Giardia Guidelines for Canadian Drinking Water Quality: Supporting Documentation

Giardia Guidelines for Canadian Drinking Water Quality: Supporting Documentation
Health
Canada
Santé
Canada
Guidelines for Canadian Drinking Water Quality:
Supporting Documentation
Protozoa: Giardia and Cryptosporidium
Prepared by the
Federal-Provincial-Territorial Committee on Drinking Water
of the
Federal-Provincial-Territorial Committee on Health and the Environment
Health Canada
Ottawa, Ontario
April 2004
This document is an updated version of the supporting document on Protozoa that was published
in July 1996. It may be cited as follows:
Health Canada (2004) Guidelines for Canadian Drinking Water Quality: Supporting
Documentation — Protozoa: Giardia and Cryptosporidium. Water Quality and Health Bureau,
Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, Ontario.
The document was prepared by the Federal-Provincial-Territorial Committee on Drinking Water
of the Federal-Provincial-Territorial Committee on Health and the Environment.
Any questions or comments on this document may be directed to:
Water Quality and Health Bureau
Healthy Environments and Consumer Safety Branch
Health Canada
Sir Charles Tupper Building, 4th Floor
2720 Riverside Drive (Address Locator 6604B)
Ottawa, Ontario
Canada K1A 0K9
Tel.: 613-948-2566
Fax: 613-952-2574
E-mail: [email protected]
Other supporting documents in the Canadian Guidelines for Drinking Water Quality can be
found on the Water Quality and Health Bureau web page at
http://www.hc-sc.gc.ca/hecs-sesc/water/dwgsup.htm.
Table of Contents
1.
Guideline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
2.
Executive Summary for the Microbiological Quality of Drinking Water . . . . . . . . . . . . . 1
2.1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
2.2
Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
2.3
Giardia and Cryptosporidium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.4
Health Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.5
Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.6
Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3.
Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3.1
Giardia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3.2
Cryptosporidium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
4.
Health Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4.1
Giardia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4.2
Cryptosporidium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
5.
Emerging Pathogenic Waterborne Protozoans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
6.
Sources and Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
6.1
Giardia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
6.2
Cryptosporidium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
7.
Analytical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
7.1
Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
7.2
Viability and Infectivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
8.
Treatm ent Te chnol ogy
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
8.1
Municipal-scale Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
8.2
Residential-scale Treatment Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
9.
Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
9.1
Giardia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
9.2
Cryptosporidium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
9.3
Balancing Risks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
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Protozoa: Giardia and Cryptosporidium (April 2004)
10.
Rationale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
11.
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Annex A: CT Tables for the Inactivation of Giardia lamblia Cysts by Chlorine, Chlorine
Dioxide, Chloramine, and Ozone at Various Temperatures . . . . . . . . . . . . . . . . . . . . . . 51
Annex B: UV Dose (IT) Table for the Inactivation of Giardia and Cryptosporidium . . . . . . . . 73
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Protozoa: Giardia and Cryptosporidium
1.
Guideline
Although Giardia and Cryptosporidium can be responsible for severe and, in some cases,
fatal gastrointestinal illness, it is not possible to establish maximum acceptable concentrations
(MACs) for these protozoa in drinking water at this time. Routine methods available for the
detection of cysts and oocysts suffer from low recovery rates and do not provide any information
on their viability or human infectivity. Nevertheless, until better monitoring data and information
on the viability and infectivity of cysts and oocysts present in drinking water are available,
measures should be implemented to reduce the risk of illness as much as possible. If the presence
of viable, human-infectious cysts or oocysts is known or suspected in source waters, or if Giardia
or Cryptosporidium has been responsible for past waterborne outbreaks in a community, a
treatment and distribution regime and a watershed or wellhead protection plan (where feasible)
or other measures known to reduce the risk of illness should be implemented. Treatment
technologies in place should achieve at least a 3-log reduction in and/or inactivation of cysts
and oocysts, unless source water quality requires a greater log reduction and/or inactivation.
2.
Executive Summary for the Microbiological Quality of Drinking Water
2.1
Introduction
The information contained in this Executive Summary applies to the microbiological
quality of drinking water as a whole. It contains background information on microorganisms,
their health effects, sources of exposure, and treatment. Information specific to protozoa is
included as a separate paragraph. It is recommended that this document be read in conjunction
with other documents on the microbiological quality of drinking water, including the supporting
document on Turbidity.
2.2
Background
There are three main types of microorganisms that can be found in drinking water:
bacteria, viruses, and protozoa. These can exist naturally or can occur as a result of
contamination from human or animal waste. Surface water sources, such as lakes, rivers, and
reservoirs, are more likely to contain microorganisms than groundwater sources, unless the
groundwater sources are under the influence of surface water.
The main goal of drinking water treatment is to remove or kill these organisms to reduce
the risk of illness. Although it is impossible to completely eliminate the risk of waterborne
disease, adopting a multi-barrier, source-to-tap approach to safe drinking water will reduce the
numbers of microorganisms in drinking water. This approach includes the protection of source
water (where possible), the use of appropriate and effective treatment methods, well-maintained
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distribution systems, and routine verification of drinking water safety. All drinking water
supplies should be disinfected, unless specifically exempted. In addition, surface water sources
and groundwater sources under the influence of surface water should be filtered.
The performance of the drinking water filtration system is usually assessed by monitoring
the levels of turbidity, a measure of the relative clarity of water. Turbidity is caused by matter,
such as clay, silt, fine organic and inorganic matter, plankton, and other microscopic organisms,
that is suspended within the water. Suspended matter can protect pathogenic microorganisms
from chemical and ultraviolet light disinfection.
Currently available detection methods do not allow for the routine analysis of all
microorganisms that could be present in inadequately treated drinking water. Instead,
microbiological quality is determined by testing drinking water for Escherichia coli, a bacterium
that is always present in the intestines of humans and animals and that would indicate faecal
contamination of the water. The maximum acceptable concentration of E. coli in drinking water
is none detectable per 100 mL.
2.3
Giardia and Cryptosporidium
Protozoa such as Giardia and Cryptosporidium are relatively large pathogenic
microorganisms that multiply only in the gastrointestinal tract of humans and other animals. They
cannot multiply in the environment, but they can survive longer in water than intestinal bacteria
and are more infectious and resistant to disinfection than most other microorganisms. Routine
methods detect only a fraction of the total number present and do not provide any information on
the viability of these organisms or their ability to infect humans. As a result, it is not currently
possible to establish maximum acceptable concentrations for Giardia and Cryptosporidium in
drinking water. Instead, the use of a multi-barrier approach to safeguard drinking water supplies
and reduce exposures to Giardia and Cryptosporidium in drinking water is recommended.
Routine water quality monitoring for E. coli is also important, as the presence of E. coli is an
indication that Giardia and Cryptosporidium could also be present. However, because Giardia
and Cryptosporidium are more resistant to disinfection, the absence of E. coli does not
necessarily mean that they are also absent.
2.4
Health Effects
The health effects of exposure to disease-causing bacteria, viruses, and protozoa in
drinking water are varied. The most common manifestation of waterborne illness is
gastrointestinal upset (nausea, vomiting, and diarrhoea), and this is usually of short duration.
However, in susceptible individuals such as infants, the elderly, and immunocompromised
individuals, the effects may be more severe, chronic (e.g., kidney damage), or even fatal. Bacteria
(such as Shigella and Campylobacter), viruses (such as Norovirus and Hepatitis A), and protozoa
(such as Giardia and Cryptosporidium) can be responsible for severe gastrointestinal illness.
Other pathogens may infect the lungs, skin, eyes, central nervous system, or liver.
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Protozoa: Giardia and Cryptosporidium (April 2004)
If the safety of drinking water is in question to the extent that it may be a threat to public
health, authorities in charge of the affected water supply should have a protocol in place for
issuing, and cancelling, advice to the public about boiling their water. Surveillance for possible
waterborne diseases should also be carried out. If a disease outbreak is linked to a water supply,
the authorities should have a plan to quickly and effectively contain the illness.
2.5
Exposure
Drinking water contaminated with human or animal faecal wastes is just one route of
exposure to disease-causing microorganisms. Outbreaks caused by contaminated drinking water
have occurred, but they are relatively rare compared with outbreaks caused by contaminated
food. Other significant routes of exposure include contaminated recreational waters (e.g., bathing
beaches and swimming pools) and objects (e.g., doorknobs) or direct contact with infected
humans or domestic animals (pets or livestock). Although surface waters and groundwater under
the influence of surface waters may contain quantities of microorganisms capable of causing
illness, effective drinking water treatment can produce water that is virtually free of diseasecausing microorganisms.
2.6
Treatment
The multi-barrier approach is an effective way to reduce the risk of illness from
pathogens in drinking water. If possible, water supply protection programmes should be the first
line of defence. Microbiological water quality guidelines based on indicator organisms (e.g., E.
coli) and treatment technologies are also part of this approach. Treatment to remove or inactivate
pathogens is the best way to reduce the number of microorganisms in drinking water and should
include effective filtration and disinfection and an adequate disinfection residual. Filtration
systems should be designed and operated to reduce turbidity levels as low as possible without
major fluctuations.
It is important to note that all chemical disinfectants (e.g., chlorine, ozone) used in
drinking water can be expected to form disinfection by-products that may affect human health.
Current scientific data show that the benefits of disinfecting drinking water (reduced rates of
infectious illness) are much greater than any health risks from disinfection by-products. While
every effort should be made to reduce concentrations of disinfection by-products to as low a level
as possible, any method of control used must not compromise the effectiveness of water
disinfection.
3.
Description
3.1
Giardia
Giardia is a small, flagellated protozoan (small single-cell organism lacking cell walls)
parasite (Phylum Protozoa, Subphylum Sarcomastigophora, Superclass Mastigophora, Class
Zoomastigophora, Order Diplomonadida, Family Hexamitidae) that inhabits the small intestines
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Protozoa: Giardia and Cryptosporidium (April 2004)
of humans and other animals. The trophozoite, or feeding stage, lives mainly in the duodenum
but is often found in the jejunum and ileum of the small intestine. Trophozoites (9–21 µm long,
5–15 µm wide, and 2–4 µm thick) have a pear-shaped body with a broadly rounded anterior end,
two nuclei, two slender median rods, eight flagella in four pairs, a pair of darkly staining median
bodies, and a large ventral sucking disc (cytostome). Trophozoites are normally attached to the
surface of the intestinal villi, where they are believed to feed primarily upon mucosal secretions.
After detachment, the binucleate trophozoites form cysts (encyst) and divide within the cyst, so
that four nuclei become visible. Cysts are ovoid, 8–14 µm long by 7–10 µm wide, with two or
four nuclei and remnants of organelles visible. Environmentally stable cysts are passed out in the
faeces, often in large numbers. A complete life cycle description and diagram can be found in a
review paper by Meyer and Jarroll (1980).
Giardia lamblia cysts can survive up to 77 days in tap water at 8°C (Bingham et al.,
1979), but survival decreases with increasing temperature (54 days at 21°C and 4 days at 37°C).
Giardia muris cysts remain viable for up to 2.8 months in river water when the temperature is
<10°C and for approximately 1 month at 15–20°C in lake water (deRegnier et al., 1989). Cysts
have no external features and are recognized by shape and visible internal morphology, as
described above. Upon ingestion of the cysts by a suitable host, excystation is triggered by acid
and enzymes in the stomach; by the time the parasite reaches the duodenum, a quadranucleate
mass of protoplasm emerges, which rapidly divides into two trophozoites from each cyst (Meyer,
1994). Colonization of the small intestine then occurs by asexual reproduction.
The taxonomy of the genus Giardia is based on the species definition proposed by Filice
(1952), who defined three species: G. duodenalis (syn. G. intestinalis, G. lamblia), G. muris, and
G. agilis, based on the shape of the median body, an organelle composed of microtubules that is
most easily observed in the trophozoite. Three species, G. ardea, G. psittaci, and G. microti,
have subsequently been described on the basis of cyst morphology and small-subunit rRNA
sequence analysis (Adam, 2001). These six species have been reported from mammals, birds,
rodents, and amphibians and are not easily distinguished. Their host preferences have been
widely debated — except for agilis, which is morphologically different, has been reported only
from amphibians, and is not regarded as infective to humans. The name Giardia lamblia is
commonly applied to isolates from humans.
Giardiasis is believed to be a zoonotic disease, although most of the evidence is
circumstantial or compromised by inadequate controls. It is known that beaver, dogs, and
muskrat can become infected with human-source G. duodenalis (Davies and Hibler, 1979;
Hewlett et al., 1982; Erlandsen and Bemrick, 1988; Erlandsen et al., 1988), but the pathogenicity
to humans for Giardia reported from birds, cattle, bears, cats, and other hosts is uncertain.
Giardia duodenalis can adapt to a variety of hosts, and, being asexual, its population
genetics are best described in terms of clonal expansion (Tibayrenc, 1993) of virulent individuals
within heterogeneous populations. Giardia duodenalis is capable of high rates of chromosomal
rearrangement (Lymbery and Tibayrenc, 1994) and may indeed be able to adapt to new hosts
(and even to in vitro cultivation) more readily than other parasites. Giardia muris from voles and
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Protozoa: Giardia and Cryptosporidium (April 2004)
mice is generally regarded not to be infective to humans, although mice can be used as an animal
model for human Giardia isolates (Byrd et al., 1994). The environmental resistance and
prolonged viability of Giardia cysts in water at low temperature, the endemic nature of Giardia
infections in humans and animals, and cross-species transmission, together with the low
infectious dose needed to establish colonization within a new host, all point towards the potential
for waterborne spread of this disease (Erlandsen, 1994). Recent research has focussed on
distinguishing human-infective Giardia from other strains or species; however, the applicability
of these methods to analysis of Giardia within water has been limited. Thus, at present, it is
necessary to consider that any Giardia cysts found in water are potentially infectious to humans
(Erlandsen, 1994). Molecular methods, such as the polymerase chain reaction (PCR), have been
successfully used to differentiate Giardia species (Mahbubani et al., 1992; Ionas et al., 1997);
however, further research is required to validate these methods.
3.2
Cryptosporidium
Cryptosporidium parvum is a protozoan parasite (Phylum Apicomplexa, Class
Sporozoasida, Subclass Coccodiasina, Order Eucoccidiorida, Suborder Eimeriorina, Family
Cryptosporidiidae) that was first recognized as a potential human pathogen in 1976 in a
previously healthy 3-year-old child. A second case occurred 2 months later in an individual who
was immunosuppressed as a result of drug therapy (Ungar, 1990). Subsequently, the disease
became best known in immunosuppressed individuals exhibiting the symptoms now referred to
as acquired immune deficiency syndrome, or AIDS (Meisel et al., 1976). The recognition of C.
parvum as a human pathogen led to increased research into the life cycle of the parasite and an
investigation of the possible vectors of transmission. Cryptosporidium has a multi-stage life
cycle, typical of enteric coccidia, that takes place in a single host and evolves in six major stages:
excystation, where sporozoites are released from an excysting oocyst; merogony, where asexual
reproduction takes place; gametogeny, the stage at which gametes are formed; fertilization of the
gamete by a microgamete to form a zygote; oocyst wall formation; and sporogony, sporozoite
formation within the oocyst (Current, 1986).A complete life cycle description and diagram can be
found in a review paper by Smith and Rose (1990).
