Fate of Cryptosporidium oocysts, Giardia cysts,and microbial indicators during wastewatertreatment and anaerobic sludge digestion

Christian Chauret, Susan Springthorpe, and Syed Sattar

Abstract: The extent of reduction in selected microorganisms was tested during both aerobic wastewater treatment andanaerobic digestion of sludge at the wastewater treatment plant in Ottawa to compare the removal of two encystedpathogenic protozoa with that of microbial indicators. Samples collected included the raw wastewater, the primaryeffluent, the treated wastewater, the mixed sludge, the decanted liquor, and the cake. All of the raw sewage sampleswere positive forCryptosporidiumoocysts andGiardia cysts, as well as for the other microorganisms tested. Duringaerobic wastewater treatment (excluding the anaerobic sludge digestion),Cryptosporidiumand Giardia were reduced by2.96 log10 and 1.40 log10, respectively.Clostridium perfringensspores,Clostridium perfringenstotal counts, somaticcoliphages, and heterotrophic bacteria were reduced by approximately 0.89 log10, 0.96 log10, 1.58 log10, and 2.02 log10,respectively. All of the other microorganisms were reduced by at least 3.53 log10. Sludge samples from the plant werefound to contain variable densities of microorganisms. Variability in microbial concentrations was sometimes greatbetween samples, stressing the importance of collecting a large number of samples over a long period of time. In allcases, the bacterial concentrations in the cake (dewatered biosolids) samples were high even if reductions in numberswere observed with some bacteria. During anaerobic sludge digestion, no statistically significant reduction wasobserved forClostridium perfringens, Enterococcussp., Cryptosporidiumoocysts, andGiardia cysts. A 1–2 log10

reduction was observed with fecal coliforms and heterotrophic bacteria. However, the method utilized to detect theprotozoan parasites does not differentiate between viable and nonviable organisms. On the other hand, total coliformsand somatic coliphages were reduced by 0.35 log10 and 0.09 log10, respectively. These results demonstrate the relativepersistence of the protozoa in sewage sludge during wastewater treatment.

Key words: Cryptosporidium, Giardia, indicators, wastewater, sludge.

Résumé: Nous avons mesuré le niveau d’élimination de certains microorganismes-cibles lors du traitement aérobie del’eau usée et de la digestion anaérobie des boues à l’usine de traitement des eaux usées d’Ottawa et ce dans le but decomparer l’élimination de deux protozoaires enkystés pathogènes avec celle des indicateurs bactériens. Les échantillonsprélevés étaient l’eau usée brute, l’effluent primaire, l’eau usée traitée, la boue mélangée, le liquide de décantion et lerésidu solide. Tous les échantillons d’eau usée brute contenaient des oocystes deCryptosporidiumet des kystes deGiardia ainsi que les autres organismes normalement recherchés. Lors du traitement en aérobiose de l’eau usée(excluant la digestion anaérobie de la boue),Cryptosporidiumet Giardia ont diminué de 2.96 log10 et 1.40 log10

respectivement. Les spores deClostridium perfringens, les comptes totaux deClostridium perfringens, les coliphagessomatiques et les bactéries hétérotrophes ont diminué d’environ 0.89 log10, 0.96 log10, 1.58 log10 et 2.02 log10

respectivement. Tous les autres microorganismes ont diminué d’au moins 3.53 log10. Nous avons constaté que leséchantillons de boue de l’usine contenaient des concentrations variables de microorganismes. Cette variabilité étaitparfois grande entre les échantillons, ce qui confirme l’importance de récolter un grand nombre d’échantillons pendantune longue période de temps. Dans tous les cas, les concentrations bactériennes des échantillons de résidus solides(biosolides déséchés) étaient élevées malgré une diminution de certaines bactéries. Lors de la digestion anaérobie desboues, il n’y a pas eu de diminution significative deClostridium perfringens, Enterococcussp., des oocystes deCryptosporidiumet des kystes deGiardia. Une diminution de 1–2 log10 a été enregistrée avec les coliformes fécaux etles bactéries hétérotrophes. Par contre, la méthode utilisée pour détecter les protozoaires n’a pas permis de distinguerles organismes viables des non-viables. Les coliformes totaux et les coliphages somatiques ont diminués de 0.35 log10

et 0.09 log10 respectivement. Ces résultats confirment la persistance relative des protozoaires dans les boues lors dutraitement des eaux usées.

