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Protozoain

Drinking Water

Document for Public Comment

Prepared by the Federal-ProvincialSubcommittee on Drinking Water

Comment Period EndsMarch 31, 1998

GIARDIA AND CRYPTOSPORIDIUMIN DRINKING WATER

What are Giardia and Cryptosporidium?

Giardia and Cryptosporidium are The symptoms of cryptosporidiosismicroscopic parasites that can be found in are similar; the most common being waterywater. Giardia causes an intestinal illness diarrhoea, abdominal cramps, nausea andcalled giardiasis or "beaver fever". headaches. These symptoms occur within 2Cryptosporidium is responsible for a similar to 25 days of infection and can last from oneillness called cryptosporidiosis. to two weeks or as long as a month.

How do these parasites cause illness? How can drinking water become

Both parasites produce cysts that arevery resistant to harsh environmental Giardia are often found in faecesconditions. When ingested they germinate, from humans, beaver, muskrat and dogs.reproduce and cause illness. After feeding, Cattle appear to be the primary source ofthe parasites form new cysts, which are then Cryptosporidium, although they have alsopassed in the faeces. Studies with human been found in humans and other animals. volunteers have shown that ingestion of only Drinking water sources becomea few of the cysts will cause illness. contaminated when faeces containing the

What are the symptoms?

Diarrhoea, abdominal cramps, gas, parasites to cause illness. Other sourcesmalaise and weight loss are the most include direct exposure to faeces of infectedcommon symptoms caused by Giardia. humans and animals, eating contaminatedVomiting, chills, headache and fever may food and accidental ingestion ofalso occur. These symptoms usually happen contaminated recreationalwithin 6 to 16 days of the initial

contact and can continue as long as a month.

contaminated with these parasites?

parasites are deposited or flushed into water. If water treatment is inadequate, drinkingwater may contain sufficient numbers of

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water. The comparative importance of these immunocompromised people in whom thevarious routes of exposure is unknown. illness could otherwise develop into a

Have these parasites been found inCanadian drinking water supplies?

Low levels of both parasites, month. Anti-diarrhoeal drugs andespecially Giardia, were detected in a rehydration therapy may be used if diarrhoeanational survey of drinking water conducted becomes severe. There are no approvedby Health Canada. Only a small fraction of drugs to fight the illness although many arethe parasites appeared to be viable. now being tested.Nevertheless, outbreaks linked to drinkingwater have been reported in severalprovinces. Their spread in swimming poolshas also been reported.

How can these waterborne illnesses beprevented?

Municipal drinking water treatment patients receiving immunosuppressive drugs. providing filtration and disinfection can For these people the symptoms are morereduce the risk of giardiasis and severe and can be life threatening. cryptosporidiosis. Protection of the rawwater supply is also beneficial. At present it is unknown whether

In the outdoors, water should be greater risk to waterborne Giardia andboiled for at least one minute before it is Cryptosporidium than the general public. used for drinking, food preparation or dental Nevertheless immunocompromisedhygiene. This will destroy Giardia and individuals should discuss these risks withCryptosporidium plus any other disease- their physicians. Those who wish to takecausing microorganisms that might be extra precautions can boil their water for 1present. Certain types of water filters can minute to kill any parasites that may beremove the parasites. present. This practice will also destroy any

Travellers to countries where the concern to these individuals. As bottledsafety of drinking water is suspect should water is not routinely monitored for Giardiaboil or disinfect and filter water that is to be and Cryptosporidium, its suitability as anused for drinking, food preparation or dental alternative to boiled tap water is unknown.hygiene.

How are these infections treated?

Giardia is usually cleared without and suspect that your symptoms may be duetreatment from healthy people within a to Giardia or Cryptosporidium, visit yourmonth. Anti-parasitic drugs are available physician and mention any exposure toand are particularly helpful to water, food or faeces that may have

persistent state.

Cryptosporidium will usuallydisappear from healthy people within a

What extra precautions canimmunocompromised people take?

Both parasites, but particularlyCryptosporidium, can pose a more seriousthreat to immunocompromised people suchas those with AIDS or cancer or transplant

immunocompromised individuals are at

other microorganisms that might be of

What should you tell your physician?

If you are suffering from diarrhoea

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been contaminated with the parasites. For more information:

What is Health Canada doing to ensurethe safety of our drinking water?

A consistent approach to improving Health)drinking water quality is provided by HealthCanada's Guidelines for Canadian DrinkingWater Quality (soon to include a guidelinefor Giardia and Cryptosporidium) which aredesigned to ensure that Canadians haveaccess to wholesome and safe drinkingwater.

Water Treatment Devices (It's Your Health)

Drinking Water Guidelines (It's Your

January 22, 1996

(formerly Issues) is one of a series of information sheetsproduced by the Health Protection Branch of Health Canada forthe public, media and special interest groups.

Aussi disponible en français.

------------------------------------------------------------------------------------------------------------------For additional copies of It's Your Health contact:Pour des copies supplémentaire de Votre santé et vous communiquez avec :

Health Canada/Santé CanadaPublicationsOttawa, Ontario K1A 0K9 Fax.: (613) 941-5366

Protozoa: Giardia and CryptosporidiumPublic Comment Document

Table of Contents

Purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Guideline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4Giardia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4Cryptosporidium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Sources and Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Giardia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Cryptosporidium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Analytical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Treatment Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Table 1. Minimum levels of disinfection and assumed log Giardia cyst removals

for various methods of filtration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Health Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Giardia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Cryptosporidium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Giardia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Cryptosporidium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Rationale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Health Canada For Public Comment(Federal–Provincial Subcommittee on Drinking Water) July 1997

Index of ANNEX A:

CT Tables for theInactivation of Giardia lamblia Cysts by

Chlorine, Chlorine Dioxide, Chloramine and Ozoneat Various Temperatures

CHLORAMINE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53CHLORINE

(a) 99.9% (3-log) inactivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34-39(b) 90% (1-log) inactivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40-45(c) 50% (0.5-log) inactivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46-51

CHLORINE DIOXIDE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52OZONE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54


July 1996(edited January 1997)

Protozoa: Giardia and Cryptosporidium

PurposeThe purpose of this document is to solicit comments on the draft Criteria

Summary on protozoa (Giardia and Cryptosporidium) in drinking water. For the pastseveral years, the Federal-Provincial Subcommittee on Drinking Water has beenassessing the available information with the intent of establishing a guideline forprotozoa in drinking water. This draft Criteria Summary recommends zero viableorganisms detectable per 1000 L, however, monitoring of drinking water is notrecommended at this time.

The Subcommittee has requested that this document be made available to thepublic and open for comment. The Subcommittee is soliciting comments on theapproach and potential costs of this proposed guideline. Comments are appreciatedwith accompanying justification, if required. In reviewing this document, key questionsyou should consider include:

• Does the water quality monitoring data accurately reflect the current situation inCanada? Are additional water quality data available?

• Is the risk assessment valid? Are there other studies that should be included inthe Criteria Summary?

• Will the proposed approach adequately protect public health? Is this a cost-effective solution?

It should be noted that this Criteria Summary will be part of a larger criteriadocument on Microbiological Quality, which will also include summaries onBacteriological Quality, Virological Quality and Guidance for Issuing and RescindingBoil Water Advisories.

GuidelineThe proposed maximum acceptable concentration (MAC) for Giardia and

Cryptosporidium in drinking water is zero viable organisms detectable per 1000 L.However, monitoring of drinking water is not recommended at this time, as the methodsavailable for the routine detection of cysts and oocysts and for the determination of theirviability and infectivity in drinking water are inadequate. If viable cysts or oocysts arepresent or suspected in treated waters and if the protozoan parasites have beenresponsible for past waterborne outbreaks in a community, a treatment regime and awatershed protection plan (where feasible) or other measures known to reduce the riskof illness should be implemented.