As a waterborne pathogen, the most important stage in the life cycle is the round, thickwalled, environmentally stable oocyst, 4–6 µm in diameter. There is sometimes a visible external
suture line, and the nuclei of sporozoites can be stained with fluorogenic dyes such as 4',6diamidino-2-phenylindole (DAPI). Cryptosporidium oocysts have been shown to survive in cold
waters (4°C) in the laboratory for up to 18 months (AWWA, 1988). Robertson et al. (1992)
reported that C. parvum oocysts could withstand a variety of environmental stresses, including
freezing (viability greatly reduced) and exposure to seawater. Upon ingestion by humans, the
parasite completes its life cycle in the digestive tract. Ingestion initiates excystation of the oocyst
and releases four sporozoites, which adhere to the epithelial surface of the gastrointestinal tract.
Conflicting evidence from electron microscopic studies has led to some disagreement as to
whether the parasite is intracellular (invasive) or extracellular (Anderson, 1982; Anderson et al.,
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Protozoa: Giardia and Cryptosporidium (April 2004)
1982; Tzipori, 1983). All stages possess a feeding organelle that is protected along with the
parasite body itself by an outer membrane. It is not certain whether the outer membrane is
derived from the host cell (intracellular) or of parasitic origin (extracellular). The sporozoite
undergoes asexual reproduction at the site and develops into a gamete. Some of these gametes
release microgametes, which fertilize other macrogametes to form zygotes. A small number of
zygotes retain a thin cell wall, which ruptures after the development of the sporozoites to aid in
maintaining the infection within the host. The majority of the zygotes develop a thick cell wall
and four sporozoites to become oocysts, which are then passed in the faeces.
The first description of Cryptosporidium was made by Tyzzer (1907), when he isolated
the organism, which he named Cryptosporidium muris, from the gastric glands of mice. Tyzzer
(1912) found a second isolate, which he named C. parvum, in the intestine of the same species of
mice. This isolate was considered to be structurally and developmentally distinct by Upton and
Current (1985). Although numerous species names have been proposed based on the identity of
the host, most isolates of Cryptosporidium from mammals, including humans, are similar to C.
parvum as described by Tyzzer (1907, 1912). Ten valid species have been recognized (Fayer et
al., 2000): C. parvum, C. muris, C. andersoni, C. felis, and C. wrairi, which infect mammals; C.
baileyi and C. meleagridis, which infect birds; C. serpentis and C. saurophilum, which infect
reptiles; and C. nasorum, which infects fish (Smith, 1990). Cryptosporidium muris has been
reported from cattle in North America and Europe as well as from camels, deer, and other
animals. Experimental infections of dogs, cats, and rabbits with C. muris have been described by
Iseki et al. (1989). Cryptosporidium parvum is generally regarded as the major species
responsible for clinical disease in humans and domestic animals, and zoonotic transmission is
possible, especially from lambs, calves, and adult cattle. Symptomatic cryptosporidiosis has been
reported from humans, cattle (common), lambs, goats, birds, horses, and monkeys. Humansource Cryptosporidium has been shown to be infective to cattle and lambs (Tzipori, 1983;
Upton and Current, 1985). Other species of Cryptosporidium, such as C. felis, C. canis, C. muris,
and C. meleagridis, have also been implicated in human infections (Katsumata et al., 2000;
Guyot et al., 2001; Pedraza-Diaz et al., 2001a,b; Yagita et al., 2001; Tiangtip and Jongwutiwes,
2002).
As many as 18 distinct genotypes of C. parvum have been proposed, including human,
bovine I, bovine II, rabbit, pig, mouse, deer, deer mouse, ferret, marsupial, opossum I, opossum
II, skunk, bear, fox, muskrat, monkey, and sheep genotypes (Chalmers et al., 2002; Xiao and Lal,
2002). The molecular analysis of C. parvum human and bovine isolates, linked to human
cryptosporidiosis outbreaks, indicates the existence of two predominantly distinct genotypes in
humans (Morgan et al., 1997; Peng et al., 1997; Spano et al., 1998; Sulaiman et al., 1998;
Widmer et al., 1998; Awad-El-Kariem, 1999; Ong et al., 1999; Caccio et al., 2000; McLauchlin
et al., 2000; Xiao et al., 2001).Genotype 1 (currently referred to as C. hominis) isolates have been
reported only in humans, while genotype 2 isolates have been reported in calves and humans
exposed to infected cattle and in materials contaminated with cattle faeces. Genotype 2 isolates
are able to infect mice and calves, whereas C. hominis isolates are not. Recent studies have
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Protozoa: Giardia and Cryptosporidium (April 2004)
identified novel C. parvum genotypes in humans. Pieniazek et al. (1999) identified two novel
Cryptosporidium genotypes in persons infected with human immunodeficiency virus (HIV): a
dog and a cat genotype. Ong et al. (2002) also identified two new C. parvum genotypes in
humans, one cervine (deer) and a not-yet-identified genotype (i.e., not been previously identified
in humans or other animals). These findings have important implications for communities whose
source water may be impacted by faeces from wildlife.
4.
Health Effects
4.1
Giardia
The prepatent period (time between ingestion of cysts and excretion of new cysts) for
giardiasis is 6–16 days (Rendtorff, 1978; Stachan and Kunstýr, 1983; Nash et al., 1987), and the
minimal infective dose can be as low as 1–10 cysts (Rendtorff, 1978; Stachan and Kunstýr,
1983), although there are large differences between isolates of the parasite in terms of their
virulence and antigenic diversity (Nash, 1994). The ID 50 (number of cysts ingested resulting in
50% of the test subjects becoming infected) was found to be 19 cysts by Rendtorff (1978)
(calculated from his data) using human-source cysts in humans, but it can be as high as 543 for
human-source Giardia in gerbils. Giardia strains that are well adapted to their hosts (e.g., by
serial passage) can frequently infect with 50 cysts or less (Hibler et al., 1987). Research with
animal models has shown that smaller inocula result in longer prepatent periods but do not
influence the resulting parasite burden (Belosevic and Faubert, 1983).
Exposure to the parasite resulted in partial or total immunity for periods of up to 21
weeks in mice (Roberts-Thomson et al., 1976; Belosevic and Faubert, 1983). Olson et al. (1994)
observed lower cyst output and greater weight gain in kittens immunized subcutaneously.
Humoral immune response is revealed by increased levels of circulating IgG and IgM antibodies
and secretion of IgA in milk, saliva, and possibly intestinal mucus. These immune products are
active in eliminating disease (Heyworth, 1988), but lasting immunity has not been demonstrated.
Very little is known about cellular immunity, but spontaneous killing of trophozoites by human
peripheral blood monocytes has been described (denHollander et al., 1988). The host–parasite
relationship is complex, and Giardia has been shown to be versatile in the expression of antigens
(Nash, 1994), so universal lasting immunity is improbable. Olson et al. (1994) showed that
potential for a vaccine exists, but infections and symptoms are only attenuated, and prevention of
infection is not feasible at this time. Symptoms include nausea, anorexia, an uneasiness in the
upper intestine, malaise, and perhaps low-grade fever or chills. The onset of diarrhoea is usually
sudden and explosive, with watery and foul-smelling stools (Wolfe, 1984). The acute phase of
the infection commonly resolves spontaneously, and organisms may disappear from the faeces.
Some patients become asymptomatic cyst passers for a period and have no further clinical
manifestations. Other patients, particularly children, suffer recurring bouts of the disease that
may persist for months (Lengerich et al., 1994). Giardiasis can be treated using a number of
drugs, including metronidazole, quinacrine, furazolidone, tinidazole, ornidazole, and nimorazole.
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Lengerich et al. (1994) evaluated hospitalization rates for severe giardiasis in the United
States. An estimated 4600 persons were hospitalized annually, a rate similar to that of shigellosis.
The median length of hospital stay was 4 days.
4.2
Cryptosporidium
Although a complete pathogenesis for C. parvum in humans has yet to be determined,
more information is becoming available through the study of both immunocompetent individuals
and AIDS patients. DuPont et al. (1995) found that 18 of 29 healthy volunteers became infected
after administration of doses of from 30 to 1 000 000 oocysts, 39% of whom were asymptomatic.
The ID50 was 132 oocysts, and 61% of the infected subjects experienced enteric symptoms. A
follow-up experiment conducted 1 year after the primary exposure demonstrated that an initial
exposure is inadequate to protect against future bouts of cryptosporidiosis (Okhuysen et al.,
1998). Although the rates of diarrhoea were similar after each of the exposures, the severity of
diarrhoea was lower after re-exposure. The ID50 of 132 oocysts compares well with an ID50 of 79
oocysts reported for a bovine strain of C. parvum in CD-1 mice (Finch et al., 1993b), although
the minimum infective dose of oocysts required to produce infection in animals ranges in
published research data from 10 to 100 oocysts (Miller et al., 1986; Ernest et al., 1987; Blewett
et al., 1993).
Cryptosporidium parvum genotypes appear to have unique virulence and infectious dose
properties. The TAMU strain of C. parvum (originally isolated from a horse) was shown to have
an ID 50 of 9 oocysts and an illness attack rate of 86%, compared with the UCP strain of C.
parvum (isolated from a cow), which had an ID50 of 1042 oocysts and an illness attack rate of
59% (Okhuysen et al., 1999; Messner et al., 2001).Virulence genes responsible for this
phenomenon are unknown (Okhuysen and Chappell, 2002). DuPont et al. (1995) found that the
prepatent period ranged from 2 to 25 days (although most occurred within 3–11 days), with
the shortest time to cyst excretion occurring with an inoculum of 1 000 000 oocysts. All
individuals recovered spontaneously. Similarly, an investigation of a Cryptosporidium infection
in travellers returning from the Caribbean indicated a prepatent period of 4–9 days (Ma et al.,
1985). The usual symptom associated with the disease is diarrhoea, characterized by very watery,
non-bloody stools. The volume of diarrhoea can be extreme, with 3 L/day being common and
with reports of up to 17 L/day (Navin and Juranek, 1984). This symptom can be accompanied by
nausea, vomiting (particularly in children), low-grade fever (below 39°C), anorexia, and
dehydration. The symptoms reported from a waterborne outbreak are diarrhoea (100%),
abdominal cramps (76%), nausea (45%), vomiting (19%), fever (14%), headache (29%), and
muscle aches (13%) (D’Antonio et al., 1985). Some protective immunity appears to develop in
infected populations. The primary mechanism of host defence appears to be cellular immunity,
although humoral immunity is also known to be involved (Janoff and Reller, 1987).
The duration of infection is dependent on the condition of the immune system (Juranek,
1995) and can be broken down into three categories: (1) immunocompetent individuals who clear
the infection in 7–14 days, (2) AIDS patients or others with severely weakened immune
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Protozoa: Giardia and Cryptosporidium (April 2004)
systems (i.e., individuals with CD4 cell counts <180 cells/mm3) who in most reported cases
never completely clear the infection (it may develop into an infection with long bouts of
remission followed by mild symptoms), and (3) individuals who are immunosuppressed
following chemotherapy, short-term depression or illness (e.g., chicken pox), or malnutrition. In
cases where the immunosuppression is not AIDS related, the infection usually clears (no cyst
excretion, and symptoms disappear) within 10–15 days of the time the immune system returns to
normal, although there have been reported cases involving children in which the infection has
persisted for up to 30 days. The sensitivity of diagnosis of cryptosporidiosis by stool examination
is low — so low that oocyst excreters may be counted as negative prematurely.
Immunocompetent individuals usually carry the infection for a maximum of 30 days. With the
exception of AIDS cases, individuals may continue to pass oocysts for up to 24 days. In an
outbreak in a day care facility, children shed oocysts for up to 5 weeks (Stehr-Green et al., 1987).
The reported rate of asymptomatic infection is believed to be low, but a report on an outbreak at
a day care facility in Philadelphia, Pennsylvania, concluded that up to 11% of the children were
asymptomatic (Alpert et al., 1986), and Ungar (1994) discussed three separate studies in day care
centres where the asymptomatic infection rate ranged from 67 to 100%. It has been suggested
that many of these asymptomatic cases were mild cases that were incorrectly diagnosed (Navin
and Juranek, 1984). In AIDS patients, Juranek (1995) showed that only 13% (5/39) of patients
with CD4 cell counts of <180 cells/mm3 had self-limiting disease, but 100% (8/8) of those with
counts >180 cells/mm3 had infections that cleared.
Infections of Cryptosporidium spp. in the human intestine are known to cause damage to
the mucosa, including villous atrophy and lengthening of the crypt (Tzipori, 1983). Most of the
pathological data available have come from AIDS patients, and the presence of other
opportunistic pathogens has made assessment of damage attributable to Cryptosporidium spp.
difficult. There is some suggestion in the literature that outbreaks of cryptosporidiosis are of
seasonal duration. This has yet to be addressed by investigation, but the seasons reported, by
country, are as follows: February–April (Great Britain), April–July (Bangladesh), May–July
(United States), and May–October (Italy) (D’Antonio et al., 1985; Baxby and Hart, 1986; Nigar
et al., 1987; Caprioli et al., 1989).
No effective antimicrobial treatment for cryptosporidiosis in adults has been approved,
although more than 120 drugs have been tested (Tzipori, 1983; O’Donoghue, 1995). The U.S.
Food and Drug Administration (FDA) recently approved Alinia™ (nitazoxanide) for treatment of
cryptosporidiosis and giardiasis in (1- to 11-year-old) children (U.S. FDA, 2002). Some progress
has been reported with furazolidone in reducing the symptoms of immunocompetent patients.
Spiramycin has apparently been used with some success in Chile and the United States, but at
this time it is not licensed for general use by the FDA (Janoff and Reller, 1987). A functional
immune system will usually eliminate symptoms and organisms spontaneously, but the
immunocompromised individual may suffer from chronic infection.
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During the Milwaukee, Wisconsin, cryptosporidiosis outbreak, investigators surveyed
285 patients with laboratory-confirmed infection. Of these, 130 were hospitalized, including 48
immunocompromised patients (MacKenzie et al., 1994).
5.
