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Aerosolization of Ebola Virus Surrogates in Wastewater Systems Kaisen Lin, and Linsey Chen Marr Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b04846 • Publication Date (Web): 26 Jan 2017 Downloaded from http://pubs.acs.org on January 27, 2017

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Environmental Science & Technology

Aerosolization of Ebola Virus Surrogates in Wastewater Systems

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Kaisen Lin and Linsey C. Marr*

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Department of Civil and Environmental Engineering, Virginia Tech, 418 Durham Hall,

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Blacksburg, Virginia 24061, United States

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*Corresponding author

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Mailing address: Department of Civil and Environmental Engineering, Virginia Tech,

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418 Durham Hall, Blacksburg, Virginia 24061, United States

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Telephone: (540) 231-6071

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E-mail: [email protected]

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Key words: Ebola, virus, bioaerosol, inhalation, wastewater

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ACS Paragon Plus Environment


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Abstract

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Recent studies have shown that Ebola virus can persist in wastewater. We evaluated the

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potential for Ebola virus surrogates to be aerosolized from three types of wastewater

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systems: toilets, a lab-scale model of an aeration basin, and a lab-scale model of

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converging sewer pipes. We measured the aerosol size distribution generated by each

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system, spiked Ebola virus surrogates (MS2 and Phi6) into each system, and determined

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the emission rate of viruses into the air. The number of aerosols released ranged from 105

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to 107 per flush from the toilets or per minute from the lab-scale models, and the total

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volume of aerosols generated by these systems was ~10-9 to 10-7 mL per flush or per

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minute in all cases. MS2 and Phi6, spiked into toilets at an initial concentration of 107

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plaque-forming units per milliliter (PFU mL-1), were not detected in air after flushing.

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Airborne concentrations of MS2 and Phi6 were ~20 PFU L-1 and ~0.1 PFU L-1,

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respectively, in the chambers enclosing the aeration basin and sewer models. The

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corresponding emission rates of MS2 and Phi6 were 547 PFU min-1 and 3.8 PFU min-1,

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respectively, for the aeration basin and 79 PFU min-1 and 0.3 PFU min-1 for the sewer

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pipes.


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TOC ART

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Introduction

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The Ebola virus disease (EVD) outbreak in West Africa that began in 2014 is the largest

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in history. It has caused 11,310 deaths as of June 2016 with a fatality rate of 53%.1 The

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particular species causing the current outbreak is Zaire ebolavirus.2, 3 EVD is transmitted

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via direct contact with blood, body fluids, and objects contaminated with body fluids.4-6

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Transmission is not thought to occur via air, water, or food, although in theory,

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aerosolization of blood and body fluids has the potential to lead to infection, as aerosol

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transmission has been demonstrated in nonhuman primates.7-10 Besides inhalation of

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aerosolized virus, contact of unprotected mucous membranes such as the eyes and mouth

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with aerosolized virus is also a potential route of transmission.

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EVD patients can produce large volumes of diarrhea that contain up to 107

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genome copies of the virus per milliliter.11-13 The World Health Organization

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recommends direct disposal of contaminated liquid waste into the sewage system without

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disinfection,14 but this guidance has raised concerns among environmental engineers

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because workers may come into close contact with wastewater,15 and as few as 10

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infectious viral particles are sufficient to infect individuals.16-18 Recent studies indicate

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that Ebola virus and surrogates can survive for at least 1 day in wastewater.19,

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addition, we have shown that at least 94% of virions in wastewater remain in the liquid

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fraction rather than partition to biosolids and material surfaces.21 Thus, the vast majority

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of Ebola virus is likely to remain mobile in wastewater systems and maintain the

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potential to be aerosolized during certain processes.