4

Description

Giardia Giardia is a small, flagellated, protozoan parasite (Phylum Protozoa, SubphylumSarcomastigophora, Superclass Mastigophora, Class Zoomastigophora, OrderDiplomonadida, Family Hexamitidae) that inhabits the small intestines of animals andhumans. The trophozoite, or feeding stage, lives mainly in the duodenum but is oftenfound 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 roundedanterior end, two nuclei, two slender median rods, eight flagella in four pairs, a pair ofdarkly staining median bodies and a large ventral sucking disk (cytostome).Trophozoites are normally attached to the surface of the intestinal villi, where they arebelieved to feed primarily upon mucosal secretions. After detachment, the binucleatetrophozoites form cysts (encyst) and divide within the cyst, so that four nuclei becomevisible. Cysts are ovoid, 8–14 µm long by 7–10 µm wide, with two or four nuclei andremnants of organelles visible. Environmentally stable cysts are passed out in thefaeces, often in large numbers. Giardia lamblia cysts can survive up to 77 days in tapwater at 8 C, but survival decreases with increasing temperature (54 days at 21 C1

and 4 days at 37 C). Giardia muris cysts remain viable for up to 2.8 months in riverwater when the temperature is <10 C and for approximately one month at 15–20 C inlake water. Cysts have no external features and are recognized by shape and visible2

internal morphology, as described above. Giardia cysts are highly resistant to chlorineand other oxidants commonly used for water treatment. Upon ingestion of the cysts bya suitable host, excystation is triggered by acid and enzymes in the stomach; by thetime the parasite reaches the duodenum, a quadranucleate mass of protoplasmemerges, which rapidly divides into two trophozoites from each cyst. Colonization of3

the small intestine then occurs by asexual reproduction.The taxonomy of the genus Giardia has recently been reviewed by Meyer, and3

the genus comprises at least four species: duodenalis (including lamblia=intestinalis),ardeae, muris and agilis, which have been reported from mammals, birds, rodents andamphibians. The name Giardia lamblia is commonly applied to isolates from humans.The most widely accepted species definition is still that proposed by Filice, who4

divided the Giardia found in warm-blooded animals into G. duodenalis and G. murisbased on the shape of the median body, an organelle composed of microtubules that ismost easily observed in the trophozoite. The species of Giardia are not easilydistinguished, and their host preferences have been widely debated — except for agilis,which is morphologically different, has been reported only from amphibians and is notregarded as infective to humans. For the purposes of this document, all Giardia foundin water are assumed to be potentially human infective (although this may not be true),because it is not possible to distinguish species in routine analysis.

Giardiasis is believed to be a zoonosis, although most of the evidence iscircumstantial or compromised by inadequate controls. It is known that beaver, dogs


5

and muskrat can become infected with human-source Giardia duodenalis, but the5–8

pathogenicity to humans for Giardia reported from birds, cattle, bears, cats and otherhosts is uncertain.

Giardia duodenalis can adapt to a variety of hosts, and, being asexual, itspopulation genetics are best described in terms of clonal expansion of virulent9

individuals within heterogeneous populations. Giardia duodenalis is capable of highrates of chromosomal rearrangement and may indeed be able to adapt to new hosts10

(and even to in vitro cultivation) more readily than other parasites. Giardia muris fromvoles and mice is generally regarded not to be infective to humans, although mice canbe used as an animal model for human Giardia isolates. The environmental resistance11

and prolonged viability of Giardia cysts in water at low temperature, the endemic natureof Giardia infections in humans and animals and cross-species transmission, togetherwith the low infectious dose needed to establish colonization within a new host, all pointtowards the potential for waterborne spread of this disease. At present, it is not12

possible to readily distinguish human-infective Giardia from other strains or species, soit is necessary to consider any Giardia spp. cysts found in water as being potentiallyinfectious to humans.12

CryptosporidiumCryptosporidium 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 in1976 in a previously healthy three-year-old child. A second case occurred two monthslater in an individual who was immunosuppressed as a result of drug therapy.13

Subsequently, the disease became best known in immunosuppressed individualsexhibiting the symptoms now referred to as Acquired Immune Deficiency Syndrome, orAIDS. The recognition of Cryptosporidium as a human pathogen led to increased14

research into the life cycle of the parasite and an investigation of the possible vectorsof 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, wheresporozoites are released from an excysting oocyst; merogony, where asexualreproduction 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.15

As a waterborne pathogen, the most important stage in the life cycle is theround, thick-walled, environmentally stable oocyst, 4–6 µm in diameter. There issometimes a visible external suture line, and the nuclei of sporozoites can be stainedwith fluorogenic dyes such as 4',6-diamidino-2-phenylindole (DAPI). Cryptosporidiumoocysts have been shown to survive in cold waters in the laboratory (4 C) for up to 18months and are even more resistant than Giardia cysts to oxidants such as chlorine16

and ozone. Robertson et al. report that C. parvum oocysts can withstand a variety of17

environmental stresses, including freezing (viability greatly reduced) and exposure toseawater. Upon ingestion by humans, the parasite completes its life cycle in the


6

digestive tract. Ingestion initiates excystation of the oocyst and releases foursporozoites, which adhere to the epithelial surface of the gastrointestinal tract.Conflicting evidence from electron microscopic studies has led to some disagreementas to whether the parasite is intracellular (invasive) or extracellular. All stages18–20

possess a feeding organelle that is protected along with the parasite body itself by anouter membrane. It is not certain whether the outer membrane is derived from the hostcell (intracellular) or of parasitic origin (extracellular). The sporozoite undergoesasexual reproduction at the site and develops into a gamete. Some of these gametesrelease microgametes, which fertilize other macrogametes to form zygotes. A smallnumber of zygotes retain a thin cell wall, which ruptures after the development of thesporozoites to aid in maintaining the infection within the host. The majority of thezygotes develop a thick cell wall and four sporozoites to become oocysts, which arethen passed in the faeces.

The first description of Cryptosporidium was made by Tyzzer, when he isolated21

the organism, which he named Cryptosporidium muris, from the gastric glands of mice.Tyzzer found a second isolate, which he named C. parvum, in the intestine of the22

same species of mice. This isolate was considered to be structurally anddevelopmentally distinct by Upton and Current. Although numerous species names23

have been proposed based on the identity of the host, all isolates of Cryptosporidiumfrom mammals, including humans, are similar to C. parvum as described by Tyzzer,22,23

although other investigators maintain that as many as five other species exist (C.parvum and C. muris, which infect mammals, C. baileyi and C. meleagridis, which infectbirds, C. crotali, infecting reptiles, and C. nasorum, infecting fish). Cryptosporidium24

muris has been reported from cattle in North America and Europe as well as fromcamels, deer and other animals. Experimental infections of dogs, cats and rabbits withC. muris have been described by Iseki et al. Cryptosporidium parvum is generally25

regarded as the major species responsible for clinical disease in humans and domesticanimals, and zoonotic transmission is possible, especially from lambs, calves and adultcattle. Symptomatic cryptosporidiosis has been reported from humans, cattle(common), lambs, goats, birds, horses and monkeys. Human-source Cryptosporidiumhas been shown to be infective to cattle and lambs. For the purposes of this18,23

document, all Cryptosporidium found in water are assumed to be potentially humaninfective (although this may not be true), because it is not possible to distinguishspecies in routine analysis.

Sources and Exposure

GiardiaGiardia is the most commonly reported intestinal parasite in North America and

in the world. Giardiasis has been shown to be endemic in humans and in over 4026,27

species of animals, with prevalence rates ranging from 1 to 90+%. The prevalence rateamong humans in Canada is typically 5–10%, but accurate rates are difficult toestimate because of the large number of asymptomatic cases and the inadequacy of28


7

reporting. Over 9000 confirmed cases of giardiasis are reported each year to theLaboratory Centre for Disease Control in Ottawa. Gyorkos reported 4.11% of29,30

112 714 (or 4110 per 100 000) stool samples to be positive for Giardia in 1984. Thenumber of cases actually notified in 1984 was only 27.8 per 100 000, suggesting thatthe real incidence of giardiasis is grossly under-reported.

The beaver has often been implicated in waterborne outbreaks of giardiasis.31–33

It is now clear, however, that other mammals, including dogs, muskrat, cattle andsheep, can also be responsible for the introduction of cysts to surface water that isused for drinking. As population pressures increase and as more human-related activityoccurs in catchment areas, the potential for faecal contamination becomes greater, andthe possibility of contamination with human sewage must always be considered.Erlandsen and Bemrick concluded that Giardia cysts in water may be derived from5

multiple sources and that epidemiological studies that focus on beavers may bemissing important sources of cyst contamination.

Despite the obvious potential for zoonotic transmission in Canada, mostwaterborne outbreaks have been traced back to human sewage contamination (e.g.,Temagami, Ontario ), although aquatic mammals have also been implicated as a34

source (e.g., Creston, B.C.; Corner Brook, Newfoundland) by circumstantialevidence. Ongerth et al. showed that there is a statistically significant relationship35,36 37

between increased human use of water for domestic and recreational purposes and theprevalence of Giardia in animals and water in the Olympic Mountains of Washington. Itis known that beaver and muskrat can be infected with human-source Giardia, and6

these animals are frequently exposed to raw or partially treated sewage in Canada. Ifaquatic mammals are contaminating lakes and rivers with human-infective Giardia, it isvery likely that they acquired the infection themselves from sewage-contaminatedwater. Beaver and muskrat undoubtedly play an important role in amplifying thecontamination of water with Giardia cysts when infected animals live in close proximityto water intakes, but human sewage is most commonly the source. Watershed12

management to control both sewage inputs and the populations of aquatic mammals inthe vicinity of water intakes is just as important to disease prevention as adequatewater treatment. It should also be remembered that, in addition to water, giardiasis canbe transmitted person-to-person via poor hygiene, food and sexual practices.

