A COMPARATIVE EVALUATION of BOAEROSOLS BETWEEN VACUUM and GRAVITY TOILETS. by Elizabeth Nyarkoa Osei a Research Dissertation

A COMPARATIVE EVALUATION OF BOAEROSOLS BETWEEN VACUUM AND GRAVITY TOILETS. by

Elizabeth Nyarkoa Osei

A research dissertation submitted in partial fulfilment of the requirements of the award of the degree of Master of Science at Loughborough University

August 2019

Supervisors: Professor M. Sohail Khan

Dr. Sola Afolabi

ACKNOWLEDGEMENTS I am grateful to God for granting me good health to complete this dissertation. I would like to express my sincerest gratitude to the following people, for their immense support and encouragement which have helped make this project a success:

• My family • My Supervisors, Professor Sohail M. Khan and Dr. Sola Afolabi • Lorraine Withers, Andrew Hay, Kris Wojcik and the entire team at Otter Vacuum Systems • Mrs Jayshree Bhuptani • Mr. Bernard Agyeman • Mr. Kobina Paintsil • The entire WEDC class of 2019.

ABSTRACT Toilet flushing is known to generate and disperse bioaerosols which can contaminate surfaces in the washroom and increase the risk of infections through contact with the contaminated surfaces or inhalation of these bioaerosols. Whilst this phenomenon is well known for gravity toilets, vacuum toilets have been hypothesized not to generate bioaerosols and have been tagged as more sanitary compared to the gravity toilet. This study investigates whether a vacuum toilet produces bioaerosols and how the bioaerosols and particle concentrations of a vacuum and a gravity toilet differ from each other. The study further looks into the reasons and behaviour of the measured concentrations based on the main difference between the toilets - their flushing mechanism. Experiments were conducted in a warehouse-office building using two identical toilet cubicles with dimensions of 1.5 meters by

1.17 meters which were constructed for the purposes of this study. 12 experimental conditions this were framed around two main toilet-use scenarios; using the toilet during an incidence of diarrhea and using the toilet when there was no diarrhea (faecal matter of solid consistency). Faecal matter was not used in this study; rather, Escherichia coli bacteria, an indicator organism for faecal contamination on surfaces and in toilet water was used in seeding the toilet bowls to represent the experimental conditions in the study. The bioaerosol concentrations in this study were measured according to the ACGIH and ASTM standards. Particle concentrations were measured using an optical particle counter.

Based on the results of this study, it can be concluded that, gravity toilets produce bioaerosols via visible splashes or overspray when faecal matter of loose or solid consistency is flushed with the lid open, but a vacuum toilet would not produce bioaerosols or visible splashes under the same conditions.

Keywords: Bioaerosols, Toilet Plume, Gravity-Flush Toilets, Vacuum Toilets, Droplet Nuclei,

Bioaerosol Sampling.

EXECUTIVE SUMMARY The aim of this research is to generate quantitative and qualitative evidence to test the hypothesis that, gravity toilets produce toilet plume which contains bioaerosols when flushed but vacuum toilets do not produce toilet plume which contains bioaerosols when flushed.

Toilet flushing is the most common source of bioaerosols in indoor settings (Darlow and Bale, 1959).

Even though the microbiological profiles of bioaerosols differ from setting to setting, bioaerosols are usually made up of high concentrations of bacteria and fungi (Zemouri et al., 2017).

Literature review alone cannot provide enough qualitative and quantitative data to compare bioaerosol and particle concentrations between gravity and vacuum toilets. It was therefore necessary to supplement data collection with experiments to generate enough quantitative data for the testing of the hypothesis of this study, and qualitative data to support the results of the hypothesis test.

The experiments for this study were conducted in a warehouse-office building. For the purpose of this study only, two identical toilet cubicles with dimensions of 1.5 meters by 1.17 meters were constructed in a large storage room on the site where the experiments were to be conducted.

The experimental conditions for this study, labelled A-L, were chosen to represent the several scenarios which may exist in an actual toilet cubicle which is left idle or in use. The conditions were then framed around two main toilet-use scenarios; using the toilet during an incidence of diarrhea and using the toilet when there was no diarrhea (faecal matter of solid consistency).

Faecal matter was not used for the experiments in this study; instead, Escherichia coli bacteria, an indicator organism for faecal contamination on surfaces and in water was used. To determine the concentration Escherichia coli that would be seeded into the toilets, a membrane filtration test was performed for successive dilutions of the E. coli broth used in this study. The procedure used for measuring bioaerosol concentrations in this study are according to the ACGIH (1999) and ASTM (2019) standards and guidelines for collecting culturable aerosolized

v bacteria unto agar plates using inertial impaction for indoor air quality investigations. A total of 56 trials were conducted across all the experimental conditions for both the gravity and vacuum toilet (28 trials for each toilet).

Line, violin, distribution and strip plots were used to observe the distribution of bioaerosol concentrations across the experimental conditions and particle concentrations across the experimental conditions and bin sizes for the two toilet types. A t-test was performed to determine the statistical differences between bioaerosol and particle concentrations at an experimental condition for the test gravity toilet and at the same condition for the test vacuum toilet. Paired t-tests (significance level 5%) were conducted for each toilet type, within pre and post seeding, pre and post flush, lid open and lid closed and first and subsequent flush experimental conditions.

For the testing of the hypothesis, separate t-tests were performed using the average bioaerosol and particle concentrations for the flush conditions. The generated p-values were then compared with the desired significance level of the t-test (p=0.05).

For the gravity toilet, the agar plates retrieved from the impactor showed growth after the incubation period, for only experimental conditions which included flushing the toilet with the lid open with no disinfection prior to flushing. For the vacuum toilet, all agar plates retrieved from the impactor for all the experimental conditions showed no growth after the 24-hour incubation period. he zero bioaerosol concentrations for the vacuum toilet implies a variance of zero making it deterministic. For the gravity toilet, 75-85% of all the particles generated in each experimental condition were of bin size 0.3 µm or less in all the three minutes of sampling. For the vacuum toilet, 70-81% of all the particles generated in each experimental condition were of bin size 0.3 µm or less in all the three minutes of sampling.

For the test gravity toilet, all surfaces tested in experimental conditions A and L (no seeding with E- coli and disinfection prior to flushing) tested negative for E-coli or total coliforms. For the

vi test vacuum toilet, all surfaces tested in experimental conditions A and L tested negative for E-coli or total coliforms.

