Inactivation and potential repair of Cryptosporidium parvum following low- and medium-pressure ultraviolet irradiation

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Water Research 37 (2003) 3517–3523

Inactivation and potential repair of Cryptosporidium parvum following low- and medium-pressure ultraviolet irradiation J.L. Zimmera, R.M. Slawsona,b,*, P.M. Huckb b

a Department of Biology, University of Waterloo, Waterloo, Ont., Canada, N2L 3G1 Department of Civil Engineering, NSERC Chair in Water Treatment, University of Waterloo, Waterloo, Ont., Canada, N2L 3G1

Received 30 December 2002; received in revised form 8 April 2003; accepted 10 April 2003

Abstract This study investigated the level of inactivation and the potential for Cryptosporidium parvum to repair following low doses (1 and 3 mJ/cm2) of ultraviolet (UV) irradiation from both low- and medium-pressure UV lamps. Cryptosporidium parvum oocysts suspended in phosphate buffered saline were exposed to UV using a bench-scale collimated beam apparatus. Oocyst suspensions were incubated at 5
C or 25
C under light and dark conditions up to 120 h (5 days) following exposure to UV irradiation, to examine photoreactivation and dark repair potential, respectively. Cryptosporidium parvum infectivity was determined throughout the incubation period using an HCT-8 cell culture and an antibody staining procedure for detection. No detectable evidence of repair was observed after incubation under light or dark conditions following either LP or MP UV lamp irradiation. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: UV radiation; Cryptosporidium parvum; DNA repair; HCT-8 cell culture; Low-pressure UV lamps; Medium-pressure UV lamps

1. Introduction The increased interest in ultraviolet (UV) technology in drinking water treatment in various jurisdictions has mainly come about due to the recently recognized ability of UV irradiation to inactivate protozoan pathogens, in particular Cryptosporidium parvum. Numerous studies using infectivity assays (e.g. [1–4]) have demonstrated that C. parvum oocysts are very susceptible to UV doses well below those being utilized or proposed for use in drinking water treatment applications [5,6].

*Corresponding author. Department of Civil Engineering, NSERC Chair in Water Treatment, University of Waterloo, Waterloo, Ont., Canada, N2L 3G1. Tel.: +1-519-8884567x6193; fax: +1-519-746-7499. E-mail addresses: [email protected] (J.L. Zimmer), [email protected] (R.M. Slawson), [email protected] (P.M. Huck).

Damage to the structure and function of DNA is the primary mechanism responsible for cell injury and loss of viability by UV radiation [7]. The most detrimental UV wavelengths (often referred to as germicidal) have been shown to be between 200 and 300 nm (UV-C range), with the maximum absorption by DNA bases occurring at 260 nm [7]. When DNA is exposed to these wavelengths, the electrons within specific bases become energized resulting in the formation of covalent links between adjacent bases. These abnormal base linkages cause various types of structural damage in DNA [7]. If significant damage occurs, DNA replication is blocked which ultimately results in cell death. Of the two UV sources predominantly used in water treatment, low-pressure (LP) and medium-pressure (MP) mercury lamps, the former have been traditionally used. The low pressure applied to the mercury gas inside LP lamps (o10 Torr) causes sharp emission lines that output at B254 nm [8]. LP lamps are considered

0043-1354/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0043-1354(03)00238-0