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In

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Current risk assessments on inhalation exposure to Ebola virus in wastewater

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systems are based mainly on data from studies of bacteria, not viruses, conducted on


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toilets and wastewater treatment plants.22 Bacterial and fungi, including pathogenic ones,

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have been detected in the surrounding air,23-32 demonstrating that wastewater treatment

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processes have the potential to generate bioaerosols. However, few studies have focused

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on aerosolization of viruses in wastewater systems.33-35 Aerosolization of viruses could be

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significantly different from aerosolization of bacteria due to dissimilarities in their size,

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structure, and surface properties. One important difference is that bacteria partition

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mainly to biosolids while viruses remain suspended in the liquid.21, 36 Even fewer studies

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have focused on aerosolization from the wastewater collection system,30 yet processes

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such as converging flows at pipe junctions and high-pressure cleaning have the potential

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to generate aerosols as well.

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The goal of this research is to characterize the aerosolization of Ebola virus

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surrogates during the regular operation and maintenance of wastewater systems. This

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study investigates flush toilets, a laboratory-scale aeration basin model, and a laboratory-

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scale sewer model. Specific objectives are to determine the size distribution of aerosols

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produced by each system and the emission rate of aerosolized Ebola virus surrogates

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spiked into the system.

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Materials and Methods

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Measurement of Aerosol Size Distribution in Model Systems. The size distribution of

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aerosols generated by flush toilets, a lab-scale aeration basin, and a lab-scale sewer model

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of converging pipes was measured inside custom-designed chambers. Aerosols were

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measured in triplicate experiments for each system using a scanning mobility particle

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sizer (TSI SMPS 3936) for particles 14-700 nm and an aerodynamic particle size



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spectrometer (TSI APS 3321) for particles 0.5-20 µm. Results were combined using the

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TSI Data Merge program to generate a composite size distribution and corresponding

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fitted function using the default parameters. Prior to measurement, each chamber was

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flushed with air from a gas cylinder passed through a high-efficiency particulate air

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(HEPA) capsule to reduce the background particle concentration to below 50 cm-3.

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Toilet-generated aerosols were characterized in a chamber fashioned by fitting a

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46 × 51 cm2 acrylic sheet over the toilet bowl. As shown in Figure S1 in the Supporting

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Information, the sheet had a 15 × 15 cm2 square cutout with a Tedlar bag sealed over it to

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allow the chamber volume to shrink as air was removed for sampling. The sheet

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supported a fan to promote mixing, three ports, and a temperature and humidity logger

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(Omega OM-EL-USB-2-LDC). The initial chamber volume of 15 L was estimated by

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filling the bowl and bag with water.

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Measurements were conducted on two commercial toilets in public restrooms at

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Virginia Tech. Both of them had a Zurn Aquaflush automatic flushing mechanism. The

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acrylic sheet was taped to the bowl to ensure airtightness, and the chamber was flushed

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with HEPA-filtered air. One liter of anaerobically-digested sludge, used to mimic the

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consistency of loose stool associated with EVD, was poured into the toilet. The sludge

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was obtained from the Christiansburg Wastewater Treatment Plant (WWTP) in Virginia,

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and its properties are shown in Table S1. The toilet was then flushed, and the aerosol size

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distribution was measured.

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The lab-scale aeration basin, shown in Figure S2, consisted of a bubble disk

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diffuser (FlexAir, 22.9 cm diameter) placed at the bottom of an 11.4 L plastic basin. The

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basin was placed in a 200 L polyethylene glove bag (Sigma AtmosBag) that served as a


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chamber, supported by a PVC frame with dimensions of 86 × 66 × 61 cm3. Three

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portable mini-fans were used to promote mixing inside the chamber

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The basin was filled with mixed liquor collected from the Christiansburg WWTP,

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and the top of the diffuser was submerged 10 cm below the liquid surface. Table S1

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shows the characteristics of the mixed liquor. HEPA-filtered air was then pumped

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through the diffuser at a flow rate of 10 L min-1, and excess air was vented to maintain a

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constant volume in the chamber. The system ran for 1 h to achieve steady-state

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conditions, confirmed by measurement of the aerosol concentration. The aerosol size

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distribution was then measured. The concentration of dissolved oxygen in the mixed

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liquor increased to 4 mg L-1 at the end of the experiment compared to 2 mg L-1 in the

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fresh mixed liquor, indicating sufficient aeration.