Mots clés: Cryptosporidium, Giardia, indicateurs, eau usée, boue.

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257

Received September 17, 1998. Revision received December 15, 1998. Accepted December 21, 1998.

C. Chauret,1 S. Springthorpe, and S. Sattar.Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine,University of Ottawa, 451 Smyth Rd., Ottawa, ON K1H 8M5, Canada.1Author to whom all correspondence should be sent at the following address: Indiana University Kokomo, 2300 South WashingtonSt., P.O. Box 9003, Kokomo, IN 46904-9003, U.S.A. (e-mail: cchauret@iuk.edu).

Introduction

Wastewater treatment processes do not remove or inacti-vate all pathogenic microorganisms. Many of the microor-ganisms are trapped in, or adsorbed to, particulates and areconcentrated in the sludge.CryptosporidiumandGiardia areprotozoan parasites which are important causes of water-borne diseases in North America and Europe. Waterborneoutbreaks of cryptosporidiosis have been recently docu-mented in North America. For instance, approximately400 000 cases of the disease were estimated to have oc-curred in Milwaukee, Wisconsin (MacKenzie et al. 1994).One of the potential sources of contamination may be waste-water discharges into sources for drinking water (Roach etal. 1993). In addition, very little is known about the occur-rence and survival of these parasites in sewage sludge. It hasbeen shown, using a model wastewater treatment system,that 99.9% removal ofG. muris cysts is achieved duringsludge digestion (van Praagh et al. 1993). However, otherstudies have foundGiardia cysts in both undigested and di-gested sludge, and there was no significant difference be-tween their occurrence in raw and treated sludge (Sykora etal. 1991; Soares et al. 1994). A more recent study by Mayerand Palmer (1996) indicated a 3 log10 reduction ofGiardiacysts and a 2 log10 reduction of Cryptosporidiumoocyststhrough the wastewater treatment process using the immuno-fluorescence method for detection. Bacterial pathogens insludge include many species ofSalmonella, Shigella, Cam-pylobacter, Yersinia, Vibrio, and pathogenicEscherichia coli(Bitton 1994). In general, pathogenic and indicator bacteriaundergo a 1–3 log10 reduction during anaerobic digestion(Bitton 1994).

Use of stabilized sewage sludge (biosolids) for land appli-cation is increasing for both economical and environmentalreasons. Such land application of materials containing largenumbers of microorganisms can, under certain conditions,contaminate surface waters or groundwaters. Risk ofgroundwater contamination from biosolids may be greatestwith viruses because of their relatively small size and pooradsorption to soil particles (Yates and Yates 1991). Viruseslikely to be present in wastewaters and anaerobically di-gested sewage sludges are polioviruses, coxsackieviruses Aand B, adenoviruses, hepatitis A and E viruses, echovirus,Norwalk virus, and reoviruses (Bitton 1994). Bacteriophageshave been used as indicators for the presence of humanpathogenic viruses (Havelaar and Hogeboom 1983).

Wallis et al. (1996) studied the prevalence ofGiardia andCryptosporidium in sewage samples in Canada and con-cluded that human-infectiveGiardia cysts are routinely de-tected in sewage in Canada. Accordingly, the main objectiveof this study was to look at the levels of reduction for a vari-ety of human pathogens, includingCryptosporidium andGiardia, and indicators before and after the anaerobic diges-tion, as well as after dewatering of sewage sludge. Bacterio-phages, as somatic coliphages, were used to indicate theinactivation of human enteric viruses. The samples were alsoexamined forCryptosporidiumoocysts,Giardia cysts, totaland fecal coliforms,Clostridium perfringens, and Entero-coccussp. In addition, reduction of these microorganismsthrough wastewater treatment was monitored. The data ob-

tained from this study could be used to (i) estimate thepathogen loading of the Ottawa River due to treated waste-water discharges, and (ii ) determine the levels ofCrypto-sporidium oocysts, Giardia cysts, and other pathogenicmicroorganisms in treated sewage sludge to be applied toland as biosolids.

