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AWWA RESEARCH
FOUNDATION
6666 West Quincy Avenue
Denver, Colorado 80235
RESEARCH REPORT
SUBJECT AREA: Water Treatment and Operations
INACTIVATION OF GIARDIA CYSTS WITH CHLORINE AT 0.5 C TO 5.0 C
by
Charles P. Hibler
Carrie M. Hancock
Leah M. Perger
John G. Wegrzyn
K. Diane Swabby
Department of Pathology, College of Veterinary Medicine and Biomedical Sciences,
Colorado State University, Fort Collins, Colorado 80523
Prepared for
American Water Works Association Research Foundation
Martin J. Alien, PhD, Project Officer
6666 West Quincy Avenue
Denver, Colorado 80235
Published by American Water Works Association
DISCLAIMER
This study was funded by the American Water Works Association Research Foundation (AWWARF).
AWWARF assumes no responsibility for the content of the research study reported in this publication, or for
the opinions or statements of fact expressed in the report. The mention of tradenames for commercial products
docs not represent or imply the approval or endorsement of AWWARF. This report is presented solely for
informational purposes.
Although the research described in this document has been funded in part by the United States
Environmental Protection Agency through a Cooperative Agreement, CR-811335-01, to AWWARF, it has not
been subjected to Agency review and therefore does not necessarily reflect the views of the Agency and no
official endorsement should be inferred.
ISBN 0-89867-401-8
Copyright 1987
by
American Water Works Association Research Foundation
American Water Works Association
Printed in U.S.
11
CONTENTS
Tables........................................................................................................................................^
Figures...................................................................................................................................... vii
Foreword.................................................................................................................................... ix
Acknowledgements........................................................................................................................ x
Executive Summary...................................................................................................................... xi
Introduction.................................................................................................................................. 1
Materials and Methods....................................................................................................................?
General............................................................................................................................?
The Animal Model............................................................................................................7
Source of Giardia Cysts...................................................................................................... 7
Collection, Cleaning and Counting Giardia Cysts....................................................................?
Preparation of Buffer.......................................................................................................... 8
Effect of Buffer and Sodium Thiosulfate on Giardia Cysts........................................................ 8
Chlorine Stock Solution..................................................................................................... 8
Determination of Free Chlorine............................................................................................ 8
Inoculation of Buffer with Chlorine...................................................................................... 9
Maintenance of Temperatures............................................................................................... 9
Equipment....................................................................................................................... 9
Statistical Analysis............................................................................................................ 9
Experimental Procedure................................................................................................................. 10
General...........................................................................................................................10
Procedures for Trials......................................................................................................... 10
Method of Determining and Evaluating Infection.................................................................... 11
Results and Discussion.................................................................................................................. 12
Sources of Giardia Cysts.................................................................................................... 12
Infectivity of the Giardia Sources.........................................................................................12
Evaluation of the Gjaidia Sources........................................................................................ 12
Effect of Buffer and Sodium Thiosulfate on Giardia Cysts....................................................... 13
Chlorine Determinations.................................................................................................... 13
CT Values for Inactivation of Giardia Cysts with Chlorine...................................................... 13
Recommendauons ........................................................................................................................16
References...................................................................................................................................38
111
TABLES
Page
Human Sources of Giardia Cysts: Cyst Production and Morphologic
Quality in Mongolian Gerbils........................................................................................ 17
2.
Minimum Dose of Human Source Giardia Cysts that would Consistently Infect
Mongolian Gerbils and the Morphologic Quality of the Cysts Produced................................. 17
3.
N (number of data points), R (correlation coefficient), Slope, Probability,
Standard Error (all in logs), Predicted CT Ranges, and Predicted Mean CT
(all in antilog) for results in which 1 to 4 animals/group and no animals/group
were infected at 0.5 C,2.5 C,5.0 C,pH 6.0,7.0 and 8.0...................................................18
4.
Interpolation of CT values for Temperatures 0.5 C to 5.0 C, pH 6.0 to 8.0............................ 19
FIGURES
Page.
Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Arithmetic plot of CT values for 0.5 C, pH 7................................................................... 20
Arithmetic plot of CT values for 2.5 C, pH 7................................................................... 21
Arithmetic plot of CT values for 5.0 C, pH 7................................................................... 22
Arithmetic plot of CT values for 0.5 C, pH 6................................................................... 23
Arithmetic plot of CT values for 2.5 C, pH 6................................................................... 24
Arithmetic plot of CT values for 5.0 C, pH 6................................................................... 25
Arithmetic plot of CT values for 0.5 C, pH 8................................................................... 26
Arithmetic plot of CT values for 2.5 C, pH 8................................................................... 27
Arithmetic plot of CT values for 5.0 C, pH 8................................................................... 28
Log/log plot of CT values for 0.5 C, pH 7 ...................................................................... 29
Log/log plot of CT values for 2.5 C, pH 7...................................................................... 30
Log/log plot of CT values for 5.0 C, pH 7 ...................................................................... 31
Log/log plot of CT values for 0.5 C,pH 6......................................................................32
Log/log plot of CT values for 2.5 C,pH 6...................................................................... 33
Log/log plot of CT values for 5.0 C, pH 6 ...................................................................... 34
Log/log plot of CT values for 0.5 C,pH 8...................................................................... 35
LogAog plot of CT values for 2.5 C, pH 8 ...................................................................... 36
Log/log plot of CT values for 5.0 C, pH 8 ...................................................................... 37
Vll
FOREWORD
This report is part of the on-going research program of the AWWA Research Foundation. The research
described in the following pages was funded by the Foundation on behalf of its members and subscribers in
particular and the water supply industry in general. Selected for funding by AWWARF's Board of Trustees, the
project was identified as a practical, priority need of the industry. It is hoped that this publication will receive
wide and serious attention and that its findings, conclusions, and recommendations will be applied in
communities throughout the United States and Canada.
The Research Foundation was created by the water supply industry as its center for cooperative research
and development. The Foundation itself does not conduct research; it functions as a planning and management
agency, awarding contracts to other institutions, such as water utilities, universities, engineering firms, and
other organizations. The scientific and technical expertise of the staff is further enhanced by industry volunteers
who serve on Project Advisory Committees and on other standing committees and councils. An extensive
planning process involves many hundreds of water professionals in the important task of keeping the
Foundation's program responsive to the practical, operational needs of local utilities and to the general research
and development needs of a progressive industry.
All aspects of water supply are served by AWWARFs research agenda: resources, treatment and
operations, distribution and storage, water quality and analysis, economics and management. The ultimate
purpose of this effort is to assist local water suppliers to provide the highest possible quality of water,
economically and reliably. The Foundation's Trustees are pleased to offer this publication as contribution
toward that end.
This research report quantifies the efficacy of free chlorine against Giardia cysts at temperatures below
5 C. Chlorine and time (CT) values were determined using an animal model to demonstrate cyst inactivation
following exposure to various concentrations of the disinfectant. The CT values can be used by utilities in
conjunction with other specific operating conditions to evaluate the biocidal effectiveness of their treatment
processes on this pathogen.
—j(
erome B. Gilbert
''Chairman, Board of Trustees
AWWA Research Foundation
^
IX
James F. Manwaring, P.E.
Executive Director
AWWA Research Foundation
ACKNOWLEDGEMENTS
Those of us engaged in this research effort could not have accomplished the goals set forth without the
time, effort, expertise and equipment provided by a number of specialists in the water industry. We are
especially indebted to all of these individuals: Kevin Gertig and Grant Jones, Class A Water Treatment
Operators, Water Production Division, City of Fort Collins; Keith Hancock, Instructor and Head of the
Department of Water and Waste Water Treatment, Larimer County Voc-Tech Center, Fort Collins, Colorado;
Kirke Martin, Director of the Water Laboratory, Colorado State University, Fort Collins, Colorado; Sue Martin,
Laboratory Supervisor, Water Quality Laboratory, City of Fort Collins; Ben Alexander, Water Production
Manager, City of Fort Collins; Dr. Dwayne Hamar and Ms. Marlene Gerlach, Chemistry Laboratory,
Department of Pathology, Colorado State University.
A portion of the funding for this research was a public service contract, administered by Colorado State
University, for analysis of municipal water filters for Giardia cysts. Support for a significant portion of the
research was, therefore, supplied by municipalities across North America.
The Project Advisory Committee consisted of Dr. Martin Alien, Project Officer, AWWARF, Denver,
Colorado; Richard Karlin, Drinking Water Section Chief, Colorado Department of Health; D. William Liechty,
Washington Department of Social and Health Services; Jack Hoffbuhr, USEPA, Region VIII, Denver, Colorado;
Dr. Robert Champlin, Professor of Civil Engineering, University of Wyoming, Laramie; and Dr. John Hoff,
Drinking Water Research Division, USEPA, Cincinnati, Ohio. The committee members provided many helpful
suggestions, and Dr. Alien's guidance kept the project running smoothly. Dr. John Hoff must receive special
acknowledgement for his patience and conscientious efforts to help us correctly accumulate, analyze and interpret
the data. His constructive suggestions greatly facilitated our progress.