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The lab-scale sewer model of converging pipes, shown in Figure S3, comprised a

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35 × 35 cm2 concrete model with two inflow open channels of diameter of 3.8 cm

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meeting at a 45º angle and exiting via a single outflow open channel with the same

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diameter. The channels were connected to hoses, and a sump pump in an 18.9 L bucket

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circulated mixed liquor through the system. The flow velocity at the outlet was 0.4 m s-1,

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and the Reynolds number was 1.8 × 104, indicative of turbulent flow and comparable to

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that of other lab-scale studies37, 38 but lower than typically found in real sewer systems.

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The section of converging pipes was enclosed in a cylindrical chamber with a plastic lid,

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creating a 10 L space above the channels, and two fans were used to promote mixing

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inside the chamber. The system was allowed to run for 30 min to reach steady state,

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confirmed by measurement of the aerosol concentration, prior to determination of the



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aerosol size distribution. HEPA-filtered air was used to make up that removed by the

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SMPS and APS during measurements.

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Aerosolization of Ebola Virus Surrogates. Emission rates of two surrogates for

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Ebola virus, MS2 and Phi6, were determined in each wastewater system. MS2 (ATCC

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15597-B1) is an unenveloped, single-stranded, RNA bacteriophage with Escherichia coli

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as its host. Phi6 (kindly provided by P. Turner of Yale University, New Haven, CT) is a

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lipid-enveloped, double-stranded, RNA bacteriophage with Pseudomonas syringae as its

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host. Although these two bacteriophages do not mimic Ebola virus’ filamentous,

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enveloped structure, they have been identified as the best available options for

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investigating the persistence and partitioning of Ebola virus in wastewater.19-21

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MS2 and Phi6 were propagated from stock suspensions according to established

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methods.39 The stocks had concentrations of 109-1011 plaque-forming units per milliliter

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(PFU mL-1). Briefly, 50 µL of MS2 stock was mixed with 200 µL of overnight cultured

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E. coli and 4.75 mL of lysogeny broth (LB) soft agar. The mixture was poured on LB

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plates and incubated at 37 °C for 24 h. Soft-agar layer was removed to a flask, and 3 mL

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of LB liquid medium was added per plate. The culture was then incubated with aeration

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at 37 °C for 4 h. MS2 in suspension was harvested by low-speed centrifugation through

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0.22 µm filters at 6,000 rpm for 1 min. For Phi6, P. syringae and tryptic soy broth (TSB)

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were used instead, and incubation took place at 25 °C.

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For toilets, 1 L of fresh anaerobically-digested sludge containing MS2 and Phi6 at

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a concentration of 107 PFU mL-1 was poured into the toilet bowl. No interference

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between MS2 and Phi6 was observed in a separate test, described in the Supporting

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Information. HEPA-filtered air was passed through the toilet chamber, and the toilet was


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then flushed. Aerosols were collected onto two 25-mm gelatin filters (SKC Inc. 225-

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9551) installed in stainless steel filter holders (Advantec 304500) at a flow rate at 2 L

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min-1 for 20 min. The inlet was positioned 10 cm above the water level in toilets. During

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the sampling process, HEPA-filtered makeup air was provided to the chamber. After

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sample collection, one gelatin filter was dissolved in 3 mL of LB for analysis of MS2,

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and one was dissolved in TSB for analysis of Phi6. Ten-fold serial dilutions ranging from

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1:10 to 1:10-9 were prepared from the gelatin-filter-derived solutions. Aliquots of 50 µL

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were used for bacteriophage quantification by plaque assay. The plates were incubated at

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37 °C and 25 °C for MS2 and Phi6, respectively, for 24 h. The number of plaques on

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each plate was counted, and bacteriophage concentrations were determined by

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multiplying the number by the dilution coefficient.

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The aeration basin and sewer models were filled with 10 L and 15 L, respectively,

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of mixed liquor containing MS2 and Phi6 at a concentration of 107 PFU mL-1. As

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described previously, steady-state conditions were established. Aerosol samples were

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then collected onto gelatin filters, as described for the toilet experiments, at a point 20 cm

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above the center of the aeration basin and 10 cm above the junction of the sewer model,

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midway between the fluid surface and the top of the chamber. Excess air was vented in

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the aeration basin and makeup air was added to the sewer model chamber to maintain

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flow balance. The interior surfaces of both chambers were cleaned using 70% ethanol

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after each experiment.