Materials and methods

Sampling site and sample typesSamples of raw sludge, thickened waste activated sludge

(TWAS), decanted liquor and cake, and raw and treated waste-waters were collected at the Robert O. Pickard Environmental Cen-tre, which is the wastewater treatment plant for the RegionalMunicipality of Ottawa–Carleton. The plant processes approxi-mately 500 000 m3/day of wastewater using a full conventionalsystem consisting of screening, primary clarification, aeration, sec-ondary clarification, and chlorination (Fig. 1). The sewage sludgeis generated from the wastewater during primary and secondarytreatment and it undergoes mesophilic anaerobic digestion (36°C ±1°C) for about 20 days. The digested sludge is then dewatered toproduce a semisolid cake (biosolids).

Sample collection and processingSludge samples (raw sludge, TWAS, decanted liquor, and cake)

were collected on a weekly basis at the Pickard plant from June 19to August 22, 1995 (n = 10). The mean solid content of the cake(32%) was determined by the Regional Municipality of Ottawa–Carleton (Stephen Cann, personal communication). During thesame period, samples (n = 4) of raw sewage as well as the primaryeffluent and treated (processed) wastewater were collected fromthe same plant. Immediately after collection, the samples were puton ice and transported to the laboratory where they were stored at4°C until used for the analyses. Bacterial and bacteriophage analy-ses were always performed within 24 h of sampling, whereas pro-tozoan analyses were completed within 7 days. The mixed sludgewas produced in the laboratory by mixing TWAS and raw sludgesamples in a 1:3 ratio (w/w) to simulate the conditions in the plant.

Bacterial countsBacterial enumerations were performed by suspending the

mixed sludge and the cake samples separately in 0.1% (w/v) poly-ethylene glycol (PEG). The samples were then mixed at 4°C on ashaker (200 rpm) for 15 min. Following shaking, the samples werediluted in 0.1% (w/v) PEG and aliquots were spread on variousagar media. The decanted liquor samples were also diluted in 0.1%PEG. Liquid samples (wastewater) were diluted and plated for enu-meration. Fecal coliforms were enumerated on mFC agar (DifcoLaboratories, Detroit, Mich.), total coliforms on mT7 (Difco Labo-ratories), heterotrophic bacteria on R2A (Difco Laboratories),Enterococcussp. on KF agar (Difco Laboratories),Aeromonassp.on Aeromonasagar base (Oxoid, Basingstoke, England), andClos-tridium perfringenslevels were as detailed previously (Chauret etal. 1995). The mFC plates, to detect fecal coliforms, were incu-bated at 44.5°C and the R2A plates were incubated at room tem-perature (20–23°C) for 4–5 days. All other incubations were at37°C.

Cryptosporidiumand Giardia analysesBoth the mixed sludge and the cake were suspended in 1%

(w/v) Tween 80 solution. The suspended mixed sludge sampleswere shaken mechanically at 4°C for 15 min. The cake sampleswere homogenized in a blender for 3 min. Following homogeniza-tion, the samples were processed by flotation, purification, fluores-cent antibody staining, and microscopic examination as outlined in

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258 Can. J. Microbiol. Vol. 45, 1999

Standard methods for the examination of water and wastewater(Anonymous 1995).Cryptosporidium oocyst and Giardia cystanalyses in wastewater samples were performed by filtering sam-ples through a 1-µm filtration cartridge as detailed inStandardmethods for the examination of water and wastewater(Anonymous1995; Chauret et al. 1995). The monoclonal antibody used was theAqua-Glo™ combo (Waterborne, Inc., New Orleans, La.), whichspecifically detectsGiardia cysts andCryptosporidiumoocystsfrom environmental samples. This method detects the parasites butdoes not assess viability. A vital stain, such as 4,6-diamidino-2-phenylindole, was not included in the procedure because it wasfound to produce too much background fluorescence, especially inbiosolid samples, and thus hindered the detection of cysts andoocysts.

Bacteriophage analysisSomatic coliphage enumerations were performed by suspending

the mixed sludge and cake samples in a 1:1 sterile mixture of 8%beef extract and 1 M NaCl (pH 9.0). The samples were then mixedat 4°C on a shaker (200 rpm) for 15 min. Following mixing,aliquots of the samples were centrifuged (2500 ×g, 5 min) to re-move bacteria and debris. These supernatants, as well as thewastewater samples, were diluted and somatic coliphages (Esche-richia coli C as the host) were determined as detailed inStandard

methods for the examination of water and wastewater(Anonymous1995).

