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 using medium- and low-pressure ultraviolet radiation

The effect of ultraviolet radiation from low- and medium-pressure mercury arc lamps on Cryptosporidium parvum oocysts was studied using a collimated beam apparatus. Experiments were conducted using parasites suspended in both filtered surface water and phosphate buffered laboratory water. Inactivation of oocysts was measured as reduction in infectivity using a CD-1 neonatal mouse model and was found to be a non-linear function of UV dose over the range of germicidal doses tested (0.8-119 mJ/cm2). Oocyst inactivation increased rapidly with UV dose at doses less than 25 mJ/cm2 with two and three log-units inactivation at approximately 10 and 25 mJ/cm2, respectively. The cause of significant leveling-off and tailing in the UV inactivation curve at higher doses was not determined. Maximum measured oocyst inactivation ranged from 3.4 to greater than 4.9 log-units and was dependent on different batches of parasites. Water type and temperature, the concentration of oocysts in the suspension, and the UV irradiance did not have significant impacts on oocyst inactivation. When compared on the basis of germicidal UV dose, the oocysts were equally sensitive to low- and medium-pressure UV radiation. With respect to Cryptosporidium, both low- and medium-pressure ultraviolet radiation are attractive alternatives to conventional chemical disinfection methods in drinking water treatment.     

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.     

Synergistic inactivation of Cryptosporidium parvum using ozone followed by monochloramine in two natural waters

The effect of sequential exposure to ozone followed by monochloramine on inactivation of Cryptosporidium parvum oocysts suspended in untreated natural surface water from two different sources was studied in bench-scale batch reactors. Animal infectivity using neonatal CD-1 mice was used to measure oocyst inactivation. A statistically significant synergistic effect on oocyst inactivation was measured in both natural water samples studied. The magnitude of the effect measured in the natural water with lower turbidity, colour, and organic carbon concentration was comparable to that previously reported for oocysts suspended in buffered de-ionized water but was reduced considerably in the natural water with higher turbidity, colour and organic carbon concentration. Synergy increased with initial pH and with the degree of ozone pre-treatment but was independent of temperature. For water treatment plants with adequate disinfectant contact times, ozone followed by monochloramine may be a practical means of achieving additional C. parvum inactivation, however, the influence of water quality characteristics should be considered.     

Inactivation of Bacillus subtilis spores with ozone and monochloramine

The inactivation kinetics of Bacillus subtilis spores with ozone and monochloramine was characterized by a lag phase followed by a pseudo-first-order rate of inactivation. The lag phase decreased and the post-lag phase rate constant increased with increasing temperature within the range investigated (1-30 degrees C for ozone, 1-20 degrees C for monochloramine). The corresponding activation energies were 46820 J/mol for ozone and 79640 J/mol for monochloramine. The CT concept was found to be valid within the concentration range investigated of 0.44-4.8 mg/l for ozone, and 3.8-7.7 mg/l as Cl(2) for monochloramine. The inactivation kinetics of B. subtilis spores with both ozone and monochloramine varied with pH within the range of pH 6-10 investigated. The fastest ozone and monochloramine inactivation rates were observed at pH 10 and 6, respectively. Different stocks of the same strain of B. subtilis spores had different resistance to ozone and monochloramine mainly because of discrepancies in the extent of the lag phase. B. subtilis spores might not be conservative surrogates for C. parvum oocysts for ozone disinfection at relatively low temperature mainly due to the spores having a lower activation energy compared to that for the oocysts. In contrast, the activation energy for monochloramine was comparable for both microorganisms but differences in the extent of the lag phase might result in the spores being overly conservative surrogates for the oocysts at relatively low temperature.     

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.     

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.     

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.     

Development of a Ct equation taking into consideration the effect of lot variability on the inactivation of Cryptosporidium parvum oocysts with ozone

Cryptosporidium parvum oocysts are prevalent in surface water and ground water under the influence of surface water, and are difficult to inactivate using free chlorine, the most common disinfectant currently used for treating drinking water. In contrast, it has been shown that ozone is a more effective disinfectant than chlorine. US EPA is currently evaluating a treatment rule, which addresses the control of C. parvum oocysts in drinking water. The use of Ct (average disinfectant concentration multiplied by characteristic contact time) values is being considered as one of the options for demonstrating adequate control of this microbial contaminant. The purpose of this study is to incorporate the variability in inactivation kinetics among different lots of oocysts and to develop a statistical model for Ct based on first-order delayed Chick--Watson inactivation kinetics. A Bayesian approach is used to estimate the delayed Chick--Watson kinetic parameters. A log-linear regression analysis is then used to represent the effect of temperature on the resulting kinetic parameters. The overall model developed in this study provides an approach for water utilities and regulatory agencies to decide on the level of safety needed when developing treatment requirements for the inactivation of C. parvum oocysts with ozone as part of broader risk assessment considerations.     

