Inactivation of Cryptosporidium parvum oocysts with ozone and free chlorine

The objective of this study is to investigate the synergy involved in the sequential inactivation of C. parvum oocysts with ozone followed by free chlorine at 1-20 degrees C. Primary ozone and free chlorine inactivation curves are characterized by an initial lag-phase, followed by one or two post-lag-phase segments, the first segment at a faster rate than the second, of pseudo-first-order inactivation. The kinetics of primary inactivation with ozone and free chlorine has a relatively strong temperature dependence, and vary both with oocyst lot and oocyst age. Synergy is observed for the sequential inactivation of C. parvum oocysts with ozone/free chlorine. Ozone pre-treatment results in the disappearance of the lag-phase and the occurrence of a secondary free chlorine inactivation curve with generally two pseudo-first-order segments, the first segment at a faster rate than the second. The kinetics of both secondary segments is significantly faster than the post-lag-phase rate of inactivation with free chlorine alone. The temperature dependence for both phases of the secondary free chlorine inactivation kinetics is weaker compared to that for primary inactivation with ozone or free chlorine. As a result, the level of synergy in sequential disinfection with ozone/free chlorine increases with decreasing temperature within the range relevant to drinking water utilities. Good agreement is found between the kinetics determined using the modified in-vitro excystation method of viability assessment and animal infectivity data recently reported in the literature for both primary inactivation with ozone, and sequential disinfection with ozone/free chlorine.     

Inactivation of Cryptosporidium parvum oocysts with ozone and monochloramine at low temperature

The rate of Cryptosporidium parvum inactivation decreased with decreasing temperature (1-20 degrees C) for ozone and for monochloramine applied alone as well as after pre-treatment with ozone. Synergy was observed at all temperatures studied for the ozone/monochloramine sequential disinfection scheme. The synergistic effect was found to increase with decreasing temperature. The inactivation rate with monochloramine after ozone pre-treatment was 5 times faster at 20 degrees C and 22 times faster at 1 degree C than the corresponding post-lag phase rates of inactivation with monochloramine at these temperatures when no ozone pre-treatment was applied. The CT required for achieving 2-logs of inactivation ranged from 11,400 mg min l-1 at 20 degrees C to 64,600 mg min l-1 at 1 degree C when monochloramine was applied alone. In contrast, the CT required for an overall sequential inactivation of 2-logs ranged from 721 mg min l-1 at 20 degrees C to 1350 mg min l-1 at 1 degree C when applying monochloramine after ozone pre-treatment. The presence of excess ammonia in the monochloramine solutions was not responsible for the synergy observed in ozone/monochloramine sequential disinfection.     

Photocatalytic inactivation of Cryptosporidium parvum with TiO(2) and low-pressure ultraviolet irradiation

This study investigated the efficacy of low-pressure ultraviolet (UV) irradiation and the synergistic effect of UV/titanium dioxide (TiO(2)) photocatalysis on Cryptosporidium parvum oocyst inactivation. At UV doses of 2.7, 8.0, and 40mJ/cm(2), oocyst inactivation was 1.3, 2.6, and 3.3log(10), respectively. Reactive oxygen species (ROS) generated by longwave UV radiation (>315nm) and TiO(2) achieved less than 0.28-log inactivation. However, the synergistic effect of germicidal (254nm) UV and TiO(2) resulted in 2-log and 3-log oocyst inactivation with 4.0 and 11.0mJ/cm(2), respectively. Therefore, using TiO(2) in combination with UV reduced the dose requirement for 3-log inactivation by 56%. An approximate 1-log decrease in inactivation of oocysts was observed with nanopure water in comparison to buffered water, whereas changes in pH from 6 to 8 had little effect on the photocatalytic inactivation of oocysts in either matrix (P>0.1).     

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

This study investigated the level of inactivation and the potential for Cryptosporidium parvum to repair following low doses (1 and 3mJ/cm(2)) 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 degrees C or 25 degrees 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.     