Emerging Pathogenic Waterborne Protozoans
Acanthamoeba are free-living amoebae found in a variety of environments, including soil,
air, and water. Acanthamoeba have been detected in a number of aquatic environments, including
chlorinated swimming pools and drinking water (Rivera et al., 1993; Vesaluoma et al., 1995;
Michel et al., 1998; Rohr et al., 1998). Although this organism is common in the environment
and is capable of infecting mammals, including humans, infections in humans are rare. Most
human infections have been associated with the use of “homemade” contact lens (saline)
solutions, which resulted in keratitis (inflammation of the cornea) (Buck et al., 2000; Seal, 2000;
Yeung et al., 2002). Since Acanthamoeba are relatively large, water filtration processes should
be efficient for their removal; however, their cyst form is resistant to typical levels of chlorine
(King et al., 1988). Water treatment processes applied for the removal/inactivation of Giardia
and Cryptosporidium should be effective against this organism. Additional concern over
Acanthamoeba stems from the fact that it may harbour opportunistic pathogens (e.g., Legionella
pneumophila, Mycobacterium avium) (Henke and Seidel, 1986; King et al., 1988; Steinert et al.,
1998; Newsome et al., 2001). Thus, if Acanthamoeba cysts are able to pass through the water
treatment process into potable water, these pathogenic bacterial symbionts could cause human
illness.
Microsporidia are spore-forming intracellular, obligate parasites that are widely
distributed in the environment. Microsporidia are opportunistic pathogens, primarily affecting
persons infected with HIV (Fournier et al., 2000; Svedhem et al., 2002). However,
immunocompetent individuals can become infected as well. Microsporidia capable of causing
human illness have not been detected in surface or potable water. This is likely due to the limited
sensitivity of existing detection methods. Since microsporidia are similar in size to large bacteria,
they should be removed by conventional coagulation/sedimentation and filtration processes;
however, no information is available regarding the efficacy of these processes in removing
microsporidia. Very little is known about the susceptibility of microsporidia to disinfectants.
However, recent research suggests that ultraviolet (UV) light disinfection is highly effective for
inactivating microsporidia (Huffman et al., 2002).
Toxoplasma gondii is an obligate, intracellular parasite that affects almost all warmblooded animals, including humans. Cats shed the oocyst form of this organism in their faeces.
Oocysts are extremely resistant to environmental conditions and appear to retain their infectivity
for several months (at temperatures of -5°C) (Dubey, 1998). Although this organism tends to
cause mild flu-like symptoms, it can be life-threatening for immunocompromised individuals and
pregnant women. Little is known about the distribution of this organism in water sources;
however, oocysts have been reported to survive for up to 17 months in tap water. In 1995, a
toxoplasmosis outbreak was reported in British Columbia, involving 110 acute cases, including
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42 pregnant women and 11 neonates (Bowie et al., 1997). This outbreak was thought to be due to
contamination of a water reservoir by (domestic and wild) cat faeces (Isaac-Renton et al., 1998;
Aramini et al., 1999). Limited information is available on the efficacy of water treatment
processes in removing or inactivating Toxoplasma gondii. However, because of its size, it should
be readily removed by conventional coagulation/sedimentation and filtration processes. In effect,
water treatment processes applied for the removal/inactivation of Giardia and Cryptosporidium
should be effective against this organism.
6.
Sources and Exposure
6.1
Giardia
Giardia is the most commonly reported intestinal parasite in North America and the
world (Farthing, 1989; Adam, 1991). Giardiasis has been shown to be endemic in humans and in
over 40 species of animals, with prevalence rates ranging from 1 to 90+%. The prevalence rate
among humans in Canada is typically 5–10%, but accurate rates are difficult to estimate because
of the large number of asymptomatic cases (Keystone et al., 1978) and the inadequacy of
reporting. Over 5000 confirmed cases of giardiasis were reported in 1999 in Canada. This
represents a significant decline from the 9000 cases that were reported in 1987. Incidence rates
have similarly declined over this period (34.44–17.7 cases per 100 000 persons) (Health Canada,
2003).
Most waterborne outbreaks have been associated with zoonotic transmission, particularly
beaver (Kirner et al., 1978; Lopez et al., 1980; Lippy, 1981; Isaac-Renton et al., 1993). It is now
clear, however, that other mammals, including dogs, muskrat, cattle, and sheep, can also be
responsible for the introduction of cysts to surface water used as sources of drinking water. As
population pressures increase and as more human-related activity occurs in catchment areas, the
potential for faecal contamination becomes greater, and the possibility of contamination with
human sewage must always be considered. Erlandsen and Bemrick (1988) concluded that
Giardia cysts in water may be derived from multiple sources and that epidemiological studies
that focus on beavers may be missing important sources of cyst contamination. Some waterborne
outbreaks have been traced back to human sewage contamination (Wallis et al., 1998). Ongerth
et al. (1995) showed that there is a statistically significant relationship between increased human
use of water for domestic and recreational purposes and the prevalence of Giardia in animals and
surface water. It is known that beaver and muskrat can be infected with human-source Giardia
(Erlandsen et al., 1988), and these animals are frequently exposed to raw or partially treated
sewage in Canada. Thus, it is likely that mammals act as a reservoir of human-infective Giardia
from sewage-contaminated water and in turn amplify concentrations of Giardia cysts in water. If
infected mammals live in close proximity to drinking water treatment plant intakes, then they
could play an important role in the waterborne transmission of Giardia. Watershed management
to control both sewage inputs and the populations of aquatic mammals in the vicinity of water
intakes is just as important to disease prevention as adequate water treatment. It should also be
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remembered that, in addition to water, giardiasis can be transmitted person to person via poor
hygiene, food handling, and sexual practices.
In Canada, Giardia cysts are commonly found in sewage and surface waters and
occasionally in drinking water. In a cross-Canada survey, Wallis et al. (1995) found that 56.2%
of 162 raw sewage samples contained Giardia cysts, ranging in concentration from 1 to 88 000
cysts/L, and 10% of 1215 raw and treated drinking water samples contained 0.001–2 cysts/L. In
samples at three sites on two rivers in the Montreal, Quebec, area, average Giardia cyst
concentrations ranged from 0.07 to 14 cysts/L (Payment and Franco, 1993). The average cyst
level in treated water prepared from the river waters was <0.002 cysts/L. Additional data from
Quebec collected by the Ministry of the Environment and Wildlife showed that 45% of polluted
and 34% of pristine water sources in the province were contaminated with Giardia cysts (total
number of samples was 71), most of which were from rivers (Barthe and Brassard, 1994).
Giardia cysts have been monitored in raw and treated drinking water in Ottawa, Ontario (Chauret
et al., 1995). Cysts were not detected in treated water but were present in 83.3% and 66.6% of
samples collected at the intakes of the two treatment plants. Concentrations ranged from <1 to 25
cysts/100 L (arithmetic means 6.0 and 5.8/100 L). Samples from the wastewater treatment plant
were also examined. The arithmetic mean concentration for Giardia in treated wastewater was 73
cysts/100 L (representing a 99.3% reduction). Between 1990 and 1996, the annual geometric
mean concentration of Giardia cysts in raw water at two treatment plants in Edmonton, Alberta,
ranged from 8 to 193 cysts/100 L (Goatcher and Fok, 2000). No cysts were detected in almost all
of the 1000-L treated water samples collected. On three occasions, one or two cysts were
detected. However, in 1997, heavy spring runoff produced record levels of 2500 cysts/100 L of
raw water. Cyst levels in treated water peaked at 34 cysts/1000 L, prompting municipal health
officials to issue a precautionary boil water advisory to immunocompromised individuals. No
increase in the number of cases of giardiasis in the Edmonton area was detected during this
period. Isaac-Renton and colleagues (Isaac-Renton et al., 1987, 1993, 1994, 1996; Ong et al.,
1996) have reported Giardia cyst contamination from a number of sites in British Columbia,
some of which experienced waterborne outbreaks of giardiasis. In a province-wide survey, cysts
were detected in 68% of raw water samples and 59% of disinfected samples. Mean cyst
concentrations in raw water samples (2.9/100 L) were greater than those from treated water
samples (2.1/100 L). The proportion of viable cysts in the raw and treated water samples was not
determined. Infectivity in Mongolian gerbils was more frequent following inoculation with raw
water concentrates than following inoculation with treated water concentrates (Isaac-Renton et
al., 1996). In another study, levels in two adjacent watersheds in the interior of the province were
determined (Ong et al., 1996). At the drinking water intake in one watershed, the geometric mean
concentration of Giardia during a 21-month period was 173 cysts/100 L (range 4.6–2215
cysts/100 L). At the intake in the other watershed, Giardia was detected in samples collected
over a 17-month period, with a geometric mean concentration of 26 cysts/100 L (range 2–114
cysts/100 L). In the Yukon, 7 of 42 drinking water samples collected over a period of 1 year at
Whitehorse contained Giardia, with concentrations ranging from 0.2 to 1.4 cysts/100 L (Roach
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et al., 1993). None of the 10 samples collected from Dawson’s drinking water contained Giardia.
In eight municipal drinking water supplies in the Atlantic region, 44% of 152 samples collected
between 1991 and 1993 contained Giardia (Wallis et al., 1995). Although most positive samples
contained less than 2.5 cysts/100 L, a few contained more than 150 cysts/100 L. Very few
published data are available from Ontario, but an outbreak of giardiasis at Temagami in the
spring of 1994 was characterized by an attack rate of 30% and cyst concentrations up to 2/L in
treated water (Wallis et al., 1998). LeChevallier et al. (1991a) found Giardia cysts in 81% of 83
raw water samples and in 17% of 83 filtered water samples from the northeastern United States.
Concentrations in raw water ranged from 0.05 to 242 cysts/L. One raw water sample from
Alberta was included in the survey, and a concentration of 4.94 cysts/L was reported.
Concentrations in finished drinking water samples ranged from 0.29 to 64 cysts/100 L.
LeChevallier et al. (1991a) also found significant positive correlations between cyst
concentration and other raw water quality parameters, such as turbidity and total and faecal
coliform densities; however, data from previous studies do not support these associations,
possibly because of early reports of waterborne giardiasis from places like Colorado, which
normally experience very low turbidities in raw water (Karlin and Hopkins, 1983). LeChevallier
et al. (1991a) concluded that cyst contamination could be modelled in terms of watershed
characteristics and that water reuse and sewage contamination were important factors in
predicting cyst concentrations.
The viability of Giardia cysts found in water is commonly assumed to be high, but
monitoring experience suggests otherwise. Cysts found in surface waters are often dead, as
shown by propidium iodide (PI) dye exclusion (approximately 50% viability was observed using
this technique during the Temagami outbreak), and water and sewage isolates infected only 9.4%
of gerbils inoculated in the Canadian survey reported by Wallis et al. (1995). It is possible that
not all of the Giardia isolates tested were actually infective to gerbils, but it is common to
observe cysts that are non-refractile under phase microscopy and that have obviously damaged
cyst walls. The immunofluorescent technique commonly used for detection is very sensitive and
frequently reveals the presence of empty cysts (“ghosts”), particularly in sewage. Observations by
LeChevallier et al. (1991b) also suggest that most of the cysts present in water are non-viable; 40
of 46 cysts isolated from drinking water exhibited “non-viable-type” morphologies (i.e., distorted
or shrunken cytoplasm).
Several outbreaks associated with public drinking water systems have occurred in British
Columbia (Penticton, 100 Mile House, Creston, Kimberley, Kelowna, Kitimat, Fernie, West
Trail/Rossland, Barriere) (Health Canada, 1975–1995; Isaac-Renton et al., 1994), Alberta
(Canmore, Banff, Morley, Exshaw, Edmonton) (Health Canada, 1975–1995), Saskatchewan
(Brightsand, Flaxcombe) (Health Canada, 1975–1995), Manitoba (Dauphin) (Federal–Provincial
Drinking Water Subcommittee, 1998), Ontario(Temagami, Muskoka/Perry Sound) (Wallis et al.,
1998), Quebec(Shenley, St. Ferreol-les-Neiges, St. Perpetue, Les Escoumins) (Health Canada,
1975–1995; Federal–Provincial Drinking Water Subcommittee, 1998), New Brunswick (St.
Quentin, Plaster Rock) (Federal–Provincial Drinking Water Subcommittee, 1998), and
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Newfoundland (Botwood, Corner Brook, Roberts Arm, Springdale, Deer Lake, Bird Cove, St.
Anthony Bight, Harbour Grace) (Health Canada, 1975–1995; Federal–Provincial Drinking Water
Subcommittee, 1998). Outbreaks associated with semi-public drinking water supplies have also
been reported in Ontario (Kingston, Peterborough, Guelph, Camp Tawingo), Quebec
(Outaouais), and Saskatchewan (Swift Current) (Health Canada, 1975–1995). In the United
States, outbreaks have been reported from 24 states (Jakubowski, 1994), especially Colorado and
New England. During the period from 1965 to 1992, 115 outbreaks were reported that resulted in
26 530 known cases of giardiasis in the United States (Moore et al., 1993; Jakubowski, 1994).
Craun (1979), in an earlier study, identified reliance on surface water, minimal treatment (usually
only chlorination), and inadequate treatment facilities as common causes of waterborne
giardiasis. Small water treatment systems that used otherwise good-quality surface water of low
turbidity seemed to be most commonly affected. A useful review of some select U.S. outbreaks
has been compiled by Lin (1985), who concluded that these and other outbreaks had been caused
by lack of filtration, improper filter operations, inadequate chlorination, cross-connections to
sewers, and drinking contaminated surface waters.
6.2
Cryptosporidium
As with giardiasis, cryptosporidiosis may be waterborne, foodborne, or transmitted
sexually or by the faecal–oral route. Reported prevalence rates of human cryptosporidiosis range
from 0.6 to 20% (Caprioli et al., 1989; Zu et al., 1992; Mølbak et al., 1993; Nimri and Batchoun,
1994), based on stool samples. A survey of 1346 Canadian patients revealed a prevalence rate of
1.25% in 1985 (Janoff and Reller, 1987), but data are limited because the disease is not
universally reportable. Infection rates for patients with AIDS are reported to be 4% in the United
States and 2.5% in Canada (Janoff and Reller, 1987; Soave and Johnson, 1988). The parasite in
immunocompetent individuals does not seem to be more prevalent among any particular age
group, although the probability of seroconversion increases with age (Kuhls et al., 1994).
Infected human hosts can excrete up to 1010 oocysts/g faeces (Smith and Rose, 1990).
Although not conclusive, existing literature suggests that livestock may be a significant
source of C. parvum in surface waters. Olson et al. (1997) reported that Cryptosporidium is
common in Canadian farm animals. It was present in faecal samples from cattle (20%), sheep
(24%), hogs (11%), and horses (17%). Oocysts were more prevalent in calves than in adult
animals; conversely, they were more prevalent in mature pigs and horses than in young animals.
Infected calves can excrete up to 107 oocysts/g faeces (Smith and Rose, 1990). Other studies have
also suggested that cattle may be a source of Cryptosporidium in surface waters. For example, a
weekly examination of creek samples upstream and downstream of a cattle ranch in the B.C.
interior during a 10-month period revealed that the downstream location had significantly higher
levels (geometric mean 13.3 oocysts/100 L, range 1.4–300 oocysts/100 L) than the upstream
location (geometric mean 5.6/100 L, range 0.5–34.4 oocysts/100 L) (Ong et al., 1996). A
pronounced spike was observed in downstream samples following calving in late February.
