Comparative Inactivation of Murine Norovirus and MS2 Bacteriophage

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Comparative Inactivation of Murine Norovirus and MS2 Bacteriophage by Peracetic Acid and Monochloramine in Municipal Secondary Wastewater Effluent Nathan Dunkin, ShihChi Weng, Kellogg J. Schwab, James McQuarrie, Kati Y. Bell, and Joseph G. Jacangelo Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b05529 • Publication Date (Web): 06 Feb 2017 Downloaded from http://pubs.acs.org on February 7, 2017

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Environmental Science & Technology

Ms. ID: es-2016-05529u

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Comparative Inactivation of Murine Norovirus and MS2 Bacteriophage by Peracetic Acid

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and Monochloramine in Municipal Secondary Wastewater Effluent

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Nathan Dunkin1, ShihChi Weng2, Kellogg J. Schwab1,2, James McQuarrie3, Kati Bell4,

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and Joseph G. Jacangelo1,2,4*

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1

Department of Environmental Health and Engineering, Bloomberg School of Public Health,

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Johns Hopkins University, Baltimore, MD, 21205, USA

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Metro Wastewater Reclamation District, Denver CO, 80229, USA

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MWH-Stantec, Pasadena, CA, 91101, USA

JHU/MWH Alliance, Johns Hopkins University, Baltimore, MD, 21205, USA

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*corresponding author: [email protected]

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Keywords: norovirus, peracetic acid, monochloramine, wastewater disinfection

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ACS Paragon Plus Environment


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TOC/ABSTRACT ART

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ABSTRACT

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Chlorination has long been used for disinfection of municipal wastewater (MWW) effluent while

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the use peracetic acid (PAA) has been proposed more recently in the United States. Previous

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work has demonstrated the bactericidal effectiveness of PAA and monochloramine in

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wastewater, but limited information is available for viruses, especially ones of mammalian origin

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(e.g. norovirus). Therefore, a comparative assessment was performed of the virucidal efficacy of

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PAA and monochloramine against murine norovirus (MNV) and MS2 bacteriophage in

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secondary effluent MWW and phosphate buffer (PB). A suite of inactivation kinetic models was

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fit to the viral inactivation data. Predicted concentration-time (CT) values for 1-log10 MS2

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reduction by PAA and monochloramine in MWW were 1,254 and 1,228 mg-min/L, respectively.

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The 1, 2, and 3-log10 model predicted CT values for MNV viral reduction in MWW were 32, 47,

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and 69 mg-min/L for PAA and 6, 13, and 28 mg-min/L for monochloramine, respectively.

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Wastewater treatment plant disinfection practices informed by MS2 inactivation data will likely

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be protective for public health but may overestimate CT values for reduction of MNV.

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Additionally, equivalent CT values in PB resulted in greater viral reduction which indicate that

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viral inactivation data in laboratory grade water may not be generalizable to MWW applications.

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INTRODUCTION Disinfection of municipal wastewater (MWW) is critical to controlling release of

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pathogenic microorganisms such as viruses into receiving waters. While there are currently no

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federal or state requirements in the United States for virus inactivation in secondary MWW

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effluents, utilities are expecting the United States Environmental Protection Agency (US EPA) to

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publish ambient water quality criteria (AWQC) for a viral indicator in the near future1.

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Furthermore, because most National Pollutant Discharge Elimination System permits regulate

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microbial contaminants at the end-of-pipe based on the AWQC, this development has led to

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renewed interest in the efficacy of various disinfection methods to inactivate viruses in MWW.

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Chlorination remains the most common method for disinfection of MWW largely due to

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its low cost and acceptance among regulatory agencies. It is well documented that free chlorine

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reacts with MWW to form a wide range of halogenated and nonhalogenated disinfection by-

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products (DBPs) of concern to human health and the environment2. Chloramines are formed

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when free chlorine reacts with available ammonia that is often present in MWW. While

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chloramines are also known to produce halogenated byproducts3, DBPs such as trihalomethanes

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(THMs) and haloacetic acids (HAAs) typically occur at lower concentrations than with free

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chlorine4, 5.

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Previous work examining monochloramine inactivation of viruses has been performed

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primarily in reagent grade water (RGW)6-10. However, these data may not be generalizable to

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MWW applications because source water quality can affect inactivation rates11. For example,

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Kahler et. al found that differences in water quality between one untreated ground water and two

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partially treated surface waters accounted for variation in CT values for 3-log10 reduction of

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coxsackievirus B5 (monochloramine CT values varied between 220-320 mg-min/L depending on


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water type), echovirus 11 (310-950 mg-min/L), and human adenovirus 2 (300-820 mg-min/L)12.