Statistical analysisStatisitical analysis (95% confidence level) using at-test fol-

lowed by a Mann–Whitney rank sum test was performed usingSigmaStat 2.0 (Jandel Scientific Software, San Rafael, Calif.). Theanalysis was made on the anaerobic sludge digestion data.

Results

Microbial reduction in wastewaterConcentrations ofCryptosporidiumoocysts andGiardia

cysts were reduced during aerobic wastewater treatment by2.96 log10 and 1.40 log10, respectively (Table 1). Less over-all reduction was observed with bothClostridium per-fringens spores and vegetative cells: 0.89 log10 and 0.96log10, respectively. The other bacteria tested, with the excep-tion of total heterotrophic bacteria, were reduced by at least3.50 log10 (Table 1). Somatic coliphage concentrations weredetermined to experience a 1.58 log10 reduction.

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Chauret et al. 259

COARSESCREENING

RAWSEWAGEPUMPING

SCREENING DEGRITTING PRIMARYCLARIFICATION

CHEMICALADDITION

AERATION SECONDARYCLARIFICATION

CHLORINATION

RAWSLUDGE

PUMPING

SCUM HOLDINGAND

PUMPING

RETURNACTIVATED

SLUDGEPUMPING

WASTEACTIVATED

SLUDGEPUMPING

WASTEACTIVATED

SLUDGETHICKENING

ANAEROBICSLUDGE

DIGESTION

DIGESTEDSLUDGE

DEWATERING

SEPTAGERECEIVING

FINALEFFLUENT

DISCHARGEDTO

OTTAWARIVER

LEGEND

SEWER

WASTEWATER PROCESS STREAM

SOLIDS HANDLING PROCESS STREAM

COARSE SCREENINGHAULED OFF SITE

FOR DISPOSAL

SCREENINGS AND GRITHAULED OFF SITE

FOR DISPOSAL

OPEN CREEK

COLLECTION

SOUTH OTTAWA COLLECTION

OTTAWACOLLECTOR

BY

PA

SS

CANOTEK SEWER

OR

LEA

NS

/CU

MB

ER

LAN

D C

OLL

EC

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Fig. 1. Schematic representation of wastewater treatment at the Robert O. Pickard Environmental Centre (wastewater treatment plantfor the Regional Municipality of Ottawa–Carleton).

Microbial reduction during sludge digestionTable 2 shows the extent of reduction in several microor-

ganisms during anaerobic sludge digestion. No statisticallysignificant reduction was observed forClostridium per-fringens, Enterococcussp., andGiardia cysts. Clostridiumoocysts were reduced by 0.30 log10, but this reduction wasnot statistically significant (P = 0.623). A 1–2 log10 reduc-tion was observed with fecal coliforms and heterotrophicbacteria (Table 2). On the other hand, total coliforms and so-matic coliphages were reduced by 0.35 log10 (P = 0.021) and0.09 log10 (P = 0.645), respectively.

Discussion

In this study,Cryptosporidiumoocysts andGiardia cystswere detected in both raw and treated wastewater. However,

the concentrations ofCryptosporidiumoocysts were verylow in treated wastewater, suggesting that wastewater dis-charges (from less than 2.5 × 107 up to 1.0 × 108 organisms/day) may not have a significant influence on levels in theOttawa River. WhereasCryptosporidiumoocysts were re-duced by 2.96 log10, Giardia cysts were inactivated by only1.40 log10 during aerobic wastewater treatment. Roach et al.(1993) also found higher levels ofGiardia cysts thanCryptosporidiumoocysts in raw and treated wastewaters inthe Yukon. However, very few cattle are present in the Yu-kon, suggesting that humans or some other animal specieswere the reservoir ofCryptosporidiumin that area. In Ot-tawa, where few cattle are present within the city, humansare also likely to be the most important reservoir. In fact, aprevious study showed that higher concentrations of oocystswere present in samples collected downstream from the City

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260 Can. J. Microbiol. Vol. 45, 1999

MicroorganismsType ofsample n

Arithmeticmean

Minimumno.

Maximumno.