Although the sample size was limited, none of the faecal specimens collected during
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Protozoa: Giardia and Cryptosporidium (April 2004)
the study was positive for oocysts. However, during a confirmed waterborne outbreak of
cryptosporidiosis in British Columbia, oocysts were detected in 70% of the cattle faecal
specimens collected in the watershed close to the reservoir intake (Ong et al., 1997). Waterfowl
can also act as a source of C. parvum. Graczyk et al. (1998) demonstrated that C. parvum oocysts
retain infectivity in mice following passage through ducks. Histological examination of the
respiratory and digestive systems at 7 days post-inoculation revealed that the protozoa were
unable to establish infection in the birds. In an earlier study (Graczyk et al., 1996), the authors
found that faeces from migratory Canada geese collected from seven of nine sites on Chesapeake
Bay contained Cryptosporidium oocysts. Oocysts from three of the sites were infectious to mice
and were identified as C. parvum. Based on these studies, it appears that waterfowl can pick up
infectious C. parvum oocysts from their habitat and can carry and deposit them in the
environment, including drinking water supplies. Although the evidence collected to date is
scarce, it appears that, unlike the case with Giardia, wild ungulates and rodents are not a
significant source of human-infectious Cryptosporidium (Roach et al., 1993; Ong et al., 1996).
Cryptosporidium oocysts have been reported in wastewater (3.3–20 000/L), surface
waters receiving agricultural or wastewater discharges (0.006–2.5/L), pristine surface water
(0.02–0.08/L), drinking water (0.006–4.8/L), and recreational water (0.66–500/L) in various
studies, as summarized by Smith (1990). Similar results were reported by Madore et al. (1987) in
the United States. Rose et al. (1991) found oocysts in 55% of 257 surface water samples at an
average concentration of 43 oocysts/L and in 17% of 36 drinking water samples at concentrations
ranging from 0.5 to 1.7 oocysts/L. In a multi-year Canada-wide survey, Wallis et al. (1995) found
that 11.1% of 162 raw sewage samples contained Cryptosporidium oocysts ranging in
concentration from 1 to 120/L, and 6.4% of 1215 raw and treated drinking water samples
contained 0.001–0.005 oocysts/L. In samples from three sites on two rivers in the Montreal area,
average Cryptosporidium oocyst concentrations ranged from <0.02 to 7/L (Payment and Franco,
1993). Cryptosporidium oocysts have been monitored in raw and treated drinking water in
Ottawa (Chauret et al., 1995). Oocysts were not detected in treated water but were present in
50% and 100% of samples collected at the intake of the two treatment plants. Concentrations
ranged from <1 to 95 oocysts/100 L (arithmetic means 4.0 and 22.3 oocysts/100 L). Samples
from the wastewater treatment plant were also examined. The arithmetic mean concentration of
Cryptosporidium in treated wastewater was 56.0 oocysts/100 L (representing a 96.8% reduction).
Between 1990 and 1996, the annual geometric mean concentration of Cryptosporidium oocysts
in raw water at two treatment plants in Edmonton ranged from 6 to 83 oocysts/100 L (Goatcher
and Fok, 2000). Oocysts were not detected in 1000 L of treated water collected at either plant. In
1997, heavy spring runoff produced record levels of 10 300 oocysts/100 L of raw water and 80
oocysts/1000 L of treated water. As a result, municipal health officials issued a precautionary
boil water advisory to immunocompromised individuals. No increase in the number of cases of
cryptosporidiosis in the Edmonton area was detected during this period. In British Columbia,
Isaac-Renton and colleagues conducted a number of studies on Cryptosporidium in drinking
water supplies. In one study, levels in two adjacent watersheds in the interior of the province
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were determined (Ong et al., 1996). At the drinking water intake in one watershed, the geometric
mean concentration of Cryptosporidium during a 9-month period was 3.5 oocysts/100 L, with a
range of 1.7–44.3 oocysts/100 L. At the intake in the other watershed, oocysts were detected in
samples collected over a 6-month period, with a geometric mean concentration of 9.2
oocysts/100 L (range 4.8–51.4 oocysts/100 L). In another study, Cryptosporidium was monitored
in the drinking water of three communities (groundwater supply, unprotected surface supply, and
protected surface supply) for 1 year (Isaac-Renton et al., 2000). Although Cryptosporidium was
not found in the groundwater, intermittent detectable levels were found in the drinking water
taken from surface supplies. In the drinking water from the unprotected supply, 71% of samples
were positive, compared with 34% of the samples from the protected supply. LeChevallier et al.
(1991a,b) reported that 87% of 83 raw surface water samples and 27% of 83 filtered water
samples taken from the northeastern United States contained Cryptosporidium oocysts.
Concentrations in raw water ranged from 0.07 to 484 oocysts/L. One raw water sample from
Alberta contained 0.34 oocysts/L. As with Giardia cysts, LeChevallier et al. (1991a,b) concluded
that water reuse and sewage contamination were important predictors of Cryptosporidium
concentrations in water.
All Cryptosporidium oocysts found in water are frequently assumed to be viable, a view
supported by the knowledge that the oocyst is highly resistant to environmental stress, but this is
probably incorrect. Viability is probably less than 100%, as shown by Smith et al. (1993), who
found that oocyst viability in surface waters is often very low. This point of view is supported
directly by LeChevallier et al. (1991b), who found that 21 of 23 oocysts in filtered waters had
“non-viable-type” morphology (i.e., absence of sporozoites and distorted or shrunken cytoplasm).
No outbreaks of cryptosporidiosis occurred in any of the municipalities included in the
LeChevallier et al. (1991b) survey. Similarly, Sorvillo et al. (1994) concluded that municipal
drinking water was not an important risk factor for cryptosporidiosis in AIDS patients residing in
Los Angeles County, California, on the basis of epidemiological data collected before and after
the introduction of filtration in a major water supply for the area. However, it should be
emphasized that although low concentrations of viable oocysts are routinely found in raw water,
they may not represent an immediate public health risk; rather, it is the sudden and rapid influx
of parasites into source waters that is likely responsible for the increased risk of infection
associated with transmission through drinking water. Environmental events such as flooding or
high precipitation can facilitate the rapid rise in oocyst concentration within a defined area of a
watershed.
Outbreaks associated with public drinking water systems have been reported in Ontario
(Kitchener–Waterloo and Collingwood) (Welker et al., 1996; Federal–Provincial Drinking Water
Subcommittee, 1998) and British Columbia (Cranbrook, Kelowna, and Chilliwack) (Welker et
al., 1996; Ong et al., 1997). Outbreaks associated with a semi-public drinking water supply and
swimming pool were reported in British Columbia (West Bank) (Health Canada, 1975–1995;
Bell et al., 1993; Meeds, 1993). Nineteen outbreaks have been reported in the United Kingdom;
seven outbreaks have been reported in the United States (Craun et al., 1998). Attack rates were
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Protozoa: Giardia and Cryptosporidium (April 2004)
typically high, ranging from 26 to 40%, and many thousands of people were affected. In addition,
there have been several outbreaks associated with swimming pools, wave pools, and lakes.
7.
Analytical Methods
7.1
Detection
Existing methodologies for routine monitoring of Giardia and Cryptosporidium are only
semi-quantitative and do not provide any information on viability or human infectivity. The U.S.
Environmental Protection Agency’s (EPA) Method 1623, for example, is one of the most widely
used methods for the simultaneous detection of Cryptosporidium and Giardia in water; however,
this method does not determine (oo)cyst viability or infectivity (U.S. EPA, 2001). Most water
samples contain few (oo)cysts, and concentration techniques are required to obtain even a small
number of (oo)cysts. Moreover, neither organism can be reliably cultured from a water sample,
although excystation and culture procedures have been established for both.
The routine analysis of protozoan parasites in water samples relies upon direct
microscopic detection after concentration of particulate matter by filtration or centrifugation.
Sample concentration is generally accomplished by filtration through a 1-µm nominal porosity
wound filter (Jakubowski and Ericksen, 1978; APHA, 1995), 2-µm absolute porosity membrane
filter (Spaulding et al., 1983; Wallis and Buchanan-Mappin, 1984; Ongerth, 1989), or 1-µm
absolute porosity polysulphone filters (Fricker and Clancy, 1998). Recovery efficiencies ranging
from 70 to 80% have been reported for the latter. Three filtration options have been validated for
use with Method 1623, including the Envirochek™ capsule filter, CrypTest™ capsule filter, and
Filta-Max™ foam filter (U.S. EPA, 2001).
If a wound or membrane filter is used for raw or treated drinking water, approximately
1000 L are pumped through the filter, and particulate matter is recovered by backflushing,
rinsing, hand washing, or machine processing (using a stomacher bag) (LeChevallier et al.,
1991a,b) and concentrated into a pellet. The background material in the pellet is then reduced by
discontinuous density gradient centrifugation using zinc sulphate, 1.0 M sucrose, or a mixture of
Percoll (Pharmacia Biotech) and sucrose. Centrifugation will cause denser particles to pass
through the density medium and form a pellet at the bottom of the tube. Theoretically, Giardia
cysts and Cryptosporidium oocysts will float on the surface of the density medium and may be
recovered by pipette. Practically, the use of density media introduces significant errors. Dead
Cryptosporidium oocysts tend to penetrate the density flotation medium and accumulate in the
pellet. Sucrose and zinc sulphate density media selectively concentrate viable oocysts (Bukhari
and Smith, 1995), whereas Percoll–sucrose concentrates empty (ghost) oocysts (LeChevallier et
al., 1995). Material recovered from the surface of the density medium is then (re)centrifuged, and
the final pellet is examined microscopically. Membrane filters offer higher recovery efficiencies,
but the amount of water that can pass through without filter clogging is small, often only 10–20
L. Membrane filters are useful, however, because they retain more material and may be dissolved
to recover cysts and oocysts (Aldom and Chagla, 1995).
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Protozoa: Giardia and Cryptosporidium (April 2004)
Immunomagnetic separation (IMS)/immunocapture represents an alternative to the use of
density-gradient flotation procedures and is increasingly being applied (McCuin et al., 2001;
Moss and Arrowood, 2001; Rimhanen-Finne et al., 2001, 2002; Sturbaum et al., 2002; Ward et
al., 2002). Typically, 10-L samples are processed, and the collected material is eluted with a
detergent and concentrated by centrifugation. The pellet is resuspended in buffer and mixed with
specific monoclonal antibodies attached to magnetized particles, also referred to as
immunomagnetic beads. The (oo)cysts are then separated from the debris in a magnetic field.
(Oo)cysts seeded into low-turbidity waters can be recovered with efficiencies of >90% (Fricker
and Clancy, 1998). Although IMS aids in reducing false positives by reducing the level of debris
on slide preparations for microscopic analysis, it is a relatively expensive procedure, with few
manufacturers supplying the immunomagnetic beads (e.g., Dynal Inc., Aureon Biosystems,
ImmuCell Inc., Miltenyi Biotech). Moreover, it has been reported that high levels of iron may
inhibit immunomagnetic separation (Yakub and Stadterman-Knauer, 2000).
The use of antibodies specific for Giardia cysts and Cryptosporidium oocysts has greatly
enhanced the probability of finding these organisms against a cluttered background and making a
positive identification. Immunofluorescent staining is usually performed by trapping a portion of
the pellet on a small membrane and rinsing antibodies through, but it can also be carried out in
centrifuge tubes or on microscope slides (Sauch, 1985; LeChevallier et al., 1991a,b; Wallis,
1994). Unfortunately, there are some algae that are very close in size and staining characteristics
to cysts and oocysts, and final identification often requires light, phase, and differential
interference microscopy in addition to immunofluorescence. Murine monoclonal antibodies are
commercially available from several manufacturers (Meridian Diagnostics Ltd., Cellabs Pty.
Ltd., Waterborne Inc.) in both direct and indirect fluorescence kits. The intercalation of the vital
dye DAPI highlights nuclei and aids identification (Grimason et al., 1994).
Detection of Giardia cysts and Cryptosporidium oocysts by immunofluorescence requires
specialized equipment and a high level of technical skill. The analysis is tedious, expensive, and
only semi-quantitative, but it has been used to confirm waterborne transmission of both parasites
in many outbreaks and is being continuously improved. Clancy et al. (1994) conducted a blind
survey (analysis of spiked wound filter samples) of 16 commercial labs in the United States and
found that recovery of Giardia cysts ranged from 0.8 to 22.3% (average 9.3%) and that recovery
of Cryptosporidium oocysts ranged from 1.3 to 5.5% (average 2.8%). In 1995, Health Canada
commissioned a similar study of commercial, government, and research laboratories in Canada,
and Giardia cyst recovery ranged from 0 to 90% (average 21%) for eight laboratories analysing
10 unknown samples. Cryptosporidium oocyst recovery ranged from 0 to 43% (average 5.3%)
for the same samples (Clancy Environmental Consultants, Inc., 1996). LeChevallier et al. (1995)
conducted a critical analysis of the immunofluorescence method and concluded that losses of
Cryptosporidium oocysts typically exceed losses of Giardia cysts and that major losses occur
during centrifugation and clarification.
Alternative techniques for detecting (oo)cysts following concentration and recovery have
been proposed. For example, flow cytometry with fluorescence activated cell sorting (FACS) has
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Protozoa: Giardia and Cryptosporidium (April 2004)
been used more and more as an alternative separation and enumeration technique (Vesey et al.,
1997; Bennett et al., 1999; Reynolds et al., 1999; Delaunay et al., 2000; Lindquist et al., 2001).
However, the specificity and sensitivity of the FACS procedure are impaired by the presence of
autofluorescent algae and cross-reaction of other organisms and particles with the monoclonal
antibodies. Recent work by Ferrari et al. (2000) has led to the development of a two-colour
FACS assay, which has addressed a number of these problems, leading to much improved
specificity. Automated cell sorting devices (e.g., ChemScan RDI) have also been used to detect
(oo)cysts and are relatively easy to operate (Rushton et al., 2000; De Roubin et al., 2002).
