Ozone Disinfection Validation Protocol - WaterRA · Ozone does not physically remove oocysts, but...

Ozone disinfection

Validation protocol

About Australian WaterSecure Innovations Ltd

Australian WaterSecure Innovations Ltd (trading as WaterSecure) was established in 2016 to oversee the implementation of national research outcomes, including the WaterVal™ program, one of the flagship outcomes developed by the Australian Water Recycling Centre of Excellence (the Centre), an independent research organisation established in 2009 by Commonwealth funding.

About WaterVal™

WaterVal™ is a framework that provides national consistency in the validation of water treatment technologies for the water industry. The framework, jointly developed by the Centre, regulators, water utilities, researchers and the private sector, is underpinned by protocols and agreed methods to validate pathogen removal by treatment technologies. The framework and protocols are applicable to a broad range of water sources, and give effect to key objectives of the Australian guidelines for water recycling and the Australian drinking water guidelines.

Acknowledgements

WaterSecure is grateful for the contributions made by Melbourne Water (John Mieog and Clare McAuliffe), the WaterVal™ Protocol Development Group, Karl Linden and Cedric Robillot, and input received from external reviewers.

Citation

WaterSecure 2017, Ozone disinfection, WaterVal validation protocol, Australian WaterSecure Innovations Ltd, Brisbane.

Date of publication

February 2017

Publisher

Australian WaterSecure Innovations Ltd Level 16, 333 Ann Street, Brisbane, Queensland 4000 www.watersecure.com.au

© Australian WaterSecure Innovations Ltd

This work is copyright. Apart from any use permitted under the Copyright Act 1968, no part of it may be reproduced for any purpose without written permission from the publisher. Requests and inquiries concerning reproduction rights should be directed to the publisher.

Disclaimer

While every effort has been made to ensure the accuracy of the information contained in this document, Australian WaterSecure Innovations Ltd cannot guarantee that it is entirely accurate and error-free.

This document is intended to be used in conjunction with the WaterVal™ framework. Australian WaterSecure Innovations Ltd does not accept any legal liability or responsibility whatsoever for any injury, loss or damage, due to or arising out of any use of this document independent of that framework.

It is the responsibility of the user to determine the suitability and appropriateness of the information and its specific application.

201702_WaterVal_Validation Protocol_Ozone Disinfection 1

Contents

1. Background and scope ........................................................................................................................ 3

2. Identification of pathogen removal mechanisms ................................................................................ 5 2.1. Protozoa ......................................................................................................................................... 5 2.2. Viruses and bacteria ....................................................................................................................... 6

3. Identification of target pathogens and surrogates .............................................................................. 7 3.1. Protozoa ......................................................................................................................................... 7 3.2. Viruses and bacteria ....................................................................................................................... 7

4. Influencing factors ............................................................................................................................... 9 4.1. Ozone exposure.............................................................................................................................. 9

4.1.1. Protozoa ............................................................................................................................ 9 4.1.2. Viruses and bacteria .......................................................................................................... 9 4.1.3. Potential alternative approaches to quantifying ozone exposure ..................................... 9

4.2. Dose–response relationship ......................................................................................................... 10 4.3. Factors influencing the ozone disinfection reaction in water ....................................................... 10

4.3.1. Ozone disinfection reactions ........................................................................................... 10 4.3.2. pH .................................................................................................................................... 11 4.3.3. Water temperature ......................................................................................................... 11 4.3.4. Dissolved organic matter concentration ......................................................................... 11 4.3.5. Inorganic ozone demand ................................................................................................. 12 4.3.6. Suspended solids / turbidity / particles in the water ....................................................... 12

4.4. Factors influencing effective exposure to dissolved ozone .......................................................... 13 4.4.1. Mixing and dispersion of ozone ....................................................................................... 13 4.4.2. Hydraulic efficiency of the contactor............................................................................... 13

5. Operational monitoring parameters.................................................................................................. 15

6. Validation method ............................................................................................................................. 16 6.1. Tier 1 ............................................................................................................................................ 17 6.2. Tier 2 ............................................................................................................................................ 17 6.3. Tier 3 ............................................................................................................................................ 18

6.3.1. Protozoa .......................................................................................................................... 19 6.3.2. Viruses and bacteria ........................................................................................................ 19

7. Data collection and analysis .............................................................................................................. 21 7.1. Cryptosporidium infectivity techniques ........................................................................................ 21

8. Critical limits and operational monitoring ......................................................................................... 23 8.1. CT set-point .................................................................................................................................. 23 8.2. Flow .............................................................................................................................................. 23 8.3. Feedwater turbidity ...................................................................................................................... 23 8.4. Ozone residual .............................................................................................................................. 24 8.5. Water temperature ...................................................................................................................... 24 8.6. Ozone CT ...................................................................................................................................... 24 8.7. Applied ozone dose ...................................................................................................................... 24 8.8. Determination of critical limits ..................................................................................................... 24 8.9. Degree of pathogen inactivation provided ................................................................................... 24

9. Method to determine the LRV for each pathogen group .................................................................. 25 9.1. Tier 1 and tier 2 ............................................................................................................................ 25

9.1.1. Protozoa .......................................................................................................................... 25 9.1.2. Viruses and bacteria ........................................................................................................ 25

10. Triggers for revalidation .................................................................................................................... 28


Glossary and abbreviations ............................................................................................................................... 29

References………………………………………………………………………………………………………………………………………………….. . 30

Tables

Table 1 Ozone disinfection system operational monitoring requirements .................................................... 15

Table 2 CT values (mg·min/L) for 0.25 to 3.0 log reduction values of protozoa by ozonation at temperatures ranging from 5 °C to 30 °C .......................................................................................... 25

Table 3 CT values for 1 to 4 log reduction values of viruses and surrogates in wastewater at pH 7.6 and a temperature of 16 °C ............................................................................................................... 26

Table 4 CT values for 1 to 4 log reduction values of viruses in wastewater at temperatures ranging from 5 °C to 30 °C .............................................................................................................................. 27

Figures

Figure 1 Morphological changes in Giardia oocysts after exposure to ozone .................................................. 5

Figure 2 Overview of validation method for ozone disinfection .................................................................... 16


1. Background and scope

Ozone disinfection in drinking water applications is recognised and well established, with appropriate regulatory guidance for high-level disinfection of a range of target organisms. Ozone disinfection has also been successfully used in wastewater applications for many years; however, in these applications, success is most often measured in terms of achieving low coliform concentrations in treated water rather than validated disinfection log credits. Ozone is also used to disinfect wastewater effluents, particularly where chlorine disinfection could negatively impact sensitive freshwater receiving environments.

This document provides guidance for validating ozone disinfection of protozoa, viruses and bacteria. A tiered approach is recommended, which is applicable to both drinking water and wastewater applications, and takes into account the potential shielding effect associated with particulate material. This protocol is largely based on the United States Environmental Protection Agency (US EPA) guidance and on work by Melbourne Water in validating the Eastern Treatment Plant ozone treatment process.

Alternative applied ozone dose methods, based on normalisation to organic content, are currently being investigated (e.g. ozone/dissolved organic carbon [DOC] and DOC/ultraviolet [UV] absorbance). However, it is not proven that these approaches can accommodate the variation in ozone demand associated with wastewaters and recycled waters derived from wastewaters while delivering consistent levels of pathogen inactivation. Our approach to ozone validation is therefore based on concentration × time (i.e. CT) exposure disinfection.