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Despite the importance of the water matrix in determining inactivation rates, there is a paucity of

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information on monochloramine inactivation of viruses in MWW.

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There is also growing interest in alternatives to chlorine-based disinfection of MWW due

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to security concerns with gaseous chlorine, general safety issues, and DBP toxicity concerns

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after effluent discharge into the environment13. As a result, there has been renewed interest in

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peracetic acid (PAA) as a disinfectant for MWW. PAA is recognized as an efficient organic

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bactericide, sporicide, fungicide, and algaecide, and has been applied in the food-processing,

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beverage, pharmaceutical, and textile industries14. PAA requires no post-treatment neutralization,

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and DBPs formed by PAA are mostly carboxylic acids that have less mutagenicity,

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carcinogenicity, and genotoxicity than halogenated DBPs produced from chlorination15, 16.

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Furthermore, laboratory and pilot studies have demonstrated PAA’s efficacy in reducing E. coli

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and fecal coliform levels to meet permit regulations in MWW effluent17, 18.

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PAA has been reported to exhibit modest inactivation of viruses in MWW, usually

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around one log, in studies employing realistic concentrations and contact times for wastewater

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treatment plant (WWTP) operations18-21. However, all published studies on PAA inactivation of

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viruses in MWW have looked exclusively at seeded or naturally occurring coliphage18-24.

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Because evidence has suggested that coliphage inactivation data may not be representative for

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mammalian viruses such as norovirus (NoV)25, 26, there is need to comparatively assess PAA

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inactivation of NoV and coliphage in MWW.

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NoV is now recognized as the number one cause of acute gastroenteritis sporadic cases

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and outbreaks globally across all age groups27. In the United States, NoVs are estimated to cause

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21 million illnesses and 71,000 hospitalizations annually28. Furthermore, despite recent



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advancements29, researchers have struggled to develop an easily reproducible cell culture model

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to propagate HuNoV in the laboratory. As a result, murine norovirus (MNV), one of the first

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NoVs to be cultured in a laboratory, has been suggested as a human NoV surrogate30. MNV is

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included in genogroup GV of norovirus31 and shares substantially greater morphological and

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genetic similarities with human NoV than coliphage32. These facts have led researchers to

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conclude that MNV is likely to be more predictive of human norovirus inactivation by

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engineered treatment systems than coliphage33, 34, though this has been difficult to prove

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experimentally due to the historical lack of cell culture system for human NoV. Additionally,

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utilizing MNV in treatment studies allows researchers to measure infectivity reduction, which is

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considered the gold standard measurement in treatment and inactivation studies35.

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Because there are no identified data in the literature on inactivation of NoV by PAA and

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chloramine in MWW, the objective of this study was to comparatively assess these disinfectants

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using MNV as a surrogate virus. Additionally, inactivation of MNV was compared to the more

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commonly employed surrogate MS2 coliphage. To our knowledge this is the first study utilizing

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a norovirus to measure infectivity reduction in wastewater by chemical oxidation.

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EXPERIMENTAL SECTION

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Water Matrices. A 0.01 M phosphate buffer solution (pH 7) and municipal secondary

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treated effluent (non-chlorinated) from a water resource recovery facility at the Metro

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Wastewater Reclamation District (Denver, CO) were used in this study. Water quality analysis of

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the secondary wastewater effluent (Table S1) was performed upon sample arrival using methods

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as previously described36.

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MNV Stock Preparation. MNV stocks were propagated in RAW 267.4 cells and concentrated as previously described37. Following concentration, MNV stocks were further


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purified by ultracentrifugation on a sucrose cushion38 in order to remove cell culture components

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that would cause oxidant demand during disinfection experiments. Briefly, filtered MNV stock

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was laid over sterile-filtered 20% sucrose solution in an Ultraclear centrifuge tube (Beckman,

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Brea, CA) and centrifuged at 95,000 g for 3 h. After centrifugation the sucrose and media were

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aspirated and the procedure repeated using the same ultracentrifuge tube in order to further

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concentrate the virus. The virus pellet was then resuspended in 600 µl of Dulbecco’s phosphate

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buffered saline (D-PBS), portioned, and stored at -80°C.