A number of molecular approaches have also been used in the detection of Giardia and
Cryptosporidium (oo)cysts. PCR, especially in association with IMS (i.e., IMS-PCR), has been
used by many groups (Deng et al., 1997, 2000; Bukhari et al., 1998; Di Giovanni et al., 1999;
Kostrzynska et al., 1999; Rochelle et al., 1999; Hallier-Soulier and Guillot, 2000; Hsu and
Huang, 2001; McCuin et al., 2001; Moss and Arrowood, 2001; Rimhanen-Finne et al., 2001,
2002; Sturbaum et al., 2002; Ward et al., 2002). PCR is highly sensitive and specific and, when
combined with other molecular biology techniques, such as restriction fragment length
polymorphism (RFLP), can be used to discriminate between species and genotypes of
Cryptosporidium. Genotype information can be used to help identify the potential host sources of
Cryptosporidium responsible for an outbreak (Morgan et al., 1997; Widmer, 1998; Lowery et al.,
2000, 2001a,b). PCR is highly sensitive (i.e., level of a single (oo)cyst), is amenable to
automation, and may permit discrimination of viable and non-viable (oo)cysts. However, several
PCR inhibitors are frequently found in water, including divalent cations and humic and fulvic
acids (Sluter et al., 1997). Despite the potential for inhibition of amplification, many PCR assays
have been developed for detection of waterborne (oo)cysts. Some of these include primers
directed at the 18S rRNA coding region (Lowery et al., 2000; Ong et al., 2002; Sturbaum et al.,
2002; Ward et al., 2002) or mRNA coding for heat shock proteins (i.e., reverse transcriptasePCR assay, or RT-PCR) (Stinear et al., 1996; Kaucner and Stinear, 1998; Griffin et al., 1999;
Gobet and Toze, 2001; Karasudani et al., 2001). Fluorescence in situ hybridization (FISH) has
also been used in the detection of Giardia and Cryptosporidium (oo)cysts. However, because of
relatively weak signals, there have been difficulties in microscopic interpretation, resulting in the
limited use of this method (Deere et al., 1998; Vesey et al., 1998; Dorsch and Veal, 2001).
7.2
Viability and Infectivity
As indicated above, routine detection methods provide no indication of (oo)cyst viability
or infectivity. Viability (but not infectivity) can be estimated by excystation. Giardia can be
excysted using acid and enzymes such as trypsin and grown in TYI-S-33 medium (Diamond et
al., 1978; Rice and Schaefer, 1981), but the excystation rate for G. duodenalis is often low.
Cryptosporidium parvum oocysts can also be excysted as a measure of viability (Black et al.,
1996). However, excystation methods have been shown to be relatively poor indicators of
Cryptosporidium oocyst viability. Neumann et al. (2000b) observed that non-excysted oocysts
recovered after commonly used excystation procedures are still infectious to neonatal mice.
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Protozoa: Giardia and Cryptosporidium (April 2004)
Oocyst viability can also be determined using mouse infectivity assays. Both parasites can
be used to infect experimental animals such as the gerbil (for Giardia) (Belosevic et al., 1983) or
neonatal CD-1 mice (for Cryptosporidium) (Finch et al., 1993b), and this technique is useful for
pilot plant studies or isolate collection; however, most analytical laboratories do not maintain
animal colonies, and the expense is high. Culturing and animal infection are therefore more
useful for research purposes, such as disinfection effectiveness, than for routine monitoring
(Delaunay et al., 2000; Korich et al., 2000; Matsue et al., 2001; Noordeen et al., 2002; Okhuysen
et al., 2002; Rochelle et al., 2002).
Various staining methods have been developed to assess (oo)cyst viability (Robertson et
al., 1998; Freire-Santos et al., 2000; Neumann et al., 2000b; Gold et al., 2001; Iturriaga et al.,
2001). Of these, the fluorogenic dyes DAPI and PI and nucleic acid stains have received the most
attention. In general, DAPI/PI give good correlation with in vitro excystation (Campbell et al.,
1992). Three classes of (oo)cysts can be identified: (1) viable (permeable to DAPI, impermeable
to PI), (2) non-viable (permeable to both DAPI and PI), and (3) quiescent or dormant
(impermeable to both DAPI and PI, but potentially viable). Neumann et al. (2000a) demonstrated
a strong correlation between SYTO-9® and SYTO-59® staining intensity with animal infectivity
of freshly isolated C. parvum oocysts. Nucleic acid dyes have also proved useful for determining
the viability and infectivity of (oo)cysts in environmental samples. Stains like SYTO-59 have
been used successfully in conjunction with FITC-labelled antibodies to determine the viability
and infectivity of (oo)cysts in water samples, because their fluorescence spectra do not overlap
with that of FITC (Belosevic et al., 1997; Bukhari et al., 2000; Neumann et al., 2000b).
However, dye permeability and excystation procedures overestimate viability and potential
infectivity of treated or disinfected oocysts (Jenkins et al., 1997).
Recent advances have facilitated the use of in vitro tissue culture assays to estimate
infectivity of oocysts in water (Di Giovanni et al., 1999; Hijjawi et al., 2001;Weir et al., 2001;
Rochelle et al., 2002). Concentrated water samples are disinfected and typically inoculated on
human illeocaecal adenocarcinoma (HCT-8) cell monolayers. After a 24- to 48-hour incubation,
the monolayer is examined for the presence of specific reproductive stages using either an
indirect antigen–antibody assay (Slifko et al., 1997) or RT-PCR (Rochelle et al., 1997). In a
comparison experiment, average percent viabilities of C. parvum oocysts less than 2 months old
were 42, 40, and 78% for tissue culture infectivity, excystation, and DAPI/PI assays, respectively
(Slifko, 1998). There are several advantages to the cell culture assay, including its high
sensitivity (i.e., single oocyst), applicability to analysis of raw and treated water samples, ease of
performance, and rapid turnaround time for results. The disadvantage of this method is that it
requires the maintenance of a cell line and is often subject to poor reproducibility among similar
samples for quantitative assessments. RT-PCR has also been applied to the direct detection of
viable Giardia and C. parvum in water concentrates (Kaucner and Stinear, 1998). When
compared with the immunofluorescence assay (IFA) DAPI/PI method, the frequency of detection
of viable Giardia increased from 24% with IFA to 69% with RT-PCR. In contrast, viable C.
parvum were detected in only 3% of samples with RT-PCR compared with 14% by IFA
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Protozoa: Giardia and Cryptosporidium (April 2004)
DAPI/PI, suggesting that other Cryptosporidium species were present in the samples. RT-PCR
possesses some disadvantages, including the need for small processed volumes, possible
inhibition by environmental constituents, inefficient extraction of RNA from (oo)cysts, and its
non-quantitative nature.
Alternative viability assays have been proposed, including FISH and nucleic acid probes
to detect 18S rRNA in Giardia and Cryptosporidium (Fricker and Clancy, 1998). The 18S
molecule is abundant in viable (oo)cysts but declines rapidly in non-viable (oo)cysts. This
method is limited by its inability to assess (oo)cyst infectivity. Further research is required to
improve the detection limit and validate the assay.
8.
Treatment Technology
The removal and inactivation of Giardia cysts and Cryptosporidium oocysts from raw
water are complicated by their small size and resistance to commonly used oxidants such as
chlorine. Cryptosporidium oocysts are more difficult to eliminate but appear to be less common
than Giardia cysts in Canadian surface waters. The detection procedure is less efficient for
Cryptosporidium oocysts, however, and the national prevalence rate may be higher than
suspected. Waterborne outbreaks of giardiasis and cryptosporidiosis have resulted both from
inadequate treatment and from improper operating procedures (Lin, 1985). The multiple-barrier
approach to treatment, including watershed or wellhead protection, optimized filtration and
disinfection, a well-maintained distribution system, and monitoring the effectiveness of treatment
(e.g., turbidity, disinfection residuals, etc.), is by far the best approach to reduce the risk of
infection to acceptable or non-detectable levels. In communities where filtration is not
economically feasible, an effective watershed protection plan, adequate disinfection, an intact
distribution system, and, possibly, recognized point-of-entry or point-of-use treatment must be
relied upon to reduce these risks. An exhaustive review of available treatment options is beyond
the scope of this document. These methods have been reviewed in water treatment manuals
prepared by UMA Engineering Ltd. et al. (1993), Health Canada (1993), and the U.S. EPA
(1991).
Effective water treatment begins with watershed management to minimize the input of
faecal contamination from human and other animal sources, by controlling aquatic mammalian
populations and locating raw water intakes as far as possible from sewage outfalls (Crockett and
Haas, 1997). The possible flooding of sewage collection and treatment systems cannot be
overlooked, and sudden increases in indicator organisms can give advance warning of problems
(e.g., the outbreak at Temagami, Ontario). Coagulation, flocculation, clarification, filtration
(including direct filtration), and post-disinfection are all commonly used to good effect in
municipal water treatment plants to remove or inactivate Giardia cysts, but problems can still
occur with Cryptosporidium oocysts (because of their small size and resistance to oxidants).
There are approximately 1000 communities in Canada, including some major cities, that use
surface water supplies and rely solely upon chlorination with varying degrees of watershed
management.
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The degree of treatment required to remove and/or inactivate (oo)cysts is dependent upon
their concentrations in source waters. For this reason, source waters should be periodically
monitored for (oo)cysts, particularly at times when concentrations are expected to be highest —
for example, following spring freshet or heavy rains — to determine appropriate levels of
treatment. For example, the U.S. EPA has proposed that if average concentrations of oocysts in
source waters are less than 0.075/L, then conventional filtration achieving 3-log reduction of
oocysts should be adequate (U.S. EPA, 2002a). Where monitoring for oocysts is not feasible, E.
coli can be used as an indicator of their presence and treatment requirements. It has been
proposed by the U.S. EPA that treatment known to achieve 3-log reduction of oocysts is
adequate, provided average concentrations of E. coli do not exceed 10 colony-forming units
(cfu)/100 mL in lakes or 50 cfu/100 mL in flowing streams.
8.1
Municipal-scale Technologies
Barring system-specific exemptions, all public (municipal) supplies should be
disinfected. A disinfectant residual should be maintained throughout the distribution system at all
times. In addition to disinfection, minimum treatment of all supplies derived from surface water
sources and groundwater impacted by surface waters should include coagulation, flocculation,
clarification, and filtration, or equivalent technologies.
The efficacy of disinfection can be predicted based on a knowledge of the residual
concentration of disinfectant, temperature, pH (for chlorine only), and contact time to first
customer. This relationship is commonly referred to as the CT concept, where CT is the product
of C (the residual concentration of disinfectant, measured in mg/L) and T (the disinfectant
contact time, measured in minutes). CT values for chlorine, chlorine dioxide, chloramine, and
ozone developed by the U.S. EPA to achieve various degrees of inactivation of Giardia are
provided in Annex A to guide water purveyors on the conditions required to achieve adequate
inactivation of Giardia. The degree of inactivation considered adequate will, of course, depend
upon the levels of Giardia in the raw water and the level of acceptable risk of illness. It is
possible to reduce the viability of Giardia cysts by as much as 99.9% using chlorination alone,
but long contact times are required. Where source waters are of high quality, either naturally or
because of an effective watershed protection programme, disinfection achieving less than a
99.9% inactivation may be sufficient. Ozone and chlorine dioxide are much better disinfectants,
but both are expensive and result in the formation of unwanted by-products (particularly chlorite,
in the case of chlorine dioxide, and bromate, in the case of ozone). Ozone is a better choice but is
unreliable when turbidity is high or variable, because cysts are protected in flocculated particles.
Chloramine should not be used as a primary disinfectant. A discussion of the effect of these
variables can be found in the water treatment manuals mentioned above or in von Huben (1991)
for chlorine, chlorine dioxide, chloramine, and ozone.
Chlorination alone does not appear practical for the inactivation of Cryptosporidium
(Finch et al., 1993a). Watershed protection followed by filtration and an intact distribution
system are at present the best available means of reducing the risk of waterborne
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Protozoa: Giardia and Cryptosporidium (April 2004)
cryptosporidiosis in treatment plants relying upon chlorination. Work carried out by Finch et al.
(1997) has shown that ozonation may be effective when used properly. These authors also
demonstrated that the use of the two disinfectants sequentially gave better results than were
obtained when either was used by itself. Chlorination followed by chloramination is more
effective than previously believed and can inactivate Cryptosporidium oocysts by up to 1.6 logs
when viability is measured by infection of mice. Chlorine following ozone or chlorine dioxide
was particularly effective. A discussion of the effects of ozonation and other water treatment
processes may be found in Smith et al. (1995).
Filtration with the aid of coagulation/flocculation followed by disinfection is the most
practical method to achieve high removal/inactivation rates of cysts and oocysts. Payment and
Franco (1993) showed that 99.998% of Giardia cysts and Cryptosporidium oocysts were
removed from heavily polluted water by full conventional treatment (flocculation, settling, preand post-disinfection with chlorine dioxide and chlorine, and filtration) at three Montreal water
treatment plants. Slow sand and diatomaceous earth filtration can also be highly effective.
Optimizing filtration is desirable to provide stable filter performance and minimize breakthrough
of cysts and oocysts. The recycling of filter backwash water containing cysts or oocysts, without
treatment, is not recommended. Pressure filters vary widely in their performance and are not as
reliable as properly operated gravity filter operations. Water types vary, however, and the choice
of the most appropriate system must be made by experienced engineers after suitable pilot
testing. An effective system of operator training and process control is essential in areas of
known contamination where the risk is high. Monitoring of raw water for cysts and oocysts is
useful for establishing prevalence, and analysis of treated water provides an indication of risk if
viability or infectivity assays are incorporated. This can be particularly important in the spring
after heavy rains or snowmelt. Useful data for process control may also be obtained by
monitoring for cysts and oocysts or for their appropriate surrogates in treated water. Abnormal
turbidity measurements or particle counts can quickly indicate a malfunction in filter
performance. LeChevallier and Norton (1992) observed that particles >5 µm and turbidity were
useful predictors of Giardia and Cryptosporidium. A 1-log removal of particles and turbidity
corresponded to a 0.66- and 0.89-log removal of cysts and oocysts, respectively.
Membrane filtration has become an (increasingly) important component of drinking water
treatment systems. Microfiltration, ultrafiltration, nanofiltration, and reverse osmosis are the
most commonly used membrane processes for microbial removal. Microfiltration membranes
have the largest pore size ($0.1 µm), while reverse osmosis membranes have the smallest pore
size ($0.0001 µm) (Taylor and Weisner, 1999). While all of these processes appear effective in
removing protozoan (oo)cysts, microfiltration and ultrafiltration are most commonly
applied/used because of their cost-effectiveness. Jacangelo et al. (1995) evaluated the removal of
G. muris and C. parvum from three source waters of varying quality using a variety of
microfiltration and ultrafiltration membranes. Microfiltration membranes of 0.1 µm and 0.2 µm
and ultrafiltration membranes of 100, 300, and 500 kilodaltons were assessed. Both
microfiltration and ultrafiltration resulted in log removals of >4.7–7.0 for G. muris and >4.4–7.0
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Protozoa: Giardia and Cryptosporidium (April 2004)
for C. parvum. Karami et al. (1999) also evaluated the effectiveness of microfiltration
membranes (0.2 µm) for removal of (oo)cysts. Average log removals of 3.3–4.4 were reported
for Giardia-sized particles, and log removals of 2.3–3.5 were reported for Cryptosporidium-sized
particles. More recently, States et al. (1999) reported absolute removal of Cryptosporidium and
Giardia by microfiltration, and Parker et al. (1999) reported a 5.3-log removal of C. parvum
using microfiltration membranes (0.2 µm). Although membrane filtration is highly effective for
microbial removal, including removal of protozoan (oo)cysts, membrane fouling (caused by
accumulation of particles, chemicals, and biological growth on membrane surfaces) and
degradation (caused by hydrolysis and oxidation) must be considered. Because the physical
characteristics of the membrane could vary during the manufacturing process by different
manufacturers, the cyst and oocyst removal efficiency for a specific membrane must be
demonstrated through challenge testing and verified by direct integrity testing (e.g., measuring
pressure loss upstream or downstream of the membrane, or assessing removal of spiked
particulates using a marker-based approach). More detailed information on filtration techniques
can be found in Health Canada’s Turbidity supporting document (http://www.hc-sc.gc.ca/hecssesc/water/publications/turbidity1/toc.htm).
