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Page 1 ofEnvironmental 26 Science & Technology

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Environmental Science & Technology

Kinetics of inactivation of waterborne enteric viruses by ozone Camille Wolf1, Urs von Gunten2,3 and Tamar Kohn1* 1

Laboratory of Environmental Chemistry, School of Architecture, Civil and Environmental Engineering (ENAC), École Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland 2

Laboratory for Water Quality and Treatment, School of Architecture, Civil and Environmental Engineering (ENAC), École Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland 3 Eawag, Swiss Federal Institute of Aquatic Science and Technology, Dübendorf, Switzerland *Corresponding author email address: [email protected] Phone: +41 21 693 0891; Fax: +41 21 +41 21 693 80 70


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1

Abstract

2

Ozone is an effective disinfectant against all types of waterborne pathogens. However, accurate and

3

quantitative kinetic data regarding virus inactivation by ozone is scarce, due to the experimental

4

challenges associated with the high reactivity of ozone towards viruses. Here, we established an

5

experimental batch system that allows tailoring and quantifying very low ozone exposures and

6

simultaneously measuring virus inactivation. Second-order ozone inactivation rate constants (kO3-virus)

7

of five enteric viruses (laboratory and two environmental strains of coxsackievirus B5: CVF, CVEnv1

8

and CVEnv2, human adenovirus: HAdV, and echovirus 11: EV) and four bacteriophages (MS2, Q, T4

9

and Φ174) were measured in buffered solutions. The kO3-Virus of all tested viruses ranged from 4.5 x 105

10

to 3.3 x 106 M-1s-1. For MS2, kO3-MS2 only depended weakly on temperature (2 – 22 °C, Ea = 22.2 kJmol-

11

1

12

viruses towards ozone decreases in the order Q > CVEnv2 > EV  MS2 > Φ174  T4 > HAdV > CVF 

13

CVEnv1. Based on the measured kO3-Virus and typical ozone exposures applied in water and wastewater

14

treatment, we conclude that ozone is a highly effective disinfectant for virus control.

) and pH (6.5 – 8.5), with an increase of kO3-MS2 with increasing pH. The susceptibility of the selected



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15

Introduction

16

Ozone (O3) has been applied to drinking water disinfection and chemical oxidation for over a century.1

17

Ozone is known to efficiently inactivate bacteria, and protozoans, including chlorine resistant

18

pathogens such as Cryptosporidium parvum oocysts, Giardia cysts and Legionella. 2–5 In addition, ozone

19

can be applied for taste and odor control, as well as for the oxidation of inorganic contaminants.6 More

20

recently, the application of ozone has been extended to wastewater treatment,7–10 where its main

21

purpose is to contribute to the abatement of micropollutants in secondary wastewater effluent and

22

the reduction of their load to the aquatic environment. Finally, ozone is becoming an integral part of

23

many potable reuse trains, where it can be applied for pre-oxidation of effluent organic matter,

24

micropollutant abatement and disinfection.11

25

Ozone has also been recognized as an effective treatment against waterborne viruses. This property is

26

particularly desirable in the context of potable reuse, where guidelines require a stringent treatment

27

goal of up to 12 log10 virus removal from raw wastewater to tap water.12 Previous studies have reported

28

inactivation data for different enteroviruses, adenoviruses and several bacteriophages.13–16 However,

29

reproducible measurements of kinetic parameters for viral inactivation remain sparse, and the

30

reported efficacy of ozone toward a given virus varies dramatically. For example, as reviewed by Finch

31

and Fairbairn (1991), the reported inactivation of poliovirus type 1 ranges from 0.5 log10 at an

32

estimated ozone exposure (ozone concentration integrated over exposure time10) of 3.9 mg*min/L (15

33

min at 0.26 mg/L ozone) to 4 log10 at an estimated exposure of only 1.7*10-4 mg*min/L (0.1 s at 0.1

34

mg/L ozone).14 In a less extreme example, Roy et al. (1981) reported a 2 log10 inactivation of

35

coxsackievirus B5 at an ozone exposure of 0.072 mg*min/L (0.48 min at 0.15 mg/L ozone),17 whereas

36

Sigmon et al. (2015) determined an ozone exposure of 0.51 mg*min/L to achieve the same extent of

37

inactivation.18

38

These differences in reported kinetic data are in part associated with different experimental setups to