We sincerely appreciate the efforts of Dr. Hibler's wife, Dr. Barbara Powers, Assistant Professor of
Radiology and Radiation Biology, Colorado State University, for taking time from her busy schedule to perform
the statistical analyses. The Animal handlers, Ms. Barbara Shear and Ms. Kelly Bellefuil kept abreast of the
job of providing gerbils for this research. Last, but not least, our sincere appreciation to the very busy
departmental secretaries, especially Ms. Esta Amen and Ms. Janice Gentz, for their expertise.
EXECUTIVE SUMMARY
Limited information is available on the amount of free chlorine and time necessary to inactivate cysts
of Giardia duodenalis at temperatures of 5 C and above, but no information is available for temperatures less
than 5 C. Many municipalities across North America obtain water from surface sources either at risk for, or
contaminated with cysts of Giardia. Late fall, winter and early spring water temperatures for most of these
municipalities are less than 5 C. Some use conventional treatment while others use direct filtration, or
filtration without coagulation, especially when the source turbidity becomes less than one nephelometric
turbidity unit. Other municipalities have such high quality water that chlorination is the only barrier used.
Filtration does not insure removal of Giardia and many existing plants do not employ a filtration system that
will effectively remove Giardia. especially in cold water, low turbidity situations.
Cold water increases the life span of Giardia cysts: temperatures less than 5 C allow survival for about
two months. The cyst wall effectively protects the organism from adverse environmental conditions, even
postponing the biocidal activity of some disinfectants. Cold temperatures slow the reaction rate of some
disinfectants, possibly by as much as 2 to 3 fold for every 10 degree decrease. Basic pH values, especially those
above pH 7.5, limit the biocidal activity of some disinfectants. Because these factors act in concert to increase
the risk for waterborne giardiasis from fall to spring, our purpose was to provide municipalities with the
information on the amount of chlorine and time necessary for inactivation of Giardia cysts at temperatures from
0.5 C to 5.0 C, pH 6 to 8.
An animal model, the Mongolian gerbil, Meriones unguiculatus. was used to determine viability of the
cysts of Giardia duodenalis following exposure to chlorine. Cysts were exposed to varying concentrations of
chlorine for different periods of time using temperatures of 0.5 C, 2.5 C and 5.0 C, at pH 6,7 and 8. Five
different human isolates of Giardia cysts were maintained in gerbils and used in the experiments. The pH was
maintained using chlorine demand-free buffer, and temperatures were maintained in water baths using ice and low
temperature incubators. The source of chlorine was reagent grade sodium hypochlorite. The data generated was
analyzed by regression analysis to obtain predicted CT values.
The pH, temperature and chlorine levels to be evaluated were established several days prior to a trial,
and the bottles containing the chlorinated buffer brought to the desired temperature. On the day of a trial cysts
were cleaned and introduced into bottles at a calculated concentration sufficient to provide 1.02 x 10^ cysts/ml of
chlorinated buffer. Available chlorine levels were measured after the introduction of cysts and at the end of a
trial. Bottles containing unchlorinated buffer also were inoculated with cysts at that temperature to serve as an
inoculum source for the positive control animals. All bottles were agitated every quarter hour to insure
continual cyst and chlorine mixing as the cysts settled. At a predetermined time, sufficient chlorinated buffer
containing cysts was removed from the bottle, the chlorine inactivated with 0.1% sodium thiosulfate, and five
gerbils intubated with 5 x 104 cysts/gerbil. At the same time, five gerbils were inoculated with sufficient
material from the unchlorinated buffer bottle to provide 50 cysts/gerbil; these animals served as positive
controls. An equal number of negative controls remained in the animal holding facility. Starting five days after
the trial, gerbils were placed in collection cages, and their pellets were collected and examined for Giardia cysts.
If gerbils did not pass cysts by day 7 they were euthanized and examined postmortem for infection.
CT values obtained for groups with 1 to 4 animals/group infected were compared with CT values for 0
animals/group infected. This data was used to calculate the predicted CT values for each temperature and pH
using a regression equation. Data from 48 trials, using 744 groups of 5 animals/group was used to calculate the
predicted CT values.
Data generated for chlorine concentrations of 0.3 mg/1 to 2.5 mg/1 was used to calculate the predicted
CT values for all pH and temperatures examined. Use of chlorine concentrations greater than 2.5 mg/1 often
produced erratic results.
XI
Data generated from groups of animals where 1 to 4 animals/group were infected is between 99.9% and
99.99% inactivation of Giardia cvsts. The mean predicted CT for pH 6,0.5 C is 185 (176-197); for pH 6,
2.5 C the mean predicted CT is 142 (141-142); for pH 6, 5.0 C the mean predicted CT is 146 (146-147). The
mean predicted CT for pH 7,0.5 C is 289 (285-295); for pH 7,2.5 C the mean predicted CT is 252 (246-256);
for pH 7, 5.0 C the mean predicted CT is 161 (159-163). For pH 8, 0.5 C the mean predicted CT is 342 (312389); for pH 8,2.5 C the mean predicted CT is 268 (250-295); for pH 8, 5.0 C the mean predicted CT is 280
(278-281).
Data generated from groups of animals where 0 animals/group were infected is greater than 99.99%
inactivation of Giardia cysts. The average predicted CT for pH 6,0.5 C is 220 (202-233); for pH 6,2.5 C the
mean predicted CT value is 175 (156-190); for pH 6, 5.0 C the mean predicted CT is 157(139-171). ForpH
7, 0.5 C the mean predicted CT is 310 (302-315); for pH 7,2.5 C the mean predicted CT is 265 (260-268); for
pH 7, 5.0 C the mean predicted CT is 166 (165-166). For pH 8,0.5 C the mean predicted CT is 425 (392475); for pH 8,2.5 C the mean predicted CT is 343 (336-353); for pH 8,5.0 C the mean predicted CT is 290
(243-367).
As is shown by the mean predicted CT values and the range of values for temperatures of 0.5 C to
5.0 C, the biocidal activity of chlorine is much less effective at pH 8. Municipalities with sources above pH 8
must realize that extrapolation of the existing data would be risky; we cannot recommend a CT value that would
be effective.
There is so much variation between sources of Giardia that it is unlikely an exact CT can be specified
for any given temperature and pH. Although efforts were made to develop CT values with human sources of
Giardia that were extremely infectious and highly adapted to gerbils, we must consider the possibility that there
may be sources that are even more infectious; hopefully these will fall within the range of CT values generated
in these trials. A primary reason for choosing CT values necessary to inactivate 99.99% of the cysts was to
encompass this possibility.
Increasing chlorine concentrations above 2.5 mg/1 to decrease time is not likely to solve the problems
for municipalities with short contact time in the distribution. The data generated for chlorine concentrations
between 2.5 and 4.0 mg/1 indicate that CT may not be a reliable indicator of inactivation. Quite possibly the
cyst wall surrounding Giardia is capable of resisting even extreme adverse environmental conditions for a short
period of time. Until more data is generated to determine if this is indeed the case, municipalities should be
prepared to use an alternative safeguard.
Systems at risk for an outbreak of waterborne giardiasis, whether filtered or unfiltered, should apply
the CT values generated from this research when temperatures are less than 5.0 C. This may necessitate either
increasing the amount of chlorine or developing additional contact time to achieve inactivation of the cysts.
Xll
INTRODUCTION
Giardiasis is the most commonly reported parasitic disease of humans in the United States (CDC,
1978). Transmission of the parasite generally is by the fecal-oral route through person-to-person contact and
accounts for the majority of cases. Unfortunately, waterborne transmission of Giardia has become increasingly
important. Giardia has been incriminated as the pathogen responsible for most of the outbreaks of waterborne
disease over the past 20-25 years. In the 15 year period from 1965 to 1980,42 outbreaks of waterborne
giardiasis were reported and since 1980 several additional outbreaks have occurred. According to statistics
compiled by Craun (1986), most waterborne outbreaks (67%) and most cases (52%) resulted from consumption
of untreated surface water or surface water with disinfection as the only treatment. Ineffective filtration was
responsible for only 5 (12%) outbreaks during the 15 year period between 1965 and 1980.
The first outbreak suspected to be caused by a waterborne source of Giardia was in Aspen, Colorado
during the 1965-66 winter tourist season. Retrospective epidemiologic survey indicated sewage contamination
of wells, although the creek also may have been contaminated (Moore et al., 1969). In 1974-75 an outbreak of
giardiasis occurred in Rome, New York (Shaw et al., 1977). During this outbreak investigators from the
National Center for Disease Control detected cysts in the water, the first direct proof that Giardia was present in
the source. In addition, concentrate from the source was introduced into specific-pathogen-free beagle dogs
maintained at Colorado State University (Hibler, et al., 1975). Some dogs became infected with Giardia from
this source; however, this proved only that the cysts were ah've and were infectious for dogs.
Aspen and Rome used unfiltered sources of water, seemingly substantiating the statistics that
waterborne giardiasis was a potential threat only for unfiltered sources. However, in the spring of 1976 an
outbreak of giardiasis occurred in a filtered water source at Camas, Washington (Kirner et al., 1978; Dykes et
al., 1980). Giardia cysts were found in the water using an improved sampling device developed by the
Environmental Protection Agency. Moreover, infected beaver were found near the intakes (Dykes, et al., 1980).