UV light disinfection is an emerging (alternative) treatment approach that appears to be
highly effective for inactivating Giardia and Cryptosporidium. Whereas earlier studies (i.e.,
those prior to 1998) (Rice and Hoff, 1981; Karanis et al., 1992; Lorenzo-Lorenzo et al., 1993;
Campbell et al., 1995) reported protozoan inactivation only at very high UV doses, recent studies
have shown that low doses can achieve substantial inactivation (Clancy et al., 1998; Bukhari et
al., 1999; Craik et al., 2000, 2001; Belosevic et al., 2001; Drescher et al., 2001; Linden et al.,
2001, 2002; Shin et al., 2001; Campbell and Wallis, 2002; Mofidi et al., 2002; Rochelle et al.,
2002). These contrasting observations are the result of in vitro viability assays, used in earlier
studies, which greatly overestimate the UV dose necessary for inactivation (Clancy et al., 1998;
Bukhari et al., 1999; Craik et al., 2000). Current studies rely on in vivo assays (e.g., neonatal
mouse model) and cell culture techniques for assessing (oo)cyst inactivation. Based on these and
other studies, the U.S. EPA developed a UV light dose table, also known as an “IT table,” which
was released in June 2003 as part of the draft “Ultraviolet Disinfection Guidance Manual” (U.S.
EPA, 2003). According to this dose table, a (low-pressure UV light) dose of 12 mJ/cm2 is
required for a 3-log inactivation of Cryptosporidium (refer to Table B.1). In contrast, a dose
greater than 140 mJ/cm2 is required to achieve the same level of inactivation for certain viruses.
Although UV light disinfection appears to be highly effective for inactivating protozoans, the
possibility of reactivation after UV light treatment must be considered. The ability of
microorganisms to repair their UV-damaged DNA (reactivate) has been reported. Belosevic et al.
(2001) observed reactivation of G. muris after treatment with relatively low doses (<25 mJ/cm 2)
of medium-pressure UV light. However, C. parvum and G. muris (oo)cysts exposed to mediumpressure UV doses of $60 mJ/cm 2 did not exhibit reactivation after treatment. Linden et al.
(2002) did not observe any reactivation of G. lamblia after exposure to UV light doses of 16 and
40 mJ/cm 2.
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Even the most sophisticated municipal treatment system cannot provide water that is
absolutely free of disease-causing microorganisms all the time. The real goal of treatment is to
reduce the presence of disease-causing organisms and associated health risks to an acceptable or
safe level. The risk of illness can be minimized by maximizing the number and efficacy of
treatment barriers present. This level of acceptability or safety may vary from community to
community and depends on many site-specific environmental, human health, and economic
conditions. For example, determinations of safety must consider the various types and, where
possible, concentrations of infectious organisms in the raw water, the seriousness of the illnesses
they cause, the degree of resistance to the illnesses in the exposed population, the extent of
disease surveillance in the community, and the available financial resources that must be shared
among drinking water treatment and other common services in a community. Nevertheless,
minimum treatment of all supplies derived from surface water sources and groundwater under the
influence of surface water should include coagulation, flocculation, clarification, and filtration, or
equivalent technologies, in addition to disinfection. Because Giardia and Cryptosporidium are
ubiquitous in surface waters in Canada and are more resistant to disinfection than most other
infectious organisms, it is desirable that treatment known to achieve at least a 99.9% reduction of
Giardia and Cryptosporidium be in place.
In the United States, the EPA has promulgated the Surface Water Treatment Rule
(SWTR) to control the presence of Giardia and viruses in public drinking water systems using
either surface water or groundwater under the influence of surface water (U.S. EPA, 1989). All
systems using filtered or unfiltered surface water must achieve at least a 99.9% (3-log) removal
and/or inactivation of G. lamblia cysts. This level of removal/inactivation is believed to reduce
the risk of waterborne giardiasis to less than 10!4 (i.e., <1 in 10 000 people infected) per year.
Under the rule, a public water system using surface water must use filtration unless it meets
certain water quality, operational, and public health standards. It is assumed that filtration
removes 99% (2-log) of Giardia cysts and that disinfection need only provide a further 90% (1log) inactivation (see Annex A). Systems using conventional treatment that are able to achieve
turbidity levels of less than 0.5 nephelometric turbidity units (NTU) in the filtered water in 95%
of samples are assumed to achieve 2.5-log removal of Giardia cysts, providing that coagulation
and flocculation conditions are optimized for turbidity removal. Disinfection in these systems
need only inactivate 68.4% (0.5-log) of Giardia cysts (see Annex A).
Recognizing that systems with very poor source water may not be adequately protected by
a 3-log reduction in Giardia cysts and that the requirements for Giardia reduction may not be
applicable to Cryptosporidium, the U.S. EPA has promulgated an Interim Enhanced Surface
Water Treatment Rule (IESWTR) (U.S. EPA, 1998). One component of this rule establishes that
systems required to filter under the SWTR must achieve a 2-log removal of Cryptosporidium.
Conventional or direct filtration plants producing water with turbidities of 0.3 NTU or less in
95% of monthly samples and in which the turbidity never exceeds 1 NTU are deemed to meet
this requirement. The Long Term 1 Enhanced Surface Water Treatment Rule (LT1ESWTR),
promulgated in January 2002, builds upon the requirements of the SWTR and the IESWTR and
Guidelines for Canadian Drinking Water Quality: Supporting Documentation
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Protozoa: Giardia and Cryptosporidium (April 2004)
is designed to strengthen microbial controls for small systems (i.e., those serving <10 000
people) that use surface water and groundwater under the influence of surface water (U.S. EPA,
2002b). The U.S. EPA recently proposed the Long Term 2 Enhanced Surface Water Treatment
Rule (LT2ESWTR), which will apply to all public water systems that use surface water or
groundwater under the influence of surface water. This rule defines system-specific treatment
requirements and assigns systems into different categories (known as bins) based on the results of
source water Cryptosporidium monitoring. For example, systems serving at least 10 000 people
with a Cryptosporidium source water concentration of >0.075/L but <1.0/L (as determined by at
least 1 year of monitoring) are classified into Bin 2. Systems within this bin category are required
to achieve a total Cryptosporidium removal/inactivation of at least 4 logs (U.S. EPA, 2002a).
The LT2ESWTR will also establish a “toolbox” of various disinfection technologies, such as
ozone and UV light, that can be used to obtain Cryptosporidium disinfection credits. Included in
the toolbox will be specific disinfection criteria, such as ozone CT products and UV IT dosages
required for certain levels of Cryptosporidium inactivation.
Drinking water technologies meeting the turbidity limits prescribed in the Guidelines for
Canadian Drinking Water Quality (Health Canada, 2004) can apply the estimated potential
removal credits for Giardia, Cryptosporidium, and enteric viruses given in Table 1. These log
reduction credits are based on the mean or median removals established by the U.S. EPA as part
of the LT2ESWTR (U.S. EPA, 2002a). Facilities that do not meet the requirements or facilities
that believe they can achieve a higher log credit than is automatically given can be granted a log
reduction credit based on a demonstration of performance.
8.2
Residential-scale Treatment Options
Minimum treatment of all semi-public and private supplies derived from surface water
sources or groundwater under the influence of surface water should include adequate filtration
(or equivalent technologies) and disinfection. Semi-public and private supplies are considered to
be residential-scale for the purposes of this document.
An array of options is available for treating source waters to provide high-quality
pathogen-free drinking water. For public systems, these include various filtration methods,
disinfection with chlorine-based compounds, or alternative technologies, such as UV light or
ozonation. Semi-public and private systems can employ many of the same technologies but on a
smaller scale, along with others, such as distillation, not used by public systems. These
technologies have been incorporated into point-of-entry devices that treat all water entering the
system or point-of-use devices that treat water only at a single location — for example, at the
kitchen tap. The commonly used drinking water disinfectants are chlorine, chloramine, chlorine
dioxide, ozone, and UV light. All of the above disinfectants are used in public systems; however,
semi-public and private systems using disinfection are more apt to rely on chlorine or UV light.
Guidelines for Canadian Drinking Water Quality: Supporting Documentation
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Protozoa: Giardia and Cryptosporidium (April 2004)
Table 1: Giardia, Cryptosporidium, and virus potential removal credits for various technologies meeting the
turbidity limits prescribed in the Gu ideline s for C ana dian Drinking Water Q uality
Cyst/oo cyst cred itc
Virus credit
3.0 log
2.0 log
Direct filtrationa
2.5 log
1.0 log
Slow sand or diatoma ceous earth
filtrationa
3.0 log
2.0 log
Micro- and ultrafiltration,
nano filtration, and reverse o smosis b
Removal efficiency demonstrated
through challenge testing and
verified by direct integrity testing
No credit for micro- and
ultrafiltration; for nanofiltration
and reverse osmosis, removal
efficiency demonstrated through
challenge testing and verified by
direct integrity testing
Technology
Conventional filtration
a
b
c
a
Conventional/direct/slow sand/diatomaceous earth filtration should be followed by free chlorination to obtain additional virus
credit.
Micro- and ultrafiltration should be followed by free chlorination for the inactivation of viruses.
Depending on (oo)cyst levels in source water, additional treatment is required using UV light, ozone, chlorine, or chlorine
dioxide.
Health Canada does not recommend specific brands of drinking water treatment devices,
but it strongly recommends that consumers look for a mark or label indicating that the device has
been certified by an accredited certification body as meeting the appropriate NSF International/
American National Standards Institute (ANSI) standard. These standards have been designed to
safeguard drinking water by helping to ensure the material safety and performance of products
that come into contact with drinking water. Certification organizations provide assurance that a
product or service conforms to applicable standards. In Canada, the following organizations have
been accredited by the Standards Council of Canada (http://www.scc.ca) to certify drinking water
devices and materials as meeting the appropriate NSF/ANSI standards:
•
•
•
•
•
Canadian Standards Association International (http://www.csa-international.org);
NSF International (http://www.nsf.org);
Underwriters Laboratories Inc. (http://www.ul.com);
Quality Auditing Institute (http://www.qai.org); and
International Association of Plumbing & Mechanical Officials (http://www.iapmo.org).
9.
Assessment
Giardia and Cryptosporidium are infectious protozoans that can be transmitted by water,
poor hygiene, sexual activities, and food. Both these organisms are enteric pathogens that cause
serious illness in immunocompetent and immunocompromised individuals. Cryptosporidiosis is
the more serious of the two because (1) most cases are symptomatic, (2) there is no effective
Guidelines for Canadian Drinking Water Quality: Supporting Documentation
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Protozoa: Giardia and Cryptosporidium (April 2004)
drug treatment for adults, and (3) the illness is capable of causing death. Cryptosporidium
parvum may be fatal in immunocompromised individuals, particularly those suffering from
AIDS.
The risk of becoming infected by protozoan cysts or oocysts in drinking water depends
upon (1) the number of viable cysts or oocysts ingested (dose), (2) the virulence or infectivity of
the ingested cysts or oocysts, and (3) the susceptibility of the host population to infection. The
dose can be estimated based upon measurements of cyst or oocyst concentrations in drinking
water and the amount of water consumed over the period of exposure. The viability of cysts or
oocysts can be estimated using dye exclusion or excystation, but, as noted above, these methods
probably provide an overestimate of true viability. Infectivity can be determined from animal or
human infection assays. Human tissue culture assays can also be used to determine
Cryptosporidium infectivity. The susceptibility of individuals in the host population varies by
age, immunological status, history of previous exposures, and other genetic and environmental
factors. These factors vary widely between individuals, so it is only meaningful to estimate the
risk to populations in terms of the number of individuals who may become infected rather than
the number of individuals who may become ill.
The application of a mathematical risk model to waterborne giardiasis and
cryptosporidiosis has been proposed by Regli et al. (1991), Rose et al. (1991), Haas and Rose
(1994), and Teunis et al. (1997), based on work by Haas (1983), Rendtorff (1978), and DuPont et
al. (1995). Using this model, the probability (Pi) of an infection resulting from the ingestion of a
single volume of liquid (V) containing µ organisms per litre can be described by a simple
exponential probability density function:
Pi = 1 ! e!rµV
(1)
where r is the fraction of ingested organisms that survive to initiate infection. The probability that
less than 1 person in 10 000 per year will become infected after exposure to pathogens in
drinking water has been proposed as an acceptable level of risk (Regli et al., 1991). This
corresponds to an acceptable daily risk of 2.75 × 10!7 or, rearranging equation 1, to an acceptable
daily intake of N organisms (N = µV), equivalent to:
N = !(1/r) ln (1 ! Pi)
(2)
The proposed exponential model makes several assumptions that introduce uncertainty
into the assessment. First, it assumes that the distribution of cysts and oocysts in water is random
(Poisson). However, it is likely that the (oo)cysts are not randomly distributed but rather occur in
clusters, either loosely associated with each other or tightly bound to or within particles (Gale,
1996). Such clustering means that most consumers will not be exposed to any pathogens, but a
small portion will be exposed to infectious doses. Models that do not account for clustering will
therefore underestimate the probability of exposure and infection. Second, as there are no
Guidelines for Canadian Drinking Water Quality: Supporting Documentation
28
Protozoa: Giardia and Cryptosporidium (April 2004)
practical procedures to determine the viability and infectivity of the small number of cysts and
oocysts recovered from drinking water, it is assumed that one cyst or oocyst is capable of causing
an infection. This assumption will therefore lead to an overestimation of the risk. Until routine
practical methods to identify human-infectious strains of Giardia and Cryptosporidium are
available, it is desirable, from a health protection perspective, to assume that all viable (oo)cysts
recovered from drinking water are infectious to humans unless evidence to the contrary exists. It
must also be remembered that the current risk assessment model assumes that errors caused by
overestimating viability are at least partially counterbalanced by poor (oo)cyst recoveries during
the detection method. Third, the model assumes a daily tap water consumption of 1.5 L per
person; for many segments of the population, however, some or most of this quantity is boiled
and therefore of no microbiological significance. For example, a study in the United Kingdom
has indicated that, on average, 89% of the tap water consumed is boiled (Gale, 1996).
Assessments that do not consider boiled tap water consumption will yield a conservative estimate
of risk of infection. Fourth, dose–response experiments use single laboratory strains; therefore, it
is unknown whether organism age, prior environmental exposures, or strain type influences
infectivity. Furthermore, the experiments are conducted on healthy humans or other animals and
do not consider the immune status of the exposed population, and they are therefore likely to
underestimate the risk. Finally, all cysts and oocysts ingested are considered to be viable and
pathogenic to humans, which is probably not true, but it is assumed that errors caused by
inefficiency in cyst and oocyst recovery during the analytical procedure are counterbalanced by
overestimates in viability and pathogenicity.