39

measure virus inactivation by ozone. For example, in experiments using bubble contactors inactivation 1 ACS Paragon Plus Environment

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may be limited by ozone transfer from the gas phase to the bulk solution.18 Compared to experiments

41

using dosing of stock solutions of dissolved ozone, bubble contactors are thus likely to report slower

42

inactivation kinetics. The biggest experimental challenge, however, is the quantification of the very

43

low ozone exposures associated with virus inactivation. Although some studies report inactivation as

44

a function of ozone residual or ozone concentration applied and time of exposure 14,15 permitting rough

45

estimates of exposure values - the majority of prior studies did not provide sufficient data to evaluate

46

virus inactivation in terms of ozone exposure. Furthermore, due to the extremely rapid kinetics of viral

47

inactivation, substantial measurement uncertainties remain even when exposures were reported. For

48

example, while both Sigmon et al. (2015)13 and Thurston et al. (2005)16 reported inactivation as a

49

function of the ozone exposure, their first measured ozone concentration was only obtained after 10

50

to 15s. However, the initial phase (≤ 10s) of the ozone decay has been identified as crucial to determine

51

ozone exposures for fast reactions.19 Experimental approaches that do not capture this initial phase

52

will result in an overestimation of ozone exposure, and by consequence to an overestimation of the

53

exposure required to achieve a given virus inactivation.

54

The experimental challenges discussed above highlight the need for a reliable experimental approach

55

to measure virus inactivation kinetics at low ozone exposures. The resulting kinetic information is of

56

particular importance with respect to the use of ozone in wastewater, where competing reactions of

57

ozone with matrix constituents (e.g., dissolved organic matter ) lead to rapid ozone consumption, and

58

hence low ozone exposures. For example, a typical specific ozone dose of 0.5 mg O 3/ mg dissolved

59

organic carbon (DOC) applied to nine different secondary wastewater effluents yielded ozone

60

exposures of 0.16 – 2.2 mg*min /L (0.2-2.8*10-3 Ms). 20 To estimate the extent of virus inactivation by

61

ozonation of wastewater, virus susceptibilities to low ozone exposures must thus be known.

62

An existing experimental approach is the use of quench-flow systems. 19 However, such experiments

63

require specialized equipment, which is not practical for use with biosafety level II material. In this

64

context, the first aim of this study was to develop and validate a simple batch method to measure

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second order virus inactivation rate constants at low ozone exposures. This method was then used to

66

explore the range of susceptibilities of different viruses toward ozone, which in turn allowed an

67

estimation of virus inactivation during wastewater ozonation.

68

Experimental Section

69

Chemicals

70

The quality and sources of all chemicals are listed in the Supporting Information (SI).

71

Virus propagation, purification and enumeration

72

Five human enteric viruses (Faulkner strain and two environmental strains of coxsackievirus B5: CVF,

73

CVEnv1 and CVEnv2; human adenovirus type 2: HAdV; and echovirus 11 Gregory strain: EV) and four

74

bacteriophages (MS2, Q, T4 and Φ174) were included in this study. The information on sources of all

75

viruses, their host cells and their propagation procedures is provided in the SI.

76

All viruses were purified using a polyethylene glycol (PEG)-chloroform method, as described

77

previously.21 For MS2, a second purification method adapted from Oksanen et al. (2012)22 using a CIM

78

monolithic column (QA 1 ml, BIA Separation) was performed to study the effect of purification on

79

inactivation. Details of the procedure are given in Table S1, SI. Virus stock solutions were kept in

80

phosphate buffered saline (PBS; 5 mM NaH2PO4, 10 mM NaCl, pH=7.5) and stored at 4°C. Phages were

81

enumerated by the double-agar-layer method as described previously21 and infective phage

82

concentrations are expressed in plaque forming units (PFU)/mL. Enteric viruses were enumerated by

83

the most probable number (MPN) assay as detailed elsewhere,23 and concentrations are expressed as

84

most probable number of cytopathic units (MPNCU)/mL.