Since the creek used as a source originated in a remote, isolated area with little human activity, and there was no
evidence of sewage contamination, beaver were promptly incriminated as the probable source of the cysts. This
was the first time a wild animal source had been implicated; the previous two outbreaks were considered to be
sewage contamination. While beaver, some other wild animal (muskrat, voles, etc.), or domestic animal (cattle,
etc.) may have been responsible for this outbreak, incrimination of beaver often resulted in limited
epidemiologic investigation of subsequent outbreaks. Frost and Harter (1980) conducted an excellent study in
Washington. Their results showed that if cases of giardiasis were thoroughly investigated, the actual source
could be more clearly defined as waterborne, day-care center, hiking, etc. The second outbreak of waterborne
giardiasis to occur in a filtered water supply was in the spring of 1977 at Berlin, New Hampshire (Lippy, 1978).
A source was not determined, but beaver were implicated as the probable cause. In both outbreaks involving
filtered water, problems with filtration and chlorination had occurred. In the fall of 1979, another outbreak of
waterbome giardiasis occurred in Bradford, Pennsylvania, an unfiltered source of water (Lippy, 1981). Between
1980-83 the frequency of outbreaks of waterbome disease increased to the highest level since 1942 (Craun,
1986). The pathogen most frequently identified was Giardia. the parasite accounting for 41 of the 126 outbreaks
in the 35 states reporting an outbreak of waterbome disease. Most of these outbreaks were small, involving
only a few individuals. For example Colorado reported about half of the outbreaks of giardiasis recorded, but
this is primarily the result of an efficient epidemiologic surveillance program. Less than 50 individuals were
involved in 19 of the 21 outbreaks reported in Colorado; likely many other states have a similar problem but do
not as yet have a surveillance program sufficient to detect these small outbreaks. Several large outbreaks
occurred in communities in Pennsylvania between 1982 and 1986. An outbreak involving over a thousand
individuals occurred in Utah. Massachusetts had an outbreak in 1985 that involved over 500 individuals, and
Nevada had an outbreak involving over 300 individuals in 1982. New Mexico had two outbreaks, each
involving over 100 individuals, but only one was reported; the other occurred on a private ranch using
ineffective filtration and inadequate disinfection (Hibler, unpublished). Most of the outbreaks between 1980-83
were in communities using unfiltered, disinfected water, but some were in communities with filtration systems
that were by-passed or malfunctioning and five occurred in communities with filtration systems that had
inadequate treatment (Craun, 1986).
The statistics on waterbome giardiasis are interesting to those of us who analyze samples of surface
water for the cysts of Giardia because the data on outbreaks when combined with the results of analyses
emphasize the potential risk for additional epidemics. During the past 12 years we have examined over 6500
samples of surface water from 325 communities in 28 of the 48 contiguous United States and elsewhere in
North America. Giardia cysts have been found in 346/1218 (28%) of the creek samples, 212/828 (26%) of the
river samples, 193/1983 (10%) of the lake samples, and 16/84 (19%) of the open spring samples examined.
Cysts have been found from alpine to subtropical environments, in all months of the year, and in pristine and
urban areas (Hibler, 1987a). The analytical procedures that laboratories currently have available for diagnosis are
no more than 50% effective, primarily because any amount of turbidity interferes with recovery of cysts (Hibler,
1987b). Repeated sampling of a negative source usually has provided positive results; therefore, it is safe to
assume that contamination is far greater than current results indicate. Most, if not all, of the surface water
sources in the United States are either contaminated with the cysts of Giardia or they are at immediate risk for
contamination.
The number of outbreaks over the past 20-25 years and the extent of contamination found in surface
water should not come as a surprise. Giardia is one of the most common parasites reported from humans
throughout the world. In our experience Giardia cysts frequently are too numerous to count in raw wastewater
and their presence in treated wastewater is not unusual. Infection has been found in a number of wild and
domestic animals, including dogs, cats, coyotes, wolves, beaver, muskrat, mice, voles, cattle, domestic sheep,
horses, elk, moose, mule deer and some wild waterfowl (black-crowned night herons), many living on, near, or
with access to surface water sources supplying communities (Hibler, unpublished). The source often is the
habitat for many of these animals and they have the need, as well as the right, to share this water. Once Giardia
becomes established in animals with aquatic habits, especially voles, muskrat and beaver, the source should be
considered contaminated and appropriate measures applied to prevent risk to the consumer. Animal control
measures can and should be applied to prevent high levels of contamination near the community water supply
intakes because beaver can produce as many as 1 x 10** cysts/animal/day and muskrat can produce near 3x10^
cysts/animal/day (Monzingo and Wegrzyn, personal communication). Animal control in lieu of adequate
treatment is hazardous and should be applied in addition to treatment. As indicated earlier, animals have the
right to share this water and extensive animal control measures are not recommended; moreover, this would be a
futile exercise.
Unfortunately, examination of wastewater treatment plant effluent for Giardia seldom is considered
despite information incriminating the source as wastewater contamination in some epidemics. Many of the
large rivers we have sampled over the past 12 years are contaminated by human source Giardia from wastewater
plants.
Host specificity of Giardia from humans and other animals is a controversial subject and probably will
continue to be controversial for some time. Filice (1952) made a detailed study of Giardia from many different
animals, concluding that the parasite from humans and most of the other animals listed previously were all the
same species and should be called Giardia duodenalis. Many investigators now refer to the parasite as Q.
duodenal is. indicating they accept the broad host range concept. However, some investigators continue to use
species designations for those parasites found in humans (G_. lamblia or Q. intestinalist and other animals (e.g.
Q. ondatrae. G_. simondi. Q. bovis. etc.). While there is an increasing amount of direct and indirect evidence to
support the broad host range concept, this does not necessarily mean that Giardia from a given animal will
readily infect another animal every time cross-transmission is attempted. However, cross-transmission studies
(Davies and Hibler, 1979; Erlandsen, et al, 1987), results of endonuclease restriction analysis of DNA (Nash and
Keistcr, 1985), results of monoclonal antibody studies with Giardia from different human and animal sources
(Stibbs, et al, 1987), waterborne outbreaks for which there is no known source other than animals (Craun,
1986) and the numerous cases of giardiasis in campers, hikers, hunters and fisherman documented the past 25-30
years are more than adequate to justify the conclusion that the G_. duodenalis cysts found in water, irrespective of
their host origin, are potentially capable of initiating an outbreak of waterbome giardiasis.
Records from the analysis of water samples done in this laboratory over the last 12 years and statistics
provided by Craun (1986) have resulted in considerable information about the water supplied to consumers in
communities across the United States. Surface water often is consumed raw; however, if the source supplies a
community (at least 15 service connections; Craun, 1986), generally there is some form of treatment. High
quality water originating in a pristine environment and/or from a protected watershed frequently is unfiltered, but
is disinfected to protect consumers from any risk of a waterborne disease. We find cysts of Giardia as frequently
in water originating in pristine and/or protected sources as in unprotected sources; protection from animal
contamination generally is not possible. If the source traverses public and/or private property, the multiple-use
concept applied by the U.S. Forest Service (Forestry, Agriculture and Recreation), as well as vested interests
(grazing of animals) effectively prevent protection of that source.
The potential for outbreaks of waterborne giardiasis in unfiltered surface water sources in the United
States is a primary concern to the United States Environmental Protection Agency (USEPA). Because of the
history of epidemics, the USEPA is establishing criteria to determine when surface water sources providing
water to communities must filter this water. Some communities oppose the construction of filtration plants
specifically to prevent the possibility of waterborne giardiasis, arguing that cost would be prohibitive to use
Giardia as the sole reason for construction. Most of those opposed to mandatory filtration provide a high quality
water originating in a pristine environment and/or a protected area. Undoubtedly variances will be granted for
communities able to demonstrate that the only barrier needed is disinfection of the water. However, many high
quality surface water sources often are subject to temperatures less than 5 C in winter months; before variances
can be granted information for the inactivation of Giardia cysts at these temperatures must be generated.
Systems currently used by communities in the United States vary from simple infiltration galleries to
sophisticated conventional treatment facilities. Slow sand filtration, direct filtration, and diatomaceous earth
filtration are all effective systems when properly designed and operated efficiently (Logsdon and Lippy, 1982).
Most infiltration galleries and filtration systems that do not use chemical coagulation are not effective. Our
experience with these latter systems has shown that only 40 to 60% of the cysts can be removed.
Unfortunately, even well-designed filtration systems are not always operated efficiently. Giardia cysts also have
been found in the effluent from conventional treatment systems, direct filtration systems, mechanical (point-ofuse) filters, and commercially manufactured units (Hibler, 1987a).
As indicated earlier, Giardia cysts have been found in raw and/or finished (filtered as well as unfiltered,
disinfected) water at all times of the year. Analysis of this data suggests a seasonal distribution with more cysts
from fall to spring; indeed, 40-45% of the raw water samples often are contaminated with cysts during these
months (Hibler, 1987a). However, during these months water supplies often are low, turbidities have stabilized
to a minimum, and cyst longevity has been increased by the lower water temperatures. Analytical procedures arc
more effective when the turbidity is low. These factors may function to increase recovery of cysts during the
winter and give a false indication of seasonal distribution.