9.1
Giardia
The risk model has been applied to Canadian Giardia data by Wallis et al. (1995). Based
on a review of human and gerbil infection studies, they proposed that a value of r = 0.0105 be
used in the model. The use of gerbil infection data may be questioned, but these data show that
different isolates have markedly different ID50 values within genetically similar hosts. Until more
human dose–response data become available, it is proposed that this value be used as the most
comprehensive estimate of the infectivity of Giardia in humans, recognizing that both host and
parasite are variable in their response.
Using equation (2), the theoretical acceptable daily intake of human-infectious Giardia
cysts can be calculated to be 2.6 × 10!5. If it is assumed that each person consumes 1.5 L of tap
water per day, then a theoretical maximum acceptable concentration (MAC) for Giardia would
be 1.7 × 10!2 cysts/1000 L. This concentration is well below the detection limits of current
methods and would require filtration of at least 60 000 L of water to detect a single cyst. A more
practical approach would be to periodically monitor the source water for cysts, especially during
times when highest cyst concentrations would be expected, and to determine the adequacy of
treatment by comparing existing treatment with published treatment guidelines (U.S. EPA, 1991;
Health Canada, 1993; UMA Engineering Ltd. et al., 1993). For example, water treatment plants
Guidelines for Canadian Drinking Water Quality: Supporting Documentation
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Protozoa: Giardia and Cryptosporidium (April 2004)
maintaining a 3-log reduction could accept raw water levels of 1.7 cysts/100 L or less and
continue to maintain an annual risk of infection of less than 1 × 10!4.
Monitoring data by Wallis et al. (1995) have shown that cyst concentrations averaging 3
cysts/1000 L in finished water, which produce a theoretical daily risk of 4.75 × 10!5 and a
theoretical annual risk of 0.0172 (17 cases in 1000), do not cause detectable outbreaks of
waterborne giardiasis. Monitoring data reported by others in the absence of outbreaks present
similar levels of Giardia cysts in treated drinking water (LeChevallier et al., 1991b; Payment and
Franco, 1993; Goatcher and Fok, 2000). Although an annual rate of 17 cases per 1000 people
may not cause noticeable levels of giardiasis, drinking water treatment officials in large centres
are understandably reluctant to be responsible for outbreaks that could potentially affect tens of
thousands of people annually. The theoretical infection rate predicted by the model is reduced to
the much lower reported illness rate from official data by several factors. These could include (1)
variable viability and infectivity of cysts, (2) variable susceptibility of the host population to
infection, (3) asymptomatic cases, (4) use of effective treatment devices in the home or other
sources of drinking water, and (5) incomplete reporting. Based on known waterborne outbreaks
of giardiasis in Canada, Wallis et al. (1996) proposed an action level of 3–5 cysts/100 L in
treated drinking water.
9.2
Cryptosporidium
The risk model described for Giardia can also be applied to Cryptosporidium, but there
are fewer data available from dose–response experiments. An excellent data set was published by
DuPont et al. (1995), which permits calculation of an r value of 0.0047 (Haas et al., 1996). This
value is close to the value for Giardia (r = 0.0105), and the resulting calculations are almost the
same after rounding. Using the dose–response data of DuPont et al. (1995), Haas et al. (1996)
determined that the theoretical acceptable daily intake of oocysts is 6.54 × 10!5. Assuming a daily
tap water consumption of 1.5 L/person, the theoretical MAC would be 4.4 × 10!2 oocysts/1000 L.
This concentration is well below the detection limits of current methods and would require
filtration of at least 23 000 L of water to detect a single oocyst. A more practical approach would
be to monitor the source water for oocysts, particularly during periods when levels would be
expected to be high, and to determine the adequacy of treatment by comparing existing treatment
with published treatment guidelines. Drinking water treatment plants using direct or conventional
filtration optimized to achieve a filtered water turbidity of <0.3 NTU can be expected to achieve
at least a 2-log removal of Cryptosporidium oocysts (Patania et al., 1995). Thus, in theory,
filtration plants could accept raw water containing 0.44 oocysts/100 L or less and maintain an
annual risk of infection of less than 1 × 10!4. Using the model, Haas and Rose (1994) estimated
that the mean concentration of Cryptosporidium in finished water during the outbreak in
Milwaukee was 1.2 oocysts/L. Perz et al. (1998) used a risk assessment model to determine the
potential role of New York City tap water in the transmission of endemic cryptosporidiosis.
Assuming a reasonable baseline concentration of 1 oocyst/1000 L, they estimated that tap water
is responsible for more than 6000 infections annually, with 99% occurring in the non-AIDS
Guidelines for Canadian Drinking Water Quality: Supporting Documentation
30
Protozoa: Giardia and Cryptosporidium (April 2004)
subgroup. The authors concluded that low-level transmission via tap water may represent an
important exposure route for endemic cryptosporidiosis. Based upon outbreak and routine
monitoring data from the United States and the United Kingdom, Haas and Rose (1995)
proposed a finished water action level of 10–30 oocysts/100 L.
9.3
Balancing Risks
Havelaar et al. (2000) recently described a quantitative risk assessment approach for
comparing the risks associated with disinfection by-products with those associated with C.
parvum infection. This group evaluated the use of disability-adjusted life-years (DALYs) as a
measure of disease burden. DALYs take into account the loss of healthy life due to mortality and
morbidity and are expressed as:
DALY = LYL + YLD
where LYL represents the number of life-years lost due to mortality and YLD represents the
number of years lived with a disability, weighted with a factor between 0 and 1 for severity of the
disability. LYL represents the product of deaths due to a particular illness/disease (d) and the
standard life expectancy at the age of death due to that illness/disease (e*), whereas YLD
represents the product of the number of persons affected by a non-lethal illness/disease (N), the
duration of the illness/disease (L), and its severity (W). Overall, for a given agent, the population
health burden is calculated as:
DALY = 3die*i + 3NiLiWi
i
i
Using this approach, Havelaar et al. (2000) compared the risks associated with ozonation of
water with those associated with C. parvum infection and determined that ozonation led to an
approximate 7-fold reduction in the median risk of C. parvum infection. This decrease was
associated with an increase in bromate concentration, along with an increase in the health burden
of renal cancer. This group concluded that the health benefits of preventing gastroenteritis in the
general population and premature death in immunocompromised patients (associated with C.
parvum infection) outweigh the health losses associated with premature death from renal cancer
(due to exposure to increased concentrations of bromate) by a factor of greater than 10 (i.e., net
benefit of 1 DALY/million person-years).
Although the DALY approach is being used by many groups, no consensus has been
reached regarding the use of a common health metric. The U.S. EPA, for example, has been
assessing quality-adjusted life-years (QALYs) as a measure of disease burden (Murphy et al.,
2000).
Guidelines for Canadian Drinking Water Quality: Supporting Documentation
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Protozoa: Giardia and Cryptosporidium (April 2004)
10.
Rationale
It is not possible at this time to establish MACs for Giardia and Cryptosporidium in
drinking water. Methods available for the detection of cysts and oocysts suffer from low recovery
rates, do not provide any information on their viability or human infectivity, and provide
temporally restricted information about potential parasite numbers. Nevertheless, until better
monitoring data and information on the viability and infectivity of cysts and oocysts present in
drinking water are available, measures should be implemented to reduce the risk of illness as
much as possible. If the presence of viable, human-infectious cysts or oocysts is known or
suspected in source waters, or if Giardia or Cryptosporidium has been responsible for past
waterborne outbreaks in a community, a treatment and distribution regime and a watershed or
wellhead protection plan (where feasible) or other measures known to reduce the risk of illness
should be implemented. Periodic monitoring of source waters for changes in cyst and oocyst
concentrations should be used to adjust treatment processes and to confirm cyst and oocyst
concentrations and the adequacy of current treatment processes. This guideline is primarily
intended to protect the health of the immunocompetent population. Immunocompromised people
may be at increased risk of illness and should discuss their risks and the need for extra
precautionary measures with their physicians.
Even the most sophisticated municipal treatment system cannot provide water that is
absolutely free of disease-causing microorganisms all the time. The real goal of treatment is to
reduce the presence of disease-causing organisms and associated health risks to an acceptable or
safe level. This level of acceptability or safety may vary from community to community and
depends on many site-specific environmental, human health, and economic conditions.
Nevertheless, minimum treatment of all supplies derived from surface water sources and
groundwater impacted by surface water should include coagulation, flocculation, clarification,
and filtration, or equivalent technologies, in addition to disinfection. Because Giardia and
Cryptosporidium are ubiquitous in surface waters in Canada and are more resistant to
disinfection than most other infectious organisms, it is desirable that treatment known to achieve
at least a 99.9% reduction of Giardia and Cryptosporidium be in place.
Many disciplines, including those responsible for source water protection, treatment plant
operation, water quality monitoring, and disease surveillance, are involved in protecting the
public from drinking water-related illnesses. Therefore, it is essential that these groups have
cooperative strategies in place, not only to control waterborne outbreaks of giardiasis or
cryptosporidiosis promptly, but also to manage effectively any incident where the
microbiological safety of the water may have been compromised.
11.
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Guidelines for Canadian Drinking Water Quality: Supporting Documentation
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Protozoa: Giardia and Cryptosporidium (April 2004)
ANNEX A:
CT Tables for the
Inactivation of Giardia lamblia Cysts by
Chlorine, Chlorine Dioxide, Chloramine, and Ozone
at Various Temperatures
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Protozoa: Giardia and Cryptosporidium (April 2004)
CHLORINE
(a) 99.9% (3-log) inactivation
Table A.1. CT values (in mg @min/L) for 99.9% inactivation of Giardia la m blia cysts by free chlorine at 0.5°C
pH
Residual
(mg/L)
6.0
6.5
7.0
7.5
8.0
8.5
9.0
<0.4
137
163
195
237
277
329
390
0.6
141
169
200
239
286
342
407
0.8
145
172
205
246
295
354
422
1.0
148
176
210
253
304
365
437
1.2
152
180
215
259
313
376
451
1.4
155
184
221
266
321
387
464
1.6
157
189
226
273
329
397
477
1.8
162
193
231
279
338
407
489
2.0
165
197
236
286
346
417
500
2.2
169
201
242
297
353
426
511
2.4
172
205
247
298
361
435
522
2.6
175
209
252
304
368
444
533
2.8
178
213
257
310
375
452
543
3.0
181
217
261
316
382
460
552
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Protozoa: Giardia and Cryptosporidium (April 2004)
Table A.2. CT values (in mg @min/L) for 99.9% inactivation of Giardia la m blia cysts by free chlorine at 5°C
pH
Residual
(mg/L)
6.0
6.5
7.0
7.5
8.0
8.5
9.0
<0.4
97
117
139
166
198
236
279
0.6
100
120
143
171
204
244
291
0.8
103
122
146
175
210
252
301
1.0
105
125
149
179
216
260
312
1.2
107
127
152
183
221
267
320
1.4
109
130
155
187
227
274
329
1.6
111
132
158
192
232
281
337
1.8
114
135
162
196
238
287
345
2.0
116
138
165
200
243
294
353
2.2
118
140
169
204
248
300
361
2.4
120
143
172
209
253
306
368
2.6
122
146
175
213
258
312
375
2.8
124
148
178
217
263
318
382
3.0
126
151
182
221
268
324
389
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Protozoa: Giardia and Cryptosporidium (April 2004)