85

Ozone production

86

Ozone gas was produced by an ozone generator (Innovatec; model CMG 3-3 or CMG 3-5, Rheinbach,

87

Germany) from pure oxygen (Carbagaz) and the resulting ozone/oxygen mixture was bubbled through

88

Barnstead Nanopure (Thermofisher) /MilliQ (Millipore) water cooled in an ice bath.24 Ozone stock 3 ACS Paragon Plus Environment

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solutions reached concentrations ranging from 0.8 to 1.25 mM. For each stock solution produced, the

90

ozone concentration was measured by direct spectrophotometry using an absorption coefficient ε260

91

= 3200 M-1 cm-1.1

92

Experimental procedures

93

Approach to produce and quantify low ozone exposures

94

To measure viral inactivation at low ozone exposures, we used an experimental setup in which the

95

ozone stability was controlled by a compound with known ozone reactivity added in excess of the

96

ozone dose. Specifically, we performed inactivation experiments in solutions containing excess

97

concentrations of trans-cinnamic acid (CA) in presence of tert-butanol (t-BuOH) to avoid any

98

interferences with hydroxyl radicals (•OH). CA was chosen because it is highly reactive towards ozone

99

(kO3-CA = 3.8 x 105 – 1.2 x 106 M-1 s-1), 25,26 it does not affect viral infectivity at concentrations used for

100

the experiments, and its reaction with O3 produces benzaldehyde with a 1:1 stoichiometry (Scheme

101

1).26 O

O OH

+

O3

kO3-CinnA

H OH

+

O

HO

+

H2O2

O

102 103

trans-cinnamic acid

Benzaldehyde

Glyoxilic acid

Scheme 1 Reaction of trans-cinnamic acid with ozone: stoichiometric production of benzaldehyde.

104 105

Once the reaction between O3 and CA is completed, the concentration of benzaldehyde can be used to

106

precisely determine the initial ozone concentration. The theoretical ozone decay resulting from the

107

reaction of ozone with CA can be modelled using three parameters: the second-order rate constant

108

for the reaction of ozone with CA (kO3-CA), the initial O3 concentration (determined from final

109

benzaldehyde concentration) and the initial CA concentration. The ozone decay curve is then

110

integrated over time to determine ozone exposure. 27

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In this experimental approach, kO3-CA, is a key parameter to estimate the ozone exposure. Therefore, it

112

is crucial to adapt kO3-CA in the ozone decay model to the applied experimental conditions (pH and 4 ACS Paragon Plus Environment


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temperature). To obtain kO3-CA, the kinetics of ozone decay in the presence of excess CA were

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determined under pseudo-first order conditions at different temperatures, using a Hi-Tech Scientific

115

SF-61DX2 stopped-flow spectrometer (TGK Scientific). The experimental procedure was modified from

116

Heeb et al (2017)28 and details are given in the SI. Based on the resulting rate constants (Table S2, SI)

117

the activation energy (Ea) was determined via the Arrhenius equation (Figure S1, SI). This, in turn,

118

allowed us to calculate kO3-CA at any selected temperature (Table S3, SI).

119

Batch ozonation experiments

120

The steps involved in obtaining a virus inactivation curve are summarized in Figure 1. Specifically, a

121

series of batch reactions was run with different initial O3:CA ratios (Fig. 1A), to establish different ozone

122

exposures. After complete depletion of O3, samples were withdrawn for benzaldehyde quantification

123

by HPLC (see SI) to determine the ozone concentration in each reactor. Additionally, residual infective

124

viruses were enumerated. Subsequently, the determined ozone concentration and the initial CA

125

concentration were used to model the ozone exposure in each reactor (Fig. 1B). Finally, the inactivation

126

in each reactor was evaluated as a function of the corresponding ozone exposure (Fig.1 C), and the

127

inactivation rate constant (kO3-Virus) was determined from a least-squares fit to the linear portion of the

128

following model:

129

𝑡

𝑁

𝑙𝑛 ( ) = − 𝑘𝑂3−𝑣𝑖𝑟𝑢𝑠 ∗ ∫0 [𝑂3 ](𝑡) 𝑑𝑡 𝑁0

(Equation 1)

130

where N and N0 are the infective virus concentrations at times t and 0, respectively, [O3](t) is the time-

131

dependent ozone concentration, and ∫0 [𝑂3 ](𝑡) 𝑑𝑡 is the ozone exposure at exposure time t.

