH 7.0 with 0.001 M phosphate from sodium salts. A
20 L volume of this buffer solution was made up for each disinfection trial,
and then ozonated to achieve the desired concentration of ozone for the
experimentation planned. Following an experiment the remaining buffer was
10
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poured into prescription bottles, autoclaved, and stored. Demand free
buffer thus generated was used to dilute virus stock, resuspend samples
pelleted by centrifugation, and to carry out adsorption experiments.
PURIFICATION OF VIRUSES AND BACTERIA
Poliovirus, Coxsackievirus and Porcine Picornavirus
Concentration and purification of the crude virus stocks was conducted
according to the procedure described by Guskey and Wolff (28) and Boardman
and Sproul (29) with minor modifications. Following 3 freeze-thaws, the
virus was concentrated tenfold by dialysis against polyethylene glycol 20,000
(PEG) which was obtained from (Fisher Scientific Company, Fair Lawn, New
Jersey). The concentrated virus was mixed with an equal quantity of tri-
fluoro-trichloro-ethane (freon) (Polar Chemicals Inc., Lewiston, Maine) and
the freon-virus mixture was homogenized to remove cellular debris in a
Sorvall Omni-Mxier (Ivan Sorvall Inc., Norwalk, Connecticut) at 14,000 rpm
for 2 minutes. The homogenized virus was then centrifuged at 4080 x g, 5°C
for 10 minutes in a fixed angle rotor (Head 870) in a Model B20A IEC
refrigerated centrifuge. The virus contained in the aqueous phase was
further purified by ultracentrifugation onto a 2 to 3 ml cushion of cesium
chloride (Sigma Chemical Company, St. Louis, Missouri) containing 0.803 g Cs
Cl/ml water. The aqueous phase was ultracentrifuged 2 hours at 131,000 x g,
4°C in an SW 27 swinging bucket rotor in a Beckman Model L5-65 preparative
ultracentrifuge (Beckman Instruments Inc., Palo Alto, California). After
removing the supernatant, the CsCl cushion was diluted in sterile triple
distilled water (TDW) to obtain the desired volume and 1.2 ml aliquots
dispensed in 5 ml rubber capped serum vials (Kimble, Toledo, Ohio); stock
was stored at -70°C until needed.
Bacteriophage f?
The bacteriophage f2 was concentrated and purified according to the
techniques described by Watson and Drewry (24) and Cooper and Zinder (25)
and modified by Gentile (26). Crude f2 stock was centrifuged at 5,000 x g
for 15 minutes in an SS-34 rotor using a Sorvall RC-2 refrigerated centrifuge
(Ivan Sorvall Co. Inc., Norwalk, Connecticut). The supernatant was poured
off and saved, the pellet resuspended in 100 ml sterile TDW and homogenized
in the Sorval Omni-Mixer for 5 minutes while surrounded by an ice bath.
Centrifugation at 5,000 x g for 15 minutes was again initiated and the super-
natant was combined with the supernatant obtained from the previous step.
Sterile (NH4)2 504 (0.3 g/ml) was added to the supernatant and the mixture
was stored overnight at 4°C to allow complete precipitation. This mixture
was then centrifuged at 15,000 x g for 20 minutes and the supernatant
discarded. The remaining pellets were washed and consolidated in sterile 28
percent (NH4)2S04 and dissolved in 27 ml of sterile TDW. The dissolved
pellets were mixed with a sterile stirring bar for one hour at 4°C.
Centrifugation of the mixture followed, and the supernatant was poured off
and saved. The pellets were resuspended in 5 ml sterile TDW, disrupted with
sonic energy (sonicated), and again centrifuged. The remaining supernatant
was combined with that from the previous step. This combined supernatant
11
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was ultracentrifuged at 20,000 x g for 10 minutes in an A-321 rotor in an
International B-60 ultracentrifuge. The new supernatant was transferred to
fresh centrifuge tubes and ultracentrifuged onto a 2 ml CsCl cushion (0.065
g/tnl) at 125,000 x g for 2 1/2 hours at 1C. The supernatant was then re-
moved from each tube and the CsCl cushions consolidated into two tubes.
These tubes were then completely filled with fresh CsCl and ultracentrifuged
at 125,000 x g for 20 hours. At this time the fluid was removed from above
and below the visible phage bands, and the concentrated phage from both tubes
were combined into one tube. Fresh CsCl was again added followed by ultra-
centrifugation at 125,000 x g for 20 hours. The remaining phage band was
diluted 1:400 in sterile TDW and dispersed in 1 ml aliquots into rubber cap
serum vials. The phage was then stored at -70°C until needed.
Escherichia coli
Unadsorbed bacterial samples were prepared from 12 to 18 hour broth
cultures. The bacteria were harvested by centrifugation at 7,500 x g for
15 minutes in a fixed angle rotor (Head 870) in a model B20A IEC refrigerated
centrifuge at 5 C. Soluble ozone demanding material was removed by washing
the harvested bacteria. Washings were done by centrifuging the sample at
7500 x g, 5°C for 15 minutes and resuspending the pellet in ozone-demand
free 10"3 M, pH 7 phosphate buffer with 0.01 M NaCl. After five washings,
the final pellet was resuspended in a small amount of buffer. A tenfold
dilution of this sample was then made in ozone-demand free buffer. Five ml
were injected into the continuous flow apparatus and initial concentrations
were then determined.
TITRATION OF VIRUSES AND BACTERIA
Poliovirus and Coxsackievirus
The plague assay procedure of Wolf and Quimby (30) was used for the
enumeration of poliovirus and Coxsackievirus. Monolayers of LLC-MK2 cells
were dispersed with 0.2 percent tryspin and suspended in approximately 50 ml
of growth medium. One 32 oz bottle was used for every 10 tissue culture
plates. After splitting an appropriate number of cell monolayers, the cell
suspensions were pooled to obtain a uniform suspension. Five ml portions of
the pooled cells were placed in 60 x 15 mm tissue culture plates (Lux
Scientific Corporation, Newbury Park, California) and incubated at 37°C in an
atmosphere of 5 percent C02 until confluent monolayers were obtained.
Tenfold dilutions of poliovirus or Coxsackievirus were made in MEM with-
out serum but with 300 units/ml penicillin G, 300 yg/ml streptomycin sulfate
and 5 yg/ml amphotericin B. The growth medium was removed from the plates
and 0.2 ml of an appropriate viral dilution placed on duplicate monolayers.
The virus was allowed to adsorb for 1/2 hour at 37°C in an atmosphere of 5
percent C02- The overlay was prepared by mixing 2 parts of a melted 2 per-
cent purified agar (Difco Laboratories, Detroit, Michigan) which was kept at
50°C, and 3 parts of 2X MEM containing 4 percent newborn calf serum, 300
units/ml penicillin G, 300 yg/ml streptomycin sulfate and 5 yg/ml ampho-
tericin B which was kept at 37°C. The overlay was mixed by inverting several
12
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times and 5 ml of overlay were placed on each plate. The overlay was allowed
to harden and the plates were incubated 84-96 hr at 37°C in an atmosphere of
5 percent C02 and 100 percent humidity.
Following incubation, the cells were fixed with 37 percent formaldehyde
(Fisher Scientific Company, Fair Lawn, New Jersey) for 15 minutes. The
overlay was then removed by aiming a jet of water at the agar dish interface.
Cell sheets were stained with 1 percent crystal violet in 95 percent ethyl
alcohol by transferring the crystal violet from one plate to the next. After
staining, the cell sheets were rinsed in a pan with continually running water,
dried and counted.
Porcine Picornavirus
Infectivity of the porcine picornavirus was determined by using a micro-
titration technique. A monolayer of MPK cells from a 32 oz prescription
bottle was dispersed as described previously. A 0.2 ml aliquot of medium
containing dispersed cells was placed in each well of a microtiter plate
(Costar, Cambridge, Massachusetts). The plates were incubated at 37°C in an
atmosphere of 5 percent C02 until the monolayer reached 50 to 70 percent
confluency.
Appropriate serial dilutions of the porcine virus were prepared in MEM
as described for propagation of MPK cells, however, no serum was added. The
growth medium was removed and a 0.1 ml aliquot of 2X dilution medium was
added to each well which was to receive an undiluted sample of porcine
picornavirus. A 0.1 ml aliquot of IX dilution medium was introduced into
each remaining well. Replicate 0.1 ml inoculums of each viral dilution were
then introduced into six wells. All control wells received an additional 0.1
ml of IX dilution medium. After incubating the plates 2 hours in an atmos-
phere of 5 percent C02> the medium was removed and replaced with 0.2 ml
aliquots of MPK growth medium. Plates were examined 4 to 6 days after
infection for CPE. The viral titers were calculated and expressed as 50
percent tissue culture infectious dose (TCIDso) (31).
Bacteriophage f2
The procedure for titration of f2 phage described by Adams (32) and
Watson and Drewry (24) was used with minor modifications. Prior to each
experiment small overlay tubes were prepared containing 3 ml of 0.7 percent
purified agar. Immediately preceding their use, the tubes were autoclaved
for 5 minutes and cooled to 50 C. Standard 100 x 15 mm plastic petri dishes
(Fisher Scientific Co.), containing nutrient agar (Difco) with 0.85 percent
NaCl, were also prepared in advance and stored at 4°C until used. These
dishes were warmed to room temperature 1 to 2 hours before needed. Phage
samples from particular trials were serially diluted in TDW containing 0.85
percent NaCl. To each overlay tube was added 0.2 ml of an 18 hour nutrient
broth (BBL) culture of £. coli followed by 1 ml of diluted virus. After
sufficient mixing, the overlay was poured onto the nutrient agar surface of
the petri dish and allowed to harden at room temperature for one half hour;
two dishes were prepared for each dilution. Incubation at 37°C then
13
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proceeded for 18 to 24 hours at which time the plaques, appearing as clear
areas in the bacterial lawn, were counted.
Escherichia coli
Two methods were utilized to determine the concentration of IE. coli.
Samples which were processed in conjunction with coliforms adsorbed to or
encased in fecal material (fecal-associated coliforms) were analyzed by the
multiple-tube fermentation technique as described in Standard Methods for the
Examination of Water and Wastewater (27). Tenfold dilutions were prepared
in saline blanks containing 0.85 percent NaCl. The concentration of E_. coli
in all other samples was determined by the dilution plate count method;
serial dilutions were prepared as indicated above. Duplicate plates contain-
ing nutrient agar (Difco) were inoculated with 0.1 ml aliquots of appropriate
dilutions and evenly spread over the surface of the plate with a bent
sterilized glass rod. Plates were inverted, incubated 24 hours at 37 C and
counted. The concentration of E_. coli was expressed as colony forming
units (CPU) per 100 ml.
Streptococcus fecal is
Concentrations of S^ fecal is was determined by the dilution plate
count method. Serial dilutions were prepared in saline blanks containing
0.85 percent NaCl. Plates of trypticase soy agar were prepared in 100 x 15
mm petri dishes (Fisher Scientific Company). Duplicate plates were inoculat-
ed as indicated above, incubated 24 hr at 37 C, counted and the concentrations
expressed as CFU/100 ml.
Fecal-Associated Microorganisms
Fecal-associated coliform levels were determined by the multiple-tube
fermentation technique described previously. Due to the presence of non-
col i form bacteria within the fecal samples, primary fermentation tubes demon-
strating gas production within 48 hours were all confirmed with 2 percent
brilliant green bile lactose broth (Difco Laboratories, Detroit, Michigan).
Titers of fecal-associated porcine picornavirus were determined by
microtitration. The titers of poliovirus were determined by plaque assay.
These procedures may be found in the section on Virus Titration.
PREPARATION OF PARTICULATE MATERIALS
Hydrated Aluminum Oxide
The adsorbent used was hydrated aluminum oxide, a product of the co-
agulation process in water treatment when Al2(SO.)o is used as the coagulant.