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‘‘germicidal’’ because of this nearly monochromatic emission which closely corresponds with the associated maximum DNA absorption wavelength of 260 nm [8]. MP UV lamps have more recently been considered an effective alternative to LP lamps. In contrast to lowpressure systems, these lamps have increased pressure on the mercury gas within the lamp (B1000 Torr) which results in an increased intensity of radiation [8]. Along with an increase in intensity, the wavelengths are broadened resulting in emissions ranging from far UV (185 nm) to infrared wavelengths (1367 nm) [9]. One potential issue associated with the application of UV technology in water treatment is the ability of microorganisms to repair UV-induced DNA damage. Some organisms have evolved a variety of repair systems that are able to counteract many forms of DNA damage. Microorganisms may possess several DNA repair processes, including photoreactivation and nucleotide excision repair [7,10]. Photoreactivation (photo repair) is one mechanism used by many organisms to reverse the damage induced by UV radiation [7]. Photoreactivation involves the use of one enzyme, DNA photolyase, which uses energy from wavelengths between 300 and 500 nm (portion of UV-A and visible light range) to directly reverse many types of DNA damage. Photoreactivation has been observed in a number of organisms with evidence of repair occurring from minutes to hours [11,12]. Photo repair may be a potential issue if UV treatment, due to space constraints, occurs prior to a process unit that allows light exposure (e.g. UV exposure prior to filtration, when filters are located in areas illuminated with fluorescent lights or windows allowing penetration of sunlight). Also, photoreactivation cannot be ruled out when water is exposed to light following distribution (e.g. consumers storing UV treated water in light exposed areas). In contrast to photoreactivation, nucleotide excision repair is a more complex repair process that involves the coordination of numerous enzymes to remove DNA damage [7]. This process is often referred to as dark repair because no visible light is required for it reaction [7]. This repair process is of most concern within drinking water treatment during distribution of UV treated water. With recent reports indicating that UV can achieve high levels of Cryptosporidium inactivation (e.g. [1– 3,13]), it is imperative that reactivation potential be evaluated. The ability of C. parvum to repair its DNA is of utmost importance in drinking water treatment to fully determine the efficiency and effectiveness of UV for inactivation. During the time this study was conducted, additional research examining other aspects of C. parvum repair were conducted [4,14–16]. The findings of these studies will be discussed in more detail with the results of this research.

2. Objective The primary objective of this work was to evaluate the potential of C. parvum to photo repair and/or dark repair following UV irradiation from both LP and MP UV irradiation, using HCT-8 cell culture infectivity assay. A second objective was to evaluate the effect of temperature on the potential repair of C. parvum if lower and higher typical distribution system temperatures (5 and 25
C) were used.

3. Materials and methods 3.1. UV unit A bench-scale collimated beam apparatus (Calgon Carbon Corp., Pittsburgh, PA) was used to irradiate the oocysts used in this study. This apparatus contained an interchangeable LP (12 W) or MP (1 kW) mercury UV lamp. The sample to be irradiated was placed on a magnetic stir plate directly below a polyvinyl chloride collimating tube (93 cm), which aids in focusing the UV beam. 3.2. UV dose calculations Irradiance was measured using a radiometer (International Light, Model IL 1700, equipped with a SED 240 UV detector, Newburyport, MA) calibrated to the standards of the US National Institute of Standards and Technology (NIST). The UV dose (mJ/cm2) was determined by multiplying the average irradiance (mW/ cm2) in the sample liquid by the irradiation time (s). Before the final determination of the UV dose from LP or MP lamps, many factors must be applied to obtain an accurate dose. The term ‘‘dose’’ rather than ‘‘fluence’’ is employed in this paper because it is more commonly used in treatment applications. Determining UV dose from a MP lamp is considerably more complex than for a LP lamp due to the polychromatic nature of the emission. Therefore, numerous factors must be applied to achieve accurate doses. The MP UV dose was determined as described previously by Bukhari et al. [13] and Zimmer and Slawson [12] and calculated using software produced by Bolton Photosciences (Ayr, Canada). Factors determined to obtain an accurate MP UV dosage include determining the variation in the irradiance across the petri dish (petri factor), the attenuation of the beam within the liquid containing the organism (water factor), the reflection of UV at the liquid surface (reflection factor=0.975), and the variation in the sensor sensitivity to wavelength (sensor factor=1.206). Also a germicidal (weighted) factor was applied to the MP dose calculation [2,17,18]. This ‘‘weighted’’ factor accounts for the

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Table 1 Inactivation of C. parvum oocysts following LP and MP UV irradiation UV lamp source

Unweighted UV dose (mJ/cm2)

Weighted UV dose (mJ/cm2)

Number of trials

Average log reduction (7std dev)

LP

1 3 1.3 4

N/Aa N/A 1 3

4 4 6 3

1.5 X3.2 X3.3 >3.6

MP a

(70.3) (70.5) (70.6) (70.7)

Not applicable, low-pressure UV doses are not weighted.