𝑡

132 133 134 135 136 137


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139 Figure 1. Overview over batch approach. (A) Viruses are inactivated in batch reactors with different O3: CA ratios, whereby CA is in excess. The closer the O3:CA ratio is to unity, the higher is the resulting ozone exposure. (B) Ozone decay is simulated based on the ozone concentration (determined by quantification of benzaldehyde) and initial CA concentration, and the ozone exposure in each reactor is calculated. The residual infective virus concentration in each reactor is quantified by an infectivity assay. (C) Inactivation is plotted versus the ozone exposure to obtain the inactivation rate constant, according to Equation 1.

140 141

Experimental solutions were freshly prepared the day of the experiment with a range of CA (90-180

142

μM) in PBS. 20 mM t-BuOH was added to scavenge •OH produced during ozonation, to ensure that

143

kinetic parameters pertained to the inactivation by ozone only. Inactivation curves for all viruses were

144

obtained at pH 6.5 and a temperature of 22°C. For MS2, additional experiments were conducted over 6 ACS Paragon Plus Environment


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a range of pH values (6.5-8.5) and temperatures (2-22°C) to assess the influence of these parameters

146

on the second order ozone inactivation rate constants. Virus ozonation was performed in 50 mL glass

147

reactors filled with the experimental solution, capped and sealed to limit ozone outgassing. Viruses

148

were added to the reactors at concentrations of 107 -109 PFU/mL (coliphages) or 105 MPNCU/mL

149

(human enteric viruses). An ozone stock solution was injected into the reactors to reach a

150

concentration of 20 to 175 μM, and the reactors were mixed (650 rpm) for 30 s. A syringe connected

151

to a 0.22 µm filter (Infochroma) was simultaneously inserted to avoid overpressure during O3 injection

152

and was removed after the injection. After 2.5 to 3 min, when O3 was completely depleted, 100 μL

153

aliquots were withdrawn, were diluted 10x into PBS containing 0.1 μM EDTA (coliphages) or in culture

154

media (human viruses), and viruses were enumerated as described above. The role of EDTA was to

155

prevent virus inactivation by a Fenton-type reaction of H2O2 (produced during the reaction of O3 with

156

CA; Scheme 1) with trace metals present in PBS.29 No inactivation of viruses by H2O2 was observed if

157

the samples were diluted in culture media. O3 decay was then modelled and integrated to determine

158

the ozone exposure in each reactor. All experiments were conducted at least in duplicate.

159

In addition to viruses, the kinetics of carbamazepine (CBZ) abatement by ozone was also measured to

160

validate the experimental system.1 For these experiments, CBZ (5 µM) was mixed with CA (150-

161

170µM) in PBS adjusted to pH 3, and containing 20 mM t-BuOH. The lower pH was used to increase O3

162

stability. CBZ concentrations were quantified by HPLC as described in the SI.

163

Quench-flow ozonation experiments

164

MS2 inactivation. To confirm the inactivation rate constants obtained in the batch system, the

165

inactivation of MS2 was additionally measured by quench-flow, using a procedure adapted from

166

Czekalski et al. (2016) Specifically, a MS2 suspension (~109 PFU/mL) in PBS at pH 6.5 and 5 mM t-BuOH,

167

was mixed at a 10:1 ratio with an ozone solution (500 μM, prepared in 1mM HCl solution, kept at 4°C)

168

in reaction loops (40 μL and 500 μL) with different flow rates, to yield a range of ozone exposures

169

(Table S4, SI). Ozone was quenched after defined contact times by mixing the sample at a 11:1 ratio

170

with 100 mM CA in Nanopure water at pH ~7. Quenched samples were collected in a sterile syringe, 7 ACS Paragon Plus Environment

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and were used to quantify MS2 infectivity by plaque assay and benzaldehyde concentrations by HPLC.

172

The residual ozone concentration and ozone exposure was determined as described previously 30, and

173

the inactivation rate constant was determined using Equation 1.

174

The initial MS2 concentration was measured in the same experimental setup in ozone-free samples

175

consisting of the MS2 solution and milliQ water at a 10:1 ratio. Similarly, the experimental ozone

176

concentration was measured at the start and end of each experiment at the shortest contact time in

177

virus-free solutions consisting of milliQ water and an O3 solution at a 10:1 volumetric ratio. The decay

178

of the ozone feed solution in the quench-flow system during the experiments (15-25 min) was less

179

than 2 %.