Giardia cysts are commonly found in sewage and surface waters andoccasionally in drinking water. Jakubowski et al. found that the concentration of cysts38

in raw sewage from 11 U.S. cities ranged from 683 to 3750 cysts/L. Rose et al. found39

Giardia cysts in 16% of 257 surface water samples at an average concentration of 3cysts/L. No cysts were detected in 36 drinking water samples. In a cross-Canadasurvey, Wallis et al. found that 56.2% of 162 raw sewage samples contained Giardia36

cysts ranging in concentration from 1 to 88 000 cysts/L, and 10% of 1215 raw andtreated drinking water samples contained 0.001–2 cysts/L. In samples at three sites ontwo rivers in the Montreal area, average Giardia cyst concentrations ranged from 0.07to 14 cysts/L. Average cyst levels in treated water prepared from the river waters were40

<0.002 cysts/L. Additional data from Quebec collected by the Ministry of the


8

Environment and Wildlife showed that 45% of polluted and 34% of pristine watersources were contaminated with Giardia cysts (total number of samples was 71), mostof which were from rivers. Between 1989 and 1993, the annual geometric mean41

concentration of Giardia cysts in raw water at two treatment plants in Edmonton rangedfrom 5 to 193 cysts/100 L and from 2 to 65 cysts/100 L, respectively. No cysts were42

detected in 1000 L of treated water collected at either plant. Hibler reported that 34%43

of 301 U.S. municipal sites were found to contain Giardia cysts in either raw or treatedwater from 1979 to 1986 and that there was a trend to more positive samples in thewinter months. LeChevallier et al. found Giardia cysts in 81% of 83 raw water samples44

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 samplefrom Alberta was included in the survey, and a concentration of 4.94 cysts/L wasreported. Concentrations in finished drinking water samples ranged from 0.29 to 64cysts/100 L. Isaac-Renton et al. has reported Giardia cyst contamination from a35,45,46

number of sites in British Columbia, some of which experienced waterborne outbreaksof giardiasis. Similar results have been reported from the Yukon and the Maritimes.47 36

Very few data are available from Ontario, but an outbreak of giardiasis occurred atTemagami in the spring of 1994 that was characterized by an attack rate of 30% andcyst concentrations up to 2 cysts/L in treated water.34

LeChevallier et al. also found significant positive correlations between cyst44

concentration and other raw water quality parameters, such as turbidity and total andfaecal coliform densities; however, data from previous studies do not support theseassociations, possibly because of early reports of waterborne giardiasis from placeslike Colorado, which normally experience very low turbidities in raw water.48

LeChevallier et al. concluded that cyst contamination could be modelled in terms of44

watershed characteristics and that water reuse and sewage contamination wereimportant factors in predicting cyst concentrations.

The viability of Giardia cysts found in water is commonly assumed to be high, butmonitoring experience suggests otherwise. Cysts found in surface waters are oftendead, as shown by propidium iodide dye exclusion (approximately 50% viability wasobserved using this technique during the Temagami outbreak), and water and sewageisolates infected only 9.4% of gerbils inoculated in the Canadian survey reported byWallis et al. It is possible that not all of the Giardia isolates tested were actually36

infective to gerbils, but it is common to observe cysts that are non-refractile underphase microscopy and that have obviously damaged cyst walls. Theimmunofluorescent technique commonly used for detection is very sensitive andfrequently reveals the presence of empty cysts (“ghosts”), particularly in sewage.Observations by LeChevallier et al. also suggest that most of the cysts present in49

water are non-viable; 40 of 46 cysts isolated from drinking water exhibited “non-viable-type” morphologies (i.e., distorted or shrunken cytoplasm).

Confirmed outbreaks of waterborne giardiasis in Canada have occurred inBritish Columbia, Alberta, Ontario, Quebec, New Brunswick and Newfoundland.Waterborne outbreaks of giardiasis have been suspected but not proven in a number of


9

other communities. Many of these communities were relying on surface water with onlychlorination for water treatment, but others had filtration plants that were not functioningproperly. In the United States, outbreaks have been reported from 24 states,50

especially Colorado and New England. During the period from 1965 to 1992, 115outbreaks were reported that resulted in 26 530 known cases of giardiasis in the UnitedStates. A number of these outbreaks occurred in Colorado, including epidemics at50,51

Vail, Estes Park, Lookout Mountain, Boulder and Aspen. Karlin and Hopkins reported48

that consumption of surface water, a lack of complete conventional treatment andimproper operation or malfunction of equipment were common causes of the outbreaks.These authors concluded that strict adherence to the multiple-barrier concept of watertreatment is essential. Craun, in an earlier study, identified reliance on surface water,52

minimal treatment (usually only chlorination) and inadequate treatment facilities ascommon causes of waterborne giardiasis. Small water treatment systems that usedotherwise good-quality surface water of low turbidity seemed to be most commonlyaffected. A useful review of some of the best-known U.S. outbreaks has been compiledby Lin. These include the very large outbreaks at Rome, New York (over 5000 cases53

were reported), and at Berlin, New Hampshire (7000 cases out of a total population of15 000). Lin concluded that these and other outbreaks had been caused by lack of53

filtration, improper filter operations, inadequate chlorination, cross-connections tosewers and drinking contaminated surface waters.

CryptosporidiumAs with giardiasis, cryptosporidiosis may be waterborne, foodborne or

transmitted sexually or by the faecal–oral route. Prevalence rates of humancryptosporidiosis range from 0.6 to 20%, based on stool samples. A survey of 134654–57

Canadian patients revealed a prevalence rate of 1.25% in 1985, but data are limited58

because the disease is not universally reportable. Infection rates for patients with AIDSare reported to be at 4% in the United States and 2.5% in Canada. The parasite in58,59

immunocompetent individuals does not seem to be more prevalent among anyparticular age group, although the probability of seroconversion increases with age.60

Cryptosporidium oocysts have been reported in wastewater (3.3–20 000/L),surface waters receiving agricultural or wastewater discharges (0.006–2.5/L), pristinesurface water (0.02–0.08/L), drinking water (0.006–4.8/L) and recreational water(0.66–500/L) by various studies as summarized by Smith. Similar results were24

reported by Madore et al. in the United States. Rose et al. found oocysts in 55% of61 39

257 surface water samples at an average concentration of 43 oocysts/L and in 17% of36 drinking water samples at concentrations ranging from 0.5 to 1.7 oocysts/L. Walliset al. found that 11.1% of 162 raw sewage samples contained Cryptosporidium36

oocysts ranging in concentration from 1 to 120/L, and 6.4% of 1215 raw and treateddrinking water samples contained 0.001–0.005 oocysts/L. In samples from three siteson two rivers in the Montreal area, average Cryptosporidium oocyst concentrationsranged from <0.02 to 7/L. Between 1989 and 1993, the annual geometric mean40

concentration of Cryptosporidium oocysts in raw water at two treatment plants in


10

Edmonton ranged from 5 to 83 and from 2 to 52 oocysts/100 L, respectively. Oocysts42

were not detected in 1000 L of treated water collected at either plant. LeChevallier etal. reported that 87% of 83 raw surface water samples and 27% of 83 filtered water44,49

samples taken from the north-eastern United States contained Cryptosporidiumoocysts. Concentrations in raw water ranged from 0.07 to 484 oocysts/L. One raw watersample from Alberta contained 0.34 oocysts/L. As with Giardia cysts, LeChevallier etal. concluded that water reuse and sewage contamination were important predictors44,49

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 environmentalstress, but this is probably incorrect. Viability is probably less than 100%, as shown bySmith et al., who found that oocyst viability in surface waters is often very low and of62

little public health significance. This point of view is supported directly by LeChevallieret al., who found that 21 of 23 oocysts in filtered waters had “non-viable-type”49

morphology (i.e., absence of sporozoites and distorted or shrunken cytoplasm). Nooutbreaks of cryptosporidiosis occurred in any of the municipalities included in theLeChevallier et al. survey. Similarly, Sorvillo et al. concluded that municipal drinking49 63

water was not an important risk factor for cryptosporidiosis in AIDS patients residing inLos Angeles County on the basis of epidemiological data collected before and after theintroduction of filtration in a major water supply for the area.