For all microorganisms the procedure used for experimentation was adapted from
Boardman and Sproul (29) with various modifications which involved the pre-
paration of adsorbed and unadsorbed samples containing approximately the same
number of microorganisms per ml. The adsorption reactor contained 0.01 M
NaCl in TDW and sufficient alum as AlpfSO.K to achieve a final turbidity,
after dilution, of 1 or 5 turbidity units, as measured by the Hellige
14
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Turbidimeter (Hellige Inc., Garden City, N.Y.) and defined as a Nephelometric
Turbidity Units (NTU), after dilution (see disinfection apparatus) depending
on the particular experiment. The concentrations of alum as AloCSO.K neces-
sary to obtain these units were 150 and 700 mg/L, respectively. A TO ml
aliquot of virus or bacterial suspension was added to the reaction beaker and
the pH simultaneously adjusted to 7.0 by the addition of 0.25 M NaHCOo>
yielding a total volume of 500 ml. The beaker was placed under a laboratory
stirring machine equipped with two bladed glass stirring rods. The suspension
was rapidly mixed for 30 seconds and then gently stirred for 20 minutes at 25
rpm to facilitate floe formation and adsorption of the organisms.
The procedure for sample preparation and adsorption was the same for all
three viruses. Virus from the frozen stock was diluted 1:10 in a sterile 0~
demand free buffer solution (0.01 M NaCl in TDW, adjusted to pH 7.0 with O.Z5
M NaHCOo). A 10 ml aliquot was extracted for the adsorption procedure as
previously described. The 1:10 virus dilution (initial concentration) was
then further diluted 1:50 so that an unadsorbed sample (for disinfection)
might be obtained containing approximately the same virus concentration as
that taken from the adsorption reactor (10:500). The adsorption process then
proceeded as previously described for 20 minutes. Five 40 ml samples were
withdrawn for centrifugation at 1,900 x g for 30 minutes at 5°C in a fixed
angle rotor (head 870) using a Model B 20 A IEC refrigerated centrifuge, in
order to separate the unadsorbed virus from the floe particles and its
adsorbed fraction. The supernatant was then poured off and titrated so that
the extent of viral adsorption could be calculated by subtracting the super-
natant titer from the initial titer. The pellets were then resuspended in
the buffer solution mentioned before. This resuspension was also titrated in
order to determine the actual recoverable adsorbed fraction. A 160 ml sample
was then homogenized in a Sorvall Omni -Mixer at 4,000 rpm for 30 seconds in
order to obtain a floe particle size range that was advantageous for experi-
mentation. An aliquot was withdrawn from this volume to be utilized as the
adsorbed sample for disinfection. Preliminary studies were conducted to
determine the concentration levels of alum as A12(S04)3 necessary to achieve
a final turbidity, after homogenization and dilution, of 1 or 5 NTU. Another
40 ml portion was extracted and recentrifuged to determine that fraction of
virus released by the homogenization process (obtained from the supernatant
titer). A titer of the homogenized sample was also taken to determine the
amount of virus that actually remained adsorbed following homogenization. A
control reactor, without the alum, was also maintained throughout the
adsorption and centrifugation steps to determine the loss of virus attributed
to experimental procedure. This was accomplished by titrating the control
sample after it was centrifuged.
For the bacterial preparation, an 80 ml volume of an 18 hour £. col i
broth culture was centrifuged at 5°C for 15 minutes at 7,500 x g in a fixed
angle rotor (head 870) using a Model B 20 A IEC refrigerated centrifuge.
The pellets were combined into one and washed five times in buffer solution
at the above speed and time duration. The washed pellet of £. col i was then
resuspended in approximately 50 ml of buffer and a 10 ml sample withdrawn for
adsorption as previously stated. This bacterial suspension was then diluted
1:50, as described for the viruses, in order to match the unadsorbed bacteria
15
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count with that of the adsorbed. A 10 ml aliquot of this dilution was used
for disinfection of unadsorbed bacteria.
A settling procedure was used to separate the floe adsorbed £. coli from
the unadsorbed £. coli. The large particle size of £. coli caused the
unadsorbed bacteria to pellet with those adsorbed on the floe particles at
the centrifuge speeds necessary to completely pellet the hydrated aluminum
oxide floe. An aliquot taken from the adsorption beaker and assayed to
determine the recovery of bacteria from the floe, therefore, contained the
unadsorbed bacteria. The 160 ml sample taken from the adsorption beaker for
homogenization, and the aliquot withdrawn and plated after homogenization to
determine the recoverable bacteria after completion of this process also
contained unadsorbed £. coli. Therefore, in order to determine the actual
extent of adsorption, the remaining volume in the adsorption beaker was
settled at 4°C. Complete settling of the floe required to obtain 1 NTU was
approximately 45 minutes. The floe used to obtain 5 NTU, however, required
24 hours for complete segregation. After settling, an assay of the super-
natant was taken to determine the concentration of unadsorbed bacteria. The
volume remaining after sample homogenization (both 1 and 5 NTU) was also
allowed to settle for 24 hours at 4 C so that the concentration of bacteria
released by homogenization could be determined from the supernatant count.
At the same time a control reactor with £. coli but without alum was set up
at 4 C for 24 hours to determine the natural die-off of _E. coli after 24
hours. This also permitted determination of the sedimentation of unadsorbed
IE. coli which would occur naturally in this time span. Correction factors
for both phenomena were applied to the supernatant concentrations obtained
above. The actual concentration of bacteria recovered from the floe was then
calculated by subtracting the corrected value obtained for the supernatant in
the adsorption beaker from the concentration of the previously assayed sample
that contained both adsorbed and unadsorbed £. coli. In the same manner, the
concentration of recoverable bacteria after homogenization was calculated
by subtracting the corrected homogenized supernatant concentration from the
homogenized sample concentration that contained both adsorbed and unadsorbed
bacteria. As was the case for viruses, an aliquot of 10 ml of the homo-
genized mixture was utilized for disinfection of the adsorbed bacteria with a
control reactor used without alum to determine the losses due to experimental
procedure.
Bentonite Clay
Bentonite clay B-235 (Fisher Scientific Co.) was used to represent the
behavior of naturally occurring particulates. A stock suspension of discrete
particles was prepared by adding clay powder to a Waring blendor containing
1 L of TDW and blending until a homogeneous mixture was obtained. The
mixture was poured into a large carboy containing 48 L of TDW equipped with
a stirring device to evenly disperse the clay particles. After a 3 hour
quiescent settling period, 8 L of clay stock were removed via a siphon sus-
pended 1/3 the height of the carboy above the base. Turbidity of the siphon-
ed fraction was determined with a Hellige Turbidimeter and the particles
were measured with a microscope that had been calibrated with a stage micro-
meter. The clay stock was transferred to a 20 L carboy and ozonated at a
high rate for sufficient time to make it demand free. Disinfection studies
16
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of the clay-associated viruses were devised to produce reaction tube particu-
late concentrations of 1 and 5 NTU as measured with a Hellige Turbidimeter
(Hellige, Inc., Garden City, NY). The 5 NTU level was found to correspond
to a bentonite concentration of about 35 mg/L.
Adsorption of the viruses to the clay was accomplished through modifica-
tions of a procedure developed by Boardman and Sproul (29). A given amount
of prepared bentonite was added to a one L beaker containing an appropriate
volume of buffer solution. A concentrated solution of the phosphate buffer
system was added to adjust for the addition of the clay stock. The virus
formed by the dilution of the thawed virus concentrate was introduced and the
beaker was placed under a laboratory sitrring machine. Using 2 bladed glass
stirrers, the suspension was mixed at 100 rpm for 30 seconds and then gently
stirred at 25 rpm for a specified period to allow for adsorption. For f£
phage, poliovirus and coxsackievirus, 30 minutes were allowed for adsorption.
At the end of the adsorption period, a 40 ml sample was centrifuged at 1800 x
g for 30 minutes in the case of f?, and 20 minutes for the poliovirus and
coxsackievirus.
The supernatant fluid was decanted and analyzed for virus. The pellet
was resuspended in buffer solution and analyzed for adsorbed virus since the
virus associated with the particulate matter would have been removed by
centrifugation.
In the case of E^ coli, an adsorption period of 20 minutes was allowed.
The mass of the bacteria prevented separation of the unadsorbed organisms
from the bentonite particles by centrifugation, thus no adsorption data were
generated for JE. col i.
Fecal Samples
A modification of the procedure supplied by Dr. John C. Hoff, Drinking
Water Research Division, Environmental Protection Agency, Cincinnati, Ohio
was used in the preparation of fecal samples (Personal Communication). One
gram of fecal material was suspended in 100 ml ozone-demand free pH 7.0
phosphate buffer by homogenizing it in a Sorval Omni-Mixer for 1/2 minute at
11,500 rpm. The suspension was allowed to settle overnight. After settling,
overnight, the supernatant was centrifuged and the pellet washed in the
manner described above for the unadsorbed bacteria. The final pellet was
resuspended in a small quantity of ozone-demand free phosphate buffer and the
turbidity determined using the Hellige Turbidimeter. The sample was then
diluted to give a 10 ml sample with a turbidity of either 29 or 145. Further
dilution of these samples within the disinfection reactor yielded turbidities
of 1 and 5, respectively. Five ml were injected into the disinfection
apparatus. Initial levels of viruses and coliforms were measured on the
samples before injection.
HEp-2 Cell-Associated Viruses
The procedure of Symons and Hoff (16) was used to obtain eel 1-associated
viruses. Confluent monolayers of HEp-2 cell cultures were infected with 20
to 40 PFU/cell of either poliovirus or coxsackievirus as previously described.
17
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Infected cell cultures were incubated 12 to 15 hours at 37°C until CPE was
just visible. Cells were removed by gentle shaking and the resultant cell
suspension centrifuged 15 minutes at 2,000 rpm in an IEC B-20A refriger-
ated centrifuge. The supernatant was discarded. The pellet was washed five
times by repeated centrifugation of 2,000 rpm for 15 minutes. A stock cell
suspension was prepared by resuspending the pellet in 15 ml of ozone demand
free PH70 phosphate buffer in .01 M NaCl so that its turbidity would produce
a final turbidity of either 1 NTU or 5 NTU after dilution in the reaction
container. A portion of the stock cell suspension was reserved to determine
the titer of the cell-associated virus before ozonation.
Presonicated HEp-2 Cell-Associated Virus
A comparison of the inactivation of HEp-2 cell-associated poliovirus was
performed utilizing duplicate portions of a sample, one of which had been
sonically treated. The initial sample was divided, and one aliquot was
treated 30 seconds at 50 kilocycles/sec with a sonic dismembrator (Fisher
Scientific, Fair Lawn, N.O.). The susceptibility to inactivation by ozone
of the sonically treated cell-associated poliovirus and cell-associated virus
not exposed to sonication was determined at various applied ozone dosages in
the Sharp disinfection apparatus.
MICROSCOPY
Light Microscopy
Photo micrographs were obtained utilizing a 35 mm camera attached to a
Phase Star, Spencer microscope (American Optical, Buffalo, New York).
Replicate samples of presonicated HEp-2 cell-associated poliovirus and HEp-2
cell-associated poliovirus which had not been sonically treated were with-
drawn after 10 seconds, 20 seconds, and 30 seconds exposure to ozone in the
Sharp continuous flow apparatus. The physical state of the remaining
cellular material at each contact time was compared to cellular material of
the initial samples which were not exposed to ozone. Initial samples were
diluted with ozone demand free buffer to a turbidity of 5 NTU to faciliate
comparison with samples obtained from the Sharp disinfection apparatus.
OZONATION AND DISINFECTION APPARATUS
Ozonation Apparatus
The ozonation apparatus was constructed as shown in Figure 1. Ozone was
generated from an extra dry grade oxygen with a Model SG 4060 ozone generator
(Union Carbide Corporation, South Plainsfield, New Jersey). Oxygen was fed
through a two-stage regulator at a working pressure of 50-100 psig.