While data indicating that Giardia cysts have a seasonal distribution may be complicated by the above
factors, analysis of water samples has shown that more filtration plants are passing cysts in the winter months.
Generally this is due to the mode of operation. Municipalities often switch from conventional treatment to
direct filtration and, often, filtration without coagulants during these months, primarily because the source of
water meets and frequently exceeds existing state and federal regulations and no need is perceived to perform any
better. Coagulation and flocculation of cold water often is complicated by turbidities less than 0.6 NTU;
chemical reactions are slower and there are fewer particles to form an effective floe. Needless to say, Giardia
cysts usually are present in this cold, low turbidity water and without coagulation seldom are more than 60%
removed (Hibler, 1987a). The risk for waterborne giardiasis increases considerably as water temperatures
decrease. Not only is longevity of the cyst increased by cold water, most disinfectants in general use need a
longer contact time in cold water to inactivate the cyst; chemical reaction rates decrease by two or three fold for
every 10 degree decrease in temperature (Weber, 1972; White, 1972).
Recently, Hoff (1986) undertook the monumental task of assembling and interpreting the published
information on the use of the CT values (disinfectant concentration (C) in milligrams/liter or ppm multiplied by
contact time (T) in minutes) required to inactivate different types of pathogens (viruses, bacteria and protozoans)
to certain levels under specified conditions as established by laboratory experimentation. The CT concept is
based on an empirical equation developed by Watson (1908) to examine the effects of changing disinfectant
concentration on rates of microbial inactivation. Watson's law is K = CnT. The terms are the same in the K =
CT except for n, the coefficient of dilution, an important addition which determines the order of the chemical
reaction. If n=l the CT value remains constant regardless of the disinfection concentration used. Therefore if
n=1, concentration of disinfectant and exposure time are of equal importance. If n>l, disinfectant concentration
is the dominant factor and if n<l exposure time is more important than disinfection concentration. As Hoff
(1986) stated, n is very important in determining the degree to which extrapolation of data from disinfection
experiments is valid. For example if n=l, CT values can be used to predict efficiency over a broad range of
disinfection concentration and exposure time.
Baumann and Ludwig (1962) used Watson's Law to illustrate its use for making disinfection
recommendations for small non-public water supplies relying only on disinfection for inactivation of pathogens
(Hoff, 1986) but the CT concept received little further attention until the Safe Drinking Water Committee
(1980) used CT as the method of comparing biocidal efficiency. Thereafter the CT concept for interpretation of
disinfection data, both for assessing comparative efficiencies of different disinfectants and expressing comparative
resistance of the different pathogens, became more widely used and now is the accepted approach (Hoff, 1986).
The USEPA has been directed by Congress to prepare new criteria for treatment of surface water sources
(Draft Criteria of Surface Water Treatment Rule; November 11,1986). As a minimum, the treatment must
include disinfection and provide a 1000 fold removal and/or inactivation (99.9%) ofGiardia lamblia cysts (=G_.
duodenal is) and 10,000 fold removal and/or inactivation (99.99%) of enteric viruses. Unfortunately the limited
data currently available for inactivation ofGiardia cysts is based on 99% inactivation and some of the
information was generated using Giardia muris. a parasite of mice that is not infectious for humans. Data
accumulated by Hoff (1986) indicates that the CT values necessary for inactivation of pathogens frequently is
specific for that pathogen. Since the proposed regulations for inactivation of Giardia cysts specify Q. lamblia
(=G_. duodenalis of human origin), data generated for G_. muris may not be applicable. Meyer and Schaefer
(1984) pointed out that interpretation of results using G_. muris must be done with caution.
As Hoff (1986) has indicated, the validity of extrapolating from CT values required for 99%
inactivation to CT values required for other levels of inactivation (in this case, the 99.9% specified for Giardia in
the draft criteria) is dependent upon the nature of the inactivation curves from which the 99% inactivation CT
value was determined. These two factors together have placed the USEPA into the unenviable position of
proposing new regulations based on an inadequate and incomplete database for inactivation of Giardia cysts.
Chick (1908) characterized the inactivation of microorganisms as a first order chemical reaction using
the disinfectant and the microorganisms as the two chemical reactants. Essentially Chick's Law proposes that
the number of organisms remaining (alive and infectious) after a period of time is a proportional constant
However, microorganisms do not behave as chemical reactants (Hoff, 1986). There may be an initial lag period
before inactivation begins for some organisms (e.g., those protected by a cyst wall), and a "tailing off of
inactivation for those organisms that have a more resistant segment of the population. Whether these are real
problems or the result of experimental conditions currently is unknown; the possibility they are real problems
must be accepted until proven otherwise.
The failure of microorganisms to conform to Chick's Law has important implications: it would be
hazardous to attempt extrapolation of the data generated for 99% inactivation of pathogens to 99.9% or 99.99%
inactivation. The requirement for 1000 fold (99.9%) inactivation of Giardia cysts specified in the Draft Criteria
of Surface Water Treatment Rule (11/11/86) is a prime example. Most of the data generated for Giardia has been
for 99% inactivation using artificial excystation as the technique to determine inactivation by disinfectants.
Jarroll, et al (1980) developed and improved the excystation procedure sufficiently to evaluate the effect of
different disinfectants on Giardia cysts and then evaluated the effects of chlorine on these protozoans (Jarroll, et
al, 1981). Similar research, comparing the effect of chlorine on Giardia cysts obtained from symptomatic and
asymptomatic individuals, was performed by Rice, et al (1982). Although excystation techniques have
improved over the years, use of G_. duodenalis (=G_. lamblia) cysts directly from a human source is fraught with
problems: clinical patients seldom are available when needed, the history of the patient seldom is known, and
cysts from different human sources seldom respond the same to the excystation procedure. More often than not
less than 90% of the cysts will excyst, necessitating a correction factor and creating the problem of interpreting
results with confidence. Nevertheless, artificial excystation is a perfectly acceptable technique and when used by
these experienced investigators generated some excellent data. Unfortunately, it is not currently possible to
obtain accurate CT values beyond 99% inactivation with the excystation technique because it is logistically
difficult to accurately count the number of excysted organisms necessary to have confidence in the results for
99.9% inactivation.
The only current alternative to develop CT values for 99.9% inactivation is to use a sensitive biologic
model, a laboratory animal highly susceptible to infection with G_. duodenalis of human origin. The most
sensitive biologic model that has been evaluated for susceptibility to Giardia cysts of human origin is the
Mongolian gerbil (Meriones unguiculatusX This model was first used by Belosevic, et al (1983) after Davies
and Hibler (1979) showed that another species of gerbil (Gerbillus gerbillus) was a reasonably good model for
human-source Giardia. After Belosevic, et al (1983) showed that the gerbil was a potentially excellent biologic
model, we began using this animal in our laboratory for production of cysts from human sources as well as
from other animals (cattle, horses, cats, etc.). The colony is specific-pathogen-free (for Giardia) and the animals
are sensitive to about 60% of the human sources of Giardia cvsts to which they have been exposed (Swabby et
al, 1987). Experimentation has shown that as few as 5 cysts will infect 100% of the animals if the human
source is well-adapted to the gerbil. Probably they are susceptible to a single cyst, but determining if a single
cyst is alive or dead is difficult and we have succeeded in infecting only 10 to 20% of the animals when a single
cyst was used.
Use of a biologic model should not be considered a panacea for the problems associated with
development of a 99.9% CT value for inactivation of Giardia cvsts. While use of an animal model is not
subject to the limitations of the excystation technique, the sensitivity of the animal to five or fewer cysts
probably will result in a CT value closer to 99.99% inactivation, a 10,000 fold reduction.
The experimental database for inactivation of Giardia cysts with the disinfectants in common use is
extremely limited and most of this data is in the range of 5 C to 25 C. DeWalle and Jansson (1983) are the
only investigators to provide any free chlorine data for temperatures lower than 5 C. They used the G_. muris
model and applied the excystation technique to generate information on the CT values necessary to inactivate
cysts with chlorine in an unbuffered raw water source. Most of their trials were at 1 C and 5 C, varying the
concentration of chlorine and evaluating inactivation through a pH range of 5 to 8.7. Unfortunately, since they
did not use chlorine demand-free water, reporting chlorine as the initial levels, interpretation of their results for
comparative purposes is difficult because there is no indication of the chlorine demand or the time span of this
demand. Use of final concentrations would have been a better approach.
As previously indicated, results from analyses of water samples indicate cysts may be more numerous
in surface water from fall to spring. Despite the factors preventing confidence in these results, there is
considerable need for information on the CT values necessary to inactivate cysts of Giardia in cold water
conditions: surface water temperatures for many communities are less than 5 C much of the year. Regulations
for water treatment cannot be enacted until information on the amount of chlorine and time necessary to
inactivate cysts in water at temperatures less than 5 C has been established.