Four waterborne outbreaks have been reported in the United Kingdom (as wellas three other suspected outbreaks), six in the United States (including Milwaukee,Wisconsin; Carrollton, Georgia; and Braun Station, Texas) and one in Canada(Kitchener–Waterloo). Attack rates were typically high, ranging from 26 to 40%, and64–68

many thousands of people were affected. In addition, there have been severaloutbreaks associated with swimming pools, wave pools and lakes. The only otherCanadian outbreak was reported from a swimming pool in British Columbia.69,70

Analytical MethodsGiardia and Cryptosporidium can be detected simultaneously, but existing

methodology is only semi-quantitative. Most water samples contain few cysts, andconcentration techniques are required to obtain even a small number of cysts oroocysts, many of which may be dead. Neither organism can be reliably cultured from awater sample, although excystation and culture procedures have been established forboth. Giardia can be excysted using acid and enzymes such as trypsin and grown inTYI-S-33 medium, but the excystation rate for G. duodenalis is often low.71,72

Cryptosporidium parvum oocysts can also be excysted and used to infect bovine kidneycells in vitro, but this technique also requires a relatively large number of oocysts and73

is too tedious for routine detection. Both parasites can be used to infect experimentalanimals such as the gerbil (for Giardia) or neonatal CD-1 mice, and this technique is74 75

useful for pilot plant studies or isolate collection; however, most analytical laboratoriesdo not maintain animal colonies, and the expense is high. Culturing and animalinfection are therefore more useful for research purposes than for routine monitoring.


11

The analysis of protozoan parasites in water and wastewater samples reliesupon direct microscopic detection after concentration of particulate matter by filtrationor centrifugation. Sample concentration is usually accomplished by filtration through a1-µm porosity wound filter or membrane, although tangential flow filters and76,77 78–80 81

even centrifugal cream separators have been used. In each method, cysts and82

oocysts are trapped along with algae, silt, organic matter and other aquatic organismseither on a filter or in a concentrated pellet. Sewage samples often contain largenumbers of cysts and oocysts and can often be analysed by centrifuge concentrating asmall sample. If a filter is used for raw or treated drinking water, approximately 1000 Lare pumped through a 1-µm wound filter, and particulate matter is recovered bybackflushing, rinsing, handwashing or machine processing (using a stomacher bag )44,49

and concentrated into a pellet. Membrane filters offer higher recovery efficiencies, butthe amount of water that can pass through without filter clogging is small, often only10–20 L. Membrane filters are useful, however, because they retain more material andmay be dissolved to recover cysts and oocysts. The background material in the pellet83

is then reduced by discontinuous density gradient centrifugation using either zincsulphate (S.G. 1.18), 1.0 M sucrose (S.G. 1.15) or a mixture of Percoll (PharmaciaBiotech) and sucrose (S.G. 1.09). Samples may be either underlaid with a long needleor pipetted carefully onto the surface of the density medium. Centrifugation will causedenser particles to pass through the density medium and form a pellet at the bottom ofthe tube. Theoretically, Giardia cysts and Cryptosporidium oocysts will float on thesurface of the density medium and may be recovered by pipette. Practically, the use ofdensity media introduces significant errors. Dead Cryptosporidium oocysts tend topenetrate the density flotation medium and accumulate in the pellet. Sucrose and zincsulphate density media selectively concentrate viable oocysts, whereas84

Percoll–sucrose concentrates empty (ghost) oocysts. The recovered material is then85

centrifuge concentrated again, and the final pellet is examined microscopically.Before specific antibodies became commercially available, the most common

detection procedure relied upon staining with Lugol’s iodine, and Giardia cysts wereidentified on the basis of shape, size and visible internal morphology (nuclei, axostyles,median body). Cryptosporidium oocysts stain very poorly with Lugol’s iodine and arevery difficult to find and identify with this method. The use of antibodies specific forGiardia cysts and Cryptosporidium oocysts greatly enhanced the probability of findingthe organisms against a cluttered background and making a positive identification.Immunofluorescent staining is usually performed by trapping a portion of the pellet on asmall membrane and rinsing antibodies through, but it can also be carried out incentrifuge tubes or microscope slides. Unfortunately, there are some algae that44,49,86,87

are very close in size and staining characteristics to cysts and oocysts, and finalidentification often requires light, phase and differential interference microscopy inaddition to immunofluorescence. Murine monoclonal antibodies are commerciallyavailable from several manufacturers (Meridian Diagnostics Ltd., Cellabs Pty. Ltd.,Waterborne Inc.) in both direct and indirect fluorescence kits. The intercalation of afluorogen with DNA highlights nuclei and aids identification, and fluorescent imaging88


12

of Cryptosporidium using a cooled charge couple device has been applied with somesuccess in the United Kingdom.89

Detection of Giardia cysts and Cryptosporidium oocysts by immunofluorescencerequires specialized equipment and a high level of technical skill. The analysis istedious, expensive and only semi-quantitative, but it has been used to confirmwaterborne transmission of both parasites in many outbreaks and is being continuouslyimproved through the efforts of the U.S. Environmental Protection Agency (EPA),Health Canada and independent researchers. The method provides a useful monitoringtool, but further improvements are necessary before the technique becomes fullyquantitative and cost-effective for independent laboratories.

Alternative techniques have been proposed, including analysis by flowcytometry, which still requires some concentration and recovery steps but automatesthe detection procedure. This method requires expensive and specialized equipment90

and is subject to error unless the identification of cysts and oocysts is verified bymicroscopy. A second alternative may be offered by immunomagnetic separation,91

although commercially available antibody-bound magnetizable particles are notavailable at present.

Quality control is still in a developmental stage for the immunofluorescenceprocedure. Internal controls should include both positive and negative samples, andantibodies must be used at the correct dilution. Clancy et al. conducted a blind survey92

(analysis of spiked filter samples) of 16 commercial labs in the United States and foundthat recovery of Giardia cysts ranged from 0.8 to 22.3% (average 9.3%) and thatrecovery of Cryptosporidium oocysts ranged from 1.3 to 5.5% (average 2.8%). HealthCanada recently commissioned a similar study of commercial, government andresearch laboratories in Canada, and Giardia cyst recovery ranged from 0 to 90%(average 21%) for eight laboratories analysing 10 unknown samples. Cryptosporidiumoocyst recovery ranged from 0 to 43% (average 5.3%) for the same samples.93

LeChevallier et al. conducted a critical analysis of the immunofluorescence method85

and concluded that losses of Cryptosporidium oocysts typically exceed losses ofGiardia cysts and that major losses occur during centrifugation and clarification.

The most cost-effective method for detection remains the analysis of wound filtercartridges, as described in Standard Methods.77

Treatment TechnologyThe removal and inactivation of Giardia cysts and Cryptosporidium oocysts from

raw water are complicated by their small size and resistance to commonly usedoxidants such as chlorine. Cryptosporidium oocysts are more difficult to eliminate butappear to be less common in Canadian surface waters. The detection procedure is lessefficient for Cryptosporidium oocysts, however, and the national prevalence rate maybe higher than suspected. Waterborne outbreaks of giardiasis and cryptosporidiosishave resulted both from inadequate treatment and from improper operatingprocedures. The multiple-barrier approach to water treatment is by far the best53

approach to elimination of these parasites and other waterborne pathogens. An


13

exhaustive review of available treatment options is beyond the scope of this document.These methods have been reviewed recently in water treatment manuals prepared byUMA Engineering Ltd. et al., Health Canada and the U.S. EPA.94 95 96

Effective water treatment begins with watershed management to minimize theinput of faecal contamination from animal and human sources, by controlling aquaticmammal populations and locating raw water intakes as far as possible from sewageoutfalls. The possible flooding of sewage collection and treatment systems cannot beoverlooked, and sudden increases in indicator organisms can give advance warning ofproblems (e.g., the outbreak at Temagami, Ontario). Pre-disinfection, clarification,coagulation, filtration (including direct filtration) and post-disinfection are all commonlyused to good effect in municipal water treatment plants to remove Giardia cysts, butproblems can still occur with Cryptosporidium oocysts (because of their small size andresistance to oxidants). There are approximately 1000 communities in Canada that relyupon chlorination alone with varying degrees of watershed management, includingsome major cities.