A small amount of oxygen was diverted before reaching the ozone gene-
rator and mixed with ozone entering the 20 L Pyrex contact vessel. The
oxygen served to increase turbulence within the vessel, thus promoting the
transfer of ozone into the liquid. Diffusion of the gas mixture was accom-
plished by a Corning immersion tube (Fisher Scientific Company, Medford,
18
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Massachusetts).
Since low levels of ozone were required for inactivation experiments, a
large portion of the ozone generated was vented directly to a kill bottle.
Neutralization of the excess ozone was accomplished by 2.5 kg sodium
thiosulfate and 50 g potassium iodide in approximately 20 L of tap water.
Aquarium diffusers were used in the kill bottle.
To minimize the ozone demand of the system, 316 grade stainless steel
(SS) tubing (1/4" O.D. x .035" wall) was used. The tubing was coupled with
316 grade SS Swagelok fittings. Diffusers and vents in the contact vessel
and kill bottle were made of glass. Sections of glass and stainless steel
were mated with Tygon tubing.
Continuous Flow Disinfection Apparatus
Experiments were conducted using the Sharp continuous flow disinfec-
tion apparatus (36) as shown diagrammatically in Figure 2. The equipment
is shown in the photograph in Figure 3. Modifications were made as necessary
to accommodate ozone as the disinfectant.
The reaction tube was made of 316 grade stainless steel (1/2" O.D. x
.035" wall) coupled with Swagelok 316 grade stainless steel unions. Holes
drilled through the center nuts of the Swagelok unions served as access
ports; these ports were covered by disposable, rubber, serum vial stoppers
(Arthur H. Thomas Company, Philadelphia, Pennsylvania). Access ports were
spaced 10 feet apart. The first port served as the sample injection port. A
10 mesh stainless steel wire grid was placed just downstream of the injection
port to aid in mixing. Upstream of the injection port, a Whitey regulating
valve was installed to control flow. Four access ports downstream of the
sample injection port were utilized for sample withdrawal. These ports were
equipped with a solenoid coils (Sporlan Valve Company, St. Louis, Missouri)
which surrounded the sampling syringes. The tubing outflow was open to the
atmosphere.
The apparatus was designed to operate under a siphon head. Since it was
essential to maintain turbulent flow in the reaction tubing a Reynolds number
greater than or equal to 3000 was maintained. Since
M - vd
Nff - '
where:
v = velocity
d = diameter = 0.036'
v = kinematic viscosity
= 1.09 x 10'5 ft2/sec @ 20gC,
a velocity greater than or equal to 0.9 fps was required. For convenience, a
velocity of one foot per second (fps) was selected and by Q = VA a flow
requirement was established. The height of the contact vessel was constantly
20
-------
-------
Figure 3. Ozonation and continuous flow apparatus
22
-------
maintained at a level that yielded a flow greater than that required through-
out the course of an experiment. A metering valve was utilized to decrease
the flow to an average velocity of one fps when the volume in the contact
vessel ranged from 20 L to greater than 14 L. It was possible to maintain
this average velocity because of the large cross sectional area of the contact
vessel. With a velocity of one fps and a distance of 10 feet between access
ports the time of transit between ports was 10 seconds. Thus, samples for
analysis were taken at contact times of 10, 20, 30, and 40 seconds.
Sample injection was accomplished in the following manner. A 5 ml
sample of organisms was injected manually into the ozonated stream utilizing
a 5 ml syringe. The sample was added over a 5 second time period at a precise
rate of 1 ml/second.
Samples for analysis were extracted manually at downstream sampling
ports. Since samples were injected over 5 seconds, a five second time period
was available for sampling. To smooth out any inaccuracies in the manual
addition of the organisms, samples were taken during the second, third, and
fourth seconds. The samples were withdrawn utilizing 5 ml syringes with 22
gauge needles. Each syringe contained 1 ml of 0.025 N sodium thiosulfate
and a 3/8" x 5/16" Nalgene star magnetic mixer (Arthur H. Thomas Company,
Philadelphia, Pennsylvania). Residual ozone was neutralized by continually
mixing the sample with thiosulfate. The mixers were agitated by valve
solenoids surrounding the sample syringes.
Injection of a 5 ml sample of organisms into the reaction tubing over a
5 second period yielded a dilution factor of 29 to 1. The dilution factor
was verified by injecting organisms into unozonated dilution water and with-
drawing samples downstream at 10, 20, and 30 seconds.
In a typical experiment the contact vessel was filled with 20 L of a
0.01 M NaCl solution prepared utilizing triple distilled water (TDW). The
solution was adjusted to 20 C and buffered to yield 10~3 M, pH 7 phosphate.
The buffer was then ozonated to achieve the desired concentration. The
ozonated buffer was siphoned through the reaction tubing at the desired
flowrate. Triplicate samples were taken at the outfall to determine ozone
residual and a 10 ml sample of buffer was taken at the 40 second sample port
to aid in determining ozone demand of the samples. (An 18 gauge needle was
necessary to obtain a 10 ml sample in the alloted sampling period). Two
samples were then injected into the reaction tubing. The first sample
contained the unadsorbed virus or bacteria. Approximately one minute after
injecting the first sample, a sample of virus or bacteria in an adsorbed state
was introduced into the reaction tubing. Samples for analysis were taken at
contact times of 10, 20, and 30 seconds. Ten ml samples were taken at the
40 second sample port for determination of ozone demand. All samples taken
at 40 seconds were immediately transferred to ozone-demand free test tubes;
samples were then assayed for survival.
Declining Residual Batch Reactor
The batch reactor with a continually declining ozone residual was as-
sembled as shown in Figure 4. This reactor was made from a 500 ml Erlenmeyer
23
-------
INJECTION POHZ.T
M 1X1
Figure 4. Batch Reactor With Declining Residual
24
-------
flask with a bottom sidearm. A silicone stopper was used to attach a stop-
cock to the sidearm for use as a sampling outlet. A short length of tygon
tube attached to a hollow glass rod was fitted to a single hole rubber
stopper. The tube was closed with a clamp during the reaction period to
prevent the ozone from escaping. Free flow of ozone at the sampling period
was obtained by removing the clamp.
_3
Following ozonation, 500 ml of TDW with 10 M phosphate buffer in NaCl
(pH 7.0 +_ 0.2) were transferred to the reactor. The reactor was placed on a
magnetic stirrer (Fisher Scientific, Fair Lawn, N.J.) for the duration of the
experiment. Triplicate aliquots of the ozonated, buffered TDW were drawn to
determine the initial ozone residual in the reactor. The cell-associated
virus sample was injected into the reactor from a 10 ml syringe. The outlet
was purged 15 seconds before the designated contact time to flush possible
residual virus from the stopcock at the sampling port. At specified contact
times, samples were collected in ozone demand free test tubes. A 5.0 ml
aliquot was used to determine ozone residual as previously described, while
3.0 ml were immediately mixed with 1.0 ml ^SpOg utilizing a Vortex Genie
(Fisher Scientific, Fair Lawn, N.J.). Surviving viruses were titrated and
expressed in pfu/ml of reactor volume.
Batch Reactor for Continuous Ozonation
The ozonation apparatus as previously described (Figure 1) was used and
the 20 L Pyrex dilution water bottle was replaced with a 2 L Pyrex reagent
bottle (see Figure 5). An outlet cut in the reagent bottle was fitted with
a silicone stopper with a stopcock. Injection of the sample was made with a
10 ml syringe through the stopper at the sampling port. Injection of the
sample directly under the fritted glass immersion tube mixed the sample.
A 1500 ml volume of TDW with 10"3 phosphate buffer in NaCl (pH 7.0 +. .2)
was ozonated to obtain a maximal ozone dosage. Following removal of tripli-
cate samples for measurement of the initial ozone concentration within the
reactor, the cell-associated virus was injected from a 10 ml syringe into
the reactor. The outflow stopcock was purged 10 to 15 seconds before with-
drawing samples at specified contact times. The samples were collected in
ozone demand free test tubes with a 5 ml aliquot to determine ozone residual
and a 3 ml aliquot, neutralized with 1 ml of Na?S_0~, to determine the surviv-
ing viral titer.
Ozone Determination
Ozone concentrations were measured according to the method of Shechter
(33) with minor modifications. Two calibration graphs were generated to
measure ozone. Curve A was utilized to measure ozone concentrations ranging
from 0.01 to 0.3 mg/L and Curve B was used to measure ozone ranging from 0.3
to 2.0 mg/L. In preparing the calibration graph for Curve A, the reference
blanks were made with TDW and neutral potassium iodide reagent. Equal
aliquots of the neutral potassium Iodide reagent were placed in the reference
blank and the standard with which it was to be compared. This served to zero
out absorbance due to the potassium iodide. This modification was not neces-
sary with the B curve. Low levels of ozone (curve A) were determined using a
25
-------
VETKJT
* Oz INL.ET
TO KILL. BOTTUE
\
BOTTLE:
SAMPL1KQ POF2T
Figure 5. Batch reactor for continuous ozonation.
26
-------
blank of 2 ml neutral potassium iodide reagent in 10 ml TDW. Absorbances
were read in a Hitachi Perkin-Elmer Model 139 UV-Vis Spectrophotometer with a
Model 139-0251 photomultiplier unit (Coleman Instruments Corporation, Maywood,
Illinois). Curve A was prepared using Hellma 2 cm quartz cells (Hellma Cells,
Jamaica, New York). Fisher brand 1 cm quartz cells (Fisher Scientific
Company, Medford, Massachusetts) were used for curve B.
The ozone demand of the unadsorbed samples from the Sharp apparatus
was found by comparing a clear buffer sample taken at 40 seconds and the 40
second unadsorbed sample. The demand of the fecal material was analyzed in a
somewhat different manner. After injection of the initial sample which was
used for organism inactivation and ozone determination, another 5 ml sample
of fecal material was injected into the apparatus and a 10 ml sample was with-
drawn at the 40 second sample port (fecal blank). This sample was combined
with 2 ml TDW and placed in the dark for 30 minutes along with the other
samples taken for ozone determination. The absorbance of this sample was
read using a TDW reference blank. Since both fecal samples withdrawn at 40
seconds contained partially oxidized fecal material, a comparison of the two
samples (fecal and ozone vs fecal) enabled determination of absorbance due
solely to the ozone present. The ozone concentration of the clear buffer
sample was compared to the ozone concentration of the adsorbed sample to
obtain ozone demand.
27
-------
SECTION 4
RESULTS AND DISCUSSION
THE EFFECT OF FECAL MATERIAL ON OZONE DISINFECTION OF COLIFORM BACTERIA
AND ENTERIC VIRUSES IN WATER.
RESULTS
The approximate concentrations of the various microorganisms within the
fecal samples prepared as discussed in materials and methods are presented in
Table 1. As will be seen later, there is an apparent discrepancy between the
concentrations presented in the fecal samples and initial concentrations of
fecal-associated organisms available for disinfection. This apparent dis-
crepancy was caused by a dilution of 1:2.9 x 10^ of the fecal samples, which
occurred during sample preparation and subsequent disinfection.
After preparing the fecal suspensions, an analysis was conducted to
determine the particle size distribution. One hundred random particles were
measured using a microscope. A stage micrometer was used to calibrate the
microscope. These data are presented in Table 2. The size of fecal particles
presented after preparation of fecal suspensions for disinfection experiments
ranged from 1 to 18 ym in size. The particle size distribution is significant
in that it approximates that which might be found after direct filtration of
drinking water. Also, particles present in wastewater effluent after activa-
ted sludge treatment are within this size range.