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Calculation of Emission Rates. Emission rates were calculated in units of PFU

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min-1. Based on mass balance, the airborne bacteriophage concentration in a well-mixed



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chamber can be described by Eq. 1. At steady-state conditions, the emission rate of Ebola

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virus surrogates can be determined from Eq. 2: ( )

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= − − + −

= − + + +

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(1) (2)

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where, is the flow rate of particle-free air, is the bacteriophage concentration in

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the inflow, which equals zero, is the flow rate of excess air that is vented, is the

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airborne bacteriophage concentration, is the flow rate of the air sampling pump, E is

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the emission rate of bacteriophage from the wastewater system, k is the aerosol wall loss

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coefficient, and V is the chamber volume. The aerosol wall loss coefficients were

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determined experimentally to be 0.123 min-1 and 0.229 min-1 for the aeration basin and

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sewer models, respectively. These values were determined using the same method

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described in our previous work.40 Additional details are provided in the Supporting

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Information.

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Results

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Aerosol Size Distributions. Aerosol production by two commercial toilets containing

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anaerobically-digested sludge, a lab-scale aeration basin containing mixed liquor, and a

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lab-scale model of converging sewer pipes containing mixed liquor was measured over

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the size range of 14 nm to 20 µm. Table 1 shows the mean and standard deviation of total

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aerosol number, total aerosol volume, and mode diameter from merged, fitted data from

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the SMPS and APS across three replicates for each system.


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The total number of aerosols generated by toilets ranged from 1.7 to 2.6 million

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per flush, and the total volume of aerosols produced was on the order of 10-9 to 10-8 mL.

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These aerosols could also be considered to be very small droplets because they contain

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water. Figure 1(a) shows an example particle size distribution measured after flushing.

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The distribution was noisy because concentrations were low. It is clear that a new mode

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below 100 nm was generated by flushing. Prior to flushing, the toilet chamber was filled

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with conditioned air from a gas cylinder that was drier than achieved after flushing.

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Relative humidity (RH) in the toilet bowl chamber rose from ~85% to ~90% after each

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flush, and the same was true for controls when no flushing occurred. Thus, it does not

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appear that aerosols went undetected due to evaporation.

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Table 1. Characteristics of aerosols generated per activity or per unit of time by flush

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toilets and lab-scale models of an aeration basin and converging sewer pipes. type of system

number of aerosols

volume of aerosols

mode of aerosols

produced

produced (mL)

produced (nm)

1.7±0.3 × 106

5.2±1.6 × 10-9

28±3

2.6±1.2 × 106

1.8±1.2 × 10-8

36±16

9.5±1.2 × 106

1.9±0.1 × 10-7

60±5

2.5±0.3 × 105

2.6±0.1 × 10-8

146±16

toilet #1 (per flush) toilet #2 (per flush) aeration basin -1

(min ) converging pipes



(min-1) 207 208

The aeration basin produced aerosols at a rate of 9.5 × 106 min-1 and 1.9 × 10-7

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mL min-1 in terms of total number and volume, respectively. The total number

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concentration inside the chamber was 275 cm-3 on average, which was about twice that in

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the toilet chamber (144 cm-3). Figure 1(b) shows an example aerosol size distribution,

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which was unimodal and centered at 63 nm, indicating that the aeration basin produced

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larger aerosols than did the toilets.

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The converging sewer pipes produced aerosols at a rate of 2.5 × 105 min-1 and 2.6

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× 10-8 mL min-1 in terms of total number and volume, respectively. This rate was 38

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times lower than produced by the aeration basin in terms of number, and 7 times lower in

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terms of volume. The example size distribution shown in Figure 1(c) appeared to be

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trimodal and included a small, broad peak near 1000 nm, or 1 µm. The sewer was the

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only one among the three wastewater systems to generate these larger aerosols, which

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help account for the larger difference in volume of aerosols than in number of aerosols

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generated compared to the aeration basin.