The efficacy of disinfection can be predicted based on a knowledge of theresidual concentration of disinfectant, temperature, pH (for chlorine only) and contacttime 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 inmg/L) and T (the disinfectant contact time, measured in minutes). CT values forchlorine, chlorine dioxide, chloramine and ozone developed by the U.S. EPA to achievevarious degrees of inactivation of Giardia are provided in Annex A. It is possible toreduce the viability of Giardia cysts by 99.9% using chlorination alone, but long contacttimes are required. Ozone and chlorine dioxide are much better disinfectants, but bothare expensive and result in the formation of unwanted by-products (particularly chlorite,in the case of chlorine dioxide). Ozone is a better choice but is unreliable when turbidityis high or variable, because cysts are protected in flocculated particles. Chloramineshould not be used as a primary disinfectant. A discussion of the effect of thesevariables can be found in the water treatment manuals mentioned above or in vonHuben for chlorine, chlorine dioxide, chloramine and ozone.97

Inactivation of Cryptosporidium oocysts by chlorination alone is impractical, butozonation may be effective when used properly. A discussion of the effects of98

ozonation and other water treatment processes may be found in Smith et al.99

Preliminary work carried out by Finch et al. has shown that chlorination followed by100

chloramination is more effective than previously believed and can inactivateCryptosporidium oocysts by up to 1.6 logs when viability is measured by infection ofmice. The use of the two disinfectants sequentially gave better results than wereobtained when either was used by itself. Ozonation followed by chloramination wasparticularly effective. Preliminary data show that ultraviolet irradiation can inactivate upto 100% of viable Cryptosporidium parvum oocysts in clean water and suggest that101

this process may be very useful for water already treated to remove particles.Filtration with the aid of coagulation/flocculation is the most practical method to

achieve high removal/inactivation rates of cysts and oocysts. Payment and Franco40


14

showed that 99.998% of Giardia cysts and Cryptosporidium oocysts were removed fromheavily polluted water by full conventional treatment (flocculation, settling, pre- andpost-disinfection with chlorine dioxide and chlorine and filtration) at three Montrealwater treatment plants. Slow sand and diatomaceous earth filtration can also be highlyeffective. Filter backflushing must be carried out regularly, and the backflush watershould not be recirculated through the treatment plant. Pressure filters vary widely intheir 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 bemade by experienced engineers after suitable pilot testing. An effective system ofoperator training and process control are essential in areas of known contaminationwhere the risk is high. Regular monitoring of raw water for cysts and oocysts is usefulfor establishing prevalence, and analysis of treated water provides an indication of riskif viability assays are incorporated. Useful data for process control may also beobtained by monitoring.

In the United States, the EPA has promulgated the Surface Water TreatmentRule to control the presence of Giardia and viruses in public drinking water systemsusing either surface water or groundwater under the influence of surface water. All102

systems using filtered or unfiltered surface water must achieve at least a 99.9% (3-log)removal and/or inactivation of Giardia lamblia cysts. This level of removal/inactivationwas believed to reduce the risk of waterborne giardiasis to less than 10 (i.e., <1 in4

10 000 people infected) per year. Under the rule, a public water system using surfacewater must use filtration unless it meets certain water quality, operational and publichealth standards. It is assumed that filtration removes 99% (2-log) of Giardia cysts andthat disinfection need only provide a further 90% (1-log) inactivation (see Annex A).Systems using conventional treatment that are able to achieve turbidity levels of lessthan 0.5 NTU in the filtered water in 95% of samples are assumed to achieve 2.5-logremoval of Giardia cysts, providing that coagulation and flocculation conditions areoptimized for turbidity removal. Disinfection in these systems need only inactivate 50%(0.5-log) of Giardia cysts (see Annex A). Recommended minimum levels of disinfectionand assumed log Giardia cyst removals by filtration method are given in Table 1.

Table 1. Minimum levels of disinfection and assumed log Giardia cyst removalsfor various methods of filtration

Treatment Assumed log removal Minimum disinfection

Conventional 2.5 0.5

Direct filtration 2.0 1.0

Slow sand filtration 2.0 1.0

Diatomaceous earth 2.0 1.0filtration


15

Recognizing that systems with very poor source water may not be adequatelyprotected by a 3-log reduction in Giardia cysts and that the requirements for Giardiareduction may not be applicable to Cryptosporidium, the U.S. EPA has proposed anEnhanced Surface Water Treatment Rule.103

Health Effects

Giardia The prepatent time for giardiasis is 6–16 days, and the minimal infective104–106

dose can be as low as 1–10 cysts, although there are large differences between104,106

isolates of the parasite in virulence and antigenic diversity. The ID (number of cysts10750

ingested resulting in 50% of the test subjects becoming infected) was found to be 19cysts by Rendtorff (calculated from his data) using human-source cysts in humans,104

but it can be as high as 543 for human-source Giardia in gerbils. Giardia strains thatare well adapted to their hosts (e.g., by serial passage) can frequently infect with 50cysts or less. Research with animal models has shown that smaller inocula result in108

longer prepatent times but do not influence the resulting parasite burden.109

Exposure to the parasite resulted in partial or total immunity for periods of up to21 weeks in mice. Olson et al. observed lower cyst output and greater weight109,110 111

gain in kittens immunized subcutaneously. Humoral immune response is revealed byincreased levels of circulating IgG and IgM antibodies and secretion of IgA in milk,saliva and possibly intestinal mucus. These immune products are active in eliminatingdisease, but lasting immunity has not been demonstrated. Very little is known about112

cellular immunity, but spontaneous killing of trophozoites by human peripheral bloodmonocytes has been described. The host–parasite relationship is complex, and113

Giardia has been shown to be versatile in the expression of antigens, so universal107

lasting immunity is improbable. Olson et al. have shown that potential for a vaccine111

exists, but infections and symptoms are only attenuated, and prevention of infection isnot feasible at this time. Symptoms include nausea, anorexia, an uneasiness in theupper intestine, malaise and perhaps low-grade fever or chills. The onset of diarrhoeais usually sudden and explosive, with watery and foul-smelling stools. The acute114

phase of the infection commonly resolves spontaneously, and organisms maydisappear from the faeces. Some patients become asymptomatic cyst passers for aperiod and have no further clinical manifestations. Other patients, particularly children,suffer recurring bouts of the disease that may persist for months. Giardiasis can be115

treated using a number of drugs, including metronidazole, quinacrine, furazolidone,tinidazole, ornidazole and nimorazole.

Lengerich et al. recently evaluated hospitalization rates for severe giardiasis115

in the United States. An estimated 4600 persons were hospitalized annually, a ratesimilar to that of shigellosis. The median length of hospital stay was four days.


16

CryptosporidiumAlthough a complete pathogenesis for Cryptosporidium spp. in humans has yet

to be determined, more information is becoming available through the study of bothimmunocompetent individuals and AIDS patients. DuPont et al. found that 18 of 29116

healthy volunteers became infected after administration of doses of from 30 to1 000 000 oocysts, 39% of which were asymptomatic. The ID was 132 oocysts, and50

61% of the infected subjects experienced enteric symptoms. This value compares wellwith an ID of 79 oocysts reported for a bovine strain of C. parvum in CD-1 mice,50

75

although the minimum infective dose of oocysts required to produce infection inanimals ranges in published research data from 10 to 100 oocysts. In the DuPont117–119

et al. study, the prepatent time ranged from 2 to 25 days (although most occurred116

within 3–11 days), with the shortest time to cyst excretion occurring with an inoculum of1 000 000 oocysts. All recovered spontaneously. Similarly, an investigation of aCryptosporidium infection in travellers returning from the Caribbean indicated aprepatent time of 4–9 days. The usual symptom associated with the disease is120

diarrhoea, characterized by very watery stools that are non-bloody. The volume ofdiarrhoea can be extreme, with 3 L/d being common and with reports of up to 17 L/d.121

This symptom can be accompanied by nausea, vomiting (particularly in children), low-grade fever (below 39 C), anorexia and dehydration. The symptoms reported from awaterborne outbreak are diarrhoea (100%), abdominal cramps (76%), nausea (45%),vomiting (19%), fever (14%), headache (29%) and muscle aches (13%). Some66

protective immunity appears to develop in infected populations. The primarymechanism of host defence appears to be cellular immunity, although humoralimmunity is also known to be involved.58

The duration of infection is dependent on the condition of the immune systemand can be broken down into (1) AIDS patients who in most reported cases nevercompletely clear the infection (it may develop into an infection with long bouts ofremission followed by mild symptoms) and (2) individuals who are immunosuppressedfollowing chemotherapy, short-term depression or illness (e.g., chicken pox) ormalnutrition. In those cases where the immunosuppression is not AIDS related, theinfection usually clears (no cyst excretion, and symptoms disappear) within 10–15 daysof the time the immune system returns to normal, although there have been reportedcases involving children in which the infection has persisted for up to 30 days. Thesensitivity of diagnosis of cryptosporidiosis by stool examination is low, so low cystexcreters may be counted as negative prematurely. Immunocompetent individualsusually 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 daycarefacility, children shed oocysts for up to five weeks. The reported rate of asymptomatic122

infection is believed to be low, but a report on an outbreak at a daycare facility inPhiladelphia concluded that up to 11% of the children were asymptomatic, and123

Ungar discusses three separate studies in daycare centres where the asymptomatic124

infection rate ranged from 67 to 100%. It has been suggested that many of theseasymptomatic cases were mild cases that were incorrectly diagnosed. In AIDS121


17

patients, Juranek showed that only 13% (5/39) of patients with CD4 cell counts of67

<180 cells/mm had self-limiting disease, but 100% (8/8) of those with counts3

>180 cells/mm had infections that cleared.3

Infections of Cryptosporidium spp. in the human intestine are known to causedamage to the mucosa, including villous atrophy and lengthening of the crypt. Most of18

the pathological data available have come from AIDS patients, and the presence ofother opportunistic pathogens has made assessment of damage attributable toCryptosporidium spp. difficult. There is some suggestion in the literature that outbreaksof cryptosporidiosis are of seasonal duration. This has yet to be addressed byinvestigation, but the seasons reported, by country, are as follows: February–April(Great Britain), April–July (Bangladesh), May–July (United States) and May–October(Italy).56,66,125,126

No effective antimicrobial treatment for Cryptosporidium spp. in humans hasbeen found, although more than 120 drugs have been tested. Some progress has18,127

been reported with furazolidone in reducing the symptoms of immunocompetentpatients. Spiramycin has apparently been used with some success in Chile and theUnited States, but at this time it is not licensed for general use by the U.S. Food andDrug Administration. A functional immune system will usually eliminate symptoms and58

organisms spontaneously, but the immunocompromised individual may suffer fromlong-term chronic infection.