The US EPA (2006) provides CT values and a log inactivation equation for ozone inactivation of Cryptosporidium, and Sigmon et al. (2015) provide experimental CT values for ozone inactivation of viruses, bacteria and appropriate surrogates. This protocol recommends the adoption of these CT values, provided the application-specific water presents a low turbidity of 0.15 nephelometric turbidity units (NTU) or below (tier 1). If the application-specific water does not meet this turbidity requirement, but it can be demonstrated that the application-specific water is consistent (in terms of ozonation efficacy) with clean water as used in the experiments that were referenced to derive the reference CT values, then the latter CT values can also be adopted (tier 2). In all other situations, and recognising the potential effect of particle shielding, this protocol recommends that a specific validation and challenge testing study be undertaken to derive CT values that can consistently achieve the target log inactivation in the application-specific water (tier 3).

Because an appropriate surrogate for ozone inactivation of Cryptosporidium has yet to be identified, validation studies must use Cryptosporidium.

Ozone is much more effective against viruses and bacteria than it is against protozoa, therefore, if protozoa inactivation is an objective, any virus and/or bacteria inactivation objectives will be incidentally achieved by meeting protozoa inactivation objectives. In such a case, the validation study will be driven by the requirements for protozoa.

This document is consistent with the WaterVal validation Protocol template (AWRCE 2015), which provides a recommended approach to validation that is based on the following nine elements:

identification of the mechanisms of pathogen removal by the treatment process unit

identification of the target pathogens and/or surrogates that are the subject of the validation study

identification of factors that affect the efficacy of the treatment process unit in reducing the target pathogen

identification of operational monitoring parameters that can be measured continually and are related to the reduction of the target pathogen

identification of the validation method to demonstrate the capability of the treatment process unit

description of a method to collect and analyse data to formulate evidence-based conclusions


description of a method to determine the critical limits, as well as an operational monitoring and control strategy

description of a method to determine the log reduction value (LRV) for each pathogen group in each specific treatment process unit performing within defined critical limits

provision of a means for revalidation or additional onsite validation where proposed modifications are inconsistent with the previous validation test conditions.

Formation of disinfection by-products and other health impacts have not been considered as part of this protocol; they should be considered case by case, depending on the proposed use of the water.


2. Identification of pathogen removal mechanisms

Ozone is a powerful oxidant and is second in oxidising strength only to the hydroxyl-free radical present in oxidant chemical disinfectants commonly used in water treatment. The US EPA disinfection criteria – the Surface water treatment rule (US EPA 1989), subsequent Stage 1 disinfectants and disinfection byproducts rule (US EPA 1998a) and Interim enhanced SWTR (US EPA 1998b) – all recognise the capability of ozone to inactivate viruses, protozoa and bacteria.

Ozone inactivates pathogens through oxidation reactions that attack and lyse cell walls, damage genetic material such as nucleic acids (purines and pyrimidines), and break carbon–nitrogen bonds, leading to depolymerisation (US EPA 1999).

2.1. Protozoa

Cryptosporidium oocysts can be infectious or non-infectious; only infectious oocysts pose a risk to public health. Ozone does not physically remove oocysts, but rather inactivates them, as measured by reduced oocyst infectivity. Consequently, selection of the reduction in the percentage, or log inactivation, of infectious oocysts before and after ozone treatment is the basis for characterising the ozone system performance. Simple oocyst counts before and after treatment are unlikely to show any reduction in numbers.

How ozone inactivates Cryptosporidium is not fully understood. Ran et al. (2010) and Widmer et al. (2002) used scanning electron microscopy to study morphological changes in oocysts and cysts (in Giardia) that were exposed to ozone. When viewed under a scanning electron microscope, oocysts/cysts are initially seen to be round and spherical (Figure 1a). Later, within minutes of exposure to ozone, the oocyst wall becomes folded and eventually ruptures (Figure 1b–d). The degree of damage to the oocysts increases as the time of exposure increases. These studies suggest that ozone oxidises the wall of the Cryptosporidium oocyst and Giardia cyst, eventually leading to rupture of the oocyst/cyst.

Loss of infectivity is observed before the oocyst/cyst wall ruptures, indicating penetration of ozone into the oocyst/cyst and damage to the genetic material in the trophozoites.

Source: Ran et al. (2010)

Figure 1 Morphological changes in Giardia oocysts after exposure to ozone


2.2. Viruses and bacteria

The oxidant properties of ozone make it a very effective disinfectant for the inactivation of viruses and bacteria in water. Viruses and bacteria are very susceptible to inactivation with ozone, and, if log reductions are being claimed for Cryptosporidium, then similar or higher log reductions will be achieved, incidentally, for viruses and bacteria.

Inactivation of viruses is defined as their inability to replicate within host cells. Ozone probably causes physical disruption of the particle, or reacts with the viral coat protein, the nucleic acid or both, so as to affect attachment or intracellular step(s) in virus replication (Roy et al. 1981).

The proteins of the virion capsid are the first site of action for virus inactivation. High concentrations of ozone can completely dissociate the capsid, permit oxidation of the virus RNA or DNA, and disrupt adsorption to the host pili. Roy et al. (1981) used poliovirus 1 (PV-1) to investigate the mechanism of enterovirus inactivation by ozone. Ozone altered two of the four polypeptide chains present in the viral protein coat of the virus; however, this did not significantly impair virus adsorption or alter the integrity of the virus particle. Damage to the viral RNA after exposure to ozone was observed, and the authors concluded that damage to the viral nucleic acid is the major mechanism of PV-1 inactivation by ozone.


3. Identification of target pathogens and surrogates

A target pathogen is the subject of a validation study and is the pathogen most resistant to the treatment process. Generally, a target pathogen should be selected from each pathogen group.

3.1. Protozoa

Cryptosporidium is generally more resistant to disinfection than Giardia. The US EPA Long term 2 enhanced surface water treatment rule toolbox guidance manual (LT2ESWTR; US EPA 2010) identifies ozone dose–response relationships for Giardia and Cryptosporidium as

Cryptosporidium log credit = 0.0397 × (1.09757)temp × CT

Giardia log credit = 1.0380 × (1.0741)temp × CT where temp stands for temperature in degree Celsius. Based on these equations, Cryptosporidium is approximately 15 to 18 times more resistant to disinfection by ozone than Giardia, within a temperature range of 16 ˚C to 25 ˚C. These values are based on work conducted in reagent-grade water and surface waters. Cryptosporidium has therefore been selected as the target protozoan pathogen for validation. No suitable surrogate has been identified for protozoa inactivation with ozone, hence Cryptosporidium needs to be used for validation studies for protozoa.

3.2. Viruses and bacteria

Sigmon et al. (2015) investigated the relative resistance of four viruses to ozone:

Poliovirus 1 (PV-1) – PV-1 is the most extensively investigated enteric viruses in the literature, and its inclusion allows comparison with previous data.

Coxsackievirus B5 (CV-B5) – Because this virus aggregates at pH values typical of secondary effluent, it may have added resistance to ozone disinfection. CV-B5 is more resistant to free-chlorine than either hepatitis A or PV-1.

Adenovirus 2 (AD-2) – AD-2 is a double-stranded DNA viruses, and may exhibit variation in its resistance to disinfection compared with single-stranded RNA or DNA viruses.

