Virus inactivation by ozone: Kinetics and influence of water quality parameters

Disinfection is an important step in (waste)water treatment to prevent the transmission of waterborne diseases. Ozone is an effective disinfectants against all types of waterborne pathogens, including viruses. While ozone has typically been used to treat drinking water, its application to wastewater (WW) has also been tested. However, in particular for virus inactivation, data regarding its efficacy remain scarce. In Switzerland, a new regulation mandates the upgrade of major WW treatment plants with an advanced treatment step such as ozonation, for the abatement of micropollutants. This measure could also result in the inactivation of enteric viruses, though to an unknown extent. There are many practical limitations to directly monitoring concentration and infectivity of enteric viruses during WW ozonation. However, inactivation may be estimated if the ozone exposure, the virus inactivation rate constants and their susceptibility to environmental parameters (pH, temperature, dissolved organic matter (measured as DOC)) are known. Alternatively inactivation may be monitored based on an “easy-to-measure” proxy. For example, the decrease in UV254 absorbance (ΔUV254) is indicative of the extent of micropollutant removal during ozonation of WW. ΔUV254 is an indirect measure of the ozone exposure, which in turn, depends on the specific ozone dose (mg O3/mg DOC). Similar proxies may exist for viruses. The objectives of this study were (1) to quantify the second order ozone inactivation rate constants (kO3-Virus) for several enteric viruses and bacteriophages in buffered solutions and assess their dependence on temperature and pH; (2) to assess the influence of DOC on inactivation in differing water matrices; and (3) to test the utility of ΔUV254 as a proxy for inactivation. To determine kO3-Virus in buffered solutions, an experimental system was developed that allows to track ozone exposure and virus inactivation accurately and simultaneously. Experiments were conducted over a temperature range of 2-22° C and a pH range of 6.5-8.5. Due to the relatively fast decay kinetics, ozone exposure in environmental water matrices were determined by a quenched-flow method. The kO3-Virus of all viruses tested spanned a narrow range of 105 to 106 M-1s-1. Q was the most susceptible, and T4 and an environmental isolate of CoxB5 were the most resistant. Interestingly, significant differences in the rate constants of differing CoxB5 isolates and its lab strain were observed. Both pH and temperature increases resulted in higher kO3-Virus values, though the effect of temperature was more pronounced. In buffered solutions, the ozone exposures to inactivate 99% of viruses were about 100x lower than those reported for ClO2 and free chlorine. Effects of DOC on kO3-Virus and correlations between ΔUV254 and virus inactivation in WW are currently under investigation. Overall, our findings demonstrate that ozone is an effective disinfectant for viruses in WW effluent.

Kinetics of inactivation of waterborne enteric viruses by ozone

Tamar Kohn, Urs von Gunten, Camille Wolf

Ozone is an effective disinfectant against all types of waterborne pathogens. However, accurate and quantitative kinetic data regarding virus inactivation by ozone is scarce, due to the experimental challenges associated with the high reactivity of ozone towards viruses. Here, we established an experimental batch system that allows tailoring and quantifying very low ozone exposures and simultaneously measuring virus inactivation. Second-order ozone inactivation rate constants (kO3-virus) of five enteric viruses (laboratory and two environmental strains of coxsackievirus B5: CVF, CVEnv1 and CVEnv2, human adenovirus: HAdV, and echovirus 11: EV) and four bacteriophages (MS2, Q, T4 and Φ174) were measured in buffered solutions. The kO3-Virus of all tested viruses ranged from 4.5 x 105 to 3.3 x 106 M-1s-1. For MS2, kO3-MS2 only depended weakly on temperature (2 – 22 °C, Ea = 22.2 kJmol-1) and pH (6.5 – 8.5), with an increase of kO3-MS2 with increasing pH. The susceptibility of the selected viruses towards ozone decreases in the order Qbeta > CVEnv2 > EV = MS2 > Φ174 = T4 > HAdV > CVF = CVEnv1. Based on the measured kO3-Virus and typical ozone exposures applied in water and wastewater treatment, we conclude that ozone is a highly effective disinfectant for virus control.