The particle size distribution in finished water from three different
direct filters (dualmedia, multimedia, and anthracite and garnet) were found
by Tate et al (34) to be virtually identical. A 99 percent removal of 2.5
to 150 ym particles was accomplished in all three cases while all particles
greater than 10 ym were removed. Effluent samples were collected prior to
disinfection and analyzed to determine the solids distribution. Rickert and
Hunter (35) found that the soluble fraction (<1 my) contained 93 percent of
the total solids in an activated sludge plant effluent. One percent of the
total solids were colloidal (1 my to 1 ym), a 5 percent supra-colloidal (1 to
100 ym) and 1 percent settleable (<100 ym). Thus, it is evident that the fecal
particle size range used in this research is within the range one might expect
to find in a field situation.
Viruses
Results of the inactivation of poliovirus (Sabin Type 1) and fecal-
associated poliovirus at a turbidity of 5 NTU are presented in Table 3.
28
-------
TABLE 1. CONCENTRATION OF MICROORGANISMS PER GRAM OF FECES (WET WEIGHT)
Microorganism
Concentration/gm
Poliovirus
Porcine Picornavirus
Coliform bacteria
5.2 x 10b PFU (7)a
1.8 x 106 TCID5Q (2)
2.9 x 105 MPN (10)
Number of determinations
TABLE 2. FECAL PARTICLE SIZE DISTRIBUTION
Fecal Suspension
Particle Distribution
Particle Size
(ym)
Infant
Porcine
Adult
1
14
13
0
2
52
56
56
4
25
21
30
5
5
6
12
7
4
3
1
9
0
0
1
18
0
1
0
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Protection by the fecal material was demonstrated at an ozone concentration
of 0.013 mg/L. At this ozone concentration, the survival of fecal-associated
poliovirus with an initial concentration of 1.3 x 101 PFU/ml was noted at 10
and 30 seconds of reaction time. On the other hand, unadsorbed poliovirus
with a titer 2.7 login PFU/mL higher than that of fecal-associated poliovirus
showed complete inactivation within 10 seconds at an ozone concentration of
0.012 mg/L.
Further work with poliovirus was discontinued due to inactivation of
poliovirus within the stored frozen fecal material. As shown in Table 4,
titer of fecal associated poliovirus ranged from 1.4 x 104 to 1.3 x 10°
PFU/gm feces after 12 days at -20°C. After 71 days, however, complete
inactivation of all poliovirus within the samples had occurred.
The storage of poliovirus at -20°C should not have resulted in the marked
decrease in titer described above. The delay which occurred during shipment
of the samples, however, allowed sufficient time for possible generation of
some viricidal agent and initiation of viral inactivation. Although the
agent responsible for inactivation was not isolated, a compound such as
ammonia may have been responsible. Ammonia is known to be a potent viricide
(36). The generation of ammonia from urea associated with the samples is
conceivable since the fecal samples were obtained from infants and may have
been in contact with urine, a source of large quantities of urea.
TABLE 4. INACTIVATION OF FECAL-ASSOCIATED POLIOVIRUS (SABIN TYPE 1) AT -20°C
Virus Concentration
(PFU/gm fecal material)
Number of Days Stored at -20 C
Sample
12
71
1
2
3
4
5
6
5
2.0 x 10
5
4.0 x 10
4
1.4 x 10
1.1 x 106
1.1 x 105
6
1.3 x 10
0
0
0
oa
'Samples 4, 5, and 6 were combined for use in experiments.
31
-------
Porcine picornavirus Type 3 (Strain ECPO-6) was also protected by
adsorption to or encasement in fecal material. Data in Table 5 show that at
an initial ozone concentration of 0.024 mg/L, 100 percent inactivation of 1.1
x 10^ TCIDt-g/ml unadsorbed porcine picornavirus was obtained while survival of
fecal associated porcine picornavirus at an initial concentration of 4.1 x
10' TCIDso/ml was demonstrated after 30 seconds of contact. Complete inacti-
vation of 1.6 x 1Q4 TCIDso/ml was accomplished at an ozone concentration of
0.22 mg/L.
A comparison of the data on the inactivation of unadsorbed poliovirus
and the unadsorbed porcine picornavirus shows that the porcine picornavirus
was significantly more resistant than was poliovirus. At a level of 0.12 rng/1
ozone "(Table 3) a four log reduction of poliovirus was accomplished. Porcine
picornavirus, however, was reduced by only two logs when the ozone concen-
tration was 0.036 mg/L
Some degree of caution should be exercised in viewing the data on the
inactivation of poliovirus and porcine picornavirus since only one run
demonstrating protection was obtained for each virus. In both instances,
however, survival of fecal-associated virus was noted after 30 seconds contact
whereas total inactivation of non-associated viruses was accomplished within
10 seconds.
Bacteria
Washed cultures of E_. coli were subjected to ozone levels ranging from
0.38 mg/L to 0.62 mg/L. Samples were removed at contact times of 10, 20, 30,
and 40 seconds and assayed for survival. These data are presented in Table
6. For a given contact time the inactivation does not appear to vary signifi-
cantly over the range of ozone levels used. However, the results do vary
with contact time. An average of 5.0, 5.8, 6.8, and 9.4 logs of reduction
were observed at 10, 20, 30, and 40 seconds, respectively. The average
ozone concentration utilized to accomplish these reductions was 0.52 mg/L.
Table 6 also contains the results of the inactivation of _S_. fecal is.
While a limited amount of work was conducted with this organism, it is
evident that S^. fecal is was more sensitive to ozone than was £. coli.
Complete inactivation of S_. fecal is within 10 seconds was accomplished at
initial ozone concentrations of 0.28 mg/L and above. At an ozone concentra-
tion of 0.13 mg/L survival was noted at 10, 20, and 30 seconds, although the
level of bacteria had been reduced by 7 logs within 30 seconds of ozonation.
Data obtained on the inactivation of £. coli and fecal associated
coliforms are presented in Table 7. The samples which were assayed for
survival were diluted tenfold to yield 1/10 and 1/100 dilutions. One ml
aliquots of the diluted samples were then placed in tubes of lactose broth.
In addition, the remaining undiluted sample was distributed equally between
5 tubes of lactose broth. Aliquots of undiluted samples normally ranged
from 0.35 to 0.50 ml. Since one ml aliquots were not available for MPN deter-
minations of surviving organisms, all MPN indices were reported as the actual
values for the amount'of sample used.
32
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Complete inactivation of non-associated E_. coli occurred at all levels of
ozone. Protection of fecal-associated coliform bacteria at a NTU of 5 was
demonstrated at 0.10 mg/L and below. A definite gradient of survival was
established during disinfection of fecal-associated coliforms since increased
levels of survival were obtained with decreasing levels of ozone. It should
be noted that the initial MPN's of the unadsorbed washed E. coli were general-
ly higher than those of the corresponding fecal-associated samples. Even
though the initial MPN's of the washed culture's were greater, the E_. coli
were completely inactivated within 10 seconds while contact times of 10 to 30
seconds or more were necessary to accomplish total inactivation of fecal-
associated coliform bacteria. Thus, it is evident that the rate of inactiva-
tion of non-associated E_. coli was more rapid than was the rate of inactiva-
tion of fecal-associated coliforms.
No protection of fecal-associated coliform bacteria was demonstrated at
1 NTU. This may have been due in part to the lower levels of bacteria pre-
sent. Total inactivation of 1.1 x 10^ MPN/100 ml at 1 NTU occurred within 10
seconds at an ozone concentration of 0.028 mg/L. Unadsorbed E_. coli at an
initial concentration of 5.9 x 10d MPN/100 ml were also completely inactivated
within 10 seconds at the same ozone concentration. Since total inactivation
of unadsorbed E_. coli and fecal-associated coliforms at 1 NTU occurred within
10 seconds, the rate of inactivation of the organisms could not be determined.
Thus, whether or not 1 NTU of fecal material afforded protection to fecal-
associated coliforms could not be ascertained. In comparison, survival was
evident at 5 NTU after subjecting 1.7 x 102 MPN/100 ml to 0.027 mg/L ozone
for 30 seconds. In fact, a log reduction of only 0.8 was accomplished during
this contact period. It would appear that the greater amount of organic
material at 5 NTU did offer protection of fecal-associated coliforms while
little or no protection was afforded at 1 NTU.
An experiment was conducted to determine whether those coliforms surviv-
ing ozonation did so because of the protective effect of the fecal material or
because they were resistant forms. E_. coli which survived ozonation when
associated with fecal material (ECF) were isolated as described in Materials
and Methods. Comparisons were then made of the inactivation rates of washed
cultures of _E. coli (ATCC 15766) and washed cultures of isolated _E. coli.
This procedure was repeated twice with a separate E_. coli isolate used in each
trial. These data may be seen in Table 8. No significant differences were
noted between the two organisms. In the first trial both E_. coli and ECF
were reduced by slightly more than 5 logs within a 30 second contact period.
In the second trial IE. coli and ECF underwent reductions of approximately 6
and 5 logs, respectively. Therefore, it would appear that survival of fecal-
associated coliform bacteria occurs as a result of protection afforded the
bacteria by the fecal material rather than some inherent resistance of the
organisms themselves.
DISCUSSION
The amount of work conducted with the poliovirus and the porcine picorna-
virus was limited because of several problems which occurred during the course
of investigation. Studies with both poliovirus and porcine picornavirus were
36
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hindered by the low levels of viruses available for disinfection after sample
preparation. There was also difficulty in obtaining fecal samples which con-
tained naturally occurring viruses, i.e. samples containing viruses which
been excreted in the feces. Difficulty in obtaining sufficient quantities
of these samples and the inactivation of poliovirus within fecal samples
stored at -20°C abruptly curtailed work with this organism.
Despite the problem in working with low levels of viruses, these levels
closely approximate the levels of organisms which might normally go through
disinfection in a wastewater treatment plant. When one considers that levels
of viruses as low as 1 TCID5Q or 2 PFU are capable of producing infection in
man under certain conditions (12), low levels of viruses should be con-
sidered significant.
The level of viruses present in domestic wastewater has been estimated
to be about 7000 PFU/L (10). Assuming an influent level of 7000 PFU/L,
Sproul (12) predicted the number of viruses that would remain after waste-
water treatment. Utilizing these data, the number of viruses which would be
subject to disinfection during secondary treatment may be determined. The
level of viruses likely to remain after trickling filtration would be
approximately 3500 PFU/L and about 700 PFU/L would be expected after treatment
with activated sludge. These figures correlate well with the range of fecal-
associated poliovirus (2.0 x 102 to 1.3 x 10^ PFU/L) which were employed dur-
ing these disinfection experiments. Fecal-associated porcine picornavirus
ranged from 1.1 x 103 to 4.1 x 104 TCID50/L.
Samples of fecal-associated coliforms were similar to fecal-associated
enteric viruses in that low levels of organisms were obtained after cleanup
of the fecal samples. In order to increase the concentration of bacteria,
the fecal solution was centrifuged at 7500 x g during the washing procedure
rather than at the lower centrifgual force. Since more material was pelleted
at this higher centrifugal force, the levels of fecal-associated coliforms and
the turbidity of the sample were increased. It should be noted, however,
that centrifugation at 7500 x g resulted in removal of non-associated, as well
as associated coliforms from the suspension. Despite this fact, protection
of fecal-associated coliforms was seen. A few of those coliforms surviving
ozonation may not have been adsorbed to or encased in fecal material.
The small ozone residual remaining after disinfection was difficult to
measure spectrophotometrically. Detection was further complicated by the
large quantity of partially oxidized fecal material present in the sample.
To alleviate this problem a blank containing partially oxidized fecal material
was used to zero out absorbance due to the fecal material. This procedure
was not always effective, however, due to the large amounts of absorbance
which had to be zeroed out.
It is uncertain whether the protection afforded the microorganisms by the
fecal material was due to complete depletion of the initial ozone in solution
or adsorption of the microorganisms to or encasement in fecal material. The
increase in inactivation with longer contact times, however, seems to indicate
that complete depletion of ozone did not occur within the 30 second contact
period.