Echovirus 11 (EV-11) – EV-11 allows comparison with the other two enteroviruses (PV-1 and CV-B5) and with previous studies.

None of these viruses conclusively exhibited more resistance to ozone than any other. All viruses were highly susceptible to ozone inactivation, with 4-log inactivation achieved at CT ≤ 1 mg·min/L at a water temperature of 16 ˚C. For validation, AD-2 is selected as the reference pathogen because it demonstrated marginally higher resistance than the other viruses tested.

Sigmon et al. (2015) also investigated a number of surrogate organisms: the phages MS2, T1, T4, PRD1 and ΦX174, and two strains of Escherichia coli (CN13 and Famp). E. coli and phage PRD1 were identified as suitable surrogate organisms because their inactivation dose–responses were directly comparable with that of the most resistant human viruses tested. Inactivation of 4-log was demonstrated for both surrogates at CT ≤ 1 mg·min/L at a water temperature of 16 ˚C.

Both E. coli and PRD1 meet the general requirements of good validation surrogate organisms because they are easy to culture up to high titres; are easy to handle, both in the laboratory and experimentally; are of little


or no risk to humans; and are quick and simple to quantify in an accurate and repeatable manner at low cost.

A key finding of the work by Sigmon et al. (2015) was the sensitivity of ozone CT quantification and the associated organism inactivation sensitivity to ozone for waters that exert a high ozone demand, such as wastewaters or recycled water derived from wastewaters. Experimentally, E. coli is better to use than PRD1 because it can be generated at high numbers in a low ozone demand stock solution, thus eliminating this source of interference from any seeded validation testing.

In summary, although there does not appear to be any one virus that is substantially more resistant to ozone than any other, it is recommended that AD-2 be used as the reference virus for ozone disinfection.

E. coli is recommended as a surrogate for virus inactivation because it is neither necessary nor practicable to use AD-2 for challenge testing. However, when preparing E. coli for challenge testing, care must be taken when preparing the stock solutions to ensure that the stock solution does not add any additional ozone demand to the water matrix being assessed. Phage PRD1 could be used; however, care should again be taken to minimise the impact of the stock solution on the ozone demand, since it could confound CT measurement and the observed inactivation dose–response relationship.


4. Influencing factors

The principal factors that influence ozone disinfection efficiency are:

ozone concentration and contact time

those that affect the ozone disinfection reaction in water, such as temperature, pH, turbidity, dissolved organic matter concentration and general ozone demand

those influencing effective exposure to dissolved ozone, such as mixing/dispersion and the hydraulic efficiency of the contactor.

In general, the highest levels of pathogen inactivation are achieved with high ozone concentrations; long contact times; high water temperature, with good mixing; and low organic and inorganic demands.

4.1. Ozone exposure

4.1.1. Protozoa

Various studies have shown that ozone inactivation of Cryptosporidium can be adequately described by a linear Chick–Watson model using a CT concept, as is commonly used for chlorine disinfection processes (e.g. Clark et al 2002, Oppenheimer et al. 2000, Rennecker et al. 1999, Rosen et al. 2004).

4.1.2. Viruses and bacteria

Similarly, various studies have shown that virus and bacteria are both highly susceptible to, and rapidly inactivated by, ozone. Studies completed by Roy et al. (1982) demonstrated that increased ozone contact time at constant ozone residual resulted in greater inactivation of a range of viruses. Roy et al. (1982) also demonstrated that increased ozone residual concentration resulted in increased inactivation of viruses at a given contact time. This indicates that the CT model also applies to ozone inactivation of viruses. The outcomes of the research by Sigmon et al. (2015) also support the use of the CT model for both clean water and effluent disinfection.

In waters with high initial ozone demand, such as wastewaters or recycled water derived from wastewater, relying on the concept of applied ozone dose has been suggested to account for the inactivation observed in the apparent absence of a measurable ozone residual. However, when the ozone demand of the water varies, the observed CT for a given ozone dose applied can vary considerably; therefore, the applied ozone dose is not considered a robust predictor of pathogen inactivation, unless the ozone demand interference is eliminated (Sigmon et al. 2015). Although organism inactivation may have been observed in the absence of measured ozone residuals in studies using applied dose models, these studies may not have been able to accurately measure ozone residuals at very short contact times in high demand water where the ozone is both consumed and decaying rapidly.

Based on the outcomes of the work by Sigmon et al. (2015), a CT model should be adopted to characterise virus and bacteria inactivation by ozone.

4.1.3. Potential alternative approaches to quantifying ozone exposure

Investigations in the United States are exploring the potential of using the applied ozone dose concept to characterise both microbial reduction and chemical reduction. In particular, ozone/DOC and DOC/UV absorbance ratios are being studied as a way of quantifying the degree of reduction/inactivation achieved. This approach may be useful in high ozone demand applications where reduction/inactivation is observed in the absence of any measureable ozone residual or CT. However, it is not proposed to adopt this approach to validate LRVs until more comprehensive work shows that this operational monitoring approach is appropriate.


4.2. Dose–response relationship

Inactivation of organisms is described using a linear Chick–Watson model for the specific disinfection regime, assuming a constant chemical residual (Li et al. 2001)

𝐿𝑜𝑔 10 (𝑁𝑂

𝑁𝐼) = −𝑘𝐶𝑡

where: NO is the initial number of organisms NI is the number of organisms that remain viable after treatment k is the pathogen inactivation rate constant (or lethality constant) C is the average concentration of ozone, or residual, in the water t is the exposure time

It has been shown that k is a function of water temperature (Clark et al. 2002, Oppenheimer et al. 2000), with the inactivation rate increasing with increasing water temperature.

Based on the known dose–response relationship, ozone CT and water temperature are the key independent variables in ozone disinfection.

4.3. Factors influencing the ozone disinfection reaction in water

4.3.1. Ozone disinfection reactions

Ozone in aqueous solution reacts with dissolved and suspended matter through two mechanisms: direct reaction of molecular ozone or indirect reaction of radical species that are formed as ozone decomposes. Although molecular ozone is highly reactive and decays rapidly in water (seconds to minutes), this decomposition is slow compared with that of the hydroxyl-free radicals, where reactions take place within micro-seconds.

Protozoa

Studies (Li et al. 2000, Oppenheimer et al. 2000) have shown that the inactivation of Cryptosporidium requires extended exposure to ozone. It is therefore most likely to be predominantly a function of the molecular ozone reaction path, given hydroxyl radicals would not be expected to be present after initial ozone application.

Viruses and bacteria

Similarly, studies (Roy et al. 1982, Sigmon et al. 2015) have shown that the disinfection of viruses and bacteria by ozone takes place very rapidly – disinfection occurs seconds after exposure to ozone. Extended exposure time (up to 120 seconds [Roy et al. 1982]) or increased ozone concentration of molecular ozone both result in increased virus inactivation. Given these observed inactivation characteristics, it is most likely that viral and bacterial inactivation are predominantly a function of the molecular ozone reaction pathway. That said, reports of inactivation in the absence of any measureable ozone residual or CT does not preclude any influence from hydroxyl radical reactions. However, an effective operational monitoring control for hydroxyl radicals has yet to be developed.

Sections 4.3.2 to 4.4.2 discuss the factors influencing ozone disinfection in the context of dissolved molecular ozone.