2018

Inactivation of waterborne viruses by ozone: Kinetics and mechanisms

Camille Wolf

The growing world population, in conjunction with climate change, cases the global water de-mand to increase. It is, therefore, crucial to protect existing water resources from chemical pollution and contamination by pathogens. An increasingly popular strategy to meet increased water demand is pota-ble water reuse, which uses wastewater effluent as the water source and treats it to such an extent that it is safe to drink. However, this practise may encompass risks for human health if the treatment train does not ensure a sufficient removal of chemical and microbial contaminants. In the context of both wa-ter and wastewater treatment, ozonation has emerged as an efficient treatment method for the control of pathogens, the abatement of organic micropollutants, and the removal of taste and odor compounds. Despite its popularity, information on the inactivation of waterborne viruses by ozone is scarce. The aim of this thesis was to investigate the kinetics and mechanisms of inactivation of water-borne viruses by ozone. In a first step, the inactivation kinetics of a suite of human viruses and commonly used surrogates (bacteriophages) were determined in well-controlled buffer systems. To this end, we developed a method to control O3 decay in batch reactors. This allowed us to measure virus inactivation as a function of low ozone exposures. The resulting inactivation rate constant (kO3-virus) differed between the virus species studied, but all kO3-virus fell within a narrow range of 105-106 M-1s-1. These high inactivation rate constant indicate that ozone is efficient to inactivate waterborne virus. Increases in temperature and pH resulted in an increased kO3-virus, though the effect was relatively minor. To determine if more complex, natural water matrices influence virus inactivation by ozone, we investigated the virucidal efficacy of ozone in two surface waters and a secondary wastewater effluent. While inactivation kinetics as a function of ozone exposure initially corresponded well to those observed in buffer solutions, the inactivation curve tailed off at higher ozone exposures. Furthermore, because it is not possible to measure virus inactivation during water treatment in real-time, we tested different if âeasy-to-measureâ proxies can be used to track virus inactivation. We determined that the applied specif-ic ozone dose, the reduction in UV254, or the abatement of carbamazepine all correlated to virus inactiva-tion, though some proxy-inactivation relationships were not universal but depended on the water type. The proxies were validated in a pilot-scale ozonation reactor treating Lake Zurich water, and were found to provide good estimates of virus inactivation. Finally, an immunostaining assay was developed to observe how ozonation affects crucial steps in the life cycle of echovirus 11, a representative of the Enterovirus genus. Specifically, we investigated if ozone alters the ability of the virus to enter the host cells, and if it prevents the viral genome from replicating. Preliminary results indicate a loss of internalization after ozone treatment as well as genome replication. In conclusion, this thesis provides new information on the efficacy and mechanisms of ozone as an inacti-vating treatment for waterborne viruses, and it delivers tools to monitor virus inactivation during water and wastewater treatment in real-time.

EPFL2019

Proxies to monitor the inactivation of viruses by ozone in surface water and wastewater effluent

Tamar Kohn, Annalisa Pavese, Urs von Gunten, Camille Wolf

Ozone treatment is an effective barrier against viral pathogens, therefore it is an integral part of many water and wastewater treatment trains. However, the efficacy of ozone treatment remains difficult to monitor, due to the lack of methods to track virus inactivation in real-time. The goal of this work was to identify easy-to-measure proxies to monitor virus inactivation during water and wastewater treatment by ozone. Proxies considered were the abatement in UV absorbance at 254 nm (UV254) and carbamazepine (CBZ), a ubiquitous organic micropollutant with a similar abatement rate constant as human viruses. The proxies, as well as the inactivation of two viruses (MS2 coliphage and coxsackievirus B5) were measured in surface water and in a secondary wastewater effluent as a function of the specific ozone dose (mgO3/mg dissolved organic carbon). Virus inactivation was rapid in both matrices, but was more efficient in surface water. This trend was also evident when inactivation was assessed as a function of the ozone exposure to account for the different ozone demand of the two water types. Both proxies, as well as the specific ozone dose, were correlated with virus inactivation. The correlations depended only weakly on the virus species, but – with the exception of CBZ abatement – differed between the two water types. Finally, predictive relationships were established using Bayesian power models, to estimate virus inactivation based on the measurement of a proxy. The models were then applied to estimate the MS2 inactivation in a pilot-scale ozone reactor that treats surface water of Lake Zurich. All proxies yielded good estimates of the actual MS2 inactivation in the pilot plant, indicating that the proxy-inactivation relationships established in the laboratory can also be applied to flow-through reactors. This study confirms that ozone is a highly effective disinfectant for viruses in both surface water and wastewater, and that the abatement of UV254 and CBZ can be used to track virus inactivation during water and wastewater treatment.