Bioaerosol concentration data for the gravity and vacuum toilet suggest that, the vacuum toilet did not produce any significant concentrations of aerosolized E. coli to be captured by the impactor. The only time the vacuum toilet seat was contaminated, were in experimental conditions where the lid was closed, and the toilet was flushed. Flushing with the lid closed contaminates the lid for both vacuum and gravity toilets and this supports the reasoning that the toilet lid may be a source of bacteria and not desirable to touch.

For the gravity toilet, subsequent flushes may continue to produce bioaerosols because, bacteria residues may remain in the toilet bowl water and the insides of the bowl after the first flush even though a reduction in the concentration of bowl water contamination by subsequent flushes and hence a proportionate decrease in bioaerosol concentrations was observed.

There is no complete vacuum; every vacuum is hypothesized to be made up of appearing and disappearing ghost particles which may interfere with real particles and contradict the contribution of real particles to any space (Sakharov, 1991). Because bacteria had been flushed through the sewer pipes, it cannot be totally overruled that the vacuum sewer pipes were contaminated with bacteria.

This could also mean that, particle in the vacuum sewer pipes may have been made up of bacteria in the sewer pipes and the ghost particles mentioned by Sakharov (1991). With this theory and the relatively low vacuum percentage used for the vacuum toilet pump, this study suggests that, the vacuum that existed in the sewer pipes contained bacteria particles which floated (against gravity) into the sampling area anytime the flush button pressed, opening the discharge valve connecting the sewer pipe to the toilet bowl. This could explain why the particle concentrations in the vacuum toilet increased immediately after flush.

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LIST OF TABLES Table 1.1 Outline of research objectives, questions and data collection methods…………………....5

Table 3.1. Experimental conditions and their relevance to the objectives of the study………………15

Table 4.1. Results for hypothesis tests conducted using the particle concentrations for the chosen experimental conditions…………………………………………………………………………………….42

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List of Figures Figure 3.1. A layout of the constructed test cubicles showing the position of the toilet bowls, sinks and shelf……………………………………………………………………………………………………...12

Figure 3.2. Gravity toilet model used in the study……………………………………………………..…13

Figure 3.3. Vacuum toilet model used in the study……………………………………………………….14

Figure 3.4. Gravity toilet bowl (left) and vacuum toilet bowl (right) seeded with only 450ml of the diluted E. coli broth solution to mimic the levels of bacteria that may be shed in a toilet bowl during an incidence of diarrhea…………………………………………………………………………………….19

Figure 3.5. Gravity toilet bowl (left) and vacuum toilet bowl (right) bowl seeded with a mixture of toilet tissue paper and 450ml of the diluted E. coli broth solution to mimic the levels of bacteria that may be shed in a toilet bowl after excreting faecal matter of solid consistency……………………………..20

Figure 3.6. SKC BioStage 400-hole impactor used for bioaerosol sampling in the study………………………………………………………………………………………………………….20

Figure 3.7. AEROTRAK™ handheld optical particle counter used for particle sampling in the study………………………………………………………………………………………………………….21

Figure 3.8. Performing a zero check on the particle counter………………………………………… ..22

Figure 3.9. Determining fluorescence in Colilert test tubes using UV light……………………………..24

Figure 4.1. Bioaerosol concentrations for the gravity toilet across all experimental conditions…………………………………………………………………………………………………….25

Figure 4.2. Bioaerosol concentrations for the vacuum toilet across all experimental conditions…………………………………………………………………………………………………… 26

Figure 4.3. Particle concentrations across the bin sizes for the gravity toilet…………………………..28

Figure 4.4 Particle concentrations across time in experimental conditions for the gravity toilet…………………………………………………………………………………………………………. 28

Figure 4.5. Particle concentrations in bin sizes across time for the gravity toilet……………………..29

Figure 4.6. The distribution of particle concentrations for the gravity toilet…………………………….29

Figure 4.7. Particle concentrations across the bin sizes for the vacuum toilet…………………………………………………………………………………………………………..31

Figure 4.8 Particle concentrations across time in experimental conditions for the vacuum toilet………………………………………………………………………………………………………….. 32

Figure 4.9. Particle concentrations in bin sizes across time for the vacuum toilet…………………….32

Figure 4.10. The distribution of particle concentrations for the vacuum toilet…………………………33

Figure 4.11. Visible splashes (circled) from the gravity toilet on the BioStage sampler (left) and the toilet seat cover (right)………………………………………………………………………………………34

Figure 4.12. Visible splashes (circled) from the gravity toilet on floor………………………………….34

Figure 4.13 Visible splashes on the seat and lid when the lid of the vacuum toilet was closed, and the toilet was flushed…………………………………………………………………………………..……35

GLOSSARY OF TERMS AND ACRONYMS ACGIH American Conference of Governmental Industrial Hygienists ASTM American Society for Testing and Materials

TABLE OF CONTENTS Acknowledgements ...... i

CERTIFICATE OF AUTHORSHIP ...... ii

RESEARCH DISSERTATION ACCESS FORM ...... iii

ABSTRACT ...... iv

EXECUTIVE SUMMARY...... v

LIST OF TABLES ...... viii

List of Figures ...... ix

GLOSSARY OF TERMS AND ACRONYMS ...... xi

CHAPTER ONE - INTRODUCTION ...... 1

1.1 Background ...... 1 1.2 Scope of research...... 2 1.4 Aim of research ...... 3 1.5 Research Objectives and Questions ...... 3 CHAPTER 2 - LITERATURE REVIEW ...... 6

2.1 Characteristics of Bioaerosols ...... 6 2.2 Toilet Plume Bioaerosols and Infectious Diseases ...... 7 2.2.2 Risk of disease transmission due to contact with surfaces contaminated by toilet flush droplets...... 7 2.2.3 Risk of air-borne transmission of diseases due to toilet plume droplet nuclei...... 8 2.3 The Effect of Residual Pathogens, Toilet Lid, Time and Distance in the Aerosolization of Pathogens by Gravity / Modern Flush Toilets ...... 8 2.3.1 The effect of residual pathogens in the toilet bowl on bioaerosol generation ...... 8 2.3.2 The effect of the toilet lid in reducing bioaerosol concentrations and the subsequent contamination of surfaces in the washroom...... 9 2.3.3 The effect of time and distance on bioaerosol generation...... 9 2.4 Particle Generation by Gravity / Modern Flush Toilets ...... 9 2.5 Vacuum Toilets and Bioaerosols ...... 9 2.6 Analysis of the Collection and Sampling Methods for Bioaerosols ...... 10 CHAPTER 3 - RESEARCH METHODOLOGY ...... 11

