Aerosolization of Ebola Virus Surrogates in Wastewater Systems

Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29

1

Environmental Science & Technology

Aerosolization of Ebola Virus Surrogates in Wastewater Systems

2 3

Kaisen Lin and Linsey C. Marr*

4 5

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

6

Blacksburg, Virginia 24061, United States

7 8 9

*Corresponding author

10

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

11

418 Durham Hall, Blacksburg, Virginia 24061, United States

12

Telephone: (540) 231-6071

13

E-mail: [email protected]

14 15 16

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

17

ACS Paragon Plus Environment


18

Abstract

19

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

20

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

21

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

22

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

23

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

24

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

25

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

26

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

27

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

28

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

29

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

30

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

31

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

32

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

33

pipes.


Page 2 of 29

Page 3 of 29

34


TOC ART

35



Page 4 of 29

36

Introduction

37

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

38

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

39

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

40

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

41

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

42

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

43

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

44

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

45

with aerosolized virus is also a potential route of transmission.

46

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

47

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

48

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

49

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

50

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

51

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

52

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

53

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

54

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

55

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

56

potential to be aerosolized during certain processes.

20

In

57

Current risk assessments on inhalation exposure to Ebola virus in wastewater

58

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


Page 5 of 29


59

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

60

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

61

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

62

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

63

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

64

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

65

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

66

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

67

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

68

to generate aerosols as well.

69

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

70

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

71

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

72

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

73

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

74

spiked into the system.

75 76

Materials and Methods

77

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

78

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

79

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

80

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

81

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



82

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

83

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

84

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

85

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

86

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

87

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

88

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

89

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

90

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

91

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

92

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

93

filling the bowl and bag with water.

94

Measurements were conducted on two commercial toilets in public restrooms at

95

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

96

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

97

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

98

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

99

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

100

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

101

distribution was measured.

102

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

103

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

104

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


Page 6 of 29

Page 7 of 29


105

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

106

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

107

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

108

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

109

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

110

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

111

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

112

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

113

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

114

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

115

fresh mixed liquor, indicating sufficient aeration.

116

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

117

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

118

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

119

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

120

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

121

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

122

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

123

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

124

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

125

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

126

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



127

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

128

SMPS and APS during measurements.

129

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

130

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

131

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

132

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

133

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

134

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

135

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

136

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

137

MS2 and Phi6 were propagated from stock suspensions according to established

138

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

139

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

140

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

141

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

142

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

143

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

144

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

145

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

146

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

147

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

148

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

149

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


Page 8 of 29

Page 9 of 29


150

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

151

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

152

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

153

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

154

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

155

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

156

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

157

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

158

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

159

each plate was counted, and bacteriophage concentrations were determined by

160

multiplying the number by the dilution coefficient.

161

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

162

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

163

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

164

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

165

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

166

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

167

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

168

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

169

after each experiment.

170

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

171

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



Page 10 of 29

172

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

173

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

174

= − − + −

= − + + +

175

(1) (2)

176 177

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

178

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

179

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

180

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

181

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

182

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

183

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

184

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

185

Information.

186 187

Results

188

Aerosol Size Distributions. Aerosol production by two commercial toilets containing

189

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

190

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

191

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

192

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

193

the SMPS and APS across three replicates for each system.


Page 11 of 29


194

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

195

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

196

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

197

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

198

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

199

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

200

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

201

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

202

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

203

appear that aerosols went undetected due to evaporation.

204 205

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

206

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

209

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

210

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

211

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

212

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

213

larger aerosols than did the toilets.

214

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

215

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

216

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

217

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

218

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

219

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

220

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

221

generated compared to the aeration basin.

222 223

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

224

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

225

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


Page 12 of 29

Page 13 of 29


226

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

227

data.

228 229

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

230

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

231

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

232

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

233

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

234

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

235

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

236

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

237

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

238

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

239

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

240

wastewater at 25 °C has been reported.41

241

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

242

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

243

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

244

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

245

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

246

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

247

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

248

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



249

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

250

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

251

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

252

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

253

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

254

greater susceptibility of enveloped viruses to inactivation.41

255

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

256

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

257

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

258

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

259

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

260

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

261

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

262

efficiencies of different microbes from different media merits further investigation.

263

264


Page 14 of 29

Page 15 of 29


265

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

266

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

267

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

268 269

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

270

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

271

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

272 273

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

274

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

275

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

276

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

277

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

278

over 2-3 hours was

Aerosolization of Ebola Virus Surrogates in Wastewater Systems

Recommend Documents