relative DNA absorbance efficiency (being 1 at 260 nm) of the polychromatic medium-pressure UV lamp emissions [2,17]. It should be noted that the unweighted UV dose does not ‘‘weigh’’ any of the wavelengths differently and is most often expressed by other researchers (e.g. [1,3,13]). Weighted doses are about 25% less (Table 1), however only weighted doses will be discussed throughout. The LP UV doses were determined as previously described [12] and calculated using software (Bolton Photosciences, Ayr, Canada). For low-pressure UV lamps the irradiance can be taken directly from the radiometer because of its calibration for the emittance wavelength of 254 nm. To accurately determine the lowpressure UV dose, a petri factor, reflection factor and water factor were applied. 3.3. C. parvum oocysts C. parvum oocysts used in this study were obtained from the Sterling Parasitology Laboratory (Tucson, AZ). The oocysts were shed from calves infected with the Iowa isolate (Harley Moon Isolate) and purified using discontinuous sucrose and cesium chloride centrifugation gradients. 3.4. Infectivity procedure The cell culture used for the infectivity of C. parvum was that of ileocecal colorectal adenocarcinoma cells (human colon cells), or HCT-8 cells (CCL-244, ATCC, Manassas, VA). These cells were maintained as described by Slifko et al. [19] and subcultured every 3–4 days. During oocyst infection, growth medium was used for the HCT-8 cells. This medium was prepared as described by Slifko et al. [19] with the addition of 50 mM glucose, 35.0 mg/mL ascorbic acid, 1.0 mg/mL folic acid, 4.0 mg/mL 4-aminobenzoic acid and 2.0 mg/mL calcium pantothonate (all obtained from Sigma-Aldrich Canada Ltd., Oakville, ON) [19,20]. To prepare the cell culture for the infectivity assay, approximately 2.5  105 HCT-8 cells were inoculated into each well of an 8 well chamber slide (Lab-Tek II, Nalge Nunc International, Naperville, IL). These cells

were grown at 37
C in 5% CO2 in a CO2 incubator (CO2 Water Jacketed Incubator, Series 2, Forma Scientific) to 60–70% confluency before infection [19]. Cryptosporidium infectivity was assessed using the focus detection method (FDM) and MPN method for enumeration [19,21]. A brief description of the method is as follows: Prior to cell culture infection, the oocysts were pre-treated with a bleach solution to surface sterilize the oocysts and enhance excystation (see modifications to this bleach pre-treatment procedure below). The sample was then serially diluted (dilution range 101 to 104) in sterile tubes containing cell culture growth media. A total volume of 500 mL was added to each chamber well. Four replicate wells were inoculated per oocyst dilution. Wells were incubated for 48 h under the same conditions as they are maintained. Following incubation cells were washed, fixed and stained as described previously [19,21]. The stain used was a fluorescein-labelled antibody specific for the reproductive stages of C. parvum (Sporo-Glo Waterborne Inc., A600-FL, New Orleans, LA). To assess the infection of C. parvum, all wells were thoroughly examined under epi-fluorescence microscopy at 400  magnification (Zeiss, Axioskop 2, equipped with barrier filters, Germany). Infection within the cell culture was determined when a cluster of C. parvum within the cell monolayer, a foci, was observed. If clustering was observed, the well was marked as positive. If no infection was observed the well was marked as negative. The number of positive wells were used to quantify the number of infectious oocysts in the sample [21]. The concentration of infectious oocysts (MPN/mL) in the sample was determined using a MPN computer program [22]. For this present study the infectivity method was modified during the bleach pre-treatment step to reduce the losses of oocysts by eliminating the centrifugation/ wash step. This modified step is as follows: The 10% bleach solution was added to the oocyst suspension at a 1:40 ratio (bleach solution:oocyst suspension) for 10 min at 4
C. After 10 min, growth media was added at a 1:10 oocyst suspension to media ratio to dilute the bleach. This modified procedure produced similar levels of infectivity compared to the original method (data not shown).