4.3.2. pH

Protozoa

Research indicates that ozone disinfection of Cryptosporidium is independent of pH within the range typically seen in water and wastewater applications. Oppenheimer et al. (2000) investigated inactivation of Cryptosporidium in natural waters with a pH ranging from 6.2 to 8.2. No statistically significant relationship was identified between the lethality constant and pH, although the researchers noted that inactivation may be improved if the pH is maintained at or below 6.5 as a result of increased ozone stability.

The pH of the water affects the ozone decomposition pathway. At high pH (above 8), ozone decomposition through a hydroxyl radical pathway is favoured. This increased radical formation results from molecular ozone decaying more rapidly, and a greater applied ozone dose is therefore required to achieve a given molecular ozone CT.


Sigmon et al. (2015) investigated the effect of pH (range 6.0 to 7.5) on ozone inactivation of viruses and surrogate organisms. No measureable impact of pH on disinfection efficacy was seen, consistent with the findings of the US EPA (1991).

4.3.3. Water temperature

Protozoa

Published ozone CT values are a function of temperature (US EPA 1991, 2006). Studies by Li et al. (2000), Owens et al. (2000) and Rennecker et al. (1999) supported this, showing that a rise in water temperature increases the rate of reaction between ozone and microorganisms. As a result, higher ozone CT values are required to achieve the same degree of inactivation as the water temperatures decreases, which is consistent with other oxidant disinfectants.

Water temperature is therefore considered to directly influence ozone disinfection efficacy and so should be monitored continuously.


Water temperatures between 16 °C and 23 °C were investigated by Sigmon et al. (2015). No measureable impact of temperature was seen on the inactivation of viruses or surrogates with ozone. Although temperature is expected to be positively correlated with the rate of ozone inactivation of viruses, as evidenced by the US EPA CT values being temperature dependent (US EPA 1991, 2006), this effect was not clearly observed at the relatively high water temperatures tested. The combination of the higher ozone doses required for recycled water applications and the very high sensitivity of viruses to ozone, and hence the rapid inactivation of viruses observed, resulted in difficulty in observing any temperature effect (Sigmon et al. 2015).

4.3.4. Dissolved organic matter concentration

Protozoa, viruses and bacteria

Molecular ozone reacts readily with DOC, particularly those compounds containing double or triple bonds, and activated aromatic rings with –OH or –NH2 functional groups.

Radicals formed during ozone decay catalyse the ozone-hydroxyl radical decay pathway. However, radicals also react with dissolved organic matter. Some organics act as scavengers of radicals, and slow down the radical decay pathway by reducing the catalytic action, whereas other compounds promote radical decay.


As such, the exact impact of dissolved organics on ozone decay can vary between waters with the same nominal dissolved organic content. Thus, the specific ozone demand and decay in the water to be treated must be characterised, as this will impact the amount of ozone needed to achieve the target CT.

Therefore, practically, the effect of DOC on the decay pathway influences the development and persistence of an ozone residual, and effects the CT that will be measured for a given applied ozone dose – that is, the CT generated by a given applied ozone dose will be lower in high demand waters than in low demand waters. The use of a CT model, with the CT based on the measured ozone residuals through the contactor over the effective contact time, therefore compensates directly for this effect, and, as such, monitoring of DOC is not required.

As discussed in Section 4.1.3, work in the United States is exploring the possibility of adopting an applied ozone dose approach normalised for DOC (e.g. ozone/DOC and DOC/UV absorbance ratios). Validation proponents could consider pursuing this approach if it is possible to (a) demonstrate a validation method based on a clear relationship between the proposed ozone dosing approach and the degree of pathogen inactivation achieved, and (b) demonstrate that an appropriate operational monitoring program can be successfully implemented, which continuously monitors the system in accordance with the validation method.

4.3.5. Inorganic ozone demand

Protozoa, viruses and bacteria

In addition to the ozone demand of organic compounds, ozone will also react with inorganic constituents, including iron, manganese, nitrite and sulfide. The reaction with these compounds is rapid and forms part of the immediate ozone demand of the water.

As noted above, changes in the immediate ozone demand will change the measured ozone CT and will thus be compensated for inherently when monitoring ozone residual. Monitoring of the chemical ozone demand itself is not required where the CT is based on measured ozone residuals.

The possible influence of inorganic compounds on ozone, which would not be picked up by DOC or UV absorbance monitoring, is a potential risk to validation methods based on ozone/DOC and DOC/UV absorbance ratios.

4.3.6. Suspended solids / turbidity / particles in the water

Pathogens in treated wastewater can be either freely suspended (dispersed) or associated with particles. Pathogens that are associated with particles may be protected from inactivation because their exposure to the disinfectant may be reduced. The extent of this particle shielding may be linked to the type of particles (e.g. flocs as compared with clay or sand particles) present in the water and to their size relative to the size of the microorganisms to be inactivated.

The effect of particle shielding on ozone disinfection efficacy is expected to have two potential implications:

Even though particle shielding may be present, it is possible that dissolved ozone, like other oxidant disinfectants in water, may diffuse through the particles and therefore still provide disinfection. However, the dose–response or disinfection efficacy for a given CT may be reduced because the ozone may be consumed through the act of diffusion.

Particle shielding may be such that some pathogens are not exposed to any dissolved ozone. In this case, these pathogens may not be susceptible to ozone disinfection. This may either limit the LRV that can be attributed to the ozone treatment process or present a risk to achieving overall microbial treatment objectives, if ozone is the final treatment step and the goal is effectively non-detection of organisms.

This validation protocol specifically addresses the issue of shielding by recommending a tiered approach. Low turbidity waters (<0.15 NTU) are not considered at risk of experiencing particle shielding, independently of the particle size distribution, and reference CT values can be directly applied. For waters above this turbidity


threshold, the same reference CT values can be applied if it can be demonstrated that the water composition does not introduce any significant shielding effect. If significant particle shielding is expected or observed, a specific validation study needs to be conducted.

Protozoa

Oppenheimer et al. (2000) studied ozone inactivation of Cryptosporidium oocysts in a variety of natural surface waters. The study included a turbidity range from 0.25 NTU to 10.6 NTU. This work used seeded oocysts and thus represents dispersed oocysts in the presence of particles. More than 220 tests were conducted. The conclusion from this study was that while elevated turbidity above 10 NTU may effect disinfection efficacy, there were insufficient data to determine the point at which the impact of turbidity on ozone disinfection efficacy becomes significant.

The potential for shielding as a result of oocysts being enmeshed within particles is also important. This effect has been studied in relation to UV disinfection of indigenous bacteria in wastewater effluents, with shielding of bacteria (typically 0.5–1 μm diameter) being associated with particles over 10 μm in diameter (Emerick et al. 1999). Cryptosporidium oocysts are relatively large, with a diameter of 3–5 μm, thus it is expected that shielding would be associated only with particles significantly larger than 10 μm.

The particle size distribution of the water to be treated should be investigated. If the particles are less than 10 μm, shielding of Cryptosporidium from ozone disinfection is not expected to be an issue.


Viruses are very small organisms and could be effected by particles in effluent applications other than membrane-filtered waters. If ozone is to be used to disinfect water of higher turbidity than the recommended threshold of 0.15 NTU (e.g. if the water has not been filtered with an upstream membrane filter), then further studies are required to determine the type of particles present and the effect that they are likely to have on disinfection efficacy and the amount of shielding that may be occurring.