38
-------
Although the results of this investigation demonstrated protection of
fecal-associated coliform bacteria and enteric viruses, the significance of
this protection is small when viewed with respect to common ozone disinfection
practices. In practice, ozone residuals of 0.1 to 0.2 mg/L and 0.4 mg/L at
the end of a contact time of about 4 minutes were maintained to disinfect
bacteria and viruses, respectively. Diaper (37) reported that an applied
dosage of 0.5 to 1.5 mg/L ozone in drinking water was necessary to obtain
these residuals which are determined after a 5 minute contact period. Other
waters might require a larger applied dosage. In comparison, the initial
ozone in solution in these studies ranged from 0.010 to 0.22 mg/L with a few
exceptions and the residual ozone, measured after 40 seconds contact, ranged
from 0.005 to 0.067 mg/L. The ozone concentrations necessary to inactivate
fecal-associated organisms were somewhat higher than those required fecal-
associated organisms. However, at some point within the range of ozone levels
employed, it was possible to achieve 100 percent inactivation of both fecal-
associated and non-associated organisms within 30 seconds.
THE EFFECT OF HYDRATED ALUMINUM OXIDE FLOC ON OZONE DISINFECTION OF
£. COL I AND VIRUSES IN WATER.
RESULTS
The range of floe particle sizes used for this experimentation from
light microscope observations was from 5 to 50 pm. Although a portion of the
particles used for 03 disinfection studies were on the high side of the 10 pm
particle size cited, use of this fraction probably provided a factor of safety
since if no protection was evident using these slightly larger particles, it
is probable that no protection would be provided by the fraction less than 10
pm.
Poliovirus
As shown in Table 9, after adsorption and subsequent centrifugation of
the adsorbed poliovirus 1, the analysis of the supernatant indicated that
approximately 1 percent of the initial concentration introduced into the
adsorption reactor was not associated with the floe. Therefore, by computa-
tion, 99 percent of the initial titer was associated with the floe. Titrata-
tion of the floe, which indicates the viruses actually recoverable from the
adsorbed fraction, yielded a recovery of 114 percent. Titration of the super-
natant, after homogenization and centrifugation, indicated that approximately
2 percent of the adsorbed virus was released by homogenization. It is evi-
dent, therefore, that the virus particles were held tightly by the floe, and
mechanical shear did not release them. Titration of the floe following
homogenization showed a 121 percent recovery of the adsorbed fraction. Virus
losses due to the adsorption and centrifugation process in the TDW control
were only about 1 percent.
The 03 concentration range investigated for poliovirus disinfection,
shown in Table 10 was from 0.002 to 0.0835 mg/1. The data indicate the
the poliovirus was afforded no protection by either 1 or 5 NTU of hydrated
aluminum oxide floe at the concentration levels of 03 utilized. At an initial
39
-------
TABLE 9. ASSOCIATION OF POLIOVIRUS (SABIN TYPE 1) WITH HYDRATED ALUMINUM
OXIDE
SYSTEM RANGE OF VIRUS VIRUS
CONCENTRATIONS9 RECOVERY
(PFU/ml) (%)
Computed virus concentration in floe
(initial-suDernatant)x m 2^ x 1Q1 to 3>5 x 1Q3 99>0 (5)
i m 1 1 a I
Actual recovery from floe (adsorbed)
a.zxio^td.gxio5 m (5)
Computed virus concentration in floe
after homogenization
(adsorbed-homogenized
x 100 1 .5 x 102 to 3.3 x 103 98 (5)
Actual recovery from floe after
homogenization
(homogeni zed-homogeni zed
_, _ supernatant) ,QO 8 n x 104 to 1 7 x 105 121
(adsorbed-homogenized x IUU b'u x 1U to l>/ x IU '^'
supernatant)
TDW control
9°"^°] x 100 1.1 x 105 to 1.3 x 105 99.3(2)
i
a 45
range of initial concentration: 2.9 x 10 to 1.7 x 10
number of trials
40
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03 concentration of 0.012 mg/L, 99.95 percent of unadsorbed poliovirus with a
concentration of 1.5 x 104 PFU/ml were inactivated after 20 seconds of 03
contact. At the same 03 concentration 99.97 and TOO percent of floe-associa-
ted poliovirus at 5 NTU with concentrations of 1.2 x 104 and 1.0 x 10^ PFU/
ml, respectively, were inactivated within 20 seconds of contact. At 1 NTU
of hydrated aluminum oxide floe at an initial 03 concentration of 0.012 mg/L
complete inactivation of 1.1 x 103 PFU/ml floe associated poliovirus was also
accomplished within 20 seconds. At 0.085 mg/L 03 complete inactivation of
3 logs of both unadsorbed and adsorbed poliovirus at 1 NTU was achieved with-
in 10 seconds.
Coxsackievirus A9
Data in Table 11 show that, based on the supernatant titer obtained fol-
lowing centrifugation, the adsorption of coxsackievirus A9 was approximately
99 percent, as was the case for the poliovirus 1. Titration of the floe
showed a recovery of 96 percent of the adsorbed virus. The virus released by
homogenization again paralleled the poliovirus, with only 2 percent of the
adsorbed fraction released. An assay of the adsorbed sample after homo-
genization yielded a recovery of approximately 100 percent indicating no
losses due to homogenization. The TDW control indicated a coxsackievirus
recovery of 94 percent following the adsorption procedure and subsequent cen-
trifugation step.
As shown in Table 12, the disinfection data for the coxsackievirus also
paralleled those of the poliovirus very closely. The 03 concentration range
was froin 0.012 to 0.081 mg/L. Again, the data did not seem to indicate a
protective effect at either 1 or 5 NTU over the concentration range investi-
gated. At an initial 03 concentration of 0.032 mg/L, 99.8 and 99.0 percent of
unadsorbed coxsackievirus with initial concentrations of 2.2 x 10^ and
9.1 x 103 PFU/ml, respectively, were inactivated after 10 seconds of 03
contact. Using the same 03 concentration and contact time, 99.8 percent of
coxsackievirus with an initial concentration of 2.4 x 10^ PFU/ml was
inactivated when associated with 1 NTU of hydrated aluminum oxide floe.
Also, at 5 NTU and 0.032 mg/L, 03 99.90 percent of floe-associated coxsackie-
virus with a titer of 9.1 x 103 PFU/ml was inactivated after 10 seconds of
contact. At an initial 03 concentration of 0.081 mg/L, complete inactivation
of 4 logs/ml of coxsackievirus both unadsorbed and adsorbed at 1 NTU of
hydrated aluminum oxide floe was accomplished within 10 seconds.
The disinfection of poliovirus and coxsackievirus appeared to be very
similar in that they were both very sensitive to the 03 concentrations used.
For both viruses the 03 concentration range investigated was less than 0.1
mg/L. No protection at either 1 or 5 NTU of hydrated aluminum oxide was
evident for either virus over the very similar 0~ concentration range used.
Bacteriophage f?
The f2 bacteriophage data in Table 13 show that the adsorption, computed
using the supernatant titer obtained following centrifugation, was 90 percent
of the initial virus introduced into the adsorption reactor. Titration of
42
-------
TABLE 11. ASSOCIATION OF COXSACKIEVIRUS A9 WITH HYDRATED ALUMINUM OXIDE
SYSTEM RANGE OF VIRUS VIRUS
CONCENTRATIONS9 RECOVERY
(PFU/ml) (%)
Computed virus concentration in floe
(initial-supernatant) x m 2_Q x 1(J2 tQ g>2 x 1Q3 99>Q (6) b
i n i "Ci s l
Actual recovery from floe (adsorbed)
1.1 xl05to3.4x,05 96 (6)
Computed virus concentration in floe
after homogenization
(ads orbed-homogenized
x 10° 1.6 x 103 to 5.3 x 103 98 (5)
Actual recovery from floe after
homogenization
(homogeni zed-homogenized
to 3.1 x 105 99.98(5)
supernatant)
TDW control
.;°;K°] x 100 - 94 (3)
aRange of initial concentration: 1.4 x 105 to 3.2 x 105
Number of trials
43
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to
1
to
44
-------
TABLE 13. ASSOCIATION OF f? BACTERIOPHAGE WITH HYDRATED ALUMINUM OXIDE
SYSTEM RANGE OF VIRUS VIRUS
CONCENTRATIONS9 RECOVERY
(PFU/ml) (%)
Computed virus concentration in floe
t) x 10° 5.0 x 104 to 1.6 x 107 90 (5)b
Actual recovery from floe (adsorbed)
xlOO 1.6xl04to8.9xl04 0.04(4)
Computed virus concentration in floe
after homogenization
(adsorbed-homogeni zed
x m 5>Q x 1(J2 t(J
adsorbed
Actual recovery from floe after
homogenization
(homogenized-homogenized . ,.
supernatant) ,QO 2.1 x 10 to 1.2 x 10 93 (3)
(adsorbed-homogenized
supernatant)
TDW control
* 100 1.5 x 107 to 4.6 x 107 19 (4)
aRange of initial concentration: 6.1 x 107 to 2.0 x 108
Number of trials
45
-------
the adsorbed fraction, however, indicated a recovery of less than 0.05 per-
cent of the phage from the hydrated aluminum oxide floe. Thus, the overall
untitratable adsorbed virus was approximately 99.95 percent. It was uncer-
tain whether the inability to recover the adsorbed virus was due to inactiva-
tion or failure to elute the phage from the floe. Therefore, in an attempt
to recover more of the virus, sonication was used at a level of sonic energy
used by Boardman and Sproul (29) for minimum phage loss. No significant
increase in recovery was observed. A loss of bacteriophage amounting to
approximately 81 percent occurred during the adsorption procedure and sub-
sequent centrifugation, as indicated by the recovery of virus from the TDW
control. The supernatant titer obtained after centrifugation of the homo-
genized floe indicated a 4 percent release of phage during this process.
Titration of the floe following homogenization showed a recovery of 93 per-
cent of the adsorbed fraction, thus indicating that 7 percent of the adsorbed
phage was unrecoverable after homogenization.
Table 14 contains the compiled data for the inactivation of f~ bac-
teriophage over a range of 03 concentrations from 0.115 to 2.18 mg/c.
Caution must be exhibited in interpreting these data since there is the pos-
sibility that some phage was still incorporated within the floe particles
and was unrecoverable. Therefore, the initial titer for the adsorbed phage
is given in two forms in Table 14. The first represents the initial titer
computed assuming viable virus was present in the floe but unrecoverable,
while the second represents the virus what was actually recovered (as
previously indicated, less than 0.05 percent minus the losses incurred by
homogenization). If the computed initial titers of adsorbed f2 bacterio-
phage are used, then no protection was evident at either 1 or 5 NTU. If the
actual titers of recoverable viruses were used, a partial protective effect
may be afforded. At an initial 03 concentration of 0.127 mg/L unadsorbed
bacteriophage with an initial concentration of 8.4 x 1C)6 PFU/ml was reduced
5.9 logs within 30 seconds. At the same Oo concentration using the initial
computed titer of 7.7 x 10 PFU/ml, a 6.7 fog reduction was obtained after
30 seconds of 03 contact when the bacteriophage was associated with 1 NTU of
hydrated aluminum oxide floe. When the actual titer of recoverable phage was
used, a 3.0 log reduction of f2 with an initial concentration of 1.5 x 103
PFU/ml was obtained after 30 seconds of 03 contact. However, the data at
both 1 and 5 NTU are inconclusive with respect to protection, since the
actual extent of inactivation of the adsorbed phage could not be determined
due to the low levels of recovery from the floe.
Escherichia coli
As previously indicated in the sample preparation section of Materials
and Methods, the adsorbed E. coli samples were not centrifuged to separate
the unadsorbed from the adsorbed bacteria. Instead settling analyses done
on a portion of both the adsorbed and homogenized samples were used to cal-
culate the unadsorbed fraction and the fraction released by homogenization.
As indicated, an IE. coli control was also established and analyzed to deter-
mine the natural inactivation and sedimentation of bacteria within 24 hours.