180 181

Second order rate constant for the reaction of CBZ with ozone. The second order rate constant for the

182

reaction of CBZ with ozone was also quantified by the quench-flow setup. For these measurements,

183

the experimental solution consisted of 5 µM CBZ and 5 mM t-BuOH in milliQ water at pH 3. The

184

experiments were conducted as described above, except that ozone was quenched by 100 mM CA

185

solution at a 11:2 volumetric ratio. Prior to the experiment, the starting concentration of CBZ was

186

measured in the same experimental setup in ozone-free solutions (CBZ solution and milliQ water at a

187

volumetric ratio of 10:1). The O3 concentrations were measured at the shortest contact time in CBZ-

188

free experiments (milliQ water and O3 solution at a 10 : 1 volumetric ratio). The decay in the ozone

189

feed solution (pH~3) throughout the experiment (40-60 min) was less than 2 %. Quenched reaction

190

mixtures were withdrawn for CBZ and benzaldehyde quantification. Details on contact times, flow

191

rates, residual ozone concentrations and the derived ozone exposures are listed in Table S5.

192

Simulations and data analysis

193

Simulations of ozone depletion were performed in R31 using the packages “caTools”32, “flux”33 and

194

“desolve”34. Statistical analyses were performed in R31 using the “ggplot2”35 and “multcomp”36

195

packages. Rate constants of the different viruses were compared by ANCOVA with Tukey’s post hoc

196

test, allowing for an α-type error of 5%. 8 ACS Paragon Plus Environment


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Results and Discussion

198

Method validation and temperature-dependence of kCA-O3

199

To validate the batch ozonation approach developed herein, it was applied to measure the kinetics of

200

the abatement of CBZ. The obtained second order rate constant kO3-CBZ was then compared to a

201

previous value from literature,37 as well as to the value measured by the quench-flow system in this

202

study. As summarized in Table 1, the kO3-CBZ values obtained in the batch and the quench-flow system

203

were within the error statistically equivalent (p-value = 0.057). The larger error in the batch system

204

may be explained by the fact that the ozone exposure is simulated, whereas in the quench-flow

205

approach it is directly measured. Furthermore, the measured kO3-CBZ values were within a factor of two

206

of the previously reported values, which is typical for such kinetic measurements. This suggests that

207

the batch ozonation approach can accurately determine reaction kinetics with ozone.

208 209

Table 1. Summary of measured and reported second-order rate constants for the reaction of CBZ with ozone and corresponding statistics. The error associated with the kO3-CBZ corresponds to the standard error.

Method

kO3-CBZ, [M-1s-1]

Adjusted R2

Batch

(5.5 ± 0.8) x 105

0.90

Quench-flow

(6.1 ± 0.1) x 105

0.99

Competition kinetics

~3 x 105

-

Condition 22 ±2°C pH = 3.00 ±0.03 22 ±2°C pH = 3.00 ±0.03 20°C pH = 7

Reference This study This study Ref. 22

210 211

The batch approach generated ozone exposures ranging from approximately 10-7 Ms (8 x 10-5 mg x min

212

L-1) to approximately 10-5 Ms (8 x 10-3 mg x min L-1). The upper end of this range is determined by the

213

requirement that the O3:CA ratio has to be below unity.

214

This batch method can be applied over a range of temperatures, as long as the ozone depletion is

215

modelled using kO3-CA for the corresponding temperature. To this end, kO3-CA was measured at different

216

temperatures, and an activation energy Ea of 21.2 kJ/mol was determined (see Figure S1 and Table S3,

217

SI for details). The kO3-CA measured at 22 °C (7.6 x 105 M-1 s-1) corresponds well to previously reported

218

values (3.8 x 105 and 1.2 x 106 M-1 s-1).25,26 9 ACS Paragon Plus Environment

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In summary, the batch ozonation approach developed herein can be applied to determine kinetic

220

parameters for ozone-sensitive substances/organisms at different temperatures. Furthermore, as the

221

method does not require any specialized equipment, it is well suited for applications in any laboratory,

222

including laboratories handling biohazardous materials.

223

MS2 inactivation kinetics by the batch method

224

Influence of virus solution conditions on inactivation

225

An important prerequisite of the batch approach used herein is that CA be the main sink for ozone.