3.1 Literature Review ...... 11 3.2 Experimental Area Setup and Toilet Testing Cubicles ...... 12 xii

3.3 Experimental Conditions ...... 14 3.4 Experimental and Sampling Procedures ...... 20 3.5 Statistical Analysis ...... 24 CHAPTER 4 - RESULTS AND DISCUSSION ...... 25

4.1 Results ...... 25 4.1.1 Bioaerosol concentrations...... 25 4.1.2 Particle Concentrations ...... 27 4.1.3 Colilert Test Results ...... 33 4.2 Discussion ...... 35 CHAPTER 5 – CONCLUSIONS ...... 44

5.1 Main Conclusions ...... 44 5.2 Limitations of the study and Recommendations for Future Work ...... 45 LIST OF REFERENCES ...... 46

Appendix A - EXPERIMENTAL SETUP...... 52

A1: Schematics of ...... 52 Appendix A: Sample Calculations for Determining the Bioaerosol Concentrations in CFU/m3. ... 55 A1: Determining the volume of air sampled...... 55 Appendix B: Table of Values for Bioaerosol Concentrations Measured Across All Experimental Conditions for the Two Toilet Types ...... 56 B1: Bioaerosol Concentrations for the Test Gravity Toilet Across All the Experimental Conditions ...... 56 B2: Bioaerosol Concentrations for the Test Vacuum Toilet Across All the Experimental Conditions ...... 57 Appendix C: Table of Values for Particle Concentrations Measured Across All Experimental Conditions for the Two Toilet Types ...... 59 C1: Particle Concentrations for the Test Gravity Toilet Across All the Experimental Conditions ...... 59 C2: Particle Concentrations for the Test Vacuum Toilet Across All the Experimental Conditions ...... 65 Appendix D: Results of Colilert Tests ...... 71 D1: Colilert Test Results for the Test Gravity Toilet Across All Experimental Conditions ...... 71 D2: Colilert Test Results for the Test Vacuum Toilet Across All Experimental Conditions ...... 72 D3: Colilert Test Results for the Already Existing Gravity and Vacuum Toilet in Use by Office Staff at the Testing Site ...... 74 Appendix E: Statistical Analysis Data ...... 76

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Appendix 6: ...... Error! Bookmark not defined. Appendix 7: ...... Error! Bookmark not defined.

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CHAPTER ONE - INTRODUCTION

1.1 Background

The prevalence of microbial components of bioaerosols such as bacteria, fungi and viruses have been linked to several respiratory and infectious diseases like pneumonia, tuberculosis, influenza, measles and gastrointestinal infections (Darlow and Bale, 1959; Roy and Milton, 2004; Srikanth, Sudharsanam and Steinberg, 2008; Verani, Bigazzi and Carducci, 2014). Bioaerosols are known to contribute to about 5-34% of indoor air pollution (Mandal and Brandl, 2011) In 2003, the World Health Organization reported that, a building’s sewage plumbing system had been implicated in the spread of the SARS virus at Amoy Gardens apartment complex in Hong Kong. The blame was particularly put on strong upward air flows and non-functional water seals in the sewage drainage systems, which allowed infectious SARS droplets to spread in the building (World Health Organization, 2010, Gormley et al., 2017). However, research shows that, even when water seals are working perfectly, aerosolization of airborne pathogens occurs on the surface of toilet bowls during flushing, leading to the subsequent spread of these pathogens (Lai et al., 2018). Toilet flushing is possibly the most common process involved in the generation of infectious bioaerosols aside coughing and sneezing (Darlow and Bale, 1959). Very high concentrations of pathogens like Shigella, Salmonella and Norovirus have been reported to be present in the faeces and vomit of some infected individuals (Thomson, 1955; Caul, 1994; Atmar et al., 2008). When a contaminated toilet is flushed, there may be a significant proportion of aerosols produced containing these pathogens (Jessen, 1955; Raabe, 1968; Gerba, Wallis and Melnick, 1975; Johnson, Lynch, et al., 2013; Verani, Bigazzi and Carducci, 2014). These bioaerosols settle on surfaces like bathroom walls, door and toilet flush handles and can be picked up by human hands into the mouth, indicating the potential of toilet plume to transmit infectious diseases when pathogens are shed in faces or vomit (Gerba, Wallis and Melnick, 1975; Johnson, Mead, et al., 2013). Bioaerosols are also known to remain airborne for long (Barker and Jones, 2005) and this increases the possibility of being inhaled into the lower respiratory tract increasing the risk of diseases (Darlow and Bale, 1959; Kalogerakis et al., 2005).

1.2 Problem Statement and Justification

Gravity toilets are the most commonly used toilets. (Johnson, Lynch, et al., 2013). Modifications on gravity toilet designs have been made by introducing pressures, sensors and removing the cistern tank in some cases to produce flush toilet designs like ultra-low flush, dual flush, pressure assisted, high efficiency and flushometer toilets. The main aim of most of these designs has been to conserve water (Atta, 2013) by increasing the flush energy, with no focus on eliminating the generation of 1 bioaerosols. This is a setback in public health engineering because, aerosolization of pathogens is known to increase with increasing flush energy (Jessen, 1955; Johnson, Lynch, et al., 2013) and modern flush toilets like the flushometer, high efficiency and the pressure-assisted high efficiency toilets have been confirmed to produce significant levels of bioaerosols (Marshall, 2012; Johnson, Lynch, et al., 2013).

Vacuum toilets are another type of toilet that was designed with the focus to conserve water. These toilets use a waste disposal mechanism where upon pushing a button, a valve opens allowing air to suck faecal matter from the toilet bowl and transporting it into sewage disposal or treatment system, under high pressure. At the same time, another valve opens for a small amount of clean water to rinse the toilet bowl (Beat Stauffer, 2019).

As opposed to gravity toilets, vacuum toilets have been advertised not to produce any detectable levels of overspray (Marshall, 2012). Till date, no investigation has been done on vacuum toilets, using the international standards of sampling bioaerosols.

Gravity toilets have been chosen to represent flush toilets because they are the most commonly used toilets (Johnson, Lynch, et al., 2013; Bathroom City, 2019) and gravity toilets are the most investigated with regards to toilet plume bioaerosols.