4.4. Factors influencing effective exposure to dissolved ozone

In addition to water quality, the design of the ozone disinfection system is important to ensure that pathogens are adequately exposed to ozone. Factors to consider are relevant for protozoa, virus and bacteria inactivation.

4.4.1. Mixing and dispersion of ozone

Efficient mixing and dispersion of the oxidant disinfectant is critical to disinfection efficacy. The ozone dosing system needs to be designed to ensure that adequate mixing and dispersion occurs.

4.4.2. Hydraulic efficiency of the contactor

Because the effective duration of exposure to dissolved ozone influences the CT experienced by the microorganisms being disinfected, the design of the ozone contactor should ensure hydraulic efficiency and prevent short-circuiting. Therefore, the hydraulic residence time characteristics of the full-scale contactor must be assessed.

The T10, or the time at which 10% of the water in the contactor has passed through the contactor, is used in CT calculation. Although computational fluid dynamic modelling may be used to design the contactor, full-scale tracer testing is recommended to characterise the hydraulic residence time and determine the hydraulic efficiency, as characterised by the ratio of T10 to the theoretical hydraulic detention time (T10/HDT), over the operating flow range of the contactor. The US EPA LT2ESWTR guidance manual (US EPA 2010) describes how to conduct a tracer test.


5. Operational monitoring parameters

Selection of operational monitoring parameters to inform the initial CT and log inactivation calculations, and to provide ongoing confirmation of operational efficacy is based on the factors that can influence ozonation efficiency. The operational monitoring parameters needed to validate log inactivation by ozone disinfection are summarised in Table 1. Requirements for collecting data for these parameters and their application in determining log inactivation are provided in the following sections.

Table 1 Ozone disinfection system operational monitoring requirements

Parameter Purpose

Target LRV(s) Used to determine the minimum CT set-point

Water temperature Used to determine the minimum CT set-point Generally incorporated with other required water quality instrumentation (ozone residual)

Water flow rate Used to determine ozone contactor hydraulic residence time for CT calculation

Water turbidity Used to confirm that the water being treated is within the validated operating envelope

Ozone residual(s) Used for CT calculation

Ozone CT Calculated by the control system to confirm that the minimum CT set-point is being achieved and to enable calculation of the actual LRV provided

Applied ozone dose Provides an early warning of potential under-dosing, which may lead to insufficient CT being achieved or identification of a fault with any of the inputs to the CT calculation


6. Validation method

This validation protocol relies on (a) the use of the US EPA CT values and a log inactivation equation for ozone inactivation of Cryptosporidium (US EPA 2006), and (b) experimental CT values for ozone inactivation of viruses, bacteria and appropriate surrogates determined by Sigmon et al. (2015). In summary, the three-tier validation method recommended in this protocol is as follows (see also Figure 2):

Under tier 1, when the application-specific water has a low turbidity of 0.15 NTU or below, use the CT tables in Section 9. Under tier 1, the validation method to determine LRVs is to calculate and confirm CT values.

Under tier 2, if the application-specific water does not meet the 0.15 NTU turbidity requirement but it can be demonstrated that the water is consistent (in terms of ozonation efficacy) with clean water as used in the experiments that were referenced to derive the reference CT tables, then the CT tables in Section 9 can be used. Under tier 2, the validation method is to demonstrate that the application-specific water is consistent with clean water in terms of ozonation efficacy, and to calculate and confirm CT values.

Under tier 3, which encompasses all other situations, and recognising the potential effect of particle shielding, a specific validation and challenge testing study should be undertaken to derive CT values that can consistently achieve the target log inactivation in the application-specific water. Because an appropriate surrogate for ozone inactivation of Cryptosporidium has yet to be identified, validation studies must use Cryptosporidium.

Figure 2 Overview of validation method for ozone disinfection

Assess feedwater quality

Turbidity< 0.15 NTU

No particle

shielding

Apply C.t.tables

Conduct specific challenge testing

study

Turbidity≥ 0.15 NTU

Apply C.t.tables

Risk of particle

shielding

Further assess feedwater quality

Tier 1 Tier 2 Tier 3


6.1. Tier 1

Under tier 1, the validation method is to calculate and confirm CT values, to apply the relevant CT tables (based on the target pathogens and temperature) and determine LRVs.

CT is a measure of the strength of the disinfectant for the time that the water and disinfectant are in contact. CT is determined by multiplying the residual disinfectant concentration (C) by the contact time (t)

𝐶𝑇 = 𝐶 × 𝑡

where: C is the residual ozone concentration in mg/L measured at, or downstream of, the point at which the

contact time is achieved t is the time, measured in minutes, that the water is in contact with ozone Various methods are available to calculate ozone CT, as detailed in the US EPA LT2ESWTR guidance manual (2010). This manual presents four methods for calculating CT in an ozone contactor:

T10 method

CSTR (continuous stirred tank reactor) method

extended T10 method

extended CSTR method.

These methods differ in the level of effort required. Selecting the appropriate method(s) to use depends on the configuration of the ozone contactor, the availability of tracer testing results, and the amount of process evaluation and monitoring that a system proponent wishes to undertake (US EPA 2010).

Because of the relatively high cost of ozone treatment, there are cost incentives to adopt more sophisticated CT methods for ozone CT calculation, and these should be investigated by validation proponents. In particular, the proponent must be aware of how the CT calculation used for the validation experiments compares with that proposed for operational monitoring to ensure that observations, or test results, from validation testing are applicable to full-scale operation.

6.2. Tier 2

Under tier 2, the validation method is to demonstrate that the application-specific water is consistent with clean water in terms of ozonation efficacy, and to calculate and confirm CT values as per tier 1. As discussed previously, the ability to use findings from other studies is dependent on demonstrating that the proposed application is comparable, with regard to the conditions (and particularly the water quality) used in the studies.

As discussed in Section 4, various water quality parameters can affect both ozone demand and ozone decay. However, adoption of the dissolved ozone CT to characterise the degree of inactivation achieved effectively captures the impact of these water quality parameters on inactivation efficacy. The effect that these parameters will have is limited to how much ozone dose is required to achieve a given inactivation target. Continuous monitoring of ozone residual concentrations and associated CT calculation, and associated ozone dose control, automatically accounts for changes in ozone demand and the ozone decay profile. Where there may be some residual solids in application-specific water (turbidity > 0.15 NTU), the only effect of potential concern is that of shielding as a result of pathogens being enmeshed within particles. Strategies need to be developed to ascertain whether shielding is significant enough to prevent the application of CT values, as described in Section 9.

A detailed analysis of particle size distribution can be conducted in the application-specific water to


demonstrate that the risk of particle shielding is negligible for the target pathogen for which LRVs are sought.

The recommended approach is to directly compare ozone disinfection efficacy in the application-specific water with the efficacy described in the prescribed CT tables in Section 9. Given that water temperature is an influencing factor on ozone inactivation rates, only data at the same water temperature should be used for relative inactivation efficacy analysis. If data are collected at a range of water temperatures, the data can be corrected to a standard temperature, which may be the minimum temperature, to pool all available data.