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3.5. C. parvum irradiation and inactivation calculations C. parvum oocysts were suspended in 0.01 M phosphate buffered saline (PBS) to obtain a concentration of approximately 106/mL or 107/mL, as determined through direct hemacytometer counts (Petroff-Hausser Counter, Hausser Scientific, Horsham, PA). The sample was vortexed and 5 mL were added directly into a 50 mm plastic petri dish (Courtesy Med/Tek, Buffalo Grove, IL). Prior to irradiation, a portion of the oocyst suspension was removed and inoculated into cell culture. This sample was used to determine the initial infectivity. With the petri dish lid removed and while constantly stirring, the oocysts were exposed to UV radiation from a LP or MP lamp. Doses of 1 and 3 mJ/cm2 were chosen for irradiation from LP UV lamps and weighted doses of 1 and 3 mJ/cm2 from MP UV lamps. These doses are lower than typically used in water treatment (16 to X40 mJ/cm2) [5,6] and were chosen to favour repair. Immediately following UV exposure, the entire sample volume was collected and placed into a sterile test tube covered with foil to prevent light exposure. A portion of the irradiated sample was removed and inoculated into cell culture to determine the level of infectivity (MPN/mL) immediately following exposure. This infectivity and the initial infectivity value determined the initial log inactivation attributed to UV exposure. Oocyst inactivation levels are expressed as log reductions. Log reduction=log (MPN/mL prior to UV irradiation C MPN/mL following UV irradiation). When no detectable infections of C. parvum occurred following irradiation, values were represented as estimates. In order to estimate the log inactivation in these instances, it was assumed that one infectious oocyst was present in the original sample. Log inactivations are shown as ‘‘X’’ when some replicate experiments showed no infection or represented as ‘‘>’’ when no infections were observed in any replicate experiments.

of the two petri dishes was exposed to light to examine potential photo repair and one was covered with foil to allow for potential dark repair. Samples were incubated at 5
C or 25
C to represent lower- and higher-end temperatures found within a typical distribution system [23]. A portion of each incubated sample was aseptically removed from each dish at several times up to 120 h following the start of incubation to determine the level of infectious organisms. 3.7. Repair control A strain of Escherichia coli (ATCC 11229, Manassas, VA) with the known ability to photo repair [11,12] was used as a positive control. This control was to determine if the light intensity was adequate for repair and that the suspension media and the petri dish could allow photoreactivating light to penetrate. E. coli (ATCC 11229) was suspended in the same PBS and petri dishes used in the C. parvum investigations. E. coli (ATCC 11229) suspensions were exposed to LP UV irradiation at a dose of 10 mJ/cm2 and then exposed to a number of fluorescent lamps to investigate the occurrence of repair. A standard plate count method was used for E. coli assessment [24]. The E. coli control results are expressed as N=N0 ; where N=CFU/mL of E. coli following irradiation, N0 =CFU/mL of E. coli prior to irradiation.

4. Results Photoreactivation of the control organism (E. coli ATCC 11229) was not observed following exposure to one or two fluorescent lamps, however, repair was evident following exposure to three 15 W fluorescent lamps (data not shown). A total of five 15 W fluorescent lamps were used for the E. coli repair control and for all C. parvum studies [11,12]. Fig. 1 shows E. coli (ATCC 1.000000

3.6. Repair conditions

light dark

0.100000

0.010000

E. coli N/No

After removing an initial aliquot to determine inactivation, the remaining UV-irradiated sample was divided and transferred into two separate plastic petri dishes (Phoenix Biomedical Products Inc., Mississauga, ON). These samples were continuously mixed in a controlled environment incubator (Innova 4230, Refrigerated Incubator Shaker, New Brunswick Scientific), which was equipped with five 15 W fluorescent grow lamps (Agro-Lite, 46 cm, Philips-Lighting Co., Somerset, NJ). The intensity of the lights at the sample surface was measured to be approximately 16,900 lux (Light Meter model LI-189 with Quantum Sensor, LI-COR Bioscience, Lincoln, NE). The samples were placed 4 cm from three over-hanging lamps and two side lamps. One

0.001000

0.000100

0.000010

0.000001 0

50

100

150

200

250

300

Time (min)

Fig. 1. E. coli (ATCC 11229) control for photoreactivation conditions following exposure to LP UV at 10 mJ/cm2. Error bars represent standard deviations of three replicate experiments.

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Table 2 Effect of light and dark incubation on infectivity of oocysts over time following LP and MP UV irradiation at 1 mJ/cm2 Lamp source

Time (h)

Log reduction (7std dev) Incubation at 25
C

LP

MPa

a b

0 24 72 120 0 24 72 120

Incubation at 5
C

Light

Dark

Light

1.5 (0.4) 2.0 (0.7) 2.5 (0.4) 2.1b X3.0 (0.3) 2.8 (0.0) 3.2 (0.0) >3.2 (0.0)

1.5 (0.4) 1.8 (0.7) 1.7 (1.1) 2.4b X3.0 (0.3) X3.2 (0.0) 3.0 (0.3) >3.2 (0.0)

1.5 1.9 1.9 2.2 3.8 3.8 >3.6 X3.8

Dark (0.4) (0.1) (0.1) (0.2) (0.9) (0.9) (1.2) (0.9)

1.5 1.9 1.7 1.8 3.8 3.4 >3.6 3.6

(0.4) (0.1) (0.2) (0.3) (0.9) (0.9) (0.6) (0.6)

MP doses are weighted. Not averaged, replicate experiment did not include sample point.