There is the need to investigate and validate the hypothesis that, vacuum toilets do not produce bioaerosols. With a focus on differences, insights from this comparative evaluation study related to toilet plume bioaerosols will be very useful to the public health engineering industry as well as companies that manufacture and sell toilet wares. If the hypothesis of this study is true, then vacuum toilets can be promoted and serve as a valuable toilet technology for improved disease prevention and control. If false, this study can would create awareness and establish the need to consider toilet designs that would prevent indoor pollution and reduce the risk of transmission of infectious and respiratory diseases. Either ways, the results of this study would contribute to the overall goals of promoting good health, ensuring clean water and sanitation and sustainable cities and infrastructure in accordance with the 3rd, 6th and 11th Sustainable Development Goals (United Nations, 2019) respectively.

1.3 Scope of research This research focuses on bioaerosol concentrations and particle concentrations for both gravity and vacuum toilets and compare them to each other by investigating whether vacuum toilets produce bioaerosols and how the bioaerosols and particle concentrations of vacuum and gravity toilets differ from each other. The study also looks into the reasons and behaviour of the particle and bioaerosol concentrations of the two toilet types based on their based on their flushing mechanism. With the 2 establishment from literature that bioaerosols can contaminate the immediate environment in a washroom when a toilet is flushed, this study also looks at the extent of contamination for the both toilet types.

This study does not look at investigating how other factors like time, humidity and temperature and distance affect bioaerosol and particle concentrations.

1.4 Aim of research The aim of this research is to generate quantitative and qualitative evidence to test the hypothesis that, gravity toilets produce toilet plume which contains bioaerosols when flushed, but vacuum toilets do not produce toilet plume which contains bioaerosols when flushed. This will be based on the comparative evaluation of bioaerosols and particle concentrations for different pre-flush and post- flush conditions for a vacuum versus a gravity toilet.

1.5 Research Objectives and Questions The objectives of this study have been structured to answer research questions as shown in Table 1.1. These objectives and research questions will subsequently be addressed by a systematic literature review and other methods and techniques described in this study. Objective 1 is to confirm whether or not vacuum toilets produce bioaerosols and that, the flushing of faecal matter may generate viable and culturable microbes which may contaminate the environment and play a role in the transmission of diseases. For a gravity toilet, Objective 2 is to determine how the concentration of particles across various diameters change when a gravity toilet is flushed and to crosscheck this finding against the results of studies which measured the particle concentrations for different flush toilets. If vacuum toilets produce bioaerosols then Objective 2 will determine the particle concentrations produced by a vacuum toilet and serve as a basis to discuss how and why the particle concentrations for the two toilet types differ or are similar. Objective 3 is to provide information on the areas in a washroom where attention must be paid to when cleaning and also to encourage washing of hands. Objective 4 is to compare with literature and Objective 5 is to determine how many flushes can completely get rid of residual microbes in the toilet bowl.

Table 1.1 Outline of research objectives, questions and data collection methods.

Data Collection No. Research Objective Research Question Method To measure and compare the 1.1 Does a vacuum toilet i.Literature Review bioaerosol concentrations across produce bioaerosols? ii.Bioaerosol different pre and post-flush sampling using a 1.2 If a vacuum toilet conditions for a vacuum and cascade impactor. 1 produces bioaerosols, how gravity toilet. do the bioaerosol concentrations differ from that of gravity toilets?

To measure and compare the 2.1 How are the particle i.Literature Review particle concentrations across concentrations and size ii.Particle counting different pre and post-flush distributions of vacuum toilets using an optical 2 conditions for a vacuum and different from that of gravity particle counter gravity toilet across different toilets? conditions.

To identify and compare which 3.1 Which places in a i.Literature Review places in a washroom are washroom are contaminated ii.Colilert Tests 3 contaminated with bioaerosols when a toilet is flushed? post-flush for both gravity and vacuum toilets. To investigate and compare the 4.1 How does closing the i.Literature Review effect of closing the lid on toilet lid affect bioaerosol ii.Bioaerosol bioaerosol and particle generation and particle sampling and concentrations for a gravity and concentrations in a Particle counting. 4 vacuum toilet. washroom when a gravity and a vacuum toilet is flushed?

To confirm the presence of 5.1 Do residual bacteria i.Literature Review 5 residual bacteria after multiple remain in the toilet bowl after ii.Colilert tests flush for vacuum toilets?

4 flushes in both the vacuum toilet and the gravity toilet.

CHAPTER 2 - LITERATURE REVIEW This chapter identifies and collects most reliable and up to date information needed to answer the research questions. For a good analysis and systematic review, a range of resources under each topic have been explored to identify similar findings and different perspectives related to a subject (McMillan and Weyers, 2011)

2.1 Characteristics of Bioaerosols Bioaerosols are generated by human and machine activities in processes such as waste disposal and recycling, biosolid land application, composting, agriculture, pharmaceutical, and biotech activities (Xu et al., 2011; Zemouri et al., 2017). Due to various indoor activities like talking, walking, sneezing, coughing, sweeping floors, washing and flushing toilets, bioaerosol concentrations are higher in indoor settings than outdoor settings (Kim, Kabir and Jahan, 2018; Chen and Hildemann, 2009). Regular indoor human activities such as talking, walking, sneezing, coughing, sweeping floors, washing and flushing toilets can produce bioaerosols. According to Darlow and Bale (1959), toilet flushing is the most common producer of bioaerosols. Different studies have defined bioaerosols using different particle distribution size ranges. According to Georgakopoulos et al. (2009) and Kim, Kabir and Jahan (2018), bioaerosols range from 0.001 µm to 100 µm. Haig et al., (2016) however reports that, bioaerosols may range up to 1 mm. Van Leuken et al. (2016) reports that, despite the differences in particle size distribution mentioned in various studies, it can be concluded from these studies that, bioaerosols are of a relatively smaller size. This smaller size coupled with their lightweight makes it easy for them to be transmitted from one environment to another. The characteristics and survival rates of bioaerosols depend on environmental conditions like temperature, humidity and air flow (Dedesko et al., 2015). These conditions play a vital role in the formation and survival of microorganisms such as bacteria and fungi and as such, have a controlling effect on how bioaerosols are dispersed and formed (Dedesko et al., 2015). The conditions under which each bioaerosol component exist is different and this makes bioaerosols very difficult to quantify (Srikanth, Sudharsanam and Steinberg, 2008). Even though the microbiological profiles of bioaerosols differ from setting to setting, bioaerosols are usually made up of high concentrations of bacteria and fungi (Zemouri et al., 2017). Most studies that have looked into the negative impacts of bioaerosols on human health (May and Harper, 1957; Darlow and Bale, 1959; Barker and Jones, 2005a; Srikanth, Sudharsanam and Steinberg, 2008; Best, Sandoe and Wilcox, 2012; Verani, Bigazzi and Carducci, 2014; Kim, Kabir and Jahan, 2018). Severson et al. (2010) has pointed out that under certain circumstances, humans may benefit from developing a healthy immune system and children particularly, can be protected from developing allergies and asthma when exposed to some microbial components of bioaerosols 6