Recovery and detection of Cryptosporidium parvum oocysts from water samples using continuous flow centrifugation

Continuous flow centrifugation (CFC) was used in conjunction with immunomagnetic separation (IMS) and immunofluorescence microscopy (IFA) and nested PCR to recover and detect oocysts of Cryptosporidium parvum and cysts of Giardia intestinalis from 10L volumes of source water samples. Using a spiking dose of 100 oocysts, nine of 10 runs were positive by IFA, with a mean recovery of 4.4+/-2.27 oocysts; when another 10 runs were analyzed using nested PCR to the TRAP C-1 and Cp41 genes, nine of 10 were positive with both PCR assays. When the spiking dose was reduced to 10 oocysts in 10L, 10 of 12 runs were positive by IFA, with a mean oocyst recovery of 3.25+/-3.25 oocysts. When 10 cysts of Giardia intestinalis were co-spiked with oocysts into 10L of source water, five of seven runs were positive, with a mean cyst recovery of x=0.85+/-0.7. When 10 oocysts (enumerated using a fluorescence activated cell sorter) were spiked into 10L volumes of tap water, one of 10 runs was positive, with one oocyst detected. For the majority of the source water samples, turbidities of the source water samples ranged from 1.1 to 22 NTU, but exceeded 100 NTU for some samples collected when sediment was disturbed. The turbidities of pellets recovered using CFC and resuspended in 10 mL of water were very high (exceeding 500 NTU for the source water-derived pellets and 100 NTU for the tap water-derived pellets). While not as efficient as existing capsule-filtration based methods (i.e., US EPA methods 1622/1623), CFC and IMS may provide a more rapid and economical alternative for isolation of C. parvum oocysts from highly turbid water samples containing small quantities of oocysts.     

Transport and fate of Cryptosporidium parvum oocysts in intermittent sand filters.

The transport potential of Cryptosporidium parvum (C. parvum) through intermittent, unsaturated, sand filters used for water and wastewater treatment was investigated using a duplicated, 2³ factorial design experiment performed in bench-scale, sand columns. Sixteen columns (dia=15 cm, L=60 cm) were dosed eight times daily for up to 61 days with 65,000 C. parvum oocysts per liter at 15°C. The effects of water quality, media grain size, and hydraulic loading rates were examined. Effluent samples were tested for pH, turbidity, and oocyst content. C. parvum effluent concentrations were determined by staining oocysts on polycarbonate filters and enumerating using epifluorescent microscopy. At completion, the columns were dismantled and sand samples were taken at discrete depths within the columns. These samples were washed in a surfactant solution and the oocysts were enumerated using immunomagnetic separation techniques.

The fine-grained sand columns (d₅₀=0.31 mm) effectively removed oocysts under the variety of conditions examined with low concentrations of oocysts infrequently detected in the effluent. Coarse-grained media columns (d₅₀=1.40 mm) yielded larger numbers of oocysts which were commonly observed in the effluent regardless of operating conditions. Factorial design analysis indicated that grain size was the variable which most affected the oocyst effluent concentrations in these intermittent filters. Loading rate had a significant effect when coarse-grained media was used and lesser effect with fine-grained media while the effect of feed composition was inconclusive. No correlations between turbidity, pH, and effluent oocyst concentrations were found. Pore-size calculations indicated that adequate space for oocyst transport existed in the filters. It was therefore concluded that processes other than physical straining mechanisms are mainly responsible for the removal of C. parvum oocysts from aqueous fluids in intermittent sand filters used under the conditions studied in this research.     

Effect of NOM and biofilm on the removal of Cryptosporidium parvum oocysts in rapid filters

Laboratory experiments were performed to evaluate the effects of biofilm and natural organic matter (NOM) on removal of Cryptosporidium parvum oocysts from water by filtration. The bench-scale rapid filters consisted of 2.54 cm ID x 30.5 cm polycarbonate plastic columns packed with 0.55 mm spherical glass beads to a depth of 25 cm and a porosity of 40%. Calcium chloride (0.01 M) served as the coagulant in most of the experiments. The oocyst removal efficiency decreased from 51 +/- 6% for a clean bed to 23 +/- 3% for the biofilm-coated bed and to 14 +/- 1% in the presence of 5 ppm of NOM. The oocyst removal for an experiment with a combination of biofilm-coated filter media and NOM was similar to that for the experiment with NOM alone (15 +/- 1%). The zeta potential values for the oocysts pre-equilibrated with NOM were significantly more negative than those obtained for untreated oocysts. This suggests that NOM enhanced the electrostatic repulsion between the oocysts and the negatively charged glass beads. Fortunately, use of alum as coagulant at a dosage sufficient to neutralize the surface charge of the NOM-coated oocysts resulted in a high removal efficiency (73 +/- 6%). Pre-equilibration of the oocysts with NOM also increased the hydrophobicity of the oocysts, but this was deemed to have a negligible effect on deposition onto the glass beads. The results of these experiments suggest that water treatment facilities treating source waters with moderate organic matter concentrations and/or employing biologically active filters have a greater potential for oocyst breakthrough and proper coagulation is critical for effective removal of oocysts in the filters.     