Detailed statistical analysis is not specifically required to prove that the application-specific water and clean or reagent-grade water have similar inactivation efficacy. However, some relevant formal statistical analysis can provide increased confidence in this finding. The LRV/CT or lethality constant may be used to compare ozone disinfection in different waters. Assuming first-order disinfection kinetics, LRV/CT is the inactivation coefficient k. The lethality constant for each data point, and the mean lethality constants and standard deviations in each type of water, can be determined, and a standard t-test can be used to determine if the two sets of data are from different distributions, based on a null hypothesis that the differences between the respective waters equals 0. The t-test is a two-tailed test, since either mean can be greater than or less than the other.

A power analysis, which is commonly used to calculate the minimum sample size required so that one can be reasonably likely to detect an effect of a given size, may subsequently be conducted to determine the level of resolution to which differences in the dose–response relationship in application-specific water quality and reagent grade water can be detected.

If a normal distribution for LRV/CT is assumed, a power analysis can be applied and a family of curves developed relating detectable differences to both N (sample size) and the range of standard deviations that are observed, and any additional variability that might be encountered. This means that, based on a given data site, the detectable difference may be calculated – for example, if there is a difference between the mean inactivation rates in application-specific test water and reagent-grade water, it is less than the detectable difference.

The difference between the mean lethality constants for the two different waters can then be determined, and the power analysis used to determine the number of paired samples required to statistically detect the difference, based on extrapolation of the dataset. If the measured difference between the two waters is much less than the detectable difference, this suggests that the differences are not real and are unlikely to be statistically significant.

6.3. Tier 3

When the impact of particle shielding is expected to be significant in the application-specific water, a specific validation and challenge testing study needs to be undertaken to derive CT values that can consistently achieve the target log inactivation in the water.

The established method for undertaking ozone disinfection experiments is the batch solution ozone test method set out in Appendix A of the US EPA LT2ESWTR guidance manual (US EPA 2010).

The first part of the experiment essentially involves generating a stock of highly concentrated ozone solution by bubbling concentrated ozone gas through chilled laboratory-grade water. In the second part of the experiment, a known ozone dose, in the form of an aliquot of the ozone stock solution, is applied to a sample of the test water that contains the target test organism.

For high ozone dose applications, such as wastewater and recycled waters derived from wastewater, the dilution effect of adding the ozone solution to the test water should be taken into account. This effect can be minimised by maximising the ozone stock solution concentration. This can be achieved by chilling the stock solution to close to 0 ˚C while bubbling high concentration ozone gas (10+ wt%) through it. Higher ozone concentrations are achieved by using oxygen-feed gas rather than air-feed gas.


6.3.1. Protozoa

Because an appropriate microbial surrogate has not been identified for Cryptosporidium inactivation by ozone, the validation study should use Cryptosporidium analysed by cell culture infectivity assay.

It is essential that the feed water has a sufficient concentration of infectious oocysts to enable accurate validation of the treatment plant’s inactivation capability. Therefore, the validation study should be undertaken using seeded oocysts, and specifically the Iowa isolate of C. parvum, to ensure consistency with previous studies.

Key requirements of the testing include the following:

Testing must be undertaken in both phosphate buffered reagent-grade water and site-specific water.

Cryptosporidium oocysts must be sourced as appropriate, with suitable quality control.

Testing must be undertaken at site-specific upper and lower water temperature bounds, or just the lower bound if temperature correction is not being sought.

Testing must occur at three discrete water temperatures covering the proposed operating range. This should include minimum, maximum and average water temperature, or just the minimum water temperature if application of a temperature correlation is not being sought.

Testing must occur at a pH representative of site-specific conditions.

Oocysts must be recovered from water samples using an immunomagnetic separation technique.

Cryptosporidium oocyst infectivity must be determined using the cell culture foci detection method.

Extensive practice is recommended to develop proficiency in applying the solution ozone test, including associated ozone residual measurement techniques. To manage the risk of unsuccessful testing at significant expense, it is recommended that experimental repeatability be achieved before testing with organisms. Characteristics of good experimental methods and procedures include stable ozone residual measurements that produce a smooth, first-order ozone decay curve with only small deviations in ozone decay rate across the duration of the test. This practice will also generate information about ozone demand and decay for the particular application or water type, and facilitate test matrix design to determine the respective ozone doses that achieve the range of CT values required to achieve the target range of LRVs. These CT values may initially be based on the prescribed tables in Section 9.

Parallel testing using seeded oocysts should be done in both reagent-grade water and site-specific water under the same experimental conditions.

Details of the recommended methods and procedures can be found in Appendix A and the standard methods section of the US EPA LT2ESWTR guidance manual (US EPA 2010).


If the ozone system is to be validated for protozoa as well as virus and or/bacteria, then it is possible to use the validation study for protozoa to incidentally obtain LRVs for virus and bacteria, given how much more susceptible they are to ozone inactivation. The approach to validating virus and bacteria disinfection capacity may then be based on demonstrating that the targeted degree of virus and bacterial inactivation is achieved when some fraction of the ozone CT required for protozoa inactivation is also reached.

It is possible to validate an ozone disinfection system only for virus LRVs if there is no specific need for protozoa LRVs. The validation approach for this scenario would be to determine the CT values required to achieve the target virus LRVs.

Based on work by Sigmon et al. (2015) the preferred test organism is E. coli, either as a surrogate for virus or as the direct target for bacteria.


It is essential that the feed water used for validation testing has a sufficient concentration of the target organism to enable accurate validation of the treatment inactivation capability. One benefit of using E. coli is that it is an indigenous organism in most site-specific waters, and observations based on indigenous organisms help in understanding particle effects on inactivation efficacy for testing in other than reagent-grade water or membrane-filtered waters. However, because of the size difference between E. coli and viruses, observations from indigenous E. coli should only be applied to bacteria validation unless further evaluation of particle association is undertaken. If insufficient indigenous E. coli is present, seeded E. coli needs to be used for the validation study.

If seeding is going to be done, the E. coli stock should be washed three times in phosphate buffered saline solution to remove background contaminants that would exert an additional artificial ozone demand that would be nonrepresentative of the filtered water being routinely disinfected.

Key requirements of the testing include the following:

E. coli must be sourced, as appropriate, with suitable quality control.

Testing must occur at three discrete water temperatures, covering the proposed operating range. This should include minimum, maximum and average water temperature, or just the minimum water temperature if application of a temperature correlation is not being sought.

Testing must occur at a pH representative of site-specific conditions.

As discussed in Section 6.3.1, it is recommended that the test methods and procedures have been practised extensively before proceeding with organism testing. Details of the recommended methods and procedures can be found in Appendix A and the standard methods section of the US EPA LT2ESWTR guidance manual (US EPA 2010).


7. Data collection and analysis

Data collected during the validation testing program must be representative and reliable. To ensure that quality data is collected:

appropriate sampling methods and techniques must be consistent with the Standard methods for the examination of water and wastewater (Rice et al. 2012).

National Association of Testing Authorities (NATA)–accredited methods must be used where available. Where NATA-accredited methods are not available, the laboratory must

o demonstrate that the method used is consistent with a standard method, where this is available

o document the method used for the analysis

o retain documentation and appropriate quality assurance data

o engage independent expert(s) to peer review and endorse the method.

field and laboratory equipment must be maintained and calibrated

limits of detection must be appropriately measured

all procedures must be completed by qualified personnel and be subject to quality assurance/quality control procedures.

The monitoring program for the validation study must ensure that the data collected are relevant and sufficient for a statistically valid analysis. The raw data and their analysis must be appended to the validation report. If data are excluded from the analysis, the rationale must be provided.