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MS2 Stock Preparation. MS2 coliphage stocks were propagated as previously

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described37. MS2 stocks were further concentrated and purified using a 100,000-Da ultra-

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membrane filter (Amicon Ultra; Millipore Corp., Bedford, MD) to increase the virus titers and

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remove soluble and low molecular weight components from the supernatant. Following initial

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concentration in the membrane (viruses were retained while low-molecular-weight components

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passed through), viral stocks were then washed three times by adding 14 ml of D-PBS into the 1

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ml of virus-containing retentate and centrifuging the membrane (4,000 g for 10 min) each time.

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Purified stocks were portioned and stored at -80°C.

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In order to ensure monodispersed MS2 virus after concentration by Amicon filtration, an

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aliquot of MS2 stock was diluted in DI water to approximately 1010 PFU/mL and analyzed by

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dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern, UK). Figure S1 shows DLS

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results that indicate MS2 remained monodispersed after Amicon filtration.

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Infectivity Plaque Assays. Enumeration of infective MNV and MS2 viral particles was

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performed using standard 10-fold dilution plaque assays. For MNV, the plaque assay previously

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described by Bae and Schwab37 was followed. MS2 (ATCC 16696-B1) bacteriophage stocks



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were generated using the double agar layer (DAL) method39 with E.coli Famp (ATCC 700891)

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bacterial host.

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Peracetic Acid and Monochloramine. Peracetic acid (PAA) stock solutions were

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obtained from commercially available concentrate (VigorOx® WWT II; PeroxyChem, PA). PAA

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residuals were measured by the modified N,N-diethyl-p-phenylene diamine (DPD; > 98%,

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Sigma-Aldrich) colorimetric method with potassium iodide40, 41. The modified DPD method was

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also used for H2O2 measurement with the addition of ammonium molybdate solution (Hach

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Company, CO) as a catalyst42.

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Monochloramine master stock solution was freshly prepared by modifying the method

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from Shang and Blatchley 43. Ammonium chloride solution was spiked with chlorine

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concentrated solution (10-15 %, Sigma-Aldrich) at a chlorine:nitrogen mass ratio of 3:1 and the

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mixture was allowed to react for one hour before use. The monochloramine master stock solution

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was then diluted to desired concentration for the disinfection experiment. The DPD colorimetric

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method was also used to measure monochloramine residuals44.

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Experimental Procedure. All viral inactivation experiments were conducted 20 °C and

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at ambient pH for MWW and pH 7.0 for PB. Sterile, chlorine demand-free 125 mL Erlenmeyer

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flasks containing an initial volume of 95 mL of water matrix, 5 mL of disinfectant, and 1 mL of

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virus cocktail comprised of concentrated, purified MNV and MS2 were employed for the

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disinfection experiments. Virus concentrations in the final volume were approximately 1x105

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PFU/mL. Flasks were continuously mixed throughout the experiment. After the water matrix and

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virus were added to the flask, 5 mL of disinfectant (PAA or NH2Cl), adjusted to achieve the final

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desired concentration, was added. Four PAA doses (0.7, 1.5, 5, and 10 mg/L) and four NH2Cl

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doses (0.5, 1, 5, 10 mg/L) were selected for evaluation.


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Sampling was performed prior to the addition of disinfectant and at pre-selected time

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points after the addition of disinfectant up to 120 minutes. At each sampling time point, 4 mL

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were removed and immediately quenched with sodium thiosulfate (600 µg/mL) for infectious

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virus analysis. For experiments with PAA, the quenching mixture also included catalase (2.5

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mg/L). Quenched samples were then portioned into 1.5 mL microcentrifuge tubes and stored at -

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80°C. Kinetic Modeling and Statistical Analyses. Disinfectant decay constants (k’) for each

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experiment were calculated by regressing measured residuals at each sample time using the least-

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squares method. First-order kinetics were assumed according to the equation: = ∙

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where,

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k’ = first-order disinfectant decay rate

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C = observed disinfectant residual (mg/L)

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C0 = initial disinfectant dose (mg/L)

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t = time from start of experiment to time of sample (min)

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Viral infectivity reduction was determined by calculating the negative log of the ratio of

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surviving organisms to initial organisms (N/N0) at sample times and fit by a series of well-

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validated inactivation models derived from the generalized inactivation rate:

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=

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(1)

= −

where,

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rd = inactivation rate

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n, m, and x = dimensionless model parameters

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k = inactivation rate constant


(2)


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N = viral density (PFU/mL)

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C = disinfectant concentration (mg/L)

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T = contact time (minutes)

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To account for the effect of disinfect decay throughout an experiment, equation (1) can be