Impact of bathers on levels of Cryptosporidium parvum oocysts and Giardia lamblia cysts in recreational beach waters

Recreational beach water samples collected on weekends and weekdays during 11 consecutive summer weeks were tested for potentially viable Cryptosporidium parvum oocysts and Giardia lamblia cysts using the multiplexed fluorescence in situ hybridization (FISH) method. The levels of oocysts and cysts on weekends were significantly higher than on the weekdays (P<0.01). Concentrations of oocysts in weekend samples (n=27) ranged from 2 to 42 oocysts/L (mean: 13.7 oocysts/L), and cyst concentration ranged from 0 to 33 cysts/L (mean: 9.1 cysts/L). For the samples collected on weekdays (n=33), the highest oocyst concentration was 7 oocysts/L (mean: 1.5 oocysts/L), and the highest cyst concentration was 4 cysts/L (mean: 0.6 cysts/L). The values of water turbidity were significantly higher on weekends than on weekdays, and were correlated with the number of bathers and concentration of C. parvum oocysts and G. lamblia cysts (P<0.04). The study demonstrated positive relationships between number of bathers and levels of waterborne C. parvum oocysts and G. lamblia cysts in recreational beach water. It is essential to test recreational waters for Cryptosporidium and Giardia when numbers of bathers are greatest, or limit the number of bathers in a recreational beach area.     

Sequential inactivation of Cryptosporidium parvum oocysts with chlorine dioxide followed by free chlorine or monochloramine.

The main objective of this study was to assess the effect of temperature (4-30 degrees C) on the inactivation kinetics of Cryptosporidium parvum oocysts with sequential disinfection schemes involving the use of chlorine dioxide as the primary disinfectant and free or combined chlorine as the secondary disinfectant in synthetic water. The synergy previously reported for sequential inactivation of C. parvum oocysts with ozone/free chlorine or ozone/combined chlorine did not occur when chlorine dioxide was used. instead of ozone, as the primary disinfectant within the temperature range (4-30 degrees C) and the pre-treatment levels investigated. Sequential ozone/chlorine dioxide and chlorine dioxide ozone experiments revealed that the lower level or absence of synergy for chlorine dioxide/free chlorine and chlorine dioxide, monochloramine was likely the result of chlorine dioxide reacting with oocyst chemical groups that are mostly different from those reacting with ozone, free chlorine, or monochloramine. The CT concept was found to be valid for the primary inactivation kinetics of C. parvum oocysts with chlorine dioxide, thus allowing the use of the simpler CT approach for the development of C. partum inactivation requirements with chlorine dioxide. General consistency was found between the secondary inactivation kinetics of C. parvum oocysts with free chlorine and monochloramine after chlorine dioxide pretreatment obtained in this study with oocyst viability determined by a modified in vitro excystation method and those reported in the literature for the same sequential disinfection schemes based on an animal infectivity assay.     

Sequential inactivation of Cryptosporidium parvum using ozone and chlorine.

Inactivation of bovine-derived C. parvum oocysts was studied at bench-scale in oxidant demand free 0.05 M phosphate buffer using free chlorine alone or ozone followed by free chlorine at temperatures of 1°C, 10°C and 22°C at pH 6. Animal infectivity using neonatal CD-1 mice was used for evaluation of oocyst viability after treatment. Kinetic models based on the linear Chick–Watson model were developed for free chlorine inactivation and ozone/free chlorine sequential inactivation for 0.4 or 1.6 log-units of ozone primary kill. At 22°C, ozone pre-treatment increased the efficacy of free chlorine for about 4–6 times depending on the level of ozone primary kills. Gross kills of the ozone/free chlorine sequential inactivation were a function of ozone primary kills and increased linearly with the free chlorine C_avgt (arithmetic average of the initial and final residual×contact time) product. Temperature was critical for both single and sequential inactivation, and the efficacy of free chlorine after 1.6 log-units of ozone primary inactivation decreased by a factor of 1.8 for every 10°C temperature decrease. Given an ozone primary kill of 1.6 log-units, the free chlorine C_avgt products required for a gross kill of 3.0 log-units were 1000, 2000 and 3300 mg min/L for 22°C, 10°C and 1°C, respectively.     

A microscope system with a dual-band filter for the simultaneous enumeration of Cryptosporidium parvum oocysts and sporozoites.