In analysing data, validation uncertainty needs to be taken into account, including biases and error in measurements, laboratory equipment, experimental design and analytical techniques. The measurement of uncertainty must be included, to the extent practicable, when attributing an LRV to the treatment process unit.

Under the ISO standard to which NATA accredits laboratories – ISO/IEC 17025-2005: General requirements for the competence of testing and calibration laboratories – accredited laboratories are required to estimate the uncertainty associated with the results they produce (known as the measurement of uncertainty). Measurement of uncertainty data must be provided when reporting analytical results. This information will show the variability in the analytical data and will assist in formulating evidence-based conclusions.

Furthermore, during validation testing, all equipment must be carefully selected and calibrated to minimise uncertainty. Measurements must be traceable to a registered standard method, where this is available.

Increasing the sample number and/or sample volume, and using more accurate and precise measuring devices will provide the best estimate of the capability of a treatment process unit to remove or inactivate pathogens.

7.1. Cryptosporidium infectivity techniques

As discussed in Section 2.1, ozone treatment does not physically remove oocysts, but rather inactivates them, as measured by reduced oocyst infectivity. In principle, similar oocyst counts are achieved before and after ozone treatment, and, therefore, it is the reduction in the percentage, or log inactivation, of infectious oocysts that must be characterised by validation studies.

Measuring Cryptosporidium infectivity is particularly important for ozone disinfection and needs to be incorporated into the design of the validation study. A variety of techniques have been used to determine C. parvum infectivity (Rochelle et al. 2004), including human volunteers, animal models, in vitro excystation and in vitro cell cultures. Human volunteer studies are neither practical nor ethical, and would not be considered for use by the water industry.

In vitro excystation has been used to assess Cryptosporidium oocyst viability. Although one of the studies used to develop the US EPA ozone CT tables relied on excystation, it is not generally regarded as a reliable method,


for a number of reasons:

Excystation rates are sensitive to excystation conditions and incubation procedures (Li et al. 2001).

Intact oocysts after excystation are infectious to mice (Neumann et al. 2000).

Comparisons of excystation to mouse assays by Finch et al. (1993) showed that the two methods were not comparable.

Evaluation of Cryptosporidium oocyst viability and infectivity found significant differences between the results obtained from four different excystation methods conducted in two laboratories (Bukhari et al. 2000). Further, when assessing the viability of ozone-treated oocysts, in vitro excystation significantly overestimated viability when compared with animal infectivity. Bukhari et al. (2000) further concluded that in vitro excystation could not be used to predict oocyst inactivation with UV or ozone.

Mouse infectivity assays have historically been the accepted technique for measuring C. parvum infectivity (traditionally considered as the ‘gold standard’) and the US EPA LT2ESWTR CT tables (US EPA 2006) were developed from experimental data derived primarily from mouse assays.

Animal infectivity studies require specialised laboratory facilities, and are extremely labour intensive and expensive (Rochelle et al. 2004). Animal infectivity testing is not available in Australia and is not routinely offered by laboratories in the United States. Cell culture is therefore the only practicable analytical technique available for Cryptosporidium infectivity.


8. Critical limits and operational monitoring

Operational monitoring is necessary to enable adequate control over the system and to continuously confirm that the system is operating within the validated operational envelope. Where operational parameters are found to be outside the validated operating envelope, the log inactivation may not be achieved, resulting in supply of water that is not fit for use. Action should be taken to bring the system back into the envelope and/or stop the supply of potentially unsuitable water.

Ozone treatment should be controlled using a CT-based approach. The CT required will be calculated based on the target log inactivation of the specified target pathogen(s) and the measured temperature of the water. The CT achieved in the ozone contactors will be calculated in accordance with US EPA methods, as discussed in Section 6.1.

As well as identifying the influencing factors for ozone inactivation outlined in Section 4, the operational parameters to be monitored are summarised in Table 1 (Section 5) and discussed in further detail below.

8.1. CT set-point

The CT set-point will be determined by an automated control system, in accordance with the following philosophy:

The target pathogen minimum LRV should be an operator set-point in the control system.

o When the target pathogen is virus and/or bacteria, because they have equivalent sensitivity to ozone, a single LRV target can be adopted for both pathogen groups.

o When the target pathogen is both virus/bacteria and protozoa, different targets may be set for the respective pathogen groups. Alternatively, if it is known that the virus/bacteria target is achievable at the minimum critical protozoa target, then only a protozoa target is required.

The water temperature will be measured online.

The plant control system will use the target pathogen minimum LRV(s) and measured water temperature to determine the minimum critical ozone CT set-point from the adopted inactivation equations or CT tables (prescribed under tier 1 and tier 2 or generated through validation studies under tier 3). If there are separate pathogen group targets, the control system can assess the limiting target requiring the highest CT and adopt this as the CT set-point.

8.2. Flow

Accurate operational flow measurements through the ozone contactors is required to determine contactor hydraulic retention time (HRT) and hence ozone CT. Each ozone contactor should be equipped with an online magnetic flow meter that monitors the flow through the contactor with high accuracy.

The flow meter is also used to confirm that the actual flow through the contactor is less than the maximum flow permissible through the contactor, ensuring that the minimum contact time is always achieved. The adopted contactor T10 to HRT ratio covers the preferred flow range.

8.3. Feedwater turbidity

The feedwater turbidity is not used directly for dose control but is used to ensure that the water quality is consistent with the validated operating range.


8.4. Ozone residual

The ozone contactor should be fitted with online ozone residual monitoring, using the approach described in the US EPA LT2ESWTR guidance manual (US EPA 2010), which is suitable for the scale of the proposed installation.

8.5. Water temperature

The temperature of the water being treated effects both ozone residual measurement by online analysers and determination of the required target CT. Water temperature measurement is required.

8.6. Ozone CT

Ozone CT should be calculated continuously by the automated plant control system.

8.7. Applied ozone dose

The applied ozone dose should be monitored continuously by the automated control system, based on the following inputs:

online ozone gas flow measurement to the respective injection systems

online ozone gas concentration measurement

online water flow measurement to each contactor

Although the ozone CT is the critical measure of the degree of inactivation provided, the applied ozone dose should be used as early warning of potential under-dosing, which may lead to insufficient CT being achieved.

8.8. Determination of critical limits

The LRV will be determined in accordance with the findings from the validation study. Prescribed LRV–CT relationships, as described in Section 6 under tier 1 and tier 2, will be used to set critical limits. Under tier 3, specific critical limits will be established, based on the outcomes of the site-specific challenge testing and validation study.

8.9. Degree of pathogen inactivation provided

The automatic control system should calculate the actual degree of pathogen inactivation provided (i.e. a real-time operational LRV) by the ozone system in accordance with the adopted inactivation relationship, based on the measured water temperature and measured ozone CT.


9. Method to determine the LRV for each pathogen group

9.1. Tier 1 and tier 2

9.1.1. Protozoa

To determine the LRV for protozoa by ozonation when no particle shielding is expected, CT values in Table 2 should be used.