226

This condition can be readily met for ozonation of chemical substances such as CBZ, which are typically

227

available at high purity. The purity of stock solutions of viruses, in contrast, is compromised by residues

228

from the culture media and host cells used during virus propagation. To ensure that impurities in the

229

virus solutions do not influence ozone inactivation kinetics, three purification approaches were

230

compared. For MS2 coliphage as the benchmark virus, we tested purification by PEG-chloroform,

231

purification by ion exchange chromatography on a monolithic column, and absence of purification

232

(simple filtration using a 0.45 μm Durapore filter, Millipore). In addition, the inactivation kinetics were

233

measured for different starting concentrations of MS2 (106-109 PFU/mL), as dilution can be used as a

234

method to reduce impurities from the stock solution.

235

Second-order inactivation rate constant for MS2 (kO3-MS2) were statically equivalent for all procedures

236

as long as purification was applied (p-value = 0.14, Table S8). In contrast, kO3-MS2 decreased significantly

237

if no purification was used (p-value = 5.3 x 10-4). In the absence of purification, the culture media

238

constituents thus likely protected the viruses from inactivation.

239

concentration did not affect kO3-MS2 to a significant extent (p-value >0.05; Table S8).

240

Replicate experiments using different MS2 stock solutions purified by the PEG-chloroform method

241

indicated that this purification procedure yielded consistent kinetic data across different viral batches

242

(Table S8 and Figure S3, SI). Therefore, the PEG-chloroform method was used for all further virus

243

inactivation experiments.

Furthermore, the initial MS2



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244

Rate constant for the inactivation of MS2 by ozone

245

Based on multiple replicate batch experiments, a kO3-MS2 of (1.9 ± 0.1) x 106 M-1 s-1 was determined at

246

pH 6.5 and 22 °C. Under the same experimental conditions, quench-flow determination of kO3-MS2

247

yielded a value of (1.0 ± 0.1) x 107 M-1 s-1 (Figure S2). This difference of about a factor of 5 in measured

248

kO3-MS2 values approximately corresponds to that of reported second-order rate constants for the

249

abatement of organic substances by ozone in different studies (e.g., reported values of kO3-CA vary by a

250

factor of 3 between measurements by quench-flow25 and by competition kinetics26). The greater kO3-

251

MS2

252

procedures. First, mixing may be less efficient in the batch experiments, which are conducted in greater

253

volumes. Second, the ozone exposures in batch experiments are simulated not measured, which may

254

introduce an error in the resulting kO3-MS2. However, despite the difference in the absolute values, both

255

methods confirm the very high susceptibility of MS2 to ozone. Furthermore, the discrepancies in kO3-

256

MS2 values

257

same virus in different studies (see introduction).

258

Effects of temperature and pH

259

To investigate temperature- and pH-dependence of virus inactivation by ozone, MS2 inactivation was

260

investigated over a pH range of 6.5-8.5 and temperature range of 2 – 22°C, roughly characteristic of

261

water and wastewater treatment (Figure 2).

obtained in the quench-flow system may be explained by the differences in the experimental

observed herein are minor compared to the vast differences in ozone susceptibilities for the

262


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Figure 2. Second-order rate constants kO3-MS2 for the inactivation of MS2 with ozone at different temperature (2, 12 and 22°C) and pH (6.5 and 8.5) conditions.

263 264

Increases in temperature and pH both accelerated inactivation, though the effects were minor (see

265

Table S9). Specifically, an increase in pH from 6.5 to 8.5 enhanced kO3-MS2 by a factor 1.4 (2 °C) to 2 (22

266

°C ), whereas a temperature shift by 10 °C resulted in a 1.2 (pH 6.5) to 1.4 (pH 8.5)-fold increase in kO3-

267

MS2.

268

ozone of 12.2 to 23.4 kJ/mol at pH 6.5 and pH 8.5, respectively (see Table S10 and Figure S4, SI). These

269

values correspond well to that previously reported by Roy et al. (1981) of 15 kJ/mol.38 Similarly low

270

activation energies for the reaction with ozone are also encountered for highly reactive organic

271

compounds (e.g., the Ea for the reaction of CA with ozone determined herein corresponds to 21.2

272

kJ/mol). Such low temperature dependence of the reaction indicates that reaction rate constants are

273

at close to the upper end of ozone reactions. In contrast, the Ea for the reaction of ozone with C.