During the Milwaukee cryptosporidiosis outbreak, investigators surveyed 285patients with laboratory-confirmed infection. Of these, 130 were hospitalized, including48 immunocompromised patients.64

AssessmentGiardia and Cryptosporidium are infectious protozoans that can be transmitted

by water, poor hygiene, sexual activities and food. Both these organisms are entericpathogens that cause serious illness in immunocompetent and immunocompromisedindividuals. Cryptosporidiosis is the more serious of the two because (1) most casesare symptomatic, (2) there is no effective drug treatment and (3) the illness is capableof causing death. Cryptosporidium parvum may be fatal in immunocompromisedindividuals, particularly those suffering from AIDS.

The risk of giardiasis and cryptosporidiosis can be predicted usingdose–response curves from human and animal infection experiments. The risk ofbecoming infected by protozoan cysts or oocysts in drinking water depends upon (1)the number of cysts or oocysts ingested (dose), (2) the viability of the cysts or oocystsand (3) the susceptibility of the host population to infection. The dose can be estimatedbased upon measurements of cyst or oocyst concentrations in drinking water andamount of water consumed over the period of exposure. The viability of cysts oroocysts can also be estimated using dye exclusion, excystation or animal infectionassays. The susceptibility of individuals in the host population varies by age,immunological status, history of exposure and other genetic and environmental factors.These factors vary widely between individuals, so it is only meaningful to calculate the


18

risk to populations in terms of the number of individuals that may become infected. Riskis therefore calculated as a probability that is applied to an exposed population, and itis assumed that host susceptibility varies over a continuum.

GiardiaThe application of mathematical risk modelling to waterborne giardiasis infection

has been proposed by Regli et al. and Rose et al. based on earlier work by Haas128 39 129

and Rendtorff. This model has been modified using data from Canadian outbreaks by104

Wallis et al. The exponential model proposed by these investigators makes several36

assumptions. First, the model assumes that the distribution of cysts in water is Poisson(random). Second, it is assumed that one cyst is capable of causing an infection andthat there is constant susceptibility to infection in the human population. Finally, allcysts ingested are considered to be viable and pathogenic to humans, which isprobably not true in most cases, but it is assumed that errors caused by inefficiency incyst recovery during the analytical procedure are counterbalanced by overestimates inviability and pathogenicity. Based on these assumptions, the probability (P) of ani

infection resulting from the ingestion of a single volume of liquid (V) containing µorganisms per litre can be described by a simple exponential probability densityfunction according to the model as:

P = 1 e (1)irµV

where r is the fraction of cysts that are ingested that survive to initiate infections. Walliset al. reviewed the data available to calculate the value of r and has proposed that a36

value of r = 0.0105 be used based on both human and gerbil infection studies. The useof gerbil infection data may be questioned, but these data show that different isolateshave markedly different ID values within genetically similar hosts. Until more human50

dose–response data become available, it is proposed that this value be used as themost comprehensive estimate of the infectivity of Giardia in humans, recognizing thatboth host and parasite are variable in their response.

The probability that less than 1 person in 10 000 per year will become ill afterexposure to Giardia in drinking water is considered an acceptable level of risk. This128

corresponds to an acceptable daily risk of 2.75 × 10 or, rearranging equation 1, to an7

acceptable daily intake of N cysts (N = µV) equivalent to:

N = 1 ln (1 P) = 0.000026 cysts (2)i

r

If it is assumed that each person consumes 1.5 L of tap water per day, then thetheoretical 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 methodsand would require filtration of about 60 000 L of water to detect a single cyst. A morepractical approach would be to monitor the source water for cysts and determine the


19

adequacy of treatment by comparing existing treatment with published treatmentguidelines. For example, water treatment plants maintaining 4-, 3- or 2-log94–96

reduction could accept raw water levels of 17, 1.7 or 0.17 cysts/100 L, respectively, andcontinue to maintain an annual risk of illness of less than 1 × 10 .4

Alternatively, if the acceptable daily probability of becoming ill after ingestingcysts is 1 × 10 , then, applying equation 2, N = 0.0095 cysts. Again, if it is assumed4

that the per capita daily ingestion of tap water is 1.5 L, the theoretical MAC for Giardiawould be 6.3 cysts/1000 L. This concentration would require filtration of about 150 L oftap water to detect a single cyst. Consequently, water treatment plants maintaining 4-,3- or 2-log reduction of cysts could process raw water with levels of 6300, 630 or 63cysts/100 L, respectively, and maintain a daily risk of illness of less than 1 × 10 .4

If the mean concentration of cysts in tap water was 6.3/1000 L, the risk ofcontracting one or more illnesses annually (P ) can be calculated as follows:A

P = 1 [1 P(N)] = 0.036 (3)A ix

where x = 365 days and P(N) is the daily risk when N = 0.0095 cysts. Thesei

assumptions predict that 36 cases of giardiasis would occur in a population of 1000over one year if the concentration of cysts was constantly 6.3 cysts/1000 L. Monitoringdata by Wallis et al. have shown that cyst concentrations averaging 3 cysts/1000 L,36

which produce a theoretical daily risk of 4.75 × 10 and a theoretical annual risk of5

0.0172 (17 cases in 1000), do not cause detectable levels of waterborne giardiasis(outbreaks of disease reported). Based on known waterborne outbreaks of giardiasis inCanada, Wallis et al. have proposed an action level of 3–5 cysts/100 L.130

The theoretical rate predicted by the model is reduced to the much lowerreported rate from official data by several factors. These could include (1) variableviability and infectivity of cysts, (2) variable susceptibility of the host population toinfection, (3) asymptomatic cases, (4) use of effective treatment devices in the home orother sources of drinking water and (5) incomplete reporting.

Monitoring data reported by others in the absence of outbreaks present similarlevels of Giardia cysts in treated drinking water. Although an annual rate of 1740,42,49

cases per 1000 people may not cause noticeable levels of giardiasis, drinking watertreatment officials are understandably reluctant to be responsible for outbreaks thatcould affect tens of thousands of people annually in larger centres.

Until better monitoring data and information on the viability and infectivity ofcysts and oocysts present in drinking water are available, measures to reduce the riskof illness as much as possible should be implemented. A treatment plant employingeffective filtration and disinfection and watershed protection (where feasible) canachieve an adequate level of protection.

CryptosporidiumThe risk model described for Giardia can also be applied to Cryptosporidium, but

there are fewer data available from dose–response experiments, and there has been


20

no opportunity to test the model under outbreak conditions. An excellent data set wasrecently published by DuPont et al., which permits a calculation of an r value of116

0.0053. This value is close to the value for Giardia (r = 0.0105), and the resultingcalculations are almost the same after rounding. An action level of 10–30 oocysts/100 Lhas been proposed by Haas and Rose ; the authors recognize that a “de minimus”131

level of risk would be substantially lower than the action level. Using thedose–response data of DuPont et al., Haas et al. have further determined that an116 132

acceptable daily intake of oocysts is 6.54 × 10 . Assuming a daily tap water5

consumption rate of 1.5 L/person, the theoretical MAC would be 4.4 × 10 2

oocysts/1000 L. Drinking water treatment plants achieving a 4-, 3- or 2-log reductioncould accept raw water containing 44, 4.4 or 0.44 oocysts/100 L, respectively, andmaintain an annual risk of illness of less than 1 × 10 .4

RationaleThe proposed MAC for Giardia and Cryptosporidium is zero viable organisms

detectable per 1000 L. This guideline is based on the following:(1) Giardia and Cryptosporidium are waterborne parasites. Ingestion of as few as

one cyst or oocyst can initiate gastroenteritis. In healthy individuals, the illness isusually self-limiting, but it can be fatal to the immunocompromised individual.Waterborne outbreaks caused by either parasite are common in Canada.