A dual band filter set was designed to allow the simultaneous viewing of two fluorochromes (fluorescein isothiocyanate (FITC) and 4',6-diamidino-2-phenylindole (DAPI)) so that Cryptosporidium oocysts and sporozoites can be viewed together. Simultaneous viewing increases accuracy and decreases examination time as compared to current Environmental Protection Agency (EPA) Method 1623 for the detection of Cryptosporidium and Giardia in water by filtration/IMS/FA (Immunomagnetic Separation/Fluorescent Antibodies). Additionally, a microscope equipped with a programmable, motorized stage, CCD camera, and display monitor was used to facilitate well slide evaluation in a quick and precise fashion ensuring complete coverage without overlap and eliminating the optical strain associated with counting hundreds of images through an eyepiece.     

A Bayesian method of estimating kinetic parameters for the inactivation of Cryptosporidium parvum oocysts with chlorine dioxide and ozone

The main objective of this paper is to use Bayesian methods to estimate the kinetic parameters for the inactivation kinetics of Cryptosporidium parvum oocysts with chlorine dioxide or ozone which are characterized by the delayed Chick-Watson model, i.e., a lag phase or shoulder followed by pseudo-first-order rate of inactivation. As the length of the lag phase (CT(lag)) is not known, Bayesian statistics provides a more accurate approach than traditional statistical methods to fitting the delayed Chick-Watson kinetics. Markov Chain Monte Carlo method is used to estimate CT(lag) and first-order rate constant values. This method is also used to estimate the minimum CT requirement (with safety factor) for 99% inactivation of C. parvum oocysts.     

Ecotoxicological assessment of pulsed ultraviolet light‐treated water containing microbial species and Cryptosporidium parvum using a microbiotest test battery

Development of a pulsed ultraviolet (PUV) light system as an alternative disinfection method for water treatment has been investigated, yet little is known about the impact of applying such emerging technologies on the natural aquatic environment. This study reports on the use of a battery of ecotoxicological tests representative of different ecological trophic levels to determine the safety and potability of PUV‐treated water containing known pathogens. Water samples containing Cryptosporidium oocysts and Escherichia coli were treated with PUV light, and the ecotoxicological safety was determined by assessing toxicity in a range of representative microbiotests and the Rainbow Trout Hepatocyte (RTH)‐149 fish cell line. Results show that PUV rapidly inactivated Cryptosporidium and the selected microbial species; greater rates of inactivation were observed at higher ultraviolet doses. PUV‐treated samples did not elicit ecotoxicological effects in the trophic levels studied. This study highlights that PUV is an effective means of disinfecting water containing pathogens such as Cryptosporidium.     

Inactivation of Giardia muris cysts using medium-pressure ultraviolet radiation in filtered drinking water

The effect of medium pressure ultraviolet radiation on Giardia muris was studied using a collimated beam apparatus with filtered surface water from the Grand River, Kitchener, Ontario, Canada. UV doses ranged from 5 to 83 mJ/cm² and resulted in 2–3 log-units of reduction in infectivity measured by a C3H/HeN mouse infectivity model. In vitro excystation and nucleic acid staining with Live/Dead BacLight^™ greatly underestimated the inactivation of Giardia when compared with animal infectivity. Medium pressure ultraviolet radiation is a potential alternative to conventional chemical disinfection methods.     

Biology, persistence and detection of Cryptosporidium parvum and Cryptosporidium hominis oocyst

Cryptosporidium parvum and Cryptosporidium hominis are obligate enteric protozoan parasites which infect the gastrointestinal tract of animals and humans. The mechanism(s) by which these parasites cause gastrointestinal distress in their hosts is not well understood. The risk of waterborne transmission of Cryptosporidium is a serious global issue in drinking water safety. Oocysts from these organisms are extremely robust, prevalent in source water supplies and capable of surviving in the environment for extended periods of time. Resistance to conventional water treatment by chlorination, lack of correlation with biological indicator microorganisms and the absence of adequate methods to detect the presence of infectious oocysts necessitates the development of consistent and effective means of parasite removal from the water supply. Additional research into improving water treatment and sewage treatment practices is needed, particularly in testing the efficiency of ozone in oocyst inactivation. Timely and efficient detection of infectious C. parvum and C. hominis oocysts in environmental samples requires the development of rapid and sensitive techniques for the concentration, purification and detection of these parasites. A major factor confounding proper detection remains the inability to adequately and efficiently concentrate oocysts from environmental samples, while limiting the presence of extraneous materials. Molecular-based techniques are the most promising methods for the sensitive and accurate detection of C. parvum and C. hominis. With the availability of numerous target sequences, RT-PCR will likely emerge as an important method to assess oocyst viability. In addition, a multiplex PCR for the simultaneous detection of C. parvum, C. hominis and other waterborne pathogens such as Giardia lamblia would greatly benefit the water industry and protect human health.     