The results of 7 trials showed a 69 percent recovery of E. coli in 24 hours
at 40C. Six percent of this recovered fraction had settTed in the 24 hour
time span. Correction factors derived from these results were applied to the
46
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supernatant values obtained from the 24 hour settling analyses on both the
adsorbed and the homogenized samples to account for losses incurred by both
natural inactivation and sedimentation. The computed £. coli adsorption onto
the floe, as shown in Table 15, based on the average supernatant titer of 2
percent following sedimentation, was 98 percent of the initial bacteria
introduced. Titration of the adsorbed fraction showed that 91 percent of the
bacteria were recoverable from the hydrated aluminum oxide floe. The super-
natant titer obtained after sedimentation of homogenized floe showed that
11 percent of the bacteria were released during this process. The computed
concentration that remained associated with the floe was, therefore, 89
percent.
The E_. coli dimensions (0.3 x 1-5 ym), as compared to the floe particles
(5 to 50 ym), indicate the possibility that more than one bacterium may
have been associated with each floe particle. In the assay technique em-
ployed for _E. coli enumeration a floe particle, possibly containing multiple
bacteria, may have produced only one colony when plated thereby indicating
a source of bacteria loss.
After sample homogenization, 11 percent of the adsorbed JE. coli may have
been more available for release when the floe particles were sheared. Titra-
tion of the floe particles following homogenization showed that 100 percent
of the adsorbed bacteria associated with the floe were recoverable. The
TDW control in this case yielded 100 percent recovery of £. coli, indicating
no losses due to the adsorption procedure.
The Oo disinfection range investigated for JE. coli, shown in Table 16,
was from 0.014 to 0.53 mg/L of 03- At an initial 0, concentration of 0.021
mg/L 99.7 percent of unadsorbed _E. col i with an initial concentration of
1.5 x 10° CFU/100 ml were inactivated after 10 seconds of 0, contact. At
the same OQ concentration, 99.8 percent of floe-associated £. coli at 1 NTU
with an initial concentration of 1.3 x 108 CFU/100 ml were also inactivated
within 10 seconds. At an initial 03 concentration of 0.040 mg/L, 4.7 log
reduction of unadsorbed £. coli were inactivated within 30 seconds when the
initial bacterial concentration was 7.7 x 107 CFU/100 ml. At the same 03
concentration 5.0 log reduction associated with 5 NTU of hydrated aluminum
oxide floe were also inactivated within 30 seconds when the initial concen-
tration of adsorbed bacteria was 7.0 x 107 CFU/100 ml. An initial 03
concentration of 0.239 mg/L resulted in complete inactivation of both unad-
sorbed and adsorbed £. coli at 5 NTU in 30 seconds with initial concentra-
tions of approximately 8 logs/100 ml. The values cited above indicate that
no protective effect was afforded the £. coli at either 1 or 5 NTU of
hydrated aluminum oxide floe over the 03 concentration range investigated
( 0.014 to 0.053 mg/L). It must also be remembered that only 0.1 ml aliquots
of E_. coli were plated. Therefore, on a colony/100 ml basis, a difference
of only one colony became a difference of 1 x 103, possibly accounting for
some of the variations in the data presented.
DISCUSSION
Hoff (17) has summarized recent virus studies involving disinfection
48
-------
TABLE 15. ASSOCIATION OF ESCHERICHIA COLI WITH HYDRATED ALUMINUM OXIDE
SYSTEM RANGE OF BACTERIAL BACTERIAL
CONCENTRATIONS9 RECOVERY
(CFU/100 ml) (%)
Computed bacterial concentration in
floe
(initial-supernatant) x m l .2 x 107 to 1.1 x 108 98 (6)b
Actual recovery from floe (adsorbed)
(adsorbed-supernatant) ,nn 8.7 x 108 to 2.3 x 109 91 (5)
computed x IUU
Computed bacterial concentration in
floe after homogenization
(adsorbed-homogenized
supernatant) 10Q 2 6 1Q7 to 2 9 x 1Q8
(adsorbed-supernatant)x IUU ^'b x IU to ^'y x IU
Actual recovery from floe after
homogenization
(homogenized-homogenized
supernatant)
TDW control
x 100 1.4 x 109 100 (1)
aRange of initial concentration: 9.6 x 108 to 2.0 x 109
Number of trials
49
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with HOC1 by stating that virus associated with inorganic particles such as
clays or A^PCH showed inactivation rates similar to or perhaps slightly
slower than freely suspended virus. This, compared to the disinfection rates
of cell-associated viruses, indicated various degrees of protection, with
some complexes exhibiting extreme resistance to disinfection. These find-
ings, in conjunction with the results obtained from this study, strongly
indicate that the lack of protection shown was directly related to the in-
organic nature of the hydrated aluminum oxide floe. Chemical reactivity was
probably minimal between the 03 and the floe particle, and it would further
appear that there was no physical interference provided by the floe. It is
evident, since some of the bacteria and viruses were contained within the
particles, that 03 easily diffused into the hydrated aluminum oxide floes
in the size ranges used in this study and inactivated these organisms as
readily as it did those attached to the surface of the floe. It can be
concluded, therefore, that no major problem with ozone disinfection of the
viruses and bacteria investigated would occur if these organisms were
associated with alum floe particles of the size which might escape filtration
of potable water supplies.
THE EFFECT OF HUMAN EPITHELIAL CARCINOMA CELLS ON OZONE DISINFECTION
OF POLIOVIRUS (SABIN TYPE I) AND COXSACKIEVIRUS A9.
RESULTS
The inactivation data from the Sharp apparatus for the HEp-2 cell-
associated poliovirus at 1 and 5 NTU are shown in Table 17. These data
indicate a protective effect by the mass of cellular material on inactiva-
tion of cell-associated poliovirus by ozone. While total inactivation of
cell-associated poliovirus at 1 NTU was demonstrated at 2.30 mg/L ozone,
it should be noted that the initial viral titer of the preparation was lower
than any preceeding or subsequent trial. Applied ozone dosages as high as
4.06 mg/L with a residual of 2.56 mg/L after 40 seconds did not result in
complete inactivation of 5 NTU suspensions of cell-associated poliovirus.
The inactivation data obtained at 1 and 5 NTU for cell-associated poliovirus
suggest there was a protection afforded the virus by the presence of cellular
material.
This protection may have been provided by competitive oxidation of the
associated cellular material with a decreased ozone residual available for
inactivation of virus. A comparison of the inactivation obtained at applied
ozone dosages of 0.65 mg/L, 2.30 mg/L, and 2.84 mg/L at both 1 and 5 NTU
indicates increased poliovirus survival at 5 NTU.
The effect of physical cohesion of the cellular material in the protec-
tion of the cell-associated poliovirus was determined by preparing replicate
samples, one of which had been sonically treated prior to ozonation in the
Sharp apparatus. As seen in Table 18, presonication resulted in increased
inactivation in each trial. Concurrent with these experiments, light
microscopy displayed definite differences in the physical state of the pre-
sonicated samples and the samples not sonically treated both before and after
ozonation. A comparison of initial 5 NTU samples of HEp-2 cell-associated
51
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poliovirus and a sonically treated replicate of the initial sample of HEp-2
cell-associated poliovirus demonstrates the effect of presonication on the
mass of cellular material. The aggregation of the initial sample in Figure 6
was dispersed to single cells as seen in Figure 7 following sonication. The
integrity of the initial cells was variable. Figure 6 indicates the presence
of both whole and partially fragmented cells in a clump while the presoni-
cated samples in Figure 7 contain only cell fragments.
HEp-2 cell-associated coxsackievirus
The inactivation of HEp-2 associated coxsackieivirus A9 at 1 and 5 NTU
is shown in Table 19. Complete inactivation of cell-associated coxsackie-
virus was not obtained at the highest ozone dosage and contact time possible
with the apparatus. In general, 1 NTU preparations exhibited enhanced inac-
tivation in comparison to 5 NTU samples, but total inactivation was not
obtained.
The lack of total inactivation concurrent with the enhanced protection
exhibited by the NTU cell-associated coxsackievirus suggests that resistance
to disinfection by ozone is associated with the presence of the cellular
material rather than some inherent property of either coxsackievirus A9 or
poliovirus I.
The applied ozone dosages cited in Table 19 represent the maximal do-
sages obtained from the ozone generator and reaction apparatus previously
described. The restricted contact times of the Sharp continuous flow dis-
infection apparatus did not enable complete inactivation of 1 NTU and 5 NTU
samples of HEp-2 cell-associated coxsackievirus or poliovirus.
Batch Reactors
Extended contact times for ozone disinfection were provided in a batch
reactor. The results presented in Table 20 indicate that the decrease in
ozone concentration did not stabilize with an extended reaction time. Total
inactivation of the HEp-2 cell-associated poliovirus was not obtained in the
system within the contact time of 75 minutes. The continued survival of
HEp-2 cell-associated poliovirus at 75 minutes dictates the necessity for an
adequate ozone residual as well as extended contact times. Although initial
ozone concentrations of 3.93 mg/L and 2.51 mg/L were used, the remaining
ozone residuals at 60 minutes were 0.10 mg/L and 0.11 mg/L, respectively.
While the inactivation data obtained at 3.93 mg/L ozone indicate the
residual was sufficient to achieve 100 percent inactivation at 75 minutes,
at 2.51 mg/L ozone the rate of inactivation was lowered with only 99.1
percent inactivation after 75 minutes. The initial titers of both trials
were comparable and would allow for the enhanced survival in either trial.
Continuous Ozonation of HEp-2 Cell-Associated Poliovirus and Coxsackievirus
in a Batch Reactor.
Continuous ozonation coupled with extended contact times in a batch
reactor resulted in total inactivation of both cell-associated poliovirus
54
-------
Figure 6. HEp-2 Cells Before Ozonation at 5
NTU
Figure 7. HEp-2 cells sonically treated before ozonation
55
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and coxsackievirus as seen in Table 21. This system enabled increased levels
of ozone in comparison to that obtained in the Sharp continuous flow system.
In conjunction with extended contact times, the smaller volume of the reac-
tion bottle enabled a higher applied dosage of ozone.
Four to five logs of cell-associated poliovirus were inactivated fol-
lowing a 2 minute contact time with initial ozone concentrations of 6.82 and
6.50 mg/L which resulted in ozone residuals of 4.70 and 5.45 mg/L after 2
minutes of reaction time. The HEp-2 cell-associated coxsackievirus exhibited
an increased survival time compared to that observed with the cell-associated
poliovirus, but at a lower initial ozone concentration and consequent lower
residual. In one trial the cell-associated coxsackievirus exhibited survival
at 10 minutes with an initial ozone concentration of 5.33 mg/L and a residual
of 2.68 mg/L after 10 minutes. However, subsequent trials of 5.19 and 4.81
mg/L initial ozone concentrations with residuals of 3.39 and 2.20 mg/L after
2 minutes showed no survival.
DISCUSSION
The compiled inactivation data obtained from each disinfection system
confirm the protective effect provided to virus by association with cellular
material. This protection requires very high ozone residuals and extended
contact times for disinfection. Some of the current practices use ozone
residuals of 0.4 mg/L after five minutes for the disinfection of viruses in
drinking water. This investigation has found that such levels are ineffec-
tive in the inactivation of HEp-2 cell-associated poliovirus and coxsackie-
virus.
Interpretation of these findings is subject to qualification by the
practical composition of the contaminants. The infected Hep-2 cells utilized
in this study were measured at 10 to 15 ytn, a size which should dictate their
removal by filtration before being subjected to disinfection by ozone in
drinking water. Therefore, the initial titers of cell-associated virus con-
tained in a 1 NTU or 5 NTU sample in this investigation would not approximate
the number of cell-associated virus present after filtration of drinking
water. Nor would the 1 NTU or 5 NTU filtrate subjected to disinfection by
ozonation of a heterogenous sample be limited to cell-associated virus. A 1
NTU sample of HEp-2 cell-associated poliovirus with an initial titer of 6.0
x 10^ PFU/ml ozonated stream exhibited 100 percent inactivation at an
initial ozone concentration of 2.30 mg/L after a 30 second contact time in
the Sharp continuous flow apparatus. This finding suggests that with de-
creased initial titers, as would be present after filtration, total inactiva-
tion by ozonation might be obtained. Further experimentation using hetero-
geneous particulate matter may elucidate the possible use of a disinfectant.