(2) The application of monitoring data to risk assessment models indicates thatan average concentration of 3 Giardia cysts/1000 L corresponds to an annual rate ofillness of 0.0172 (i.e., 17 cases in 1000 exposed people). Based on comparableprevalence and infectivity data, it is assumed that Cryptosporidium presents a similarlevel of risk.

(3) Current standard methods for the routine detection of the parasites in watersuffer from low recovery rates, and practical methods for determining their viability andhuman infectivity are not available. Therefore, the routine monitoring of drinking waterfor cysts and oocysts is not recommended at this time. If viable cysts or oocysts arepresent or suspected in treated waters and if the parasites have been responsible forpast waterborne outbreaks in a community, a treatment regime and a watershedprotection plan (where feasible) or other measures known to reduce the risk of illnessshould be implemented.

References

1. Bingham, A.K., Jarroll, E.L., Jr. and Meyer, E.A. Giardia sp.: physical factors ofexcystation in vitro, and excystation vs. eosin exclusion as determinants of viability.Exp. Parasitol., 47: 284–291 (1979).

2. deRegnier, D.P., Cole, L., Schupp, D.G. and Erlandsen, S.L. Viability of Giardia cystssuspended in lake, river, and tap water. Appl. Environ. Microbiol., 55: 1223–1229


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(1989).

3. Meyer, E.A. Giardia as an organism. In: Giardia from molecules to disease. R.C.A.Thompson, J.A. Reynoldson and A.J. Lymbery (eds.). CAB International, Cambridge,UK. pp. 3–13 (1994).

4. Filice, F.P. Studies on the cytology and life history of a Giardia from a laboratory rat.Univ. Calif. Publ. Zool., 57: 53–146 (1952).

5. Erlandsen, S.L. and Bemrick, W.J. Waterborne giardiasis: sources of Giardia cystsand evidence pertaining to their implication in human infection. In: Advances in Giardiaresearch. P.M. Wallis and B.R. Hammond (eds.). University of Calgary Press, Calgary.pp. 227–236 (1988).

6. Erlandsen, S.L., Sherlock, L.A., Januschka, M., Schupp, D.G., Schaefer III, F.W.,Jakubowski, W. and Bemrick, W.J. Cross-species transmission of Giardia spp.:inoculation of beavers and muskrats with cysts of human, beaver, mouse and muskratorigin. Appl. Environ. Microbiol., 54: 2777–2785 (1988).

7. Davies, R.B. and Hibler, C.P. Animal reservoirs and cross-species transmission ofGiardia. In: Waterborne transmission of giardiasis. W. Jakubowski and J.C. Hoff (eds.).EPA 600/9-79-001, U.S. Environmental Protection Agency. pp. 104–126 (1979).

8. Hewlett, E.L., Andrews, J.S., Ruffier, J. and Schaefer III, F.W. Experimental infectionof mongrel dogs with Giardia lamblia cysts and cultured trophozoites. J. Infect. Dis.,145: 89–93 (1982).

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32

ANNEX A:

CT Tables for theInactivation of Giardia lamblia Cysts by

Chlorine, Chlorine Dioxide, Chloramine and Ozoneat Various Temperatures


33

CHLORINE

(a) 99.9% (3-log) inactivation

Table A.1. CT values for 99.9% inactivation of Giardia lamblia cysts by freechlorine at 0.5 C

Residual(mg/L)

pH

6.0 6.5 7.0 7.5 8.0 8.5 9.0

<0.4 125 156 191 227 274 322 371

0.6 135 169 207 246 297 350 401

0.8 142 179 219 259 314 370 424

1.0 149 188 229 271 329 387 442

1.2 154 195 238 280 341 402 458

1.4 158 201 245 289 352 414 471

1.6 162 206 252 296 361 425 483

1.8 166 211 258 303 370 436 494

2.0 169 216 263 309 378 445 504

2.2 173 220 268 314 385 453 514

2.4 175 224 273 320 392 461 522

2.6 178 227 278 325 398 469 530

2.8 181 231 282 329 404 476 538

3.0 183 234 286 334 410 482 545


34

Table A.2. CT values for 99.9% inactivation of Giardia lamblia cysts by freechlorine at 5 C

Residual(mg/L)

pH

6.0 6.5 7.0 7.5 8.0 8.5 9.0

<0.4 88 111 135 161 194 228 263

0.6 95 120 147 174 210 248 284

0.8 101 127 155 184 223 262 300

1.0 105 133 162 192 233 274 313

1.2 109 138 168 198 241 284 324

1.4 112 142 174 204 249 293 334

1.6 115 146 178 210 256 301 342

1.8 118 149 183 214 262 308 350

2.0 120 153 186 219 267 315 357

2.2 122 156 190 223 273 321 364

2.4 124 158 193 226 277 327 370

2.6 126 161 196 230 282 332 375

2.8 128 163 199 233 286 337 381

3.0 130 166 202 236 290 342 386


35


Residual(mg/L)

pH

6.0 6.5 7.0 7.5 8.0 8.5 9.0

<0.4 66 83 101 121 146 171 197

0.6 72 90 110 131 158 186 213

0.8 76 95 117 138 167 197 225

1.0 79 100 122 144 175 206 235

1.2 82 104 126 149 181 214 243

1.4 84 107 130 153 187 220 251

1.6 86 110 134 157 192 226 257

1.8 88 112 137 161 197 232 263

2.0 90 115 140 164 201 237 268

2.2 92 117 143 167 205 241 273

2.4 93 119 145 170 208 245 278

2.6 95 121 148 173 212 249 282

2.8 96 123 150 175 215 253 286

3.0 97 124 152 177 218 257 290


36


Residual(mg/L)

pH

6.0 6.5 7.0 7.5 8.0 8.5 9.0

<0.4 46 55 67 83 100 118 136

0.6 49 59 72 90 109 128 147

0.8 52 63 77 95 115 136 155

1.0 54 66 80 99 121 142 162

1.2 56 68 83 103 125 147 168

1.4 58 70 86 106 129 152 173

1.6 60 72 88 109 133 156 177

1.8 61 74 90 111 136 160 181

2.0 62 76 92 113 139 163 185

2.2 63 77 94 115 141 166 188

2.4 64 78 96 117 144 169 192

2.6 65 80 97 119 146 172 194

2.8 66 81 99 121 148 175 197

3.0 67 82 100 122 150 177 200


37


Residual(mg/L)

pH

6.0 6.5 7.0 7.5 8.0 8.5 9.0

<0.4 34 42 49 66 75 88 101

0.6 37 46 53 70 80 95 109

0.8 39 49 56 73 84 101 115

1.0 40 51 59 76 87 105 120

1.2 42 53 61 78 89 109 124

1.4 43 55 63 80 91 112 128

1.6 44 56 64 82 93 116 131

1.8 45 57 66 84 95 118 134

2.0 46 59 67 85 97 121 137

2.2 47 60 69 86 98 123 139

2.4 48 61 70 88 100 125 142

2.6 48 62 71 89 101 127 144

2.8 49 63 72 90 102 129 146

3.0 50 63 73 91 103 131 148


38


Residual(mg/L)

pH

6.0 6.5 7.0 7.5 8.0 8.5 9.0

<0.4 23 29 35 44 50 57 69

0.6 25 31 38 47 54 62 74

0.8 26 33 41 49 56 66 79

1.0 28 34 42 51 58 69 82

1.2 29 36 44 52 60 71 85

1.4 29 37 45 54 62 74 87

1.6 30 38 47 55 63 76 90

1.8 31 38 48 56 64 77 92

2.0 31 39 49 57 65 79 93

2.2 32 40 50 58 66 81 95

2.4 33 41 51 58 67 82 97

2.6 33 41 51 59 68 83 98

2.8 33 42 52 60 69 85 100

3.0 34 42 53 61 70 86 101


39

(b) 90% (1-log) inactivation

Table A.7. CT values for 90% inactivation of Giardia lamblia cysts by free chlorineat 0.5 C

Residual(mg/L)

pH

6.0 6.5 7.0 7.5 8.0 8.5 9.0

<0.4 42 52 64 76 91 107 124

0.6 45 56 69 82 99 117 134

0.8 47 60 73 86 105 123 141

1.0 50 63 76 90 110 129 147

1.2 51 65 79 93 114 134 153

1.4 53 67 82 96 117 138 157

1.6 54 69 84 99 120 142 161

1.8 55 70 86 101 123 145 165

2.0 56 72 88 103 126 148 168

2.2 58 73 89 105 128 151 171

2.4 58 75 91 107 131 154 174

2.6 59 76 93 108 133 156 177

2.8 60 77 94 110 135 159 179

3.0 61 78 95 111 137 161 182


40

Table A.8. CT values for 90% inactivation of Giardia lamblia cysts by free chlorineat 5 C