Studies that have investigated the negative effects of bioaerosols on human health fail to provide a proper assessment on the risk of bioaerosols using traditional culture techniques (Kim, Kabir and Jahan, 2018). According to Kim, Kabir and Jahan (2018), this is due to the complexity of microorganisms and the absence of valid bioaerosol exposure standards as well as bioaerosol dose/effect relationship.

Due to limited data and significant variability in the potential health effects between different types of bioaerosols, standards for bioaerosols based on health risk assessments are still impractical (Lee et al., 2012). The Environment Protection Agency (EPA) of the US and the World Health Organization (WHO) do not have any specific guidelines for bioaerosol concentration. In the case of Korea, the maximum allowable concentration of total bacterial bioaerosol is 800 CFU/m3 (Ministry of Environment, Republic of Korea, 2010).

2.2 Toilet Plume Bioaerosols and Infectious Diseases Many research works have recognized the potential risk of toilet plume bioaerosols in transmitting infectious diseases (Gerba, Wallis and Melnick, 1975; Johnson et al., 2013; Kim, Kabir and Jahan, 2018) but none of these works have been able to establish the specific role bioaerosols play in the development and worsening of disease symptoms (Douwes et al., 2003). Two prominent suggestions that cut across reviewed literature are the risk of disease transmission due to contact with surfaces contaminated by flush droplets and the risk of air-borne transmission of diseases due to inhalation of droplet nuclei (Johnson, Mead, et al., 2013). According to Lai et al. (2018) and Flores et al. (2011), flushing the toilet may increases the risk of infection for toilet users through primary exposure to infectious bioaerosols via direct inhalation. Secondary exposure may occur via contact with contaminated surfaces.

2.2.2 Risk of disease transmission due to contact with surfaces contaminated by toilet flush droplets. Several studies have commented on how far toilet flush droplets can travel to settle on surfaces within the washroom. Usually, flushing events will highly contaminate the surfaces closest to the toilet such as the toilet bowl rim and the seats (Barker and Jones, 2005a; Best, Sandoe and Wilcox, 2012). Findings from a study conducted by Cowling et al. (2013) report that, large droplets settle within 1-2 meters and are highly likely to contaminate surfaces such as door handles, banisters, and flush handles. Barker and Jones (2005) have also mentioned that, droplets can contaminate surfaces in a range of 1 meter. Pathogenic counts of 106 and more have been recorded by Newsom (1972) in areas likely to be touched by the hands (e.g. wash-basins, toilet seats). This suggests that, there is the potential for ingesting pathogens when an individual touches these contaminated surfaces in the

7 toilet and does not wash their hands. Hutchinson (1956) confirmed this by reporting infections by direct ingestion of bacteria. He however stated that a high degree of bacterial ingestion is required for infections by direct ingestion. According to Newsom (1972), an ingestion of 106 pseudomonads, 104 E. coli, and 105-106 of the food-poisoning Salmonellas is required to cause an infection.

2.2.3 Risk of air-borne transmission of diseases due to toilet plume droplet nuclei. Some of the droplets produced when a toilet is flushed dry up to form droplet nuclei (Johnson, Lynch, et al., 2013; Johnson, Mead, et al., 2013) and can remain airborne for as long as 60 minutes after flushing (Barker and Jones, 2005a). Production of droplet nuclei bioaerosols during toilet flushing has been shown for a variety of gravity-flush toilet types and microorganisms (Johnson, Lynch, et al., 2013).

Wells (1934) defines droplet nuclei as residuals of larger droplets whose water content has largely or completely evaporated. According to Wells (1934), droplet nuclei are of sufficiently small size that instead of settling on surfaces, they will remain airborne and be carried on air currents due to the force of gravity. Due to their low settling velocities, droplet nuclei can stay suspended in the atmosphere for a long time, spreading over a wide area within a short time (Darlow and Bale, 1959; Kalogerakis et al., 2005). Barker and Jones (2005) reported that, droplet nuclei toilet plume aerosols are capable of encapsulating microorganisms as large as bacteria. According to Jessen (1955), Darlow and Bale (1959) and Barker and Jones (2005), these microorganisms can remain viable for extended periods while airborne.

Findings from Johnson, Lynch, et al. (2013) indicate that, the risk of airborne disease transmission through toilet plume droplet nuclei bioaerosols has not been generally appreciated. According to their study, this may be due to ‘the difficulty in distinguishing epidemiologically between contact transmission that may have occurred via contact with large droplets or contaminated surfaces as opposed to airborne transmission that may have occurred via inhalation of droplet nuclei.’

2.3 The Effect of Residual Pathogens, Toilet Lid, Time and Distance in the Aerosolization of Pathogens by Gravity / Modern Flush Toilets 2.3.1 The effect of residual pathogens in the toilet bowl on bioaerosol generation A significant proportion of pathogenic bacteria and viruses may still remain in a toilet bowl even after 3 to 7 multiple flushes in a row (Gerba, Wallis and Melnick, 1975; Yahya, Straub and Gerba, 1992; Barker and Jones, 2005a; Lai et al., 2018) and these pathogens can be disseminated into the air with subsequent flushes. Even though a single flush can reduce the concentration of pathogens in the toilet bowl, some pathogens attach themselves to the toilet bowl and get washed down by subsequent flushes into the water (Barker and Jones, 2005a).

2.3.2 The effect of the toilet lid in reducing bioaerosol concentrations and the subsequent contamination of surfaces in the washroom. The findings of Darlow and Bale (1959) and Best et al., (2012) indicate that, closing the lid of a toilet before flushing is effective at reducing bioaerosol concentrations and droplets that cause surface contamination. This assertion is however contradicted in Barker and Jones (2005). Barker and Jones (2005) confirmed from their work that, closing the toilet lid had little effect in reducing the number of bacteria released into the air because, the bioaerosols are small enough to escape through the space between the toilet porcelain rim, seat and lid.