From the time that we began analyzing water samples for Giardia we have been asked "will chlorine
kill Giardia?" Initially we had to respond with "Yes, but we do not know how much is needed, or how much
time is necessary." After Jarroll, et al (1981) and Rice, et al (1982) developed CT values for inactivation of
cysts from 5 C to 25 C we could respond to these questions with more confidence. Following outbreaks in
several large communities the last 3 to 4 years, requests for analyses of surface water increased considerably,
accompanied by a tremendous number of questions regarding the effect of chlorine on these cysts. Municipal
authorities, public health authorities, engineers and water treatment plant operators needed to know how much
chlorine and contact time was necessary to inactivate the cysts. Unfortunately, rarely were we asked these
questions until cysts had been found in the distribution system of a community (often during the peak of the
winter tourist season) when the temperature of the water was near freezing.
These questions and problems prompted us to initiate research on the amount of chlorine and time
necessary to inactivate the cysts of Giardia between 0.5 C and 5.0 C, over a pH range of 6 to 8. We opted to
use the animal model as an indicator for inactivation because the model is susceptible to 5 or fewer viable G_.
duodenalis cysts if a well-adapted human source is used. This would provide considerable confidence that
inactivation could be detected between 99.9 and 99.99%, at least equal to and probably greater than the level
specified by the proposed regulations. Since these regulations specify G_. Iambiia (G_. duodenalis of human
origin) we did not feel that development of CT values with G_. muris. a species infectious for a limited number
of rodents, would be considered acceptable.
MATERIALS AND METHODS
GENERAL
Mongolian gerbils, Meriones unguiculatus. were used as indicators to determine the amount of
chlorine and time necessary to inactivate cysts of Giardia duodenalis of human origin at temperatures of 0.5 C,
2.5 C and 5.0 C, pH 6,7 and 8. Five different human isolates of Giardia cysts were perpetuated in gerbils and
used in these experiments. The pH was maintained using chlorine demand-free buffer. Temperatures were
stabilized in water baths using ice and low temperature incubators. The source of chlorine was reagent grade
sodium hypochlorite. Free chlorine was measured either with a Gilford Stasar II spectrophotometer or a Wallace
and Tieman amperometric titrator.
THE ANIMAL MODEL
Research by Belosevic, et al (1983) and three years experience with the Mongolian gerbil in our
laboratory has shown that the animal is extremely susceptible to Giardia infection from human or other animal
sources. The original breeding pairs used to provide offspring for use in this study were obtained from
Tumblebrook Farms, West Brookfield, Massachusetts. All were treated with metronidazole at 6 mg/adult gerbil
for five days and then examined for the protozoan commensals, Trichomonas sp. and Endamoeba sp. If the
gerbils were determined free of these commensals by stool examination for Endamoeba and by a culture
technique for Trichomonas. as well as postmortem examination of randomly-selected individuals, they were used
as breeding pairs. This original specific-pathogen-free colony was expanded to provide breeding pairs to produce
the offspring necessary for experiments. Rigid standards of cleanliness were maintained within the breeding
colony; only animal caretakers were permitted in the colony facilities. Offspring from the colony were used at 5
to 7 weeks of age.
SOURCE OF GIARDIA CYSTS
Ten human source Giardia infections were established in gerbils during the course of this study, but
only five sources (designated HI through H5) adapted sufficiently to produce at least 5 x 10 ^ cysts/gerbil/hour.
At least 85% of the cysts obtained from gerbils infected with these sources were alive and considered to be
morphologically excellent during peak cyst production on days 6 to 8 post-infection. Before use in the
experiments each source was further challenged to determine if 5 cysts/gerbil would consistently initiate an
infection. Each source was used to challenge ten 5 to 7 week old gerbils with a calculated dose of 5 cysts/gerbil.
The day that cyst production began, the day of maximum cyst production, maximum cyst production/gerbil, and
the morphologic quality of the cysts produced was determined for each isolate.
COLLECTION, CLEANING AND COUNTING
GIARDTA CYSTS
Cysts produced by positive control animals from each trial were used for the next trial. For one to two
days before the trial, fecal output over a 5 to 8 hour period was obtained from gerbils temporarily housed in
collection cages with screen mesh flooring of two openings/cm. The cage contained 100 to 150 ml of distilled
water below the screen. All pellets collected in the water were macerated through 60 and 100 mesh screens and
the material suspended in distilled water. On the day of a trial, centrifuge tubes containing 25 mis of this "semiclean" suspension were underlaid with twenty ml of 1.09 specific gravity ZnSO4 (underlay technique) and
centrifuged at 380 g for 5 to 8 minutes. The water above the interface between these two liquids, the interface,
and about 5 ml below the interface was gently vacuumed through a 5 micrometer nucleopore membrane.
Giardia cysts on the membrane were washed into a beaker with distilled water. The volume was adjusted to 50
ml and a 50 microliter sample removed. This 50 microliters was added to 1 ml of distilled water and a 50
microliter aliquot extracted from this dilute suspension for counting. Four replicate counts of cysts were
performed and if the results of these counts were close and consistent, the average was taken for extrapolation to
total number of cysts in 50 ml of distilled water. Cysts were then concentrated for introduction into the
chlorinated buffer solutions. After concentration, a 50 microliter aliquot was taken to evaluate the morphologic
quality of the cysts: to determine the percentage alive versus those that were dead or dying. Live, infectious
cysts possess a cytoplasm that is essentially clear (hyaline) when viewed by phase contrast and/or bright-field
microscopy whereas those that are dead and/or dying have a coagulated appearance to the cytoplasm and
intracellular organelles are easily detected. This procedure correlates extremely well with the fluorogenic dyes,
fluorescein diacetate and propidium iodide, used to determine cyst viability (Schupp and Erlandsen, 1987a;
Schupp, et al., 1987b).
PREPARATION OF BUFFER
Freshly-prepared solutions of 0.01 M Certified A.C.S. dibasic sodium phosphate and 0.01M Reagent
Analyzed monobasic sodium phosphate were combined at ratios necessary to prepare solutions at pH 6,7 or 8.
The pH of buffer was established to within less than 0.05 with an ALTEX Selection 5000 Ion Analyzer that
was calibrated daily. Buffer was sterilized and random samples analyzed for pH after sterilization.
EFFECT OF BUFFER AND SODIUM THIOSULFATE ON GIARDIA CYSTS
Three groups of five 5 to 7 week old gerbils were inoculated with cysts cleaned by the ZnSO4 underlay
technique that had been maintained in pH 6,7 and 8 buffer for 24 hours. Five gerbils were inoculated with
cysts from the same cleaned source that had been maintained in 1% sodium thiosulfate for 24 hours. Each gerbil
in each group was given 5 x 10 3 cysts. The onset of cyst production, maximum cyst production/gerbil,
duration of cyst production and quality of cysts was compared with a group of five controls each given 5x10^
cysts from the original material cleaned by the ZnSO4 underlay technique and maintained in distilled water.
CHLORINE STOCK SOLUTION
Reagent grade sodium hypochlorite (5% solution) was selected as the source of free chlorine and the
liquid was dispensed with an Eppendorf digital pipette.
DETERMINATION OF FREE CHLORINE
Initially, determination of free chlorine was performed with a Gilford Stasar H spectrophotometer using
the DPD colorimetric method as outlined in Standard Methods for the Examination of Water and Wastewater.
16th Edition. However, results with the spectrophotometer became erratic within three months; thereafter all
determinations were performed with a Wallace and Tieman amperometric titrator. All free chlorine
measurements were made by two individuals and the variations were always less than 0.1 mg/1. Accuracy of
chlorine measurements was periodically checked with U.S.E.P.A. Water Pollution Quality Control Samples and
Performance Evaluation Samples. All results were well within the 95% confidence limits given.
Available chlorine was determined before and after trials as a quality control, and readings were
performed by two technicians. If a loss greater than 0.1 mg/1 of free chlorine occurred in the course of a trial,
the data generated for that pH and temperature was eliminated and the trial repeated at a later date.
8
INOCULATION OF BUFFER WITH CHLORINE
Initially, 500 ml amber-colored narrow-mouthed bottles were used in the trials. Five replicate bottles
of buffer were established for each chlorine level desired at that temperature and pH, and chlorine dispensed into
each bottle; however, inoculation of small amounts of chlorine into small bottles was unsatisfactory and the
inability to compare different holding times from the same bottle because of small volume also was
unsatisfactory. After the first three months of effort, 2 liter narrow mouth bottles were used. Bottles were filled
with chlorinated buffer and the chlorine was adjusted two days before a trial.
MAINTENANCE OF TEMPERATURES
Temperatures of 5.0 C and 2.5 C were maintained with ice and warm water in water baths inside low
temperature incubators while 0.5 C was maintained with a shaved ice-water bath. Temperature was examined
every 15 minutes with an ASTM thermometer.
EQUIPMENT
Accuracy and reliability of the equipment used to measure pH, the spectrophotometer, and the Wallace
and Tierman amperometic titrator used in these trials was evaluated by comparison with known standards and
with comparable equipment in use elsewhere on the campus of Colorado State University, in use by the city of
Fort Collins, or in use by the Larimer County Vocational-Technical Center.