274

parvum, a more ozone-resistant pathogen, is significantly higher (51.7 kJ/mol).39

275

Due to the rather modest effect of temperature and pH, additional viruses were only tested at a single

276

temperature and pH condition (6.5 and 22 °C). The somewhat lower pH compared to that encountered

277

in wastewater was chosen to increase ozone stability throughout the experiment, and thus reduce a

278

source of variability. Given its low pH dependence, however, kO3-virus measured at pH 6.5 is still

279

representative of water and wastewater treatment conditions.

This temperature dependence of kO3-MS2 indicated an activation energy of MS2 inactivation by



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280

Inactivation of other viruses by ozone

281

After validation of the batch method and optimization of the experimental parameters using MS2, the

282

batch method was applied to eight additional bacteriophages and enteric viruses. Representative

283

inactivation curves are shown in Figure 3, and the resulting second order inactivation rate constants

284

(kO3-virus) are summarized in Table 2.

285

The investigated viruses spanned a range of genome types (CV, EV, MS2 and Q: single-stranded RNA;

286

X174: single-stranded DNA; HAdV and T4: double-stranded DNA) and lengths, as well as different

287

protein contents and capsid configurations. Despite this diversity, all second-order inactivation rate

288

constants were of similar magnitude, spanning a range from 4.5 x 105 to 3.3 x 106 M-1s-1 (Table 2). These

289

high rate constants confirm the high ozone efficiency for virus inactivation. Nevertheless, minor yet

290

statistically significant differences were observed among the ozone reactivities of the different viruses.

291

Interestingly, the biggest differences were found among closely related viruses. For example, one of

292

the most susceptible (CVEnv2) and the most resistant viruses (CVEnv1 and CVF) are representatives of

293

the same virus strain (coxsackievirus B5). Similarly, MS2 was more resistant than its closely related

294

counterpart Q. The causes underlying these differences are currently not understood and are the

295

subject of ongoing investigations.

296


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297

Figure 3. Log inactivation of all selected viruses as a function of the ozone exposure (22°C, pH = 6.5). Each panel contains data from at least two independent experiments.



298 299

Page 18 of 26

Table 2. Second-order rate constants for the inactivation of the selected viruses by ozone (kO3-virus), with corresponding standard errors and adjusted R2.

Viruses MS2 Qβ ΦX174 T4 HAdV EV CVF CVEnv1 CVEnv2

kO3-virus , [M-1 s-1] (1.9 ± 0.2) x 106 (3.3 ± 0.7) x 106 (1.2 ± 0.1) x 106 (1.3 ± 0.1) x 106 (9.0 ± 1.2) x 105 (1.9 ± 0.2) x 106 (4.4 ± 1.4) x 105 (4.9 ± 0.7) x 105 (2.5 ± 0.6) x 106

R2 0.73 0.50 0.86 0.83 0.63 0.69 0.27 0.66 0.56

300 301

Interestingly, the ozone reactivities of the viruses tested herein exhibited a similar trend as observed

302

by Sigmon et al. (2015), namely MS2>EV>T4>HAdV ≈ X174>CVF. Higher stability of CVF compared to

303

other enteric viruses tested was also observed for disinfection by free chlorine, whereas this trend did

304

not apply to disinfection by chloramine (Table 3).

305 306 307 308

Table 3. Required oxidant exposures to inactivate, EV, HAdV and CVF by 2 log10. Ct values for ozone were determined in this study (Table S10). Ct values for free chlorine and chloramine were determined by Cromeans et al. (2010) in buffer at pH 7 and 5 °C 40

Virus

Ozone, [Ms] / [mg min L-1]

Free chlorine [Ms] / [mg min L-1]

Chloramine [Ms] / [mg min L-1]

EV

~2.4 x 10-6/ 1.9 x 10-3

~7.8 x 10-4 / 0.7

~1.1 / 940

HAdV

~5.1 x 10-6/ 4.1 x 10-3

~3.4 x 10-5 / 0.3

~0.9 / 795

CVF

~1.0 x 10-5/ 8.0 x 10-3

~4.75 x 10-3 /4.2

~0.7 / 590

309 310 311

Finally, the high kO3-virus determined in this study imply that hydroxyl radicals (•OH), which are typically

312

produced during ozonation, do not significantly contribute to the inactivation of viruses of water and

313

wastewater. The ratio of the •OH to the O3 exposures (Rct)41 typically corresponds to ~10-6 to 10-8