Development of a Ct equation for the inactivation of Cryptosporidium oocysts with ozone

Cryptosporidium parvum, a protozoan parasite, has been implicated in a number of waterborne disease outbreaks. It is difficult to inactivate using free chlorine, but appears to be easily inactivated by ozone. Therefore, the US EPA has promulgated the Interim Enhanced Surface Water Treatment Rule, which for the first time, addresses the control of C. parvum in drinking water. The use of Ct (concentration of disinfectant in mg/L times, time in minutes) values is being considered as one of the options for controlling this organism. This paper proposes a Ct equation, based on first order kinetics, to provide guidance to drinking water utilities for the application of ozone for controlling C parvum oocysts in drinking water. The equation, which provides mean estimates of inactivation, was developed using standard statistical techniques and currently available field and bench scale data. In addition, the possibility of using a statistically conservative upper bound Ct value in order to insure an appropriate safety factor is explored.     

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.     

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.     

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.     

Inactivation kinetics of adenovirus serotype 2 with monochloramine

The effect of pH (6-10), temperature (10-30 degrees C), disinfectant concentration (1-11mg/l as Cl(2)), and ammonia nitrogen-to-chlorine molar ratio (1.3-52) on the inactivation kinetics of adenovirus serotype 2 with monochloramine was investigated by performing batch-reactor experiments with synthetic 0.01M buffer (phosphate or borate) solutions. The inactivation kinetics was independent of monochloramine concentration and ammonia nitrogen-to-chlorine molar ratio but had strong pH dependence, with the rate of inactivation decreasing with increasing pH. The kinetics at pH 6 and 8 were consistent with pseudo-first-order kinetics, while curves at pH 10 were characterized by a lag phase followed by a pseudo-first-order phase. The rate of inactivation increased with increasing temperature-activation energies of 56.5kJ/mole (pH 8) and 72.6kJ/mole (pH 10). The results obtained in this study revealed that monochloramine disinfection might not generally provide adequate control of adenoviruses in drinking water at high pH and low temperature.     

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.     

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.     

Synergistic inactivation of Cryptosporidium parvum using ozone followed by free chlorine in natural water

The synergistic effect of sequential exposure to ozone followed by free chlorine on inactivation of Cryptosporidium parvum oocysts suspended in natural waters was studied in bench-scale batch reactors. Animal infectivity using neonatal CD-1 mice was used to measure oocyst inactivation. The synergistic effect measured in two alkaline (pH 8.1) natural waters was statistically significant but was considerably smaller than previously reported in buffered de-ionized water at pH 6.0. Temperature, ozone primary treatment level, and water type did not have measurable impacts on the synergistic effect. Efforts to increase the synergistic effect by reducing the pH from 8 to 6 by acid addition were unsuccessful. In the two low alkalinity (pH 6.0) natural waters tested, the measured synergistic effect was greater than in the alkaline waters, but was still less than that measured previously in buffered de-ionized water. It was concluded that the synergistic effect reduction in the natural waters tested was due in part to alkalinity and in part to other unidentified water quality characteristics. Sequential treatment with ozone followed by free chlorine may only be a feasible strategy for achieving synergistic C. parvum inactivation credit for water treatment facilities with natural waters having a low pH (near 6.0).     

Modeling Cryptosporidium parvum oocyst inactivation and bromate in a flow-through ozone contactor treating natural water

A reactive transport model was developed to simultaneously predict Cryptosporidium parvum oocyst inactivation and bromate formation during ozonation of natural water. A mechanistic model previously established to predict bromate formation in organic-free synthetic waters was coupled with an empirical ozone decay model and a one-dimensional axial dispersion reactor (ADR) model to represent the performance of a lab-scale flow-through ozone bubble-diffuser contactor. Dissolved ozone concentration, bromate concentration (in flow-through experiments only), hydroxyl radical exposure and C. parvum oocyst survival were measured in batch and flow-through experiments performed with filtered Ohio River water. The model successfully represented ozone concentration and C. parvum oocyst survival ratio in the flow-through reactor using parameters independently determined from batch and semi-batch experiments. Discrepancies between model prediction and experimental data for hydroxyl radical concentration and bromate formation were attributed to unaccounted for reactions, particularly those involving natural organic matter, hydrogen peroxide and carbonate radicals. Model simulations including some of these reactions resulted in closer agreement between predictions and experimental observations for bromate formation.     

Effect of turbulent gas-liquid contact in a static mixer on Cryptosporidium parvum oocyst inactivation by ozone

Static mixers may be used to dissolve gaseous ozone in water treatment facilities in order to provide protection against the waterborne parasite Cryptosporidium parvum. The objective of this study was to determine the effect of a brief exposure to turbulent gas-liquid mixing conditions in a static mixer on inactivation of C. parvum oocysts by ozone. Inactivation measured in an ozone contacting apparatus that employed a static mixer for ozone dissolution was compared to predictions based on a previously published kinetic model of C. parvum inactivation by dissolved ozone in gently stirred batch reactors. Although initial contact in the static mixer had no immediate effect on the oocysts, a 20% increase in the rate of inactivation during subsequent contact with dissolved ozone was observed. Increasing the degree of turbulence within the static mixer did not yield additional inactivation. Use of static mixers for dissolution of ozone in drinking water treatment systems may provide limited enhancement of C. parvum inactivation by dissolved ozone.