STATISTICAL ANALYSIS
The chlorine concentrations and time values selected and used for each temperature and pH were
analyzed using regression analysis with time as the fixed variable to generate predicted values for chlorine
concentration. All of the time and chlorine values were converted to logarithms and the best fit linear regression
line calculated through these values. The data is presented on an arithmetic format with mean predicted CT
illustrated, and on a logarithmic format with the mean predicted CT and the 95% confidence intervals illustrated.
The coefficient of correlation, slope, probability, and standard error are presented in tabular format.
CT values generated using groups of animals in the 1 to 4 animals/group infected category and for 0
animals/group infected category were analyzed separately to obtain predicted CT values for each category. The
data is presented as the range of predicted CT values and the predicted mean CT. The data generated for the 1 to
4 animals/group infected category provides values between 99.9% and 99.99% inactivation. The data generated
for the 0 animals/group infected category is greater than 99.99% inactivation.
Data also was analyzed comparing all chlorine concentration and time values generated in the trials
versus use of data with chlorine concentrations of 0.3 mg/1 to 2.5 mg/1 to determine if chlorine concentration
and time were of equal importance or if there was a "lag period" before cysts were inactivated.
EXPERIMENTAL PROCEDURE
GENERAL
The pH, temperature and chlorine levels to be evaluated were established several days prior to a trial and
bottles containing chlorinated buffer brought to the desired temperature. On the day of a trial, cysts were cleaned
and introduced into bottles at a calculated concentration sufficient to provide 1.02 x 1(P cysts/ml of chlorinated
buffer. Bottles containing unchlorinated buffer were inoculated with cysts at that temperature and pH to serve
as a source for the positive controls. All bottles were agitated every 15 minutes to insure continual suspension
of cysts. At a predetermined time, sufficient chlorinated buffer was removed from the bottle to inoculate five
gerbils with 5.1 x 10^ cysts/gerbil and the chlorine inactivated with 0.1% sodium thiosulfate; at the same time,
five gerbils were inoculated with sufficient material from the buffer only bottle to provide 50 cysts/gerbil as
positive controls. An equal number of negative controls remained in the animal holding facility. Starting five
days after the trial, gerbils were placed in collection cages, pellets collected, and examined for Giardia cysts.
Initially they were examined as a group and then individually. If gerbils did not pass cysts by day 7 they were
euthanized and examined postmortem for infection.
The purpose in using 5.1 x 10^ cysts/gerbil instead of 5 x 10^ cysts/gerbil was to compensate for
losses in the syringe, gavage tube and on the sides of centrifuge tube from which the inoculum was withdrawn.
PROCEDURES FOR TRIALS
As indicated in the materials and methods section, initially 500 ml amber-colored small mouth bottles
of chlorinated buffer were used in trials. Bottles were inoculated with a cyst concentration sufficient to provide
1.02 x 10^ Giardia cysts/ml. At a predetermined time for that chlorine level, 250 ml were poured into a beaker
containing sufficient sodium thiosulfate to make a final solution of 0.1%. This suspension was then equally
distributed into five 50 ml tubes and centrifuged at 380 g for 5 minutes. The supernatant in each tube was
siphoned to the 2 ml level and 48 mis of distilled water added to replace most of the buffer. The distilled water
suspension of cysts was again centrifuged and the supernatant removed, leaving 0.5 ml of cyst concentrate in
each tube. This concentrate was drawn into a tuberculin syringe and introduced into each gerbil's stomach with a
gavage needle. The purpose in removing buffer and replacing the solution with distilled water was to prohibit
any possible compromise of the pH in a gerbil's stomach by buffer. Although pre-trial infections never
substantiated any interference by buffer, the procedure was considered necessary.
After each group of five challenge animals had been exposed, 50 ml of cyst suspension from the
unchlorinated bottle of buffer was poured into a 50 ml centrifuge tube, the material centrifuged for 5 minutes at
380 g, siphoned to 2 ml, and the cyst concentrate resuspended with 48 mis of distilled water. Each positive
control animal received 1 ml of this suspension, a dose previously calculated to provide 50 cysts/gerbil. No
effort was made to account for loss of cysts or dead cysts because gerbils were highly susceptible to fewer than
50 cysts. Negative controls animals for each trial were examined on day 6 after a trial.
The 2 liter bottles used after the first quarter of trials were of sufficient volume to provide enough cysts
for 4 groups of 5 gerbils/group. The material was prepared for inoculation as previously indicated. Four CT
values, two prior to the CT predicted for inactivation and two after the CT predicted for inactivation, were
selected and the 250 ml aliquots of cyst suspension necessary to expose 5 gerbils at each time interval were
removed for use at that predetermined time. As in the previous trials, 5 positive control gerbils were inoculated
after every 5 challenge animals.
The experimental protocol was designed to stagger trials for any given pH and temperature over a period
of several months and to use at least two separate human sources. This was done in an effort to compensate for
the variations to be expected in the source of cysts, quality of the cysts, sources of gerbils, cleaning techniques,
age of the cysts, etc.
10
METHOD OF DETERMINING AND EVALUATING INFECTION
Gerbils given 50 or more cysts generally begin low cyst production at 4.5 days. On day 5 postinoculation positive controls and chlorine-exposed groups were placed in collection cages and the pellets
examined for cysts. As indicated previously, they were examined initially as a group and then individually.
Presence or absence of cysts was detected by direct microscopic examination of the fecal suspension. Negative
animals or negative groups, and those passing only rare to occasional cysts/animal were re-examined on day 6
and euthanized then or on day 7. At post-mortem the proximal 25 mm of the small intestine of each animal
was scraped and examined microscopically for trophozoites ofGiardia.
11
RESULTS AND DISCUSSION
SOURCES OF GIARDIA CYSTS
Ten human-source Giardia isolates obtained from local hospitals were established in gerbils during the
course of these trials, but five isolates were considered unsatisfactory for use. Of the five unsatisfactory
isolates, two did not adapt well to the animals and cyst production was less than 1 x 10 4 cysts/gerbil/hour on
days 6 to 8; two isolates produced approximately 5 x 10 ^ cysts/gerbil/hour on days 6 to 8, but less than 80%
were considered to be alive based upon microscopic evaluation; and one isolate could not be maintained in
gerbils after the second passage.
The five satisfactory isolates were designated as sources HI through H5 and used for the research. Cyst
production and morphologic quality of the cysts produced by gerbils is given in Table 1. Two to three sources
were used to generate data for all temperatures and pH.
Use of morphologic quality as a means of determining the percentage of cysts alive versus those that
are dead requires considerable experience with cysts of Giardia and is not recommended for use by investigators
who do not work regularly with the cyst stage of the parasite. A few investigators currently are developing
fluorogenic dye exclusion techniques to better quantify the percentage of live cysts present in a population being
prepared for use in experiments where the percentage alive versus those that are dead will affect the results of the
experiment. Schupp and Erlandsen (1987a) and Schupp, et al (1987b) have shown that fluorescein diacetate is
incorporated only into live cysts and propidium iodide is incorporated into dead cysts. This is a rapid and
inexpensive test which should be used by investigators not accustomed to looking at cysts regularly, at least
until they are aware that a dead cyst has a coagulated appearance to the cytoplasm whereas the live cyst has an
essentially clear cytoplasm. The two procedures correlate very well. Unfortunately, with either procedure there
is a period of 1 to 2 hours that dying cysts cannot be accurately categorized as dead or alive (Schupp, et al.,
1987b; Hibler, 1987b).
INFECTIVITY OF THE GIARDIA SOURCES
For sources HI through H5, cleaned cysts from the specified source were counted, diluted to a calculated
dose of 5 cysts/ml and inoculated into ten 5 to 7 week old gerbils. Results of the minimum dosage experiments
are given in Table 2.
Efforts to determine if gerbils were uniformly susceptible to as few as 5 cysts/gerbil were complicated
by our inability to select aliquots containing exactly five cysts from a highly diluted population of cysts;
consequently, as is indicated in Table 2, the dose range based on counts of the dilute material indicated that some
animals received as few as 2 cysts while others received as many as 12 cysts. Hoff (1985) performed a similar
experiment using G_. muris and mice, concluding that about 5 cysts was the 50% infective dose. There is no
way to determine if all of the cysts are alive, the exact number of live cysts introduced, or the number that
become lost in the milieu of ingesta and unable to attach to the mucosa.
EVALUATION OF THE GTARDIA SOURCES
Throughout the course of these trials each source was continually monitored and compared. Onset of
cyst production by the positive controls, day of maximum cyst production, and the morphologic quality of cysts
produced by gerbils infected with each source was an excellent indicator of the results to be expected from
animals inoculated with chlorine-exposed cysts. Sources HI, H3 and H5 were well-adapted to gerbils; the
number and morphologic quality of cysts produced in the positive control groups was always consistent. Our
results with H3, as well as the results of other investigators (van Roodselaar and Wallis, 1987), indicate that it
is extremely resistant to inactivation with disinfectants. However, results from the positive control groups
12
infected with sources H2 and H4 often were erratic if only 80 to 85% of the cysts used in the trial were
determined to be alive. If less than 85% were alive, positive control animals did not begin cyst production as
soon as positive control animals infected with sources HI, H3 or H5, and maximum cyst production occurred at
least a day later than these other sources. These observations suggested that data generated using sources H2 and
H4 should not be used to calculate CT values for inactivation of the cysts. However, statistical analysis of the
data comparing results obtained with HI, H3 or H5 versus results obtained with H2 and H4 performed by Hoff
(personal communication) and by us did not reveal any significant difference in predicted CT values between
sources.