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Figure 1. Examples of aerosol size distributions generated by model wastewater systems:

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(a) flush toilet; (b) aeration basin; (c) converging sewer pipes. The blue curve represents

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background particles present before aerosol generation began. The red curve represents


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composite data from SMPS and APS. The black curve represents the fit to the composite

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data.

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Virus Concentrations. Figure 2 shows the concentrations of MS2 in the chamber

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air and mixed liquor in the aeration basin and sewer models. The average airborne

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concentrations of MS2 were 15 and 21 PFU L-1, respectively. Airborne concentrations of

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MS2 in the aeration basin model were slightly higher than in the sewer model, although

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this result is contingent upon the size of the experimental chamber: 200 L for the aeration

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basin and 10 L for the sewer pipes. Over the course of the experiments, which lasted 2-3

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hours each, the MS2 concentration in the mixed liquor decreased by ~0.1 log10 to an

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average of 7.3 × 106 PFU mL-1 in the aeration basin model and 8.5 × 106 PFU mL-1 in the

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sewer model. These decreases are likely to due to physical adsorption and biological

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inactivation of the virus, as observed in other studies of MS2 in wastewater and sludge. A

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T90 value (i.e., time to reach 90% inactivation) of 121 hours for MS2 in unpasteurized

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wastewater at 25 °C has been reported.41

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Figure 3 shows the concentrations of Phi6 in the chamber air and mixed liquor in

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the aeration basin and sewer models. Compared to MS2, the airborne concentration of

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Phi6 was much lower, only ~0.1 PFU L-1 in both models. Furthermore, the concentration

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of Phi6 decreased substantially in the mixed liquor over the course of the experiment, by

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0.8 log10 in the aeration basin and 2.6 log10 in the converging sewer pipes model. The

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Phi6 concentration decreased more rapidly than that of MS2 because Phi6 has a higher

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adsorption and inactivation rate in wastewater than does MS2.41 Thus, over time,

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relatively less Phi6 was available for aerosolization. The decrease in Phi6 concentration



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in our sewer model is larger compared to that in earlier studies on the persistence of Phi6

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in wastewater and the inactivation of Ebola virus in sterilized wastewater.19 The

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difference may be due to the effect of heat inactivation inside the sump pump used for

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flow circulation, as Phi6 is believed to be more sensitive to higher temperature.20 The

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finding that heat inactivation only applies to Phi6 is consistent with the general finding of

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greater susceptibility of enveloped viruses to inactivation.41

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Another reason for the difference between MS2 and Phi6 is that the aerosolization

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efficiencies of MS2 and Phi6 differ. In a separate experiment (described in the

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Supporting Information), the aerosolization efficiencies of MS2 and Phi6 from a

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nebulizer were compared. The result indicated that MS2 was aerosolized ~2 times more

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efficiently compared to Phi6. The difference could be due to the smaller size of MS2

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(~25 nm vs. ~80 nm) and/or absence of a lipid envelope. How this result applies to

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aerosolization of Ebola virus from wastewater is not known. Variability in aerosolization

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efficiencies of different microbes from different media merits further investigation.

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Figure 2. MS2 concentrations in air and corresponding mixed liquor from which they

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were generated in lab-scale models of an aeration basin and converging sewer pipes with

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MS2 at an initial concentration in mixed liquor of 107 PFU mL-1.

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Figure 3. Phi6 concentrations in air and corresponding mixed liquor from which they

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were generated in lab-scale models of an aeration basin and converging sewer pipes with

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Phi6 at an initial concentration in mixed liquor of 107 PFU mL-1.

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Virus Emission Rates. Table 2 presents the emission rates of aerosolized Ebola

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virus surrogates from the wastewater systems. Airborne MS2 and Phi6 were not detected

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with toilet flushing. These rates represent averages over the 20-min sampling period. The

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instantaneous rate would be higher at first and then decrease over time due to inactivation

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of virus. Differences over time are expected to be relatively small because viral decay

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over 2-3 hours was

Aerosolization of Ebola Virus Surrogates in ... - ACS Publications

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