However, one must conclude from these data that if one infected cell was
present in the heterogeneous particulate material in the filtered water it
is possible that virus would survive 0.4 mg/L of ozone after 5 minutes of
contact time.
58
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59
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THE EFFECT OF BENTONITE CLAY ON THE OZONE DISINFECTION OF E.. COL I AND
VIRUSES IN WATER.
Poliovirus
The results of five poliovirus clay adsorption trials are illustrated in
Table 22. Adsorption levels of approximately 10 percent were attained with
poliovirus and bentonite clay at pH 7.0. Virus recoveries from the clay
and TDW systems averaged 86 to 77 percent, respectively.
Clay particle size used in this work ranged from 3 to 8 ym, a range
which is comparable to that of finished water from a filtration plant. These
observations were determined with a light microscope.
Complete inactivation of poliovirus was accomplished by using a rela-
tively low ozone dosage, as presented in Table 23. Initial virus concen-
trations of 8.0 x 10' to 7.4 x 10^ PFU/ml, either unadsorbed or at 1 and 5
NTU of bentonite clay, were inactivated within 10 seconds at 0.21 mg/L of
ozone. The data indicate that about 0.03 mg/L would have produced complete
inactivation of about two logs of adsorbed or unadsorbed virus. Comparison
of the unadsorbed virus versus 5 NTU adsorbed trials indicate no or a slight
protection against the disinfection of poliovirus when it is adsorbed to
bentonite clay.
Coxsackievirus
Adsorption of the Coxsackievirus to the bentonite clay stock averaged
13 percent of the virus added to the clay reactor, as shown in Table 24. A
30 percent increase in virus titer was noted in both the clay and TDW systems.
Disinfection trials with Coxsackievirus produced data similar to those
obtained with poliovirus. Table 25 shows the Coxsackievirus results, 3
obtained with an initial virus concentration of 2.0 x 102 to 1 .0 x 10 PFU/
ml of ozonated stream. At 0.144 mg/L of ozone complete inactivation was
achieved within 10 seconds for the unadsorbed, 1 NTU and 5 NTU trials. The
data indicate that an initial ozone concentration of about 0.03 mg/L would
have produced the same level of inactivation. As with poliovirus, the
comparison of the unadsorbed versus 5 NTU adsorbed trials indicates an
apparent lack of protection by adsorption of Coxsackievirus to bentonite
clay. Coxsackievirus adsorption levels at pH 7.0 were only slightly greater
than those obtained with poliovirus. No protection at either 1 or 5 NTU of
bentonite clay was shown.
f Bacteriophage
Adsorption studies presented in Table 26 indicate that the f~ virus
associated with bentonite clay under the conditions examined. The bentonite-
associated fo bacteriophage averaged about 1 percent of the clay system phage
concentration. Although a low adsorption level, this represented an adsorbed
titer range of 2.4 x 103 to 2.2 x 10$ PFU/ml of ozonated stream in the dis-
infection system. Recovery of f2 from the clay system showed an apparent
60
-------
TABLE 22. ASSOCIATION OF POLIOVIRUS (SABIN TYPE 1) WITH BENTONITE CLAY
SYSTEM
Virus stock
Adsorption to clay
adsorbed
1QO
supernatant + adsorbed
Recovery from clay reactor
supernatant + adsorbed .. ,nn
initial / 25bx IUU
Recovery from TDW reactor
control inr.
initial / 25Qb x 10°
RANGE OF VIRUS
CONCENTRATIONS
(PFU/ml)
8.3 x 105 to 1.6 x 106
3.0 x 103 to 4.5 x 103
VIRUS
RECOVERY
10 (5)'
3.1 x 104 to 5.7 x 104 86 (5)
2.3 x 103 to 2.2 x 104 77 (5)
number of trials
dilution of virus stock in the reactor
61
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62
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TABLE 24. ASSOCIATION OF COXSACKIEVIEUS A9 WITH BENTONITE CLAY
RANGE OF VIRUS VIRUS
CONCENTRATIONS RECOVERY
SYSTEM (PFU/ml) (%)
Virus stock 1.5 x 106 to 4.7 x 106
Adsorption to clay 6.4 x TO3 to 2.9 x 104 13 (7)a
adsorbed
supernatant + adsorbed
x 100
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supernatant + adsorbed 1nn
initial / 25&x IUU
Recovery from TDW reactor 6.3 x 103 to 2.3 x 104 130 (3)
control 1QQ
initial / 250^ x IUU
anumber of trials
dilution of virus stock in the reactor
63
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TABLE 26. ASSOCIATION OF f BACTERIOPHAGE WITH BENTONITE CLAY
SYSTEM
Virus stock
Adsorption to clay
adsorbed
x TOO
supernatant + adsorbed
Recovery from clay reactor
supernatant + adsorbed ,nn
initial / 25bx IUU
RANGE OF VIRUS VIRUS
CONCENTRATIONS RECOVERY
(PFU/ml) (%)
8.8 x 108 to 2.3 x 109
2.8 x 105 to 3.9 x 106 1 (13)'
1.6 x 107 to 3.3 x 108 173 (8)
Number of trials
Dilution of virus stock in reactor
65
-------
73 percent increase in virus concentration. This substantial increase in
titer shown over many trials suggests a breakup of fo aggregates during the
adsorption procedure. Of the two clay system components (supernatant and
adsorbed) the phage increase was noted in the supernatant fraction. Floyd
and Sharp (38) have investigated the aggregation of poliovirus and reovirus
caused by dilution in water and by varying conditions of salinity and :pH,
They reported that aggregation may be a function of virus concentration, pH,
or the presence of particular ions in solution. Throughout the f~ virus
experimentation, salt concentration (0.01 M NaCl) and pH (7.0) remain
constant. Bentonite clay contains exchangable, surface bound cations which
may have influenced a break up of aggregrates. Unfortunately, inconclusive
data were,obtained with the TDW control, so it is uncertain whether the
presence of bentonite clay was responsible for the increase.
Inactivation data for the f~ phage are shown in Table 27. Initial
ozone concentrations of 0.25 to 6.50 mg/L resulted in complete inactivation
of unadsorbed phage in concentrations of 2.6 x 103 to 2.2 x 105 PFU/ml with-
in 10 seconds. Survival was noted at 10 seconds when the ozone level was
reduced to 0.061 mg/L, however, 30 seconds exposure to that concentration
was sufficient to completely inactivate the phage.
The inactivation levels of f2 associated with bentonite were less than
those determined in the unadsorbea system. In the presence of the clay,
initial virus concentrations of 2.4 x 103 to 2.2 x 105 PFU/ml consistently
showed survival when exposed for 10 seconds to 0.25 to 0.50 mg/L of ozone
with about the same residual ozone concentrations. Further, a 2.2 mg/L
initial ozone concentration was insufficient to completely inactivate the
phage within 10 seconds. From these results it appears that contact time is
more significant than ozone concentration as a parameter in the disinfection
of adsorbed f2 bacteriophage. Protection of the phage by adsorption is
also suggested.
Escherichia coli
The association of Escherichia coli with bentonite was not quantita-
tively determined. Separation of the bentonite-associated and freely sus-
pended £. coli was hindered by the relative size of the two components. The
dimensions of £. coli (0.3 x..1.5 ym) as compared to the clay particles (3 to
8 ym) indicate that association from other than a surface phenomenon would
have been unlikely.
As presented in Table 28, similar inactivation data were obtained for
the unadsorbed versus bentonite-associated £. coli. In both cases, initial
bacteria concentrations of 9.4 x 10' to 3.6 x 10a CFU/ 100 ml were reduced
at least three logs within 10 seconds exposure to ozone dosage of 0.038 to
0.53 mg/L. Complete inactivation was not achieved within 10 seconds, but
was consistently complete within 30 seconds over the range of ozone dosages
studies. Disinfection trials conducted with the unadsorbed E^. coli versus
£. coli adsorbed to 5 NTU of bentonite indicate a lack of protection by
the clay. Here, as in the f2 data, contact time apears to be the dominant
disinfection parameter.
66
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DISCUSSION
The virus adsorption levels obtained throughout this research were
significantly lower than those reported by Boardman and Sproul (29), whose
procedures were utilized with modifications.
Stagg, et^ al.(39) reported that association of MS-2 bacteriophage with
bentonite clay was influenced by the titer of unadsorbed viruses, clay
concentration, cation concentration, temperature, stirring rate, and the
presence of soluble organics. While our experiments were devised to
minimize the above variables, some uncertainties remain, such as the use of
a bentonite suspension as an adsorbant which had been subjected to preozona-
tion. Nevertheless, regardless of percentage, the adsorption levels were
consistent for each organism studied over numerous trials.
'A detailed review of research relating turbidity to disinfection was
compiled by Hoff (17). Included in the report were studies of the effects of
inorganic and organic turbidity on disinfection of poliovirus type 1 with
hypochlorous acid (HOC!). The results showed that virus adsorbed to bento-
nite exhibited inactivation rates similar to, or perhaps slightly slower
than, freely suspended virus. Similar first order inactivation curves were
generated with the adsorbed and unadsorbed poliovirus. Conversely, organic
turbidity represented by cell debris had a pronounced protective effect on
associated virus. Inactivation rates slowed as exposure time increased,
indicating the existence of various degrees of protection and some extremely
resistant complexes. The report concluded that turbidity is not an ideal
measure of the disinfectability of water since interference with disinfection
depends more on the types of turbidity present than the number of turbidity
units.
The results obtained in this study using poliovirus and coxsackievirus
tend to support the above findings. Comparison of adsorbed versus unadsorbed
inactivation data reveals the lack of protection offered the virus by the
inorganic clay particles. Evidently no physical interference was provided
by the bentonite, nor would chemical reactivity between the ozone and the
demand free clay particles be anticipated. In addition, due to the sensiti-
vity of the adsorbed virus to low concentrations of ozone, it appears that
ozone diffuses easily to the clay particles, inactivating the adsorbed virus
at a rate that is approximately the inactivation rate for unadsorbed virus.
Microorganisms have been found to associate with clay particles of a
size that may escape conventional filtration plants and enter potable water
supplies. Poliovirus (Sabin type 1), coxsackievirus A9, f2 bacteriophage,
and Escherichia coli associated with bentonite were inactivated by ozone
at rates similar to or, as is believed the case with f2» at slightly
slower rates than freely suspended organisms.
A comparison of various inactivation data for unadsorbed organisms used
in this study is shown in Table 29. In selecting the data an attempt was
made to include only those where some survival was noted after 30 seconds of
contact and where the residual was measured after 40 seconds. While the
69
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70
-------
data do not lend themselves to easy comparison it appears that the order of
increasing resistance to disinfection was j^. coli, poliovirus 1 and Coxsackie-
virus A9. This ranking was based on comparing JE_. coli,polio and Coxsackie at
0.017 mg/L, 0.015 mg/L and 0.016 mg/L ozone at the start of the experiment.
The corresponding inactiyations obtained were 5.2 x 10' CFU/100 ml, 1.4 x
104 PFU/ml, and 2.2 x 102 PFU/ml in 30 seconds.
OTHER REPORTS BASED ON THIS RESEARCH
Additional published material, based on research conducted under this
grant includes the following:
Theses
1. Howser, Diana M. The Effect of Fecal Material on Ozone
Disinfection of Coliform Bacteria and Enteric Viruses in
Water.
2. Walsh, Douglas S. The Effect of Hydrated Aluminum Oxide
Floe on Ozone Disinfection of Bacteria and Viruses in Water.