Log10

reduction

Cryptosporidiumoocystsa Raw 4 4.77×103 8.30×102 1.25×104

Primary 4 5.06×103 <1.25×102 1.65×104 NRb

Treated 4 5.20 ×100 <5.00×100 2.08×101 2.96

Giardia cystsa Raw 4 8.25×103 1.00×103 2.10×104

Primary 4 6.37×103 8.30×102 1.54×104 0.11Treated 4 3.29×102 1.88×101 7.50×102 1.40

Total coliformsc Raw 4 3.84×107 6.90×106 1.20×108

Primary 4 8.50×107 8.00×106 4.00×108 NRTreated 4 3.20×102 2.00×101 1.00×103 5.08

Fecal coliformsc Raw 4 1.86×107 1.00×106 1.10×108

Primary 4 6.43×106 1.00×106 1.40×107 0.46Treated 4 1.93×103 7.00×101 1.00×104 3.98

Enterococcussp.c Raw 4 2.25×105 9.70×104 3.90×105

Primary 4 1.91×105 2.50×104 4.00×105 0.07Treated 4 6.50×100 3.00×100 1.00×101 4.54

Clostridium perfringenstotal countsc Raw 4 8.85×104 3.30×102 4.10×105

Primary 4 2.76×104 3.20×103 1.00×105 0.51Treated 4 9.75×103 1.80×102 5.60×104 0.96

Clostridium perfringenssporesc Raw 4 1.70×104 1.00×104 2.10×104

Primary 4 6.63×103 2.20x103 1.30×104 0.41Treated 4 2.18×103 1.10×102 5.00×103 0.89

Aeromonassp.c Raw 4 4.24×107 1.00×107 1.10×108

Primary 4 3.02×107 1.20×107 5.90×107 0.15Treated 4 1.26×104 3.00×102 3.00×104 3.53

Total heterotrophic bacteriac Raw 4 2.75×109 1.23×108 1.11 x1010

Primary 4 1.34×109 1.54×108 4.40×109 0.31Treated 4 2.64×107 3.10×105 1.39×108 2.02

Somatic coliphagesd Raw 4 7.06×104 1.00×103 2.00×105

Clarification 4 4.82×104 3.00×103 1.40×105 0.17Treated 4 1.87×103 9.60×101 3.00×103 1.58

Note: The samples analyzed were the raw wastewater, the primary effluent, and the treated wastewater.aOocysts or cysts·100 L–1.bNo reduction.cCFU·100 mL–1.dPlaques·100 mL–1.

Table 1. Extent of reduction in selected microorganisms during aerobic wastewater treatment at the wastewater treatmentplant in Ottawa, Canada (June–August 1995).

of Ottawa than in samples collected upstream from Ottawa(Chauret et al. 1995).

High-rate anaerobic digestion of municipal sludge at 35°Cwith a detention time of 14–15 days produces a 1 or 2 log10reduction of indicator bacteria,Salmonellasp., and entero-viruses (Berg and Berman 1989; Soares et al. 1994). In thepresent study, the anaerobic digestion of the sludge at thePickard plant also produced a 1–2 log10 reduction of someindicator bacteria (fecal coliforms and heterotrophic bacte-ria). However, the reductions observed in the levels of totalcoliforms and somatic coliphages was less than one log10.Moreover, there was no apparent reduction inEnterococcussp. andClostridium perfringens. Clostridium perfringensisan anaerobic spore-forming bacterium that can withstandstresses such as highly reduced environmental conditions.Therefore, it is likely to survive anaerobic sewage sludge di-gestion. On the other hand, Kuchenrither and Benefield(1983) demonstrated that fecal streptococci (Enterococcussp.) were almost entirely inactivated after 20 days of deten-tion in anaerobic sewage sludge digesters. It may be prema-

ture to draw any conclusion on the apparent resistance ofEnterococcussp. in this study and further analyses might berequired.

As previously observed (Soares et al. 1994; Sykora et al.1991),Giardia cysts did not appear to be reduced by the an-aerobic digestion process. However, the current standardmethod used to enumerateGiardia cysts (andCryptospo-ridium oocysts) allows only for the detection of the cysts oroocysts without any indication regarding viability. There-fore, cyst or oocyst viability was not evaluated in this study.Gavaghan et al. (1993) reported a 99.9% loss inG. muriscysts viability (measured by an in vitro excystation method)during anaerobic digestion in a model wastewater treatmentplant. In the present study, it might be that intact, butnonviable, cysts were detected or enumerated and that thenumber of cysts detected in the cake was an overestimationof the number of viable cysts. The present study noted asmall (nonsignificant) reduction (0.30 log) in the concentra-tions of Cryptosporidium oocysts following anaerobicsludge digestion. As withGiardia, the analysis forCryptos-

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Chauret et al. 261

Microorganism Process stage nArithmeticmean

Minimumno.