Table 3 Effect of light and dark incubation on infectivity of oocysts over time following LP and MP UV irradiation at 3 mJ/cm2 Lamp source

Time (h)

Log reduction (7std dev) Incubation at 25
C

LP

MPa

a b

0 24 72 120 0 24 72

Incubation at 5
C

Light

Dark

Light

>3.4 (0.8) >3.4 (0.8) X3.4 (0.8) N/Ab >3.2 (0.02) >3.2 (0.02) >3.2 (0.02)

>3.4 (0.8) >3.4 (0.8) X3.4 (0.8) N/Ab >3.2 (0.02) >3.2 (0.02) >3.2 (0.02)

>3.0 >3.0 >3.0 >3.0 >3.8 >3.8 >3.8

Dark (0.0) (0.0) (0.0) (0.0) (0.9) (0.9) (0.9)

>3.0 >3.0 >3.0 >3.0 >3.8 >3.8 >3.8

(0.0) (0.0) (0.0) (0.0) (0.9) (0.9) (0.9)

MP doses are weighted. Samples not taken at 120 h.

11229) photo repair over time at 25
C following LP UV exposure at 10 mJ/cm2. Photo repair at 25
C was evident within 30 min and reached maximum levels of repair at 240 min. Photo repair was also evident following light exposure at 5
C and was detectable after 120 min exposure to photoreactivating light (not shown). Dark repair of E. coli (ATCC 11229) was not observed. This provided a negative control. Following UV irradiation the initial log reduction of C. parvum was determined as previously described. Table 1 presents the log inactivation of C. parvum following LP and MP irradiation at doses of 1 and 3 mJ/ cm2. Averages with standard deviations are shown. Except at the lower dose (1 mJ/cm2) with LP UV, at least 3.2 log inactivation was observed in all cases. Following irradiation oocysts were incubated in the light or dark at temperatures of 25
C or 5
C to investigate repair. Table 2 shows the results of the experiments investigating repair potential of C. parvum following LP UV at a dose of 1 mJ/cm2 and a weighted

MP dose of 1 mJ/cm2. Time zero represents infectivity determined immediately following irradiation and prior to incubation under repair conditions. Results from replicate experiments (n ¼ 2) with standard deviations are shown. As observed, log reductions remained steady or increased slightly with incubation time, indicating that recovery of C. parvum infectivity was not occurring following exposure to light or dark conditions up to 120 h (5 days). Table 3 summarizes results following LP and MP UV irradiation following a dose of 3 mJ/cm2. As can be seen, following each experiment the log inactivation was X3 log. Restoration of infectivity was not observed following light or dark incubation at 25
C or 5
C.