3. Boyce, Douglas S. The Effect of Bentonite Clay on Ozone
Disinfection of Bacteria and Viruses in Water.
Copies can be obtained from:
Fogle Library
University of Maine
Orono, Maine 04473
Publications
1. Sproul, O.J., M.A. Emerson, D.M. Howser, D.S. Boyce,
D.S. Walsh and C.D. Buck. "Effect of Particulate Matter on
Virus Inactivation by Ozone." Proceedings Annual Meeting
American Water Works Association, 1978 (presented at
Annual Meeting, June, 1978).
2. Howser, D.M., M.A. Emerson, D.S. Walsh, O.J. Sproul and
C.E. Buck. "Ozone Inactivation in Cell and Fecal Associated
Bacteria and Virus." Presented at Water Pollution Control
Federation Anaheim Conference, Cnaheim, California,
October 1978. (submitted to JWPCF for publication).
Papers in Preparation
1. Sproul, O.J., M.A. Emerson, D.M. Howser, D.S. Boyce, D.S.
Walsh and C.E. Buck. Effect of Particulate Matter on Virus
Inactivation by Ozone.
71
-------
2. Emerson, M.A., O.J. Sproul and C.E. Buck. Ozone Inactivation
of HEp2 Cell-Associated poliovirus and Coxsackievirus.
3. Walsh, D.S., O.J. Sproul and C.E. Buck. Ozone Disinfection
of Bacteria and Viruses Associated with Hydrated Aluminum
Oxide Floe.
4. Boyce, D.S., O.J. Sproul and C.E. Buck. Ozone Disinfection
of Bacteria and Viruses Associated with Bentonite Clay.
72
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LITERATURE CITED
1. Nupen, E.M., "Viruses in Renovated Waters", Viruses in Water, Berg, G.,
et al., ed., American Public Health Association, Inc. Washington, D.C.
(1976).
2. Long, W.N. and Bell, F.A., Jr., "Health Factors and Reused Waters",
American Water Works Association 6i (4): 220 (1972).
3. Craun, G.F., McCabe, L.J. and Hughes, J.M., "Waterborne Disease Outbreaks
in the U.S. 1971-1974", J. Am. Water Works Assn. 68 (8): 420 (1976).
4. "Engineering Evaluation of Virus Hazard in Water:, Committee on Environ-
mental1 Quality Management, Berger, B.B., Chmn., Jour. San. Eng. Div.,
ASCE, 96 (Sal): 111 (1970).
5. Geldreich, E.E., "Origins of Microbial Pollution in Streams", Trans-
mission of Viruses by the Water Route, Berg, G., ed., John Wiley and
Sons, New York (1967).
6. Kelly, S. and Sanderson, W.W., "Density of Enteroviruses in Sewage", J.
Water Pollution Control Fed. 32, 1269 (1960).
7. Cookson, J.T., Jr., "Virus and Water Supply", J. Amer. Water Works Assn.
66 (12): 707 (1974).
8. Berg, G., "An Integrated Approach to the Problem of Viruses in Water:,
Proceedings of the National Specialty Conference on Disinfection, July
1970. ASCE, New York, New York (1971).
9. Gerba, C.P., Wallis, C. and Melnick, J.L., "Viruses in Water: The
Problem, Some Solutions", Environmental Science and Technology 9^ (13):
1122 (1975).
10. Clark, N.A., Berg, G., Kabler, P.W. and Chang, S.L., "Human Enteric
Viruses in Water: Source Survival and Removability", Advances in Water
Pollution Research, Vol. 2, MacMillan Company, New York (1962).
11. Metcalf, T.G., "Indicators for Viruses in Natural Waters", Water Pollu-
tion Microbiology, Vol, 2, Mitchell, R., ed., John Wiley and Sons, New
York (1978).
12. Sproul, O.J., "Removal of Viruses by Treatment Processes", Viruses in
Water, Berg, G., et al., ed., American Public Health Association, Inc.,
Washington, D.C. (1976).
73
-------
13. Plotkins, S.A. and Katz, M., "The Minimal Infective Dose", Transmission
of Viruses by the Hater Route, Berg, G., ed., John Wiley and Sons, New
York (1967).
14. Berg, G., "Reassessment of the Virus Problem in Sewage and in Surface
and Renovated Waters", Progress in Water Technology, Jenkins, S.H., ed.,
Pergammon Press, New York (1973).
15. Schaub, S.A. and Sorber, C.A., "Viruses on Solids in Water", Viruses in
Water, Berg, G., et al., ed., American Public Health Association, Inc.,
Washington, D.C. (1976).
16. Symons, J.M. and Hoff, J.C.i "Rationale for Turbidity Maximum Contami-
nant Level", Proceedings AWWA Water Quality Technology Conference,
Atlanta, Georgia (1975).
17. Hoff, J.C., "The Relationship of Turbidity to Disinfection of Potable
Water", Evaluation of the Microbiology Standards for Drinking Water,
Hendricks, C., ed., EPA-570/9-78-OOC (1978).
18. "Ozone, Chlorine Dioxide, and Chloramines as Alternatives to Chlorine
for Disinfection of Drinking Water: State-of-the-Art", Symons, J.M.,
ed., Water Supply Research, Office of Research and Development, U.S.
Environmental Protection Agency, Cincinnati, Ohio (1977).
19. Page, T., Harris, R.H., and Epstein, S.S., "Drinking Water and Cancer
Mortality in Louisiana", Science 193, 55 (1976).
20. Kuzma, R.J., Kuzma, D.M. and Buncher, C.R., "Ohio Drinking Water Source
and Cancer Rates", Am. J. of Public Health 67 (8): 725 (1977).
21- Majumdar, S.B. and Sproul, O.J., "Technical and Economic Aspects of
Water and Wastewater Ozonation: A Critical Review", Water Res. 8,
253 (1974).
22. Venosa, A.D., "Ozone as a Water and Wastewater Disinfectant: A litera-
ture Review", Ozone in Water and Wastewater Treatment, Evans, F.L., ed.,
Ann Arbor Science Publishers, Inc., Michigan (1972).
23. Lawrence, J. and Cappelli, P.P., "Ozone in Drinking Water Treatment: A
Review", The Science of the Total Environment 7_ (2): 99 (1977).
24. Watson, J.T., and Drewry, W.A., "Adsorption of f2 Bacteriophage by
Activated Carbon and Ion Exchange Resins", Civil Engineering, The Uni--
versity of Tennessee, Research Series No. 11, 23 (1971).
25. Cooper, S. and Zinder, N.D., "The Growth of RNA Bacteriophage: The Role
of DNA Synthesis", Virology 18, 405 (1962).
26. Gentile, D.M., Unpublished Procedure (1975).
74
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27. Standard Methods for the Examination of Water and Wastewater, 14th ed.,
American Public Health Assoc. Inc., Washington, D.C. (1975).
28. Guskey, I.E. and Wolff, D.A., "Concentration and Purification of Polio-
virus by Ultrafiltration and Isopycnic Centrifugation", Appl, Micro-
biology 24 (1): 13 (1972).
29. Boardman, G.D. and Sproul, O.J., "Adsorption as a Protective Mechanism
for Waterborne Viruses", Completion Report, Project A-030-ME, Land and
Water Resources Institute, University of Maine, Orono (1977).
30. Wolf, K. and Quimby, M.C., "Fish Virology: Procedures and Preparation of
Materials for Plaquing Fish Viruses in Normal Atmosphere", United States
Department of the Interior, US ISSN 0071-6493, FDL-35 (1973).
31. Karber, G., "Beitrag zur Kollektiven Behandlung Pharmakologischer
Reihenversuche", Arch. Exptl. Path. Pharmakol., 162. 480 (1931).
32. Adams, M.H., Bacteriophage, Interscience Publishers, New York (1969).
33. Shechter, H., "Spectrophotometric Method for Determination of Ozone in
Aqueous Solutions", Water Research 7., 729 (1973).
34. Tate, C.H., Lang, J.S. and Hutchinson, H.L., "Pilot Plant Tests of
Direct Filtration", J. Am. Water Works Assn. £9 (7): 343 (1977).
35. Rickert, D.A. and Hunter, J.V., "Rapid Fractionation and Materials
Balance of Solids Fractions in Wastewater and Wastewater Effluent", ;
Jour. Water Poll. Cont. Fed. 39_ (9): 1475 (1967).
36. Ward, R.L. and Ashley, C.S., "Identification of the Vircidal Agent in
Wastewater Sludge", Appl. Environ. Microbiol. _33_, 860 (1977).
37. Diaper, E.W.J., "Disinfection of Water and Wastewater using Ozone",
Disinfection Water and Wastewater. Johnson, D.J., ed., Ann Arbor
Science Publishers, Inc., Michigan (1975).
38. Floyd, R. and Sharp, D.G., "Aggregation of Poliovirus and Reovirus by
Dilution in Water", Appl, Environ. Microbiol. 35 (6): 1084 (1978).
39. Stagg, C.H., Wallis, C., and Ward, C.H., "Inactivation of Clay Associa-
ted Bacteriophage MS-2 by Chlorine", Appl, Environ. Microbiol. ^3_ (2):
385 (1977).
75
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/2-79-089
3. RECIPIENT'S ACCESSION" NO.
4. TITLE AND SUBTITLE
EFFECT OF PARTICULATES ON OZONE DISINFECTION OF
BACTERIA AND VIRUSES IN WATER
5. REPORT DATE
August 1979 (Issuing Date)
6. PERFORMING ORGANIZATION CODE
6UTHORIS)
tis J. Sproul, Charles E. Buck, Maura A. Emerson,
Douglas Boyce, Douglas Walsh, and Diana Howser
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Civil Engineering
University of Maine Orono
Orono, Maine 04401
10. PROGRAM ELEMENT NO.
1CC824. SOS 2, Task 10
11. CONTRACT/GRANT NO.
R804587
12. SPONSORING AGEN.CY NAME AND ADDRESS
Municipal Environmental Research Laboratory-Cin.,OH
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
3VERED
14. SPONSORING AGENCY CODE
EPA/600/14
15. SUPPLEMENTARY NOTES
Project Officer:
John C. Hoff (513) 684-7331
16. ABSTRACT
This research was initiated in order to determine the effect of particulates on
ozone disinfection of enteric bacteria and viruses adsorbed to or incorporated into
particulate materials such as fecal material, HEp-2 cells, aluminum oxide floe and
bentonite clay. Microorganisms used included fecal colifqrms, poliovirus (Sabin
Type 1), Coxsackievirus A9, porcine picorna-virus Type 3 (Strain ECPO-6) and f? bac-
teriophage.
The results indicate that the encasement or adsorption of enteric bacteria and
viruses in fecal material, both human and porcine, and HEp-2 cells protects these
microorganisms from a concentration of ozone and contact time that would normally
inactivate the bacteria and viruses in an unadsorbed or free state. HEp-2 cells
gave the greatest amount of protection for the cell-associated poliovirus and Cox-
sackievirus studied. It was necessary to maintain a concentration of 5.33 to 4.81
mg/L ozone to inactivate the cell-associated Coxsackievirus in 5 to 10 minutes.
Hydrated aluminum oxide floe and bentonite clay afforded little or no protection to
the Escherchia coli. poliovirus and Coxsackievirus adsorbed to these particles over
that of the microorganisms in the free state. The f? bacteriophage adsorbed to
bentonite clay particles was inactivated at a slower rate than the freely suspended
phage.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS C. COS AT I Field/Group
Ozone, Enteroviruses, Polioviruses,
Coxsackieviruses, Escherichia coli,
Disinfection, Microorganism control
(water), Water treatment chemicals,
Potable water, Water supply, Turbidity,
Particles, Protection
Coliforms
13 B
13. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
86
20. SECURITY CLASS (This page)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
76
4 U.S. GOVERNMENT PRINTING OFFICE: 1979 -6 57-146/5470
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