Maximumno.

Log10

reduction t-Test

Cryptosporidiumoocysts Mixed sludgea 10 5.29×102 <2.50×101 3.81×103

Decanted liquorb 10 1.41×101 <1.67×100 4.17×101

Cakec 10 2.65×102 <2.50×101 5.40×102 0.30 NSSDd (P=0.623)

Giardia cysts Mixed sludge 10 4.41×102 <2.50×101 1.18×103

Decanted liquor 10 7.75×101 <1.67×100 1.22×102

Cake 10 1.28×103 <2.50×101 2.82×103 NRe NSSD (P>0.05)

Total coliforms Mixed sludge 10 1.31×1010 1.52×109 1.00×1011

Decanted liquor 10 6.89×105 1.37×105 3.50×106

Cake 10 5.85×109 1.73×107 8.93×1010 0.35 SSDf (P=0.021)

Fecal coliforms Mixed sludge 10 1.59×109 2.10×108 8.50×109

Decanted liquor 10 4.44×105 1.10×105 2.60×106

Cake 10 4.41×107 4.32×106 1.18×108 1.56 SSD (P<0.001)

Enterococcussp. Mixed sludge 10 3.88×107 1.90×107 1.08×108

Decanted liquor 10 1.15×105 2.70×104 6.10×105

Cake 10 9.81×107 2.88×106 5.62×108 NR NSSD (P=0.076)

Clostridium perfringens Mixed sludge 10 1.68×107 2.80×106 4.00×107

Decanted liquor 10 5.15×106 <1.0 9.10×106

Cake 10 1.37×108 3.74×107 2.65×108 NR NSSD (P>0.05)

Total heterotrophic bacteria Mixed sludge 10 5.04×1011 8.70×109 3.90×1012

Decanted liquor 10 2.07×1010 2.25×107 2.00×1011

Cake 10 6.97×109 3.50×108 4.26×1010 1.86 SSD (P<0.001)

Somatic coliphages Mixed sludge 10 1.74×106 1.24×105 8.60×106

Decanted liquor 10 2.50×104 1.75×103 1.18×105

Cake 10 1.41×106 2.51×105 4.38×106 0.09 NSSD (P=0.645)aOrganisms·100 g–1 wet sludge.bOrganisms·100 mL–1.cOrganisms·100 g–1 wet sludge; counts corrected to account for dewatering.dNo statistically significant difference.eNo reduction.fStatistically significant difference.

Table 2. Extent of reduction in selected microorganisms during the anaerobic sludge digestion at the wastewater treatment plant inOttawa, Canada.

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262 Can. J. Microbiol. Vol. 45, 1999

poridium oocysts does not differentiate between viable andnonviable oocysts.

The levels ofGiardia cysts in the mixed sludge rangedfrom <250 to 11 800 cysts·100 g–1. These results are similarto Sykora et al. (1991), who reported cyst concentrations insludge in the range of 50 – 30 000 cysts·kg–1. The concen-trations of Cryptosporidiumoocysts in the mixed sludgeranged from <250 to 3 810 oocysts·100 g–1. Whitmore andRobertson (1995) found that 10% ofCryptosporidiumwereviable after 18 days in a mesophilic anaerobically digestingsludge, and that oocysts would survive for at least 30 days ina sludge-treated soil. These results warrant further researchon the risk associated with land application ofCryptosporidium-contaminated sludge.

Soares et al. (1994) found 3.29 × 104 and 1.61 × 103 en-teric viruses·kg–1 of undigested and digested sewage sludge,respectively (90.1% removal). However, the reduction ofcoliphages obtained in this study was only 18.97% (0.09log10). Most viruses are likely to adsorb to the solids in sew-age. Therefore, the potential for transmission of viral dis-eases through land application of biosolids also requiresfurther evaluation.

Acknowledgements

This study was supported by funding from the RegionalMunicipality of Ottawa–Carleton and by the Ontario Minis-try of Environment and Energy (Environmental YouthCorps). The assistance of the staff at ROPEC is gratefullyacknowledged. They helped with all aspects of sampling andalso provided Fig. 1. We also thank Ranu Sharma, RobinStewart, and Marc Lafleur for their technical help.

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