Interaction between Cryptosporidium oocysts and water treatment coagulants

The electrokinetic properties of gamma-irradiated Cryptosporidium oocysts in the presence of coagulants (ferric chloride and alum) and coagulant aids (DADMAC based cationic polyelectrolytes) have been studied. The zeta potential of the oocysts was unaffected by the addition of ferric chloride at all pH values (3-10) studied. Addition of alum resulted in reversal of the oocysts charge, which suggests that the initial stage in the coagulation process leading to floc formation proceeds via the adsorption of hydrolysed aluminium species. The cationic polyelectrolyte Magnafloc LT35 was adsorbed onto iron flocs at doses of 0.1 mg/L even against an electrostatic barrier. The cationic polyelectrolyte only adsorbed and caused charge reversal at the oocyst surface at around 0.4 mg/L, suggesting a lower affinity for this surface. These results indicate that the oocysts, unlike inorganic colloidal materials such as metal oxides, appear to possess a lower surface density of active or charged sites. The lower density of sites, combined with the rapid precipitation of iron salts, may be responsible for the lack of specific adsorption of either hydroxylated ferric species or primary iron hydroxide particles on the oocysts. Further, this suggests that a process of sweep flocculation, where oocysts are engulfed in flocs during coagulation and floc formation, is the more likely mechanism involved. By comparison, it is likely that the specific interaction of hydrolysed aluminium species with the oocysts surface would result in a stronger link at the oocyst-floc interface and that the flocculation process may initially proceed via charge neutralisation.     

Evaluation and optimization of a reusable hollow fiber ultrafilter as a first step in concentrating Cryptosporidium parvum oocysts from water.

Experiments with a small-scale hollow fiber ultrafiltration system (50,000 MWCO) was used to characterize the filtration process and identify conditions that optimize the recovery of Cryptosporidium parvum oocysts from 2 L samples of water. Seeded experiments were conducted using deionized water as well as four environmental water sources (tap, ground, Arkansas river, and Rio Grande river; 0-30.9NTU). Optimal and consistent recovery of spiked oocysts was observed (68-81%), when the membrane was sanitized with a 10% sodium dodecyl sulfate (SDS) solution and then blocked with 5% fetal bovine serum (FBS).     

Settling velocity of Cryptosporidium parvum and Giardia lamblia

Understanding transport behavior of Cryptosporidium parvum oocysts and Giardia lamblia cysts (together referred to as (oo)cysts) in overland flow is important for beneficial uses of receiving water bodies. Like sediment, (oo)cysts are subjected to deposition once they are present in overland flow or low flow environments like reservoirs, wetlands and sedimentation basins. The objectives of this paper are to present the theory and experiment to determine the free settling velocity (v(s)) of (oo)cysts and to compare experimental settling velocities to estimates using Stokes' law. A settling experiment was designed to quantify the v(s) of (oo)cysts in an aqueous column. C. parvum oocysts used were spherical with average diameter (+/-1SD) of 6.6+/-1.1 microm. G. lamblia cysts were oval shaped (average eccentricity = 1.48+/-0.19) with average size of 11.8 +/-1.3 microm. Average densities were 1009 kg m(-3) for C. parvum oocysts and 1013 kg m(-3) for G. lamblia cysts. Observed experimental settling velocities are 0.27 microm s(-1) and 0.67 microm s(-1) for C. parvum and G. lamblia, respectively. Estimated average settling velocities using Stokes' law were 0.36 microm s(-1) for C. parvum and 0.84 microm s(-1) for G. lamblia. R-squared values of the observations from the settling experiments with the Stokes' law estimation are 0.87 and 0.88 for G. lamblia and C. parvum, respectively. Our results suggest that Stokes' law can be used to estimate settling velocities of (oo)cysts. Qualitatively, the low settling velocities indicate that (oo)cysts will very slowly settle out of suspension.     

Distribution of Cryptosporidium oocysts in a water supply

Cryptosporidium has emerged as a significant waterborne pathogen. Methods have been developed for analysis of this organism using immunofluorescence counts. In this paper, we report on the results of a one year sequence of weekly samples in the water supply of a relatively high-quality water system in the Northwest United States. The results show consistency with the Poisson distribution, indicating that, at least for high-quality water, the theoretical minimum variability can be attained. This will assist in the interpretation of results from diverse systems contemplated under future monitoring activities.