Gordon R. Finch

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.

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.

Nucleic acid stains as indicators of Cryptosporidium parvum oocyst viability

We developed nucleic acid dye staining methodology for untreated, heat-treated and chemically inactivated C. parvum oocysts. The nucleic acid staining was compared to in vitro excystation and animal infectivity using split samples of oocysts. Among the nucleic acid stains tested, SYTO-9, hexidium and SYTO-59 stained the oocysts consistently, and the staining was related to the infectivity of the oocysts to neonatal CD-1 mice but not to in vitro excystation. The nucleic acid viability assay was used to determine log-inactivations of the oocysts after treatment with ozone, chlorine, chlorine dioxide and combinations of different chemical disinfectants, and was found to indicate log-inactivation levels similar to that of animal infectivity. A combined immunofluorescence-nucleic acid staining assay was developed for the oocysts of C. parvum and this assay will be invaluable for the detection and viability of oocysts in the laboratory and in environmental samples.

Comparison of Animal Infectivity and Nucleic Acid Staining for Assessment of Cryptosporidium parvum Viability in Water

ABSTRACT Cryptosporidium parvum oocysts were stained with the fluorogenic dyes SYTO-9 and SYTO-59 and sorted by flow cytometry in order to determine whether the fluorescent staining intensity correlated with the ability of oocysts to infect neonatal CD-1 mice. Oocysts that did not fluoresce or that displayed weak fluorescent intensity when stained with SYTO-9 or SYTO-59 readily established infections in mice, whereas those oocysts that fluoresced brightly did not. Although fluorescent staining profiles varied among different batches of oocysts, a relative cutoff in fluorescent staining intensity that correlated with animal infectivity was observed for all batches.

Dose—response of Escherichia coli in ozone demand-free phosphate buffer

Abstract The intention of this investigation was to determine the mean response of E. coli at predetermined ozone dose levels. By using replicated, randomized, independent batch experiments, precise estimates of the response could be obtained. Particular care was taken in preparing the bacteria and glassware to eliminate effects caused by extraneous ozone demand. A total of 57 batch experiments were conducted at bench-scale using ozone doses of 4.4, 11.45, 200 and 800 μg l −1 at contact times of 30, 60 and 120 s. The logarithm of the E. coli survival ratio was plotted as a function of applied ozone dose, utilized ozone and contact time. Contact time was found to have some effect for intermediate doses of ozone, but the effects were small relative to those associated with the utilized ozone dose. It was also found that the disinfection reaction was not first-order with respect to the surviving bacteria concentration and two distinct stages were observed; an initial rapid inactivation stage followed by a slower inactivation stage. Regression analysis of the E. coli response on the logarithm of the dose parameter is typical of dose-response models for water and wastewater disinfection. However, this model form was inadequate when fitted to the data of this study as a result of a significant tailing effect in the log-log dose-response plots. It was postulated that the observed dose-response could be explained by chemical kinetic theory and that the dose-response was affected by the disinfection reaction rates which were dependent upon the surviving number of bacteria and the residual ozone concentration. For practical application, it was found that an adequate dose-response model for 0–99.99% reduction of E. coli could be developed using the logarithm of utilized ozone as the dose function.

Sequential disinfection of Cryptosporidium parvum by ozone and chlorine dioxide

Abstract A two step disinfection approach was evaluated for control of Cryptosporidium parvum using bench‐scale experiments in 0.05 M phosphate buffer at pH 8 and 22 °C. Sequential application of ozone and chlorine dioxide was evaluated where ozone was applied first followed by chlorine dioxide. Infectivity in neonatal CD‐1 mice was used to assess oocyst viability after disinfection. The sequential treatment of oocysts by ozone followed by chlorine dioxide resulted in additional inactivation of C. parvum due to the synergism of the two disinfectants. The inactivation kinetics for chlorine dioxide were modeled following preconditioning with ozone at a given level using the Integral‐Hom model which takes the disinfectant decay into account. These preliminary findings indicate that sequential disinfection with ozone followed by chlorine dioxide may have potential in controlling waterborne parasites.

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 degrees C, 10 degrees C and 22 degrees 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 degrees 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(avg)t (arithmetic average of the initial and final residual x 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 degrees C temperature decrease. Given an ozone primary kill of 1.6 log-units, the free chlorine C(avg)t products required for a gross kill of 3.0 log-units were 1000, 2000 and 3,300 mgmin/L for 22 degrees C. 10 degrees C and 1degrees C, respectively.

Nucleic acid stains as indicators of Giardia muris viability following cyst inactivation.

A reliable viability assay for Giardia is required for the development of disinfection process design criteria and pathogen monitoring by water treatment utilities. Surveys of single-staining nucleic acid dyes (stain dead parasites only), and double-staining vital dye kits from Molecular Probes (stain live and dead parasites) were conducted to assess the viability of untreated, heat-killed, and chemically inactivated Giardia muris cysts. Nucleic acid staining results were compared to those of in vitro excystation and animal infectivity. Nucleic acid stain, designated as SYTO-9, was considered the best among the single-staining dyes for its ability to stain dead cysts brightly and its relatively slow decay rate of visible light emission following DNA binding. SYTO-9 staining was correlated to animal infectivity. A Live/Dead BacLight was found to be the better of 2 double-staining viability kits tested. Logarithmic survival ratios based on SYTO-9 and Live/Dead BacLight were compared to excystation and infectivity results for G. muris cysts exposed to ozone or free chlorine. The results indicate that SYTO-9 and Live/Dead BacLight staining is stable following treatment of cysts with chemical disinfectants.

Factors influencing the infectivity of Giardia muris cysts following ozone inactivation in laboratory and natural waters

Abstract Factors affecting the inactivation of Giardia muris cysts using ozone were examined using 48 independent, experimentally designed trials in a bench-scale, batch reactor. Cohorts of 5 male, inbred C3H/HeN mice were used to establish the significance of temperature, turbidity, pH, ozone dose and contact time on G. muris cyst infectivity. Temperature, pH, applied ozone dose and the ozone dose × pH and temperature × pH interactions were found to be statistically significant ( P ⩽ 0.05) in ozone demand-free 0.05 M phosphate buffer. Contact time in demand-free buffer was significant up to 2 min ( P ⩽ 0.05), but not beyond. When experiments were done using natural surface waters, contact times up to 5 min were required for inactivation, indicating that caution must be used in the application of laboratory water results to real waters. It was found that residual and utilized ozone both had important influences in G. muris cyst inactivation. It was more difficult to achieve 2 or 3 log inactivation of G. muris cysts in the natural waters at 22°C than at 5°C.

Disinfection kinetics of heterotrophic plate count bacteria in biologically treated potable water

Abstract The disinfection kinetics of biologically and non-biologically treated drinking waters were evaluated at bench-scale using process water from a pilot plant employing pre-ozonation and biologically active carbon (BAC) contactors. The Chick-Watson kinetic model did not describe the inactivation of native heterotrophic plate count bacteria (R2A agar, 7 d at 20°C) with decreasing rates of inactivation seen at extended contact times in all process streams. The parameters were estimated for the Hom kinetic model using a non-linear least squares method. The estimates of k , n , and m parameters for the Hom model using the kinetic data from the biologically and non-biologically treated waters were found to be statistically different between the two waters for both free chlorine and preformed monochloramine. However, if sufficient contact time was provided, the overall inactivation was the same in the control and biologically-treated streams. The observed kinetic differences were thought to be due to a highly resistant subgroup within the heterotrophic plate count bacteria population in each water.