2.3.3 The effect of time and distance on bioaerosol generation. There is limited information on how bioaerosol concentrations vary with respect to the sampling time and the horizontal distance from the sampler to the center of the toilet bowl. Although there is the hypothesis that the highest bioaerosol concentrations occur at the distance and time closest to the flush, the experiments conducted by Knowlton, (2017) proved otherwise.

2.4 Particle Generation by Gravity / Modern Flush Toilets Research work done on the characterization of initial droplet size distribution after flushing some modern flush-toilets found out that, a typical toilet flush produces up to 145,000 aerosol particles (made up of water droplets and aerosolized pathogens), and more than 99% these particles are smaller than 5 μm in diameter (Johnson, Lynch, et al., 2013). This study however made the use of monodisperse fluorescent polymer particles rather than actual microbes. Knowlton et al., (2018) who seeded the toilet with bacteria, also measured particle concentrations before and after flushing and concluded that, majority of the particles produced when loose faecal waste is flushed are 0.3 μm in diameter. Knowlton also suggested that, the particles produced included microorganisms remaining in the toilet from previous use or from faecal waste. Toilets with greater energy flushes have been proved to generate higher concentrations of airborne bacteria and particles as compared to toilets with lower flush energy (Jessen, 1955; Darlow and Bale, 1959; Johnson, Lynch, et al., 2013).

2.5 Vacuum Toilets and Bioaerosols All literature reviewed in this study have only discussed aerosolization of pathogens from gravity, siphon, gravity assisted and flushometer toilets. Till date, no work has investigated bioaerosols from vacuum toilets according to the international standards (ACGIH, 1999) for sampling indoor air for bioaerosols. ACGIH (1999) and ASTM (2019) standards and guidelines for collecting culturable aerosolized bacteria unto agar plates using inertial impaction for indoor air quality investigations. Literature review identified one unpublished work (Marshall, 2012) which tested for overspray on two vacuum toilet sand other gravity-assisted toilets by arranging petri dish around the toilet on the seat, floor and wall and flushing each of the toilets after seeding with E.coli bacteria. The findings of this test concluded that the vacuum toilets had ‘non-detectable levels of overspray.’ The traditional indoor 9

AGCIH and ASTM guidelines for sampling for viable bioaerosols in indoor air were not used in this study.

2.6 Analysis of the Collection and Sampling Methods for Bioaerosols The traditional sampling techniques for bioaerosols that have been used and widely discussed in reviewed literature and in the specifications of the afore mentioned regulatory bodies are impaction, impingement, and ﬁltration (Reponen et al., 2011; Xu et al., 2011; Environment Agency, 2018).

The impaction technique employs inertial forces to collect bioaerosols unto a collection surface, usually a Petri-dish of the appropriate media which has been loaded into an impactor (Environment Agency, 2018). The sampler used for impaction usually has perforated holes and with the aid of a vacuum pump, air is drawn through these holes at a constant rate. Once this air hits the petri dish, it is forced to change direction building inertia in the microorganisms and causing them to be attached unto the petri dish media.

Different types of impactors have been used in measuring bioaerosol concentrations. High-flow impactors can sample as much as 100-700 litres of air per minute and are known to affect the actual bioaerosol concentration when used at lower airflow ranges because, they result in the desiccation of microorganisms (An, Mainelis and Yao, 2004; Yao and Mainelis, 2006; Zhen et al., 2009). The Biostage and Anderson cascade impactor brands attach microorganisms unto agar media and have been used to measure bacterial and fungal bioaerosol concentrations in various studies (Chang and Hung, 2012; Knowlton et al., 2018). Studies which have compared the performance of these two cascade impactor brands to high-flow impactors show that cascade impactors have higher efficiency in bioaerosol collection and the bioaerosols collected can be cultured (Yao and Mainelis, 2006; Zhen et al., 2009).

Cyclones are another design of impactors that are known to operate at higher rates as compared to most of the other impactors (ACGIH, 1999) and have been used in sampling fungal and bacterial bioaerosols (Gorny, Dutkiewicz and Krysinska-Traczyk, 1999; Roberts et al., 2008). Of all the impactor designs, cyclones are the least efficient and poorest choice for measuring bioaerosol concentrations because, they are known to damage bioaerosols (Yao and Mainelis, 2006, Zhen et al., 2009). Generally, all impactor designs are known to cause the loss of bioaerosols within the impactor especially when the bioaerosols get attached to the impactor instead of the Petri dish media, or there is incomplete attachment unto the collection media (Marple and Willeke, 1976; ACGIH, 1999). Impingement uses the same technique used by impaction to sample bioaerosols except that for impingement, bioaerosols are collected in a liquid medium (ACGIH, 1999, Environment Agency, 2018). After a sufficient volume of air has been sampled by an impinger, the collected liquid is processed by using molecular techniques or plating the liquid unto agar. Impingers are usually used 10 for sampling viral bioaerosols because, their design makes it possible to capture bioaerosols as small as 0.5 µm (May and Harper, 1957). Even though impingers have been used to collect bioaerosols in few studies, there has been no valid comparison of the effectiveness of impingers to the other sampling techniques.

CHAPTER 3 - RESEARCH METHODOLOGY

3.1 Literature Review Literature review provided rich learning and was the first step in exploring the research topic, defining the research problem and weighing the relevance of conducting this research. The literature review provided continued throughout the period of this research because new resources related to the research topic were discovered at all the stages of progress of this study, and this helped in writing up the dissertation. As discussed in Chapter 2, there is very limited information on vacuum toilets and their connection to bioaerosols. This is an indication that, the literature review alone cannot provide enough qualitative and quantitative data to compare bioaerosol and particle concentrations between gravity and vacuum toilets. It was therefore necessary to supplement data collection with

11 experiments to generate enough quantitative data for the testing of the hypothesis of this study, and qualitative data to support the results of the hypothesis test.

3.2 Experimental Area Setup and Toilet Testing Cubicles The experiments for this study were conducted in a warehouse-office building. For the purpose of this study only, two identical toilet cubicles with dimensions of 1.5 meters by 1.17 meters were constructed in a large storage room on the site where the experiments were to be conducted. As shown in figure 3.1, each of the cubicle was fitted with a lockable door, shelf and a hand-washing sink of the dimensions and model to represent the structure of a standard toilet cubicle in homes and public settings.