5. Discussion Consistent with findings from previous studies (e.g. [1–3]), the results of this work demonstrate that

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C. parvum oocysts are very susceptible to low doses of UV when evaluated by infectivity assays. Although findings of various researchers are not consistent in the levels of inactivation occurring, considerable oocyst inactivation is generally observed below doses of 10 mJ/ cm2 (e.g. [2,3]). Tailing was observed in the inactivation data as the dose increased. As observed by other researchers (e.g. [2,25,26]), this may be attributed to the high initial concentration of organisms used in this study. In the present study higher levels of oocyst inactivation were evident following MP UV irradiation than following LP irradiation. This difference was more notable at the lower (1 mJ/cm2) dose (Table 1). The difference in inactivation may be attributed to using weighted MP UV doses as opposed to unweighted doses (used by most other researchers). Unweighted MP doses are about onethird higher than weighted MP UV doses (Table 1) and therefore may have contributed to higher levels of DNA damage. To determine whether C. parvum could self-repair following UV irradiation, it was important to investigate conditions that favour reactivation, such as low UV doses, various temperatures and longer detention times. Appropriate light conditions through the use of positive controls for photoreactivation were also determined for this repair study. Following incubation in the light and dark up to 120 h (5 days) no photo or dark repair of C. parvum was observed following LP UV irradiation. This portion of the work involving LP UV confirms reports by Shin et al. [4], Oguma et al. [15] and Morita et al. [14]. Following LP UV irradiation Shin et al. [4] determined that no increases in oocyst infectivity occurred up to 4 h incubation under light and dark conditions, as assessed using MDCK cell culture. Oguma et al. [15] and Morita et al. [14] observed continuous genomic repair of the DNA damage in the light and dark using the molecular technique endonuclease sensitive site (ESS) assay, but no measurable increases in infectivity up to 24 h using animal models. Although Oguma et al. [15] and Morita et al. [14] observed continuous DNA repair at the genomic level following exposure to LP UV, any potential genomic repair in oocysts in the present study was not reflected in an increase in infectivity with a longer incubation time up to 120 h (5 days) under light and dark conditions. To date, no studies have been performed to investigate photo repair of C. parvum following MP UV exposure. Under the conditions of this study no detectable increases in oocyst infectivity were observed, indicating a lack of photo repair ability in C. parvum following MP UV irradiation. Following MP UV irradiation at higher doses (10, 60 and 240 mJ/cm2) Belosevic et al. [16] observed no dark repair of C. parvum up to 17 days of incubation, as assessed using an

animal model. Even following lower doses of MP UV irradiation in this present study, C. parvum did not show any dark repair potential up to 120 h (5 days). Recent studies have demonstrated that irradiation with MP UV can inhibit repair from occurring due to the wavelength emissions from these lamps [12,27]. C. parvum photoreactivation and dark repair potential was assessed at 5
C and 25
C. Due to the fact that no repair was observed, temperature had no effect on repair potential.

6. Conclusions This study confirms through infectivity assay, that C. parvum oocysts are very sensitive to low UV doses from both LP and MP UV sources. No detectable evidence of light or dark repair of C. parvum oocysts, as assessed through infectivity using HCT-8 cells, was observed following LP or MP UV, at doses of 1 and 3 mJ/cm2 when temperature (5
C and 25
C) and detention time (up to 5 days) were considered. Because the UV doses used in this study (1 and 3 mJ/ cm2) were well below doses currently used in drinking water treatment (16 to X40 mJ/cm2), it is unlikely that doses currently used in treatment would allow for any repair. Due to the fact that no evidence of repair (as measured by infectivity) was observed, this phenomenon does not need to be considered when setting UV doses for the inactivation of this organism.

Acknowledgements Thank you to Calgon Carbon Corp. for the use of the collimated beam unit. Funding for this project was provided by the Natural Sciences and Engineering Research Council of Canada (NSERC) in the form of a Collaborative Research and Development (CRD) grant and an Industrial Research Chair at the University of Waterloo. For current Chair partners see www.civil.uwaterloo.ca/watertreatment.

References [1] Clancy JL, Bakhari Z, Hargy TM, Bolton JR, Dussert BW, Marshall MM. Using UV to inactivate Cryptosporidium. J AWWA 2000;92(9):97–104. [2] Craik SA, Weldon D, Finch GR, Bolton JR, Belosevic M. Inactivation of Cryptosporidium oocysts using mediumand low-pressure ultraviolet radiation. Wat Res 2001;35:1387–98. [3] Mofidi AA, Baribeau H, Rochelle PA, De Leon R, Coffey BM, Green JF. Disinfection of Cryptosporidium parvum

ARTICLE IN PRESS J.L. Zimmer et al. / Water Research 37 (2003) 3517–3523

[4]

[5]

[6]