314

during lake water and wastewater ozonation, respectively.19 This implies that in order for •OH to play

315

a significant role in inactivation, its reactivity toward viruses would have to exceed that toward ozone

316

by a factor of 106 to 108. However, rate constants for •OH previously measured in our laboratory 15 ACS Paragon Plus Environment

Page 19 of 26


317

correspond to 7.0 x 109 M-1 s-1 for MS2, 4.0 x 109 M-1 s-1 for HAdV and 1.7 x 109 M-1 s-1 for X174,42

318

whereas those for inactivation by ozone were only 3-4 orders of magnitude lower (Table 2). The

319

contribution of hydroxyl radicals to inactivation during ozonation can thus be considered negligible.

320

Estimation of extent of virus inactivation during wastewater ozonation

321

As highlighted above, inactivation of viruses by ozone differ both between and within different virus

322

serotypes and families. However, these differences may not be relevant in the context of water and

323

wastewater treatment, as the applied ozone exposures are generally large and the inactivation of

324

viruses by ozone is efficient. Based on the kO3-virus determined herein, we estimated inactivation for

325

specific ozone doses from 0.25-1 mg O3/ mg dissolved DOC. Based on previously determined

326

relationships between specific ozone doses and ozone exposures in secondary wastewater effluent,

327

the corresponding ozone exposure during wastewater treatment can be estimated as 6 x 10 -6 to 8 x

328

10-3 Ms, 43 which is in the upper range or above the ozone exposures applied in this study (10 -7 – 10-5

329

Ms). Figure 4 shows the calculated extent inactivation of CVF (most resistant human virus studied;

330

Table 2) and MS2 in a wastewater as a function of the specific ozone dose. At the lowest specific ozone

331

dose of 0.25 mg O3/mg DOC, the inactivation of CVF is minor, whereas MS2 exceeds 4 log10 inactivation.

332

This example illustrates that viruses may escape inactivation if low specific ozone doses are applied to

333

wastewater. Furthermore, it indicates that at low specific ozone doses, MS2 may not be a good

334

surrogate for enteric viruses. At specific ozone doses ≥ 0.3 mg O3/mg DOC, both viruses are estimated

335

to be inactivated by ≥ 9 log10. Typical specific ozone doses for enhanced wastewater treatment for

336

micropollutant abatement with ozone are in the order of 0.5-0.6 mg O3/mg DOC. 20,44,45 Under such

337

conditions, an efficient inactivation of viruses can be expected. We caution, however, that these

338

estimates are based on inactivation rate constants derived from the linear regression of the

339

inactivation curves shown in Figure 3. The linear relationship between ozone exposure and inactivation

340

may break down at higher exposures, as some studies have reported tailing inactivation curves.46

341

Furthermore, a recent study has shown that flocs present in wastewater effluent can protect

342

microorganisms from inactivation by ozone,30 and this factor remains to be tested for viruses. Overall, 16 ACS Paragon Plus Environment


Page 20 of 26

343

ozone thus has a high potential as a wastewater disinfectant for viruses, but this potential should be

344

confirmed during ozonation in real wastewater systems. This is done in an ongoing study in our

345

laboratory.

Figure 4. Calculated inactivation of MS2 and CVF by ozone in wastewater as a function of the specific ozone dose (pH = 7, 22°C, kO3-MS2 = 1.94 x 10-6, kO3-CV = 4.4 x 10-5). (*) indicates value greater than 15 log reduction.

346

347

Supporting Information

348

The Supporting Information is available free of charge on the ACS Publications website at

349

http://pubs.acs.org. Additional information on chemicals, viruses and host cells; MS2 purification by

350

monolith liquid chromatography; determination of the activation energies of MS2 and CA

351

degradation by ozone; HPLC analyses; quench-flow experiments; comparison of MS2 inactivation in

352

batch and quench-flow systems; effect of purification, pH and temperature on MS2 inactivation.

353

Acknowledgements

354

This study was funded by the Swiss National Science Foundation (grant number 205321_169615).

355

Elisabeth Salhi, Virginie Bachmann, Ursula Schönenberger and Caroline Gachet Aquillon are

356

acknowledged for help in the laboratory and Michèle Heeb for assistance with the stopped-flow. 17 ACS Paragon Plus Environment

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357

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