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substituted into equation (2) and the resulting expression can be integrated to derive the

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specialized inactivation models employed in this study, which included the Chick-Watson45,

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log = − ( )! ∙ "(, $ % )

(4)

the Power Law47,

log =

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(3)

the Incomplete Gamma Function (IGF) Hom46,

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log = − ( − )

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+

&'()*()∙ ( - )∙ ./0 1 , − ()

(5)

and the Hom-Power Law48,

log = −

&'(3*()∙

/+!4 ./0 8 ! ∙7(, )∙ 5, 6

()

(6)

These models were fit to experimental data using the least squares method. Data points

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below the sensitivity limit of the assay were excluded from the analysis. Model fit was assessed

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by comparing the residual sum of squared errors (SSE). Partial F-tests were performed to assess

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significance of additional parameters in more highly parametrized models (α = 0.05). Model

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errors were checked for normality using the Shapiro-Wilk statistical test. Modeling and statistical

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analysis were performed in Microsoft Excel and R (www.r-project.org).

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RESULTS AND DISCUSSION Kinetic modeling of virus inactivation data. Temporal profiles of viral infectivity

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reduction by monochloramine and PAA in both MWW and PB are shown for MS2 and MNV in

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Figure 1. Inactivation models employed in this study were fit to the viral infectivity reduction

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data to account for the fact that the concentration-time (CT) concept is in fact a reduction of the

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CnT parameter found in the generalized inactivation rate equation, where n is not always equal to

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1. Data were segmented by virus, water type, and disinfectant type, and the best-fit model for

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each scenario is presented in Table 1 with associated parameters. Modeling results for non-

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selected models along with Shapiro-Wilk test statistics for all model residuals are provided in the

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Supporting Information. The table shows that the IGF Hom model provided the best fit to the

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viral infectivity reduction data for six of the eight virus-water type-disinfectant scenarios. The

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IGF’s ability to account for virus tailing in experimental data likely contributed to the

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effectiveness of the Hom-type models.

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Examples of experimental data with associated best-fit model are provided in Figure 2 for

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visual observation of fit. Doses of 1 mg/L NH2Cl and 1.5 mg/L PAA were selected for

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presentation because of their relevance to typical wastewater treatment plant operations

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(WWTP). Model fits for all scenarios are shown in Figure 3 where observed infectivity reduction

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is plotted versus model-predicted infectivity reduction. Together, Figures 2 and 3 demonstrate

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that selected models were able to effectively represent experimental data.

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Effect of water matrix on viral infectivity reduction. The effect of water matrix on

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viral infectivity reduction can be examined through application of best-fit models. After 120

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minutes of contact time, predicted MS2 reduction in MWW at a high monochloramine dose of

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15 mg/L was 1.1 log PFU/mL, while predicted reduction at a high PAA dose of 10 mg/L was



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only 0.7 log PFU/mL. Higher infectivity reductions were observed in PB: a monochloramine

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dose of 1 mg/L in PB achieved a predicted reduction of 1.8 log PFU/mL after 120 minutes, while

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a monochloramine dose of 15 mg/L achieved a predicted reduction of 4.1 log PFU/mL over the

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same contact time. Likewise, PAA doses of 1.5 and 10 mg/L in PB achieved predicted infectivity

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reductions of 1.2 and 3.4 log PFU/mL, respectively, after 2 hours of contact time.

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Similar trends were observed for MNV, where infectivity reductions were higher in PB

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than in MWW. Predicted infectivity reductions for MNV by monochloramine after 10 minutes of

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contact time at an initial dose of 1 mg/L were 1.9 log PFU/mL and 5.2 log PFU/mL in MWW

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and PB, respectively. Predicted infectivity reductions by PAA after 30 minutes of contact time at

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an initial dose of 1.5 mg/L were 1.2 and 3.0 log PFU/mL in MWW and PB, respectively.

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Previous studies have reported that inactivation of viruses can vary significantly between

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natural waters and reagent grade water (RGW). Researchers have hypothesized that turbidity in

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complex environmental waters may protect virions from disinfectants11 via shielding, adsorption,

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or enhanced aggregation49. One study reported that differences in water quality in three source

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waters influenced chlorine CT values for 3-log10 reduction of viruses that varied between for 2.6

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– 7.9 mg-min/L for coxsackievirus B5 (CVB5), 0.6 – 1.2 mg-min/L for echovirus 11 (E11), and

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Comparative Inactivation of Murine Norovirus and MS2 Bacteriophage

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