[7] [8] [9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

with polychromatic UV light. J AWWA 2001;93(6): 95–109. Shin G, Linden KG, Arrowood MJ, Sobsey MD. Lowpressure UV inactivation and DNA repair potential of Cryptosporidium parvum oocysts. Appl Environ Microbiol 2001;67(7):3029–32. DVGW. German association on gas, water technical standard W 294: UV systems for disinfection in drinking water supplies—requirements and testing. DVGW Standards and Guidelines, Bonn, Germany. 1997. Norwegian Institute of Public Health (Nasjonalt Folkehelseinstitutt). 2002. http://www.fhi.no/tema/drikkevann/ uvinfo-en.html [accessed 13 May 2003]. Friedberg EC, Walker G, Siede W. DNA and mutagenesis. Washington, DC: ASM Press; 1995. Bolton JR. Ultraviolet Applications Handbook. Bolton Photosciences Inc., Ayr, ON, 2001. Linden KG, Mofidi AA. Measurement of UV irradiance: tools and considerations. In Proceedings of the AWWA’s Water Quality Technology Conference, AWWA, Tampa, FL, 1999. Thoma F. Light and dark in chromatin repair: repair of UV-induced lesions by photolyase and nucleotide excision repair. EMBO J 1999;18(23):6585–98. Sommer R, Lhotsky M, Haider T, Cabaj A. UV inactivation, liquid-holding recovery, and photoreactivation of E. coli O157 and other pathogenic E. coli strains in water. J Food Prot 2000;63(8):1015–20. Zimmer JL, Slawson RM. Potential repair of E. coli DNA following exposure to UV radiation from both mediumand low-pressure UV sources used in drinking water treatment. Appl Environ Microbiol 2002;68:3293–9. Bukhari Z, Hargy TM, Bolton JR, Dussert B, Clancy JL. Medium-pressure UV for oocyst inactivation. J AWWA 1999;91(3):86–94. Morita S, Namikoshi A, Hirata T, Oguma K, Katayama H, Ohgaki S, Motoyama N, Fujiwara M. Efficacy of UV irradiation in inactivating Cryptosporidium parvum oocysts. Appl Environ Microbial 2002;68:5387–93. Oguma K, Katayama H, Mitani H, Morita S, Hirata T, Ohgaki S. Determination of pyrimidine dimers in E. coli and Cryptosporidium parvum during UV light inactivation, photoreactivation, and dark repair. Appl Environ Microbiol 2001;67:4630–7. Belosevic M, Craik SA, Stafford JL, Neumann NF, Kruithof J, Smith DW. Studies on the resistance/reactiva-

[17]

[18]

[19]

[20]

[21]

[22] [23]

[24]

[25]

[26]

[27]

3523

tion of Giardia muris and Cryptosporidium parvum oocysts exposed to medium-pressure ultraviolet radiation. FEMS Microbiol Lett 2001;204:197–203. Bolton JR, Linden KG. Determination of fluence (UV Dose) in bench-scale collimated beam apparatus for monochromatic and broadband UV lamps. In Proceedings of the AWWA’s Water Quality Technology Conference, AWWA, Nashville, TN, 2001. Linden KG, Darby JL. Estimating effective germicidal dose form medium pressure UV lamps. J Environ Eng 1997;123:1142–9. Slifko TR, Friedman D, Rose JB, Jakubowski W. An in vitro method for detecting infectious Cryptosporidium oocysts with cell culture. Appl Environ Microbiol 1997;63(9):3669–75. Upton SJ, Tilley M, Brillhart DB. Effect of selected medium supplements on in vitro development of Cryptosporidium parvum in HCT-8 cells. J Clin Microbiol 1995; 33(2):371–5. Slifko TR, Huffman DE, Rose JB. A Most-ProbableNumber Assay for enumeration of infectious Cryptosporidium parvum oocysts. Appl Environ Microbiol 1999;65(9):3936–41. Klee AJ. 1996. Most Probable Number Calculator, Version 4.04. USEPA, Cincinnati, OH. Geldreich EE. Microbial quality of water supply in distribution systems. Boca Raton, FL: CRC Press, Inc.; 1996. APHA, AWWA, and WEF. Method #9215. In: Clesceri LS, Greenberg AE, Eaton AD, editors. Standard Methods for the Examination of Water and Wastewater. 20th ed. Washington DC: United Book Press Inc, 1998. Hassen A, Mahrouk M, Ouzari H, Cherif M, Boudabous A, Damelincourt JJ. UV disinfection of treated wastewater in a large-scale pilot plant and inactivation of selected bacteria in a laboratory UV device. Bioresour Technol 2000;74:141–50. Blatchley ER, Dumoutier N, Halaby TN, Levi Y, Laine JM. Bacterial responses to ultraviolet irradiation. Wat Sci Technol 2001;43:179–86. Oguma K, Katayama H, Ohgaki S. Photoreactivation of E. coli after low- or medium-pressure UV disinfection determined by an endonuclease sensitive site assay. Appl Environ Microbiol 2002;68:6029–35.