We must assume that the cysts in a surface water source are all alive and potentially infectious for
susceptible individuals consuming that water. CT values should be established to encompass this probability;
moreover we must also consider that there may be sources of cysts in the environment that are more resistant
than the sources used to establish the CT values in these trials. Human-muskrat-gerbil and beaver-gerbil sources
occasionally were used, but this data is not included because the experiments were designed to use human
sources. The human-muskrat and beaver sources adapted well to gerbils, and greater than 95% of the cysts were
alive, but the results of a few trials indicated they were less resistant to chlorine than the sources used to
generate data.
EFFECT OF BUFFER AND SODIUM THIOSULFATE ON GIARDIA CYSTS
Cleaned cysts maintained in buffer at pH 6,7 and 8, and in 1% sodium thiosulfate for 24 hours did not
lose their infectivity for gerbils as compared to controls. Initial cyst production, day of maximum cyst
production, and duration of cyst production was not measurably different
CHLORINE DETERMINATIONS
Cleaned cysts of Giardia did not demand a measurable amount of the available chlorine. Generally the
available chlorine measured at the end of a trial was within + 0.01 mg/1 of the original value; seldom did losses
exceed 0.05 mg/1. If loss of free-chlorine was greater than 0.1 mg/1, the trial was repeated.
CT VALUES FOR INACTIVATION OF GIARDIA CYSTS WITH CHLORINE
The data used to calculate CT values for inactivation of Giardia cysts with chlorine at temperatures
0.5 C, 2.5 C and 5.0 C, pH 6, 7 and 8 was generated in 48 experimental trials using 744 groups of 5
animals/group (3720 gerbils). Each animal in the chlorine exposure groups was inoculated with a calculated
dose of 5 x 10^ Giardia cysts that had been exposed to a specified concentration of chlorine over a period of
time. An equal number of positive control animals was inoculated with a calculated dose of 50 Giardia cysts
that had not been exposed to chlorine. All positive control animals used in these trials became infected. If all
positive control animals did not become infected, the trial was repeated. Five negative control animals were
used in each trial. These animals were not exposed to Giardia cysts and none became infected. All 5 human
source isolates were used in these trials, and two to three sources were used to generate CT values at each pH and
temperature. Data generated with all 5 of these sources was used to calculate the predicted CT values at each
temperature and pH. Results are presented in Table 3 and Figures 1 through 18. Figures 1 through 9 are in
arithmetic format and Figures 10 through 18 in logarithmic format The 95% confidence intervals are presented
only in the logarithmic format Each data point on the figures represents 5 gerbils, each inoculated with 5 x
10^ cysts, or a total of 25 x 10^ chlorine-exposed cysts to generate the results for that point. In the initial trial
for all temperatures at any pH, time values for specified chlorine concentrations were selected that would
encompass the CT and provide a basis for further trials; these "working points" are not illustrated in the figures.
13
Table 3 includes the coefficient of correlation (R), slope, probability, and standard error. CT values are
expressed as the predicted range, and predicted mean CT for animal groups in which 1 to 4 animals were infected
and for animal groups in which none were infected. Data generated for CT values where 1 to 4 animals/group
were infected represents the "break-point", a chlorine concentration and time value where 99.9% to 99.99% of
the cysts were inactivated by chlorine (Table 3). Data generated for CT values where none of the animals/group
were infected includes the range and means for greater than 99.99% inactivation of the cysts.
Chlorine concentrations used in these trials ranged from 0.3 to greater than 4.0 mg/1 for all
temperatures examined at pH 6,7 and 8. However, the only chlorine concentrations used to calculate predicted
CT values were those in the range of 0.3 to 2.5 mg/1. Throughout the course of these trials we observed that
use of chlorine concentrations above 2.5 mg/1 often produced erratic and unpredictable results, suggesting a "lag
period" before complete inactivation was achieved at temperatures in the 0.5 C to 5.0 C range (Figures 1
through 18). This prompted a comparison using regression analysis of: (1) Data generated for all chlorine
concentrations; (2) Data generated with chlorine concentrations of 0.3 to 2.5 mg/1; and (3) Data generated for
chlorine concentrations of 2.5 mg/1 or greater. For most of the temperatures at any pH, use of data generated
with chlorine concentrations of 0.3 to 2.5 mg/1 resulted in a higher coefficient of correlation and a slope very
close to 1, indicating that chlorine concentration and time were of equal importance for concentrations up to 2.5
mg/1 (Table 3). When all of the chlorine concentration values were used for analysis, the coefficient of
correlation was lower, and the slope varied from 0.5 to 0.9, indicating that time was somewhat more important
than chlorine concentrations. When the data using chlorine concentrations of 2.5 mg/1 or greater was analyzed,
the slope varied from 0.15 to 0.64 for all temperatures at pH 6 and pH 8, definitely indicating that time was
more important than chlorine concentration. These same results were obtained with all temperatures at pH 7,
but changes in the slope were not as significant as the changes at pH 6 and pH 8.
Since these trials were not specifically designed to evaluate the possibility of a "lag period" before
inactivation it would be inappropriate of us to make this interpretation; the database for chlorine concentrations
greater than 2.5 mg/1 is too limited for a valid statistical comparison. Moreover, the predicted CT values were
not appreciably different when data using all chlorine concentrations was compared with chlorine concentrations
of 0.3 mg/1 to 2.5 mg/1. However, the purpose of a cyst wall on Giardia or any other protozoan is to protect
the organism from adverse environmental conditions, at least for a short period of time, and a "lag period" before
complete inactivation is achieved should not be unexpected for the chlorine concentrations used in these trials.
Although the predicted CT values were calculated using only data generated for chlorine concentrations
of 0.3 mg/1 to 2.5 mg/1, the mean predicted CT curves illustrated in Figures 1 through 18 were extended above
2.5 mg/1 of chlorine concentration to illustrate the erratic nature of the data. Until more information becomes
available, we must caution public health agencies, engineers and municipal water treatment operators that
increasing chlorine concentrations to reduce contact time may not be a prudent solution. Use of predicted CT
values obtained in these trials should be restricted to final chlorine concentrations of 0.3 mg/1 to 2.5 mg/1.
As was expected, higher CT values are necessary for the inactivation of Giardia cysts at temperatures
between 0.5 C and 5.0 C than at temperatures above 5.0 C. If municipalities do not have a filtration system
capable of removing 99.9% of the cysts, and the source is either contaminated with Giardia cysts or the source is
at risk for contamination with Giardia cysts, these municipalities must either increase the concentration of
chlorine or increase contact time (e.g. prechlorination or storage) to arrive at a CT necessary for inactivation of
the cysts when temperatures are less than 5.0 C.
We are aware that most sources of surface water seldom will be at the exact temperature and pH used to
generate the experimental data. To facilitate use of the data from 0.5 C to 5.0 C, pH 6 to 8, the predicted CT
values have been interpolated and presented in tabular format (Table 4). Individuals using these interpolated
values should be cognizant that these are mathematical interpolations and not experimental values.
The species of chlorine primarily responsible for biocidal activity is HOC1 (Weber, 1972; White,
1972). At pH 6 about 96% of the chlorine is in this form. This is reduced to about 75% at pH 7. Above pH
7.5 the chemical equilibrium shifts very quickly to the OCL" form. At pH 8 about 23% of the chlorine is in
the HOC1 form and at pH 9 less than 4% is in this form. The reduced rate of chemical reaction at lower
14
temperatures, when combined with a considerable loss of biocidal efficiency at a pH above 7.5, resulted in very
high CT values predicted for inactivation of cysts at pH 8. Indeed, we experienced considerable difficulty to even
establish predicted CT values for temperatures at pH 8. Our confidence in these results are best exemplified by
the range of CT values predicted for inactivation and the broad 95% confidence intervals (Table 3, Figures 1618). The decrease in biocidal efficiency of chlorine above pH 8 is so great that we cannot recommend
extrapolation of the current CT values for water sources with a higher pH.
Investigators performing research in laboratory conditions can effectively control physical and chemical
parameters; they cannot control variation in living organisms. The differences observed betweeen the human
sources maintained in the gerbils during the course of this study was not unexpected; we have observed similar
differences between other human and/or animal sources (Swabby et al, 1987). The most obvious interpretation
of the differences is that the human sources did not adapt equally well to the animals, resulting in a shift of the
predicted CT values. However, the differences could be inherent in the source; likely a number of strains or
variants are present in human and/or animal populations. Use of 5 human sources in this study resulted in
predicted CT values with broad ranges of inactivation at some temperatures, especially at pH 8. However, use
of several sources is extremely important; use of one source without a comparison to other sources could result
in predicted CT values lower than needed to insure inactivation of cysts. Rice et al (1982) showed a definite
difference in the inactivation of cysts between symptomatic and asymptomatic human sources. This difference
may not have been the result of symptomatic versus asymptomatic, but a difference between sources. The
purpose in using 5 human sources and performing numerous replicate trials in this study was to encompass the
probability that differences in susceptibility similar to what Rice, et al (1982) observed likewise occurs among
cyst populations contaminating sources of surface water, hopefully the ranges predicted for inactivation of these
cysts will include the most infectious sources present in the environment.