Figure 3.1. A layout of the constructed test cubicles showing the position of the toilet bowls, sinks and shelf.

One of the cubicles was fitted with a new floor standing mount type of the Armitage Shanks Contour 21 Plus S0437 gravity toilet model (figure 3.1). This toilet model is dual flush with 6/4 litre flush. According to the manufacturer, it also features a rimless design for homogenous water distribution during flushing and antimicrobial surfaces to create the most hygienic environments possible (Armitage Shanks, 2018). The anti-microbial coating provides a hygienic environment by inhibiting soiling and reducing the opportunity for bacteria to survive. The gravity toilet was then connected to

12 a cold-water supply with a minimum pressure of 1 bar. The Contour 21 gravity toilet used in this study had a seat cover, lid and was flushed using a finger push button fitted to the cubicle wall.

Figure 3.2. Gravity toilet model used in the study.

The other cubicle was fitted with a new, wall-hanging mount type of the TO662PO JetsTM Jade vacuum toilet model (figure 3.2) and connected to a cold-water supply with minimum and maximum pressures of 2 and 4 bars respectively. The vacuum pressure at the pump was 40%. The TO662PO JetsTM Jade vacuum toilet used in this study flushes with 0.8 litre upon pressing the finger -push button fitted to the wall and had a seat cover and a lid. An illustration of the schematics for the piped water connections, pressures and positions of toilet bowls and cisterns for the two toilets types are included in Appendix A.

Figure 3.3. Vacuum toilet model used in the study.

3.3 Experimental Conditions The experimental conditions for this study, labelled A-L, were chosen to represent the several scenarios which may exist in an actual toilet cubicle which is left idle or in use. The conditions were then framed around two main toilet-use scenarios; using the toilet during an incidence of diarrhea and using the toilet when there was no diarrhea (faecal matter of solid consistency). Based on this, 12 different conditions were generated for the conduction of experiments on the gravity and vacuum toilet. Table 3.1 shows the experimental conditions in this study and the data each condition would produce to answer the research questions and achieve the objectives of this study. Faecal matter was not used for the experiments in this study; instead, Escherichia coli bacteria, an indicator organism for faecal contamination on surfaces and in water was used.

Table 3.1. Experimental conditions and their relevance to the objectives of the study.

Experimental Condition Label Relevance to the study

Toilet used as installed in new state, not seeded To determine the background with E. coli, not flushed and with lid open. bioaerosol and particle concentrations of a new, unused gravity and vacuum A toilet and to confirm that the new toilet is devoid of any bacteria which may interfere with the results of the study. Toilet bowl seeded with 450ml of diluted E. coli To determine the background broth solution, not flushed and with lid open. bioaerosol and particle concentrations when a gravity and a vacuum toilet is used and left unflushed during an B incidence of diarrhea. To also investigate the potential for bioaerosols to contaminate surfaces in the toilet cubicle under this condition. Toilet bowl seeded with 450ml of diluted E. coli To determine the bioaerosol broth solution, flushed and with lid open. and particle concentrations when a gravity and vacuum toilet is used and flushed with the lid open after an incidence C of diarrhea. To also investigate the potential for bioaerosols to contaminate surfaces in the toilet cubicle under this condition. Immediate 2nd flush with lid still open right after 1st To confirm the presence of flush in Experimental Condition C, toilet not seeded residual bacteria in the toilet D with E. coli. bowl when the gravity and vacuum toilet is flushed a 2nd

time after use, with the lid open, during an incidence of diarrhea. To also determine the bioaerosol and particle concentrations and confirm the potential of residual bacteria contaminate surfaces in the toilet cubicle when the gravity or vacuum toilet is flushed under this condition. Immediate 3rd flush with lid still open right after 2nd To confirm the presence of flush in Experimental Condition D, toilet not seeded residual bacteria in the toilet with E. coli. bowl when the gravity and vacuum toilet is flushed the 3rd time after use, with the lid open, during an incidence of diarrhea. To also determine the E bioaerosol and particle concentrations and confirm the potential of residual bacteria contaminate surfaces in the toilet cubicle when the gravity or vacuum toilet is flushed under this condition. Toilet bowl seeded with 450ml of diluted E. coli To investigate the role of the broth solution, flushed and with lid closed toilet lid in facilitating or reducing the spread of bioaerosols when a gravity and F a vacuum toilet is used and flushed with the lid closed during an incidence of diarrhea.

Toilet bowl seeded with a mixture of toilet tissue To determine the background paper and 450ml of diluted E. coli broth solution, bioaerosol and particle not flushed and with lid open. concentrations when a gravity and a vacuum toilet is used (excreting faecal matter of solid G consistency) and left unflushed To also investigate the potential for bioaerosols to contaminate surfaces in the toilet cubicle under this condition. Toilet bowl seeded with a mixture of toilet tissue To determine the bioaerosol paper and 450ml of diluted E. coli broth solution, and particle concentrations flushed and with lid open. when a gravity and vacuum toilet is used (excreting faecal matter of solid consistency) and H flushed with the lid open. To also investigate the potential for bioaerosols to contaminate surfaces in the toilet cubicle under this condition. Immediate 2nd flush with lid still open right after first To confirm the presence of flush in Experimental Condition H, toilet not seeded residual bacteria in the toilet with E. coli and tissue paper mixture. bowl when the gravity and vacuum toilet is flushed a 2nd time after use (excreting faecal matter of solid consistency), with the lid open. I To also determine the bioaerosol and particle concentrations and confirm the potential of residual bacteria contaminate surfaces in the toilet cubicle when the gravity or vacuum toilet is flushed under this condition.

Immediate 3rd flush with lid still open right after 2nd To confirm the presence of flush in Experimental Condition I, toilet not seeded residual bacteria in the toilet with E. coli and tissue paper mixture. bowl when the gravity and vacuum toilet is flushed the 3rd time after use (excreting faecal matter of solid consistency), with the lid open. J To also determine the bioaerosol and particle concentrations and confirm the potential of residual bacteria contaminate surfaces in the toilet cubicle when the gravity or vacuum toilet is flushed under this condition. Toilet bowl seeded with a mixture of toilet tissue To investigate the role of the paper and 450ml of diluted E. coli broth solution, toilet lid in facilitating or flushed and with lid closed. reducing the spread of bioaerosols when a gravity and K a vacuum toilet is used (excreting faecal matter of solid consistency) and flushed with the lid closed. Toilet disinfected and cleaned with Sodium To investigate the effectiveness

Hypochlorite bleach and flushed twice with lid L of disinfection in eliminating open. residual bacteria and reducing bioaerosols.