Residual(mg/L)

pH

6.0 6.5 7.0 7.5 8.0 8.5 9.0

<0.4 29 37 45 54 65 76 88

0.6 32 40 49 58 70 83 95

0.8 34 42 52 61 74 87 100

1.0 35 44 54 64 78 91 104

1.2 36 46 56 66 80 95 108

1.4 37 47 58 68 83 98 111

1.6 38 49 59 70 85 100 114

1.8 39 50 61 71 87 103 117

2.0 40 51 62 73 89 105 119

2.2 41 52 63 74 91 107 121

2.4 41 53 64 75 92 109 123

2.6 42 54 65 77 94 111 125

2.8 43 54 66 78 95 112 127

3.0 43 55 67 79 97 114 129


41

Table A.9. CT values for 90% inactivation of Giardia lamblia cysts by free chlorineat 10 C

Residual(mg/L)

pH

6.0 6.5 7.0 7.5 8.0 8.5 9.0

<0.4 22 28 34 40 49 57 66

0.6 24 30 37 44 53 62 71

0.8 25 32 39 46 56 66 75

1.0 26 33 41 48 58 69 78

1.2 27 35 42 50 60 71 81

1.4 28 36 43 51 62 73 84

1.6 29 37 45 52 64 75 86

1.8 29 37 46 54 66 77 88

2.0 30 38 47 55 67 79 89

2.2 31 39 48 56 68 80 91

2.4 31 40 48 57 69 82 93

2.6 32 40 49 58 71 83 94

2.8 32 41 50 58 72 84 95

3.0 32 41 51 59 73 86 97


42

Table A.10. CT values for 90% inactivation of Giardia lamblia cysts by freechlorine at 15 C

Residual(mg/L)

pH

6.0 6.5 7.0 7.5 8.0 8.5 9.0

<0.4 15 18 22 28 33 39 45

0.6 16 20 24 30 36 43 49

0.8 17 21 26 32 38 45 52

1.0 18 22 27 33 40 47 54

1.2 19 23 28 34 42 49 56

1.4 19 23 29 35 43 51 58

1.6 20 24 29 36 44 52 59

1.8 20 25 30 37 45 53 60

2.0 21 25 31 38 46 54 62

2.2 21 26 31 38 47 55 63

2.4 21 26 32 39 48 56 64

2.6 22 27 32 40 49 57 65

2.8 22 27 33 40 49 58 66

3.0 22 27 33 41 50 59 67


43


Residual(mg/L)

pH

6.0 6.5 7.0 7.5 8.0 8.5 9.0

<0.4 11 14 16 22 25 29 34

0.6 12 15 18 23 27 32 36

0.8 13 16 19 24 28 34 38

1.0 13 17 20 25 29 35 40

1.2 14 18 20 26 30 36 41

1.4 14 18 21 27 30 37 43

1.6 15 19 21 27 31 39 44

1.8 15 19 22 28 32 39 45

2.0 15 20 22 28 32 40 46

2.2 16 20 23 29 33 41 46

2.4 16 20 23 29 33 42 47

2.6 16 21 24 30 34 42 48

2.8 16 21 24 30 34 43 49

3.0 17 21 24 30 34 44 49


44


Residual(mg/L)

pH

6.0 6.5 7.0 7.5 8.0 8.5 9.0

<0.4 8 10 12 15 17 19 23

0.6 8 10 13 16 18 21 25

0.8 9 11 14 16 19 22 26

1.0 9 11 14 17 19 23 27

1.2 10 12 15 17 20 24 28

1.4 10 12 15 18 21 25 29

1.6 10 13 16 18 21 25 30

1.8 10 13 16 19 21 26 31

2.0 10 13 16 19 22 26 31

2.2 11 13 17 19 22 27 32

2.4 11 14 17 19 22 27 32

2.6 11 14 17 20 23 28 33

2.8 11 14 17 20 23 28 33

3.0 11 14 18 20 23 29 34


45

(c) 50% (0.5-log) inactivation

Table A.13. CT values for 50% inactivation of Giardia lamblia cysts by freechlorine at 0.5 C

Residual(mg/L)

pH

6.0 6.5 7.0 7.5 8.0 8.5 9.0

<0.4 21 26 32 38 46 54 62

0.6 22 28 35 41 49 58 67

0.8 24 30 37 43 52 62 71

1.0 25 31 38 45 55 65 74

1.2 26 32 40 47 57 67 76

1.4 26 33 41 48 59 69 79

1.6 27 34 42 49 60 71 81

1.8 28 35 43 50 62 73 82

2.0 28 36 44 51 63 74 84

2.2 29 37 45 52 64 76 86

2.4 29 37 46 53 65 77 87

2.6 30 38 46 54 66 78 88

2.8 30 38 47 55 67 79 90

3.0 31 39 48 56 68 80 91


46


Residual(mg/L)

pH

6.0 6.5 7.0 7.5 8.0 8.5 9.0

<0.4 15 18 23 27 32 38 44

0.6 16 20 24 29 35 41 47

0.8 17 21 26 31 37 44 50

1.0 18 22 27 32 39 46 52

1.2 18 23 28 33 40 47 54

1.4 19 24 29 34 41 49 56

1.6 19 24 30 35 43 50 57

1.8 20 25 30 36 44 51 58

2.0 20 25 31 36 45 52 60

2.2 20 26 32 37 45 53 61

2.4 21 26 32 38 46 54 62

2.6 21 27 33 38 47 55 63

2.8 21 27 33 39 48 56 63

3.0 22 28 34 39 48 57 64


47


Residual(mg/L)

pH

6.0 6.5 7.0 7.5 8.0 8.5 9.0

<0.4 11 14 17 20 24 29 33

0.6 12 15 18 22 26 31 36

0.8 13 16 19 23 28 33 38

1.0 13 17 20 24 29 34 39

1.2 14 17 21 25 30 36 41

1.4 14 18 22 26 31 37 42

1.6 14 18 22 26 32 38 43

1.8 15 19 23 27 33 39 44

2.0 15 19 23 27 33 39 45

2.2 15 19 24 28 34 40 46

2.4 16 20 24 28 35 41 46

2.6 16 20 25 29 35 42 47

2.8 16 20 25 29 36 42 48

3.0 16 21 25 30 36 43 48


48


Residual(mg/L)

pH

6.0 6.5 7.0 7.5 8.0 8.5 9.0

<0.4 8 9 11 14 17 20 23

0.6 8 10 12 15 18 21 25

0.8 9 10 13 16 19 23 26

1.0 9 11 13 17 20 24 27

1.2 9 11 14 17 21 25 28

1.4 10 12 14 18 22 25 29

1.6 10 12 15 18 22 26 30

1.8 10 12 15 19 23 27 30

2.0 10 13 15 19 23 27 31

2.2 11 13 16 19 24 28 31

2.4 11 13 16 20 24 28 32

2.6 11 13 16 20 24 29 32

2.8 11 13 16 20 25 29 33

3.0 11 14 17 20 25 30 33


49


Residual(mg/L)

pH

6.0 6.5 7.0 7.5 8.0 8.5 9.0

<0.4 6 7 8 11 12 15 17

0.6 6 8 9 12 13 16 18

0.8 6 8 9 12 14 17 19

1.0 7 8 10 13 14 18 20

1.2 7 9 10 13 15 18 21

1.4 7 9 10 13 15 19 21

1.6 7 9 11 14 16 19 22

1.8 8 10 11 14 16 20 22

2.0 8 10 11 14 16 20 23

2.2 8 10 11 14 16 21 23

2.4 8 10 12 15 17 21 24

2.6 8 10 12 15 17 21 24

2.8 8 10 12 15 17 22 24

3.0 8 11 12 15 17 22 25


50


Residual(mg/L)

pH

6.0 6.5 7.0 7.5 8.0 8.5 9.0

<0.4 4 5 6 7 8 10 11

0.6 4 5 6 8 9 10 12

0.8 4 5 7 8 9 11 13

1.0 5 6 7 8 10 11 14

1.2 5 6 7 9 10 12 14

1.4 5 6 8 9 10 12 15

1.6 5 6 8 9 10 13 15

1.8 5 6 8 9 11 13 15

2.0 5 7 8 9 11 13 16

2.2 5 7 8 10 11 13 16

2.4 5 7 8 10 11 14 16

2.6 6 7 9 10 11 14 16

2.8 6 7 9 10 11 14 17

3.0 6 7 9 10 12 14 17


51

CHLORINE DIOXIDE

Table A.19. CT values for inactivation of Giardia.

Water Temperature, C

LogInactivation 1 5 10 15 20 25

0.5 10 4.3 4 3.2 2.5 2

1.0 21 8.7 7.7 6.3 5 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


52

CHLORAMINE

Table A.20. CT values for inactivation of Giardia.



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


53

OZONE

Table A.21.CT Values for inactivation of Giardia.



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

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