Table A.3. CT values (in mg @min/L) for 99.9% inactivation of Giardia la m blia cysts by free chlorine at 10°C
pH
Residual
(mg/L)
6.0
6.5
7.0
7.5
8.0
8.5
9.0
<0.4
73
88
104
125
149
177
209
0.6
75
90
107
128
153
183
218
0.8
78
92
110
131
158
189
226
1.0
79
94
112
134
162
195
234
1.2
80
95
114
137
166
200
240
1.4
82
98
116
140
170
206
247
1.6
83
99
119
144
174
211
253
1.8
86
101
122
147
179
215
259
2.0
87
104
124
150
182
221
265
2.2
89
105
127
153
186
225
271
2.4
90
107
129
157
190
230
276
2.6
92
110
131
160
194
234
281
2.8
93
111
134
163
197
239
287
3.0
95
113
137
166
201
243
292
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Protozoa: Giardia and Cryptosporidium (April 2004)
Table A.4. CT values (in mg @min/L) for 99.9% inactivation of Giardia la m blia cysts by free chlorine at 15°C
pH
Residual
(mg/L)
6.0
6.5
7.0
7.5
8.0
8.5
9.0
<0.4
49
59
70
83
99
118
140
0.6
50
60
72
86
102
122
146
0.8
52
61
73
88
105
126
151
1.0
53
63
75
90
108
130
156
1.2
54
64
76
92
111
134
160
1.4
55
65
78
94
114
137
165
1.6
56
66
79
96
116
141
169
1.8
57
68
81
98
119
144
173
2.0
58
69
83
100
122
147
177
2.2
59
70
85
102
124
150
181
2.4
60
72
86
105
127
153
184
2.6
61
73
88
107
129
156
188
2.8
62
74
89
109
132
159
191
3.0
63
76
91
111
134
162
195
Guidelines for Canadian Drinking Water Quality: Supporting Documentation
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Protozoa: Giardia and Cryptosporidium (April 2004)
Table A.5. CT values (in mg @min/L) for 99.9% inactivation of Giardia la m blia cysts by free chlorine at 20°C
pH
Residual
(mg/L)
6.0
6.5
7.0
7.5
8.0
8.5
9.0
<0.4
36
44
52
62
74
89
105
0.6
38
45
54
64
77
92
109
0.8
39
46
55
66
79
95
113
1.0
39
47
56
67
81
98
117
1.2
40
48
57
69
83
100
120
1.4
41
49
58
70
85
103
123
1.6
42
50
59
72
87
105
126
1.8
43
51
61
74
89
108
129
2.0
44
52
62
75
91
110
132
2.2
44
53
63
77
93
113
135
2.4
45
54
65
78
95
115
139
2.6
46
55
66
80
97
117
141
2.8
47
56
67
81
99
119
143
3.0
47
57
68
83
101
122
146
Guidelines for Canadian Drinking Water Quality: Supporting Documentation
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Protozoa: Giardia and Cryptosporidium (April 2004)
Table A.6. CT values (in mg @min/L) for 99.9% inactivation of Giardia la m blia cysts by free chlorine at 25°C
pH
Residual
(mg/L)
6.0
6.5
7.0
7.5
8.0
8.5
9.0
<0.4
24
29
35
42
50
59
70
0.6
25
30
36
43
51
61
73
0.8
26
31
37
44
53
63
75
1.0
26
31
37
45
54
65
78
1.2
27
32
38
46
55
67
80
1.4
27
33
39
47
57
69
82
1.6
28
33
40
48
58
70
84
1.8
29
34
41
49
60
72
86
2.0
29
35
41
50
61
74
89
2.2
30
35
42
51
62
75
90
2.4
30
36
43
52
63
77
92
2.6
31
37
44
53
65
78
94
2.8
31
37
45
54
66
80
96
3.0
32
38
46
55
67
81
97
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Protozoa: Giardia and Cryptosporidium (April 2004)
(b) 90% (1-log) inactivation
Table A.7. CT values (in mg @min/L) for 90% inactivation of Giardia la m blia cysts by free chlorine at 0.5°C
pH
Residual
(mg/L)
6.0
6.5
7.0
7.5
8.0
8.5
9.0
<0.4
46
54
65
79
92
110
130
0.6
47
56
67
80
95
114
136
0.8
48
57
68
82
98
113
141
1.0
49
59
70
84
101
122
146
1.2
51
60
72
86
104
125
150
1.4
52
61
74
89
107
129
155
1.6
52
63
75
91
110
132
159
1.8
54
64
77
93
113
136
163
2.0
55
66
79
95
115
139
167
2.2
56
67
81
99
118
142
170
2.4
57
68
82
99
120
145
174
2.6
58
70
84
101
123
148
178
2.8
59
71
86
103
125
151
181
3.0
60
72
87
105
127
153
184
Guidelines for Canadian Drinking Water Quality: Supporting Documentation
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Protozoa: Giardia and Cryptosporidium (April 2004)
Table A.8. CT values (in mg @min/L) for 90% inactivation of Giardia la m blia cysts by free chlorine at 5°C
pH
Residual
(mg/L)
6.0
6.5
7.0
7.5
8.0
8.5
9.0
<0.4
32
39
46
55
66
79
93
0.6
33
40
49
57
68
81
97
0.8
34
41
49
58
70
84
100
1.0
35
42
50
60
72
87
104
1.2
36
42
51
61
74
89
107
1.4
36
43
52
62
76
91
110
1.6
37
44
53
64
77
94
112
1.8
38
45
54
65
79
96
115
2.0
39
46
55
67
81
98
118
2.2
39
47
56
68
83
100
120
2.4
40
48
57
70
84
102
123
2.6
41
49
58
71
86
104
125
2.8
41
49
59
72
88
106
127
3.0
42
50
61
74
89
108
130
Guidelines for Canadian Drinking Water Quality: Supporting Documentation
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Protozoa: Giardia and Cryptosporidium (April 2004)
Table A.9. CT values (in mg @min/L) for 90% inactivation of Giardia la m blia cysts by free chlorine at 10°C
pH
Residual
(mg/L)
6.0
6.5
7.0
7.5
8.0
8.5
9.0
<0.4
24
29
35
42
50
59
70
0.6
25
30
36
43
51
61
73
0.8
26
31
37
44
53
63
75
1.0
26
31
37
45
54
65
78
1.2
27
32
38
46
55
67
80
1.4
27
33
39
47
57
69
82
1.6
28
33
40
48
58
70
84
1.8
29
34
41
49
60
72
86
2.0
29
35
41
50
61
74
88
2.2
30
35
42
51
62
75
90
2.4
30
36
43
52
63
77
92
2.6
31
37
44
53
65
78
94
2.8
31
37
45
54
66
80
96
3.0
32
38
46
55
67
81
97
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Protozoa: Giardia and Cryptosporidium (April 2004)
Table A.10. CT values (in mg @min/L) for 90% inactivation of Giardia la m blia cysts by free chlorine at 15°C
pH
Residual
(mg/L)
6.0
6.5
7.0
7.5
8.0
8.5
9.0
<0.4
16
20
23
28
33
39
47
0.6
17
20
24
29
34
41
49
0.8
17
20
24
29
35
42
50
1.0
18
21
25
30
36
43
52
1.2
18
21
25
31
37
45
53
1.4
18
22
26
31
38
46
55
1.6
19
22
26
32
39
47
56
1.8
19
23
27
33
40
48
59
2.0
19
23
28
33
41
49
59
2.2
20
23
28
34
41
50
60
2.4
20
24
29
35
42
51
61
2.6
20
24
29
36
43
52
63
2.8
21
25
30
36
44
53
64
3.0
21
25
30
37
45
54
65
Guidelines for Canadian Drinking Water Quality: Supporting Documentation
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Protozoa: Giardia and Cryptosporidium (April 2004)
Table A.11. CT values (in mg @min/L) for 90% inactivation of Giardia la m blia cysts by free chlorine at 20°C
pH
Residual
(mg/L)
6.0
6.5
7.0
7.5
8.0
8.5
9.0
<0.4
12
15
17
21
25
30
35
0.6
13
15
18
21
26
31
36
0.8
13
15
18
22
26
32
38
1.0
13
16
19
22
27
33
39
1.2
13
16
19
23
28
33
40
1.4
14
16
19
23
28
34
41
1.6
14
17
20
24
29
35
42
1.8
14
17
20
25
30
36
43
2.0
15
17
21
25
30
37
44
2.2
15
18
21
26
31
38
45
2.4
15
18
22
26
32
38
46
2.6
15
18
22
27
32
39
47
2.8
16
19
22
27
33
40
48
3.0
16
19
23
28
34
41
49
Guidelines for Canadian Drinking Water Quality: Supporting Documentation
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Protozoa: Giardia and Cryptosporidium (April 2004)
Table A.12. CT values (in mg @min/L) for 90% inactivation of Giardia la m blia cysts by free chlorine at 25°C
pH
Residual
(mg/L)
6.0
6.5
7.0
7.5
8.0
8.5
9.0
<0.4
8
10
12
14
17
20
23
0.6
8
10
12
14
17
20
24
0.8
9
10
12
15
18
21
25
1.0
9
10
12
15
19
22
26
1.2
9
11
13
15
18
22
27
1.4
9
11
13
16
19
22
27
1.6
9
11
13
16
19
23
28
1.8
10
11
14
16
20
23
29
2.0
10
12
14
17
20
24
29
2.2
10
12
14
17
21
25
30
2.4
10
12
14
17
21
25
31
2.6
10
12
15
18
22
26
31
2.8
10
12
15
18
22
26
32
3.0
11
13
15
18
22
27
32
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Protozoa: Giardia and Cryptosporidium (April 2004)
(c) 68.4% (0.5-log) inactivation
Table A.13. CT values (in mg @min/L) for 68.4% inactivation of Giardia la m blia cysts by free chlorine at 0.5°C
pH
Residual
(mg/L)
6.0
6.5
7.0
7.5
8.0
8.5
9.0
<0.4
23
27
33
40
46
55
65
0.6
24
28
33
40
48
57
68
0.8
24
29
34
41
49
59
70
1.0
25
29
35
42
51
61
73
1.2
25
30
36
43
52
63
75
1.4
26
31
37
44
54
65
77
1.6
26
32
38
46
55
66
80
1.8
27
32
39
47
56
68
82
2.0
28
33
39
48
55
70
83
2.2
28
34
40
50
59
71
85
2.4
29
34
41
50
60
73
87
2.6
29
35
42
51
61
74
89
2.8
30
36
43
52
63
75
91
3.0
30
36
44
53
64
77
92
Guidelines for Canadian Drinking Water Quality: Supporting Documentation
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Protozoa: Giardia and Cryptosporidium (April 2004)
Table A.14. CT values (in mg @min/L) for 68.4% inactivation of Giardia la m blia cysts by free chlorine at 5°C
pH
Residual
(mg/L)
6.0
6.5
7.0
7.5
8.0
8.5
9.0
<0.4
16
20
23
28
33
39
47
0.6
17
20
24
29
34
41
49
0.8
17
20
24
29
35
42
50
1.0
18
21
25
30
36
43
52
1.2
18
21
25
31
37
45
53
1.4
18
22
26
31
38
46
55
1.6
19
22
26
32
39
47
56
1.8
19
23
27
33
40
48
58
2.0
19
23
28
33
41
49
59
2.2
20
23
28
34
41
50
60
2.4
20
24
29
35
42
51
61
2.6
20
24
29
36
43
52
63
2.8
21
25
30
36
44
53
64
3.0
21
25
30
37
45
54
65
Guidelines for Canadian Drinking Water Quality: Supporting Documentation
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Protozoa: Giardia and Cryptosporidium (April 2004)
Table A.15. CT values (in mg @min/L) for 68.4% inactivation of Giardia la m blia cysts by free chlorine at 10°C
pH
Residual
(mg/L)
6.0
6.5
7.0
7.5
8.0
8.5
9.0
<0.4
12
15
17
21
25
30
35
0.6
13
15
18
21
26
31
36
0.8
13
15
18
22
26
32
38
1.0
13
16
19
22
27
33
39
1.2
13
16
19
23
28
33
40
1.4
14
16
19
23
28
34
41
1.6
14
17
20
24
29
35
42
1.8
14
17
20
25
30
36
43
2.0
15
17
21
25
30
37
44
2.2
15
18
21
26
31
38
45
2.4
15
18
22
26
32
38
46
2.6
15
18
22
27
32
39
47
2.8
16
19
22
27
33
40
48
3.0
16
19
23
28
34
41
49
Guidelines for Canadian Drinking Water Quality: Supporting Documentation
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Protozoa: Giardia and Cryptosporidium (April 2004)
Table A.16. CT values (in mg @min/L) for 68.4% inactivation of Giardia la m blia cysts by free chlorine at 15°C
pH
Residual
(mg/L)
6.0
6.5
7.0
7.5
8.0
8.5
9.0
<0.4
8
10
12
14
17
20
23
0.6
8
10
12
14
17
20
24
0.8
9
10
12
15
18
21
25
1.0
9
11
13
15
18
22
26
1.2
9
11
13
15
19
22
27
1.4
9
11
13
16
19
23
28
1.6
9
11
13
16
19
24
28
1.8
10
11
14
16
20
24
29
2.0
10
12
14
17
20
25
30
2.2
10
12
14
17
21
25
30
2.4
10
12
14
18
21
26
31
2.6
10
12
15
18
22
26
31
2.8
10
12
15
18
22
27
32
3.0
11
13
15
19
22
27
33
Guidelines for Canadian Drinking Water Quality: Supporting Documentation
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Protozoa: Giardia and Cryptosporidium (April 2004)
Table A.17. CT values (in mg @min/L) for 68.4% inactivation of Giardia la m blia cysts by free chlorine at 20°C
pH
Residual
(mg/L)
6.0
6.5
7.0
7.5
8.0
8.5
9.0
<0.4
6
7
9
10
12
15
19
0.6
6
8
9
11
13
15
18
0.8
7
8
9
11
13
16
19
1.0
7
8
9
11
14
16
20
1.2
7
8
10
12
14
17
20
1.4
7
8
10
12
14
17
21
1.6
7
8
10
12
15
18
21
1.8
7
9
10
12
15
18
22
2.0
7
9
10
13
15
18
22
2.2
7
9
11
13
16
19
23
2.4
8
9
11
13
16
19
23
2.6
8
9
11
13
16
20
24
2.8
8
9
11
14
17
20
24
3.0
9
10
11
14
17
20
24
Guidelines for Canadian Drinking Water Quality: Supporting Documentation
68
Protozoa: Giardia and Cryptosporidium (April 2004)
Table A.18. CT values (in mg @min/L) for 68.4% inactivation of Giardia la m blia cysts by free chlorine at 25°C
pH
Residual
(mg/L)
6.0
6.5
7.0
7.5
8.0
8.5
9.0
<0.4
4
5
6
7
8
10
12
0.6
4
5
6
7
9
10
12
0.8
4
5
6
7
9
11
13
1.0
4
5
6
8
9
11
13
1.2
5
5
6
8
9
11
13
1.4
5
6
7
8
10
12
14
1.6
5
6
7
8
10
12
14
1.8
5
6
7
8
10
12
14
2.0
5
6
7
8
10
12
15
2.2
5
6
7
9
10
13
15
2.4
5
6
7
9
11
13
15
2.6
5
6
7
9
11
13
16
2.8
5
6
8
9
11
13
16
3.0
5
6
8
9
11
14
16
Guidelines for Canadian Drinking Water Quality: Supporting Documentation
69
Protozoa: Giardia and Cryptosporidium (April 2004)
CHLORINE DIOXIDE
Table A.19. CT values (in mg @min/L) for inactivation of Giardia, pH 6.0– 9.0
Water temperature (°C)
Log
inactivation
#1
5
10
15
20
25
0.5
10
4.3
4.0
3.2
2.5
2.0
1.0
21
8.7
7.7
6.3
5.0
3.7
1.5
32
13
12
10
7.5
5.5
2.0
42
17
15
13
10
7.3
2.5
52
22
19
16
13
9
3.0
63
26
23
19
15
11
Guidelines for Canadian Drinking Water Quality: Supporting Documentation
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Protozoa: Giardia and Cryptosporidium (April 2004)
CHLORAMINE
Table A.20. CT values (in mg @min/L) for inactivation of Giardia, pH 6.0– 9.0
Water temperature (°C)
Log
inactivation
#1
5
10
15
20
25
0.5
635
365
310
250
185
125
1.0
1270
735
615
500
370
250
1.5
1900
1100
930
750
550
375
2.0
2535
1470
1230
1000
735
500
2.5
3170
1830
1540
1250
915
625
3.0
3800
2200
1850
1500
1100
750
Guidelines for Canadian Drinking Water Quality: Supporting Documentation
71
Protozoa: Giardia and Cryptosporidium (April 2004)
OZONE
Table A.21. CT values (in mg @min/L) for inactivation of Giardia
Water temperature (°C)
Log
inactivation
#1
5
10
15
20
25
0.5
0.48
0.32
0.23
0.16
0.12
0.08
1.0
0.97
0.63
0.48
0.32
0.24
0.16
1.5
1.5
0.95
0.72
0.48
0.36
0.24
2.0
1.9
1.3
0.95
0.63
0.48
0.32
2.5
2.4
1.6
1.2
0.79
0.60
0.40
3.0
2.9
1.9
1.43
0.95
0.72
0.48
Guidelines for Canadian Drinking Water Quality: Supporting Documentation
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Protozoa: Giardia and Cryptosporidium (April 2004)
ANNEX B:
UV Dose (IT) Table for the
Inactivation of Giardia and Cryptosporidium
Guidelines for Canadian Drinking Water Quality: Supporting Documentation
73
Protozoa: Giardia and Cryptosporidium (April 2004)
Table B.1. UV dose (mJ/cm 2) requirements for up to 3-log (99.9% ) inactivation of Cryptosporidium and
Giardia (U.S. EPA, 2003)
Log inactivation
Microorgan ism
0.5
1
1.5
2
2.5
3
3.5
4
Cryptosporidium
1.6
2.5
3.9
5.8
8.5
12
–
–
Giardia
1.5
2.1
3.0
5.2
7.7
11
–
–
Guidelines for Canadian Drinking Water Quality: Supporting Documentation
74
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