Development of inactivation data for cysts of Giardia is an extremely difficult and time-consuming
procedure, irrespective of whether investigators use the artificial excystation technique or an animal model.
Reliable procedures for cultivation of Giardia to the cyst stage have not been developed, necessitating use of a
human or animal source to produce viable cysts in the numbers necessary for experimental purposes. Extraction
of these cysts from fecal specimens requires considerable mechanical and chemical manipulation to obtain a
suspension of cysts sufficiently "pure" that they will not consume a measurable amount of the disinfectant and
thereby compromise the results. Any amount of manipulation probably is somewhat detrimental, resulting in
reduced viability of the final suspension of cysts. Cysts must be properly collected, carefully cleaned and used
immediately to limit this detrimental effect of cleaning.
Both techniques have limitations peculiar to the technique. Investigators using an animal model must
expect variations as a result of age differences between animals within a group and between groups.
Investigators using the excystation technique must expect differences in the excystation media and the response
of different sources of cysts to that specific media; age and/or quality of the excystation media will result in
erratic excystation results, even with the same source of cysts. Investigators using an animal model must be
aware that all sources do not adapt equally well to the model. Predicted CT values for inactivation of the cysts
will be higher or lower, depending on the source. Investigators using excystation are faced with a similar
problem, leading to the same end result some human or animal sources provide a high percentage of
excystation, at least 90%, while others will provide only 30-40% excystation, necessitating use of a conversion
factor (correction factor) to account for the cysts from that source (control cysts) that will not excyst in the
excystation media. This problem may be inherent in the source and not in the technique because our experience
both with the animal model and excystation indicates that human sources that adapt well to the animal will
provide excellent excystation results while sources that do not adapt well to the animal provide poor excystation
results. Investigators using either procedure must evaluate several sources of cysts and replicate the observations
on each source a sufficient number of times to have confidence in the results, otherwise the CT values predicted
for inactivation will be inadequate to protect the consumer.
15
RECOMMENDATIONS
Many municipalities using surface water as a source have developed facilities designed to accommodate
the physical, chemical and biological properties unique to that source and provide effective treatment of the
water. However, if the source is contaminated with the cysts of Giardia. the treatment may not be adequate to
prevent the risk of waterborne giardiasis, even though water quality meets or even exceeds existing regulations.
If filtration is not adequate to remove 99.9% of the Giardia cysts or treatment is considered inadequate to prevent
a risk of waterborne giardiasis, municipalities using chlorine as the biocidal barrier will need to introduce
sufficient chlorine and/or increase the time of contact with chlorine to inactivate at least 99.9% of the Giardia
cysts present in that water. The method by which this is to be accomplished is contingent upon the properties
unique to that source and the physical layout of the facility.
Public health agencies, engineers and municipal water treatment operators can use Figures 1 through 9
to arrive at a CT value necessary for inactivation of Giardia cysts if temperatures and pH are close to these
values. Likely the temperature and pH of the water will be between the exact levels studied, necessitating
interpolation. To facilitate this application, the CT values have been calculated and presented in matrix format
(Table 4). If the temperature or pH of the water is between the interpolated values given, use the value for the
lower temperature and higher pH.
Individuals responsible for establishing CT values should not attempt to increase chlorine
concentrations above 2.5 mg/1 to arrive at the CT necessary for inactivation. The Giardia cyst wall may be
capable of resisting external stimulus for a short period of time, especially at lower temperatures; increasing
chlorine to decrease time of contact would be risky. Municipalities with short contact times and/or with a pH
above 7.5 must consider other options, such as storage, to obtain a CT necessary to insure inactivation.
As is shown by the mean predicted CT values and the range of CT values for temperatures of 0.5 C to
5.0 C, the biocidal activity of chlorine is much less effective at pH 8 (Table 3, Figures 7-9 and 16-18).
Municipalities with sources above pH 8 must realize that extrapolation of the existing data would be risky. We
cannot recommend a CT value that would be effective.
16
Table 1. Human Sources of Giardia Cvsts: Cyst Production and Morphologic Quality in Mongolian Gerbils.
Human
Source
Day Cysts
Produced
Days Maximum
Production
Maximum Cyst
Production*
Morphologic Quality
of Cysts
HI
5
5-5.5 x 104
6-8
>95% alive
H2
5
7-7.5 x 104
6-8
85-90% alive
H3
5
0.9-1.0 x 105
6-8
>98% alive
H4
5
5-6 x 104
6-8
80-90% alive
H5
5
0.8-1 x 105
6-8
>98% alive
* Cysts/gerbil/hour over an 8 hour period.
Table 2. Minimum Dose of Human Source Giardia Cysts that would Consistently Infect Mongolian Gerbils
and the Morphologic Quality of the Cysts Produced.
Human
Source
Number
Animals
Number
Infected
Average
# Cysts/Gerbil
Dose
Range/Gerbil
Morphologic
Quality of Cysts
HI
10
10
5.4
3-8
>98% alive
H2
10
8
4.2
3-9
80-85% alive
H3
10
10
6.7
3-9
>98% alive
H4
10
8
7.4
2-12
80% alive
H5
10
10
6.8
4-12
>98% alive
17
oo
0.997
0.995
0.991
0.998
0.993
0.992
0.888
0.962
0.950
0.912
0.854
0.930
1-4
None
1-4
None
1-4
None
1-4
None
1-4
None
1-4
None
14
15
14
15
10
10
15
17
17
13
2.5
5.0
0.5
2.5
5.0
7
7
8
8
8
0.946
0.937
1-4
None
12
10
5.0
6
15
20
0.998
0.859
1-4
None
7
16
2.5
6
0.5
0.979
0.948
1-4
None
14
20
0.5
6
7
R
Animals
Infected
N
Temp
(Q
pH
0.993
1.299
1.121
1.037
1.159
1.137
0.984
0.998
0.977
0.980
1.020
0.975
1.004
0.876
0.997
0.881
1.068
0.917
Slope
<.001
<.001
<.001
<.001
<.003
<.001
<.001
<.001
<.001
<.001
<.001
<.001
<.001
<.001
<.001
<.001
<.001
<.001
Probability
0.133
0.090
0.083
0.108
0.140
0.083
0.027
0.034
0.027
0.017
0.028
0.025
0.089
0.106
0.018
0.124
0.051
0.075
Std.
Error
273-281
243-367
250-295
336-353
312-389
392^75
159-163
165-166
246-256
260-268
285-295
302-315
146-147
139-171
141-142
156-190
176-197
202-233
Predicted
CT Range
280
290
268
343
342
425
161
166
252
265
289
310
146
157
142
175
185
220
Predicted
MeanCT
Table 3. N (number of data points), R (correlation coefficient), Slope, Probability, Standard Error (all in logs), Predicted CT Ranges, and Predicted
Mean CT (all in antilog) for results in which 1 to 4 animals/group and no animals/group were infected at 0.5 C, 2.5 C, 5.0 C, pH 6.0,7.0 and 8.0.
Table 4. Interpolation of CT Values for Temperatures 0.5 C to 5.0 C, pH 6.0 to 8.O.*
PH
Degrees
Centigrade
6.0
6.5
7.0
7.5
8.0
0.5
185
237
289
316
342
1.0
174
227
280
302
324
1.5
164
217
271
288
305
2.0
153
207
261
274
287
2.5
142
197
252
260
268
3.0
143 '
188
234
252
270
3.5
144
180
216
244
273
4.0
144
171
197
237
275
4.5
145
163
179
229
278
5.0
146
154
161
221
280
* If the temperature or pH of the water is between the interpolated values given, use the value for the lower
temperature and the higher pH.
Individuals responsible for establishing CT values should not attempt to increase chlorine
concentrations above 2.5 mg/1 to arrive at the CT necessary for inactivation. The Giardia cyst wall may be
capable of resisting external stimulus for a short period of time, especially at lower temperatures; increasing
chlorine to decrease time of contact would be risky. Municipalities with short contact times and/or with a pH
above 7.5 must consider other options, such as storage, to obtain a CT necessary to insure inactivation.
19
p
in
>
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CHLORINE Cmg/l)
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01
IU
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01
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0
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TEMPERATURE 5.O°CI pH
FIGURE 6
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- 5/5 Animals infected
TEMPERATURE 2.5°C, pH 8
to
00
LL
W
[£
UJ
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4.5-
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TIME [hours)
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12 13
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• = O/5 Animals infected
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TEMPERATURE 5.O°C, pH J3
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TEMPERATURE O.5°C, pH 7
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O.3-
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•EMPERATURE 2.5°C, pH 7
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FIGURE 12
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TIME CminutesJ
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TEMPERATURE O.5°CJ pH 6
FIGURE 13
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TIME Cminutes)
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FIGURE 15
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TIME Cminutes)
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60 8O1OO
= O/5 Animals infected
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= 5/5 Animals infected
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FIGURE 16
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CT\
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FIGURE 17
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1P-7C-90526-8/87-TC
ISBN 0-89867-4G