Table 2 CT values (mg·min/L) for 0.25 to 3.0 log reduction values of protozoa by ozonation at temperatures ranging

from 5 °C to 30 °C

Log10 inactivation 5 C 10 C 15 C 20 C 25 C 30 C

0.25 4.0 2.5 1.6 1.0 0.6 0.39

0.5 7.9 4.9 3.1 2.0 1.2 0.78

1.0 16 9.9 6.2 3.9 2.5 1.6

1.5 24 15 9.3 5.9 3.7 2.4

2.0 32 20 12 7.8 4.9 3.1

2.5 40 25 16 9.8 6.2 3.9

3.0 47 30 19 12 7.4 4.7

Source: US EPA (2006), Table IV.D-3 In addition, the US EPA provides an equation to determine LRVs as a function of water temperature (°C) and ozone CT (mg·min/L) between indicated values:

Cryptosporidium log reduction value = 0.0397 × (1.09757)temp × CT where temp is temperature in degree Celsius The CT values for ozone are based on analyses by Clark et al. (2002), using data from Li et al. (2000) and Rennecker et al. (1999), with additional procedures to develop confidence bounds. The US EPA applied lower 90% confidence bounds to these predictive equations, in recognition of the inherent variability and uncertainty in inactivation experiments, and to ensure that water authorities operating at a given CT value are likely to achieve at least the corresponding log inactivation level in the CT table.


The US EPA published CT values for virus inactivation by ozone in the Surface water treatment rule (SWTR) (US EPA 1989). The SWTR was developed for drinking water applications, and the experimental data that were used to develop the CT tables were extrapolated from experiments conducted by Roy et al. (1982) in reagent-grade water, with a factor of safety of 3 applied. The Guidelines for validating treatment processes for pathogen reduction (Victorian Department of Health 2010) queried the use of US EPA drinking water criteria for wastewater applications.

Sigmon et al. (2015) published CT values for specified log inactivation levels of viruses and surrogates in

wastewater at pH 7.6 and 16 C (Table 3). These data indicate that all organisms were generally inactivated to 4-log at a CT of no more than 1.0 mg·min/L. Some variance was recorded in multiple batch experiments for AD-2, which suggests that it may be slightly more resistant than the other microorganisms tested; however,


there is no suggestion of any outliers, with all microorganisms showing similar sensitivity.

Table 3 CT values for 1 to 4 log reduction values of viruses and surrogates in wastewater at pH 7.6 and a

temperature of 16 °C

Microorganism

LRV

1 2 3 4

Escherichia coli 0.48 0.65 0.82 0.98

Coxsackievirus B5 0.32 0.51 0.71 0.90

Poliovirus 1 0.47 0.58 0.68 0.78

Adenovirus 2 (batch 1) 0.40 0.51 0.70 0.77

Adenovirus 2 (batch 2) 0.40 0.51 0.90 1.10

ɸx174 0.33 0.45 0.57 0.69

PRD1 0.43 0.63 0.83 1.00

All viruses 0.56 0.81 1.07 1.32

Source: Sigmon et al. (2015) The two batch tests for AD-2 show some variance, and both sets of data can be considered valid. It is therefore proposed to apply the higher CT to the development of the overall virus CT on the following basis:

apply a 20% safety factor to the maximum virus CT at LRV of 1-log (PV-1) –

120% × 0.47 mg·min/L = 0.56 mg·min/L

Apply a 20% safety factor to the maximum virus CT at LRV of 4-log (AD-2) –

120% × 1.10 mg·min/L = 1.32 mg·min/L

apply linear interpolation to determine the CT values required for LRVs of 2-log and 3-log.

The last row of Table 3 summarises the proposed conservative CT values as a function of LRVs of viruses by ozonation. The proposed CT values are quite conservative compared with the US EPA guidance at the same water temperature – they are around 180% higher at LRV of 2-log and 120% higher at LRVs of 3-log and 4-log. The difference between the CT values of the US EPA and Sigmon et al. (2015) arises from a combination of the following:

The US EPA relationship is non-linear, with the difference in CT between LRVs of 3-log and 4-log being less than that between LRVs of 2-log and 3-log. This increasing inactivation efficacy is not commonly observed or expected.

The US EPA relationships appears to be linear from LRVs of 3-log and 2-log back to the origin, whereas the proposed CT relationship is offset – that is, some CT is required before any LRV is observed.

The CT values determined by Sigmon et al. (2015) for specific pathogens and the conservative values proposed

for all viruses were established based on data collected at a temperature of 16 C. The US EPA SWTR (US EPA 1991) provides the following relationship

𝐿𝑅𝑉 = 2.1744 × 1.0726𝑡𝑒𝑚𝑝 × 𝐶𝑇

where temp stands for temperature in degree Celsius.

Based on this relationship, CT values for temperatures other than 16 C can be extrapolated from the CT values

at 16 C, adjusted for temperature using the equation

𝐶𝑇(𝑡𝑒𝑚𝑝) = 𝐶𝑇(16°𝐶) × 1.0726(16−𝑡𝑒𝑚𝑝) The recommended CT values for inactivation of viruses (and bacteria) by ozonation are summarised in Table 4.


Table 4 CT values for 1 to 4 log reduction values of viruses in wastewater at temperatures ranging from 5 °C to

30 °C

Log10 inactivation 5 C 10 C 15 C 20 C 25 C 30 C

1 1.21 0.85 0.60 0.42 0.30 0.21

2 1.75 1.23 0.87 0.61 0.43 0.30

3 2.31 1.63 1.15 0.81 0.57 0.40

4 2.85 2.01 1.42 1.00 0.70 0.49


10. Triggers for revalidation

Validation is the process of testing a treatment unit process under a known range of operational conditions, representative of an ‘operational envelope’ under which it is known that the certain level of pathogen reduction will be occurring. If the normal operating conditions within the plant change so that they are no longer consistent with those selected during the validation, then the unit process will need to be revalidated to ensure that disinfection is occurring in accordance with the assigned log credits. Changes that may trigger a validation will be those linked to the factors influencing the efficacy of ozone disinfection and may include:

water temperature below the validated minimum

turbidity above the validated maximum, or changes in water quality and potential for particle shielding

changes to the ozone contact tank that affect the hydraulic retention time or mixing efficacy. Although these should not effect the ability of ozone to disinfect the water, they may effect the retention time in the tank and hence change inputs to the CT calculations

need to increase LRVs.


Glossary and abbreviations

AD-2 adenovirus 2 CT Disinfection residual concentration (C, in mg/L), multiplied by contact time (t, in

minutes) at the point of residual measurement; a measure of disinfection effectiveness.

DNA deoxyribonucleic acid

DOC dissolved organic carbon

HDT theoretical hydraulic detention time Determined by dividing the volume of a process unit (contactor) by the peak hourly flow rate.

LRV log reduction value

A log10 reduction value is used in the physical–chemical treatment of water to characterise the removal or inactivation of microorganisms such as bacteria, protozoa and viruses (1-log10 = 90% or a 10-fold reduction, 3-log10 = 99.9% or a 1000-fold reduction, and so on).

LRV = log10 (N0) – log10 (N), where N0 = concentration of infectious microorganisms before treatment and N = concentration of infectious microorganisms after treatment.

LT2ESWTR long term 2 enhanced surface water treatment rule NATA National Association of Testing Authorities

NTU nephelometric turbidity units

ozone residual The level of ozone remaining in the water after the initial dosing.

PV-1 poliovirus 1 RNA ribonucleic acid

T10 contact time

The detention time experienced by 90% of the water passing through a process unit (contactor).

tracer study An experimental procedure for estimating hydraulic properties of the distribution system, such as contact time.

USEPA United States Environmental Protection Agency

UV ultraviolet


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