To determine the concentration Escherichia coli that would be seeded into the toilets, a membrane filtration test was performed for successive dilutions of the E. coli broth used in this study. After series of incubation and colony counting, the resulting concentration of E. coli in the broth was determined to be 7 x 109 Colony Forming Units (CFU) per ml. 10ml of the broth was then taken and diluted in bottles of 900ml sterile Ringus solution (of ¼ strength) to be used for the experimental conditions. Depending on the experimental condition for each toilet type, the toilet bowl was either left unseeded, seeded with 450 ml of the diluted E. coli broth solution or seeded with a mixture of toilet tissue paper 18 and the 450ml of the diluted E. coli broth solution. This implies that, the concentration of bacteria used for seeding in an experimental condition was about 35 x107 CFU per 5ml. Experimental conditions where the toilet was seeded with only 450ml of the diluted E. coli broth solution (figure 3.2) mimicked the levels of bacteria that may be shed in a toilet bowl during an incidence of diarrhea. 10g of anal cleansing tissue paper was soaked in 450 ml of the diluted E. coli broth solution and this mixture was used to seed the toilet bowl in some experimental conditions (figure 3.3) to mimic the shedding of bacteria in faecal matter which has some form of solid consistency. Experimental condition L was performed on the gravity and the vacuum toilet after all the other conditions had been performed. This is because in the actual scenario of toilet use, toilets are only flushed, and not disinfected or cleaned after every visit. Hence, seeding trials without prior disinfection is the best way to represent what happens when an individual uses the toilet. Of all the conditions, C, D, H and I were the closest representation of what happens when an individual uses the toilet.

3.4 Experimental and Sampling Procedures The procedure used for measuring bioaerosol concentrations in this study are according to the ACGIH (1999) and ASTM (2019) standards and guidelines for collecting culturable aerosolized bacteria unto agar plates using inertial impaction for indoor air quality investigations. Bioaerosol concentrations were measured using the SKC BioStage single cascade impactor shown in figure 3.5 below.

Figure 3.6. SKC BioStage 400-hole impactor used for bioaerosol sampling in the study.

This device meets the ACGIH and NIOSH recommendations for sampling indoor bioaerosols for culturable bacteria using a single stage sampler (Lonon, 1998; ACGIH, 1999). According to the NIOSH requirements, a sampling pump which can meet the flow requirements of the BioStage cascade impactor (28.3 L/min) should be used with the sampler. The BioLite+ pump was used.

Before bioaerosol sampling was done, the BioLite pump was first calibrated to 28.3L/min. The calibration was done by placing an empty 90mm petri dish into the BioStage impactor, closing it firmly, connecting the impactor to the pump via tubing and turning on the pump to sample air for 3 minutes. According to the manufacturer (SKC Inc., 2019), sampling using the BioLite pump should not exceed 5 minutes. The pump calibration was repeated three times and each time, the air flow was recorded. For every trial condition, the bioaerosol concentration was then measured by replacing the empty petri dish a with 90mm m-FC (Catalogue Number 96961-500G-F from Sigma Aldrich) agar plate in the BioStage impactor, turning on the pump and sampling the air for 3 minutes.

Particle concentrations were measured using the AEROTRAK™ handheld optical particle counter (Model 9306) shown in figure 3.7 below. This device had been calibrated by the manufacturer with an airflow of 5000L per 29 hours so automatically, setting the device to record particle concentrations per minute implied an air volume of 2.83 litres. The particle counter was also set to warm up for 15 seconds before every minute of sampling and provided particle counts per m3 for particles with diameters 0.3 μm, 0.5 μm, 1 μm, 3 μm, 5 μm and 10 μm. Before the actual particle concentrations were measured, a zero check was performed on the particle counter by attaching a zero filter to the inlet nozzle of the device and starting the device to purge for two minutes. The zero check was completed after all particle sizes provided counts of 0 as shown in figure 3.8.

Figure 3.7. AEROTRAK™ handheld optical particle counter used for particle sampling in the study.

Figure 3.8. Performing a zero check on the particle counter.

For every experimental condition, the BioStage impactor was unscrewed, cleaned thoroughly with bleach wipes after which a fresh 90mm m-FC plate was placed in it. The BioStage impactor was then screwed and placed in its sampling position. The particle counter was first started to warm up for 15 seconds and then depending on the condition, the toilet was either flushed or left unflushed and the BioStage sampler and pump were turned on to sample the air for 3 minutes. As the BioStage impactor was sampling, the particle counter was also sampling and providing particle counts every minute. The sampling was done for three minutes because, longer sampling times can destroy viable microbes. After the 3 minutes, both the bioaerosol samplers and particle counters were turned off. The agar plates were then taken out of the samplers and labelled and left at room temperature for 4 hours before incubation. The data for the particle concentrations across the bin sizes were also printed out, since the device was compatible with a printer. Three replicates were performed for each experimental condition for the gravity and the vacuum toilet. New agar plates were used for each replicate. The 6 litre button is the full flush for the dual flush gravity toilet, and this was used for all experimental conditions which required flushing for the gravity toilet.

To confirm whether surfaces in the toilet cubicle which were contaminated with E. coli bioaerosols after each experimental condition, Colilert tests were performed by swabbing cotton sticks on the toilet flush button, sink tap, toilet cubicle door handle, toilet seat top, lid, floor and the insides of the toilet bowl to check for the presence of the E. coli that was seeded in to the toilet bowl. The swabbed cotton sticks were put in the prepared Colilert tubes (IDEXX Laboratories Inc., 2017) and a stirring action was used to transfer the samples on the swab sticks into the tubes. The swab sticks were then removed, and the Colilert tubes were closed tightly and shaken vigorously. Colilert tests were

22 performed on the water in the toilet bowl for each condition by taking 1ml of the toilet bowl water to confirm the presence of E. coli.

A total of 56 trials were conducted across all the experimental conditions for both the gravity and vacuum toilet (28 trials for each toilet). All Colilert tests had a blank where one of the tubes was filled with 10 ml sterilized water and a comparator tube filled with 10ml of the diluted E. coli broth solution used in this study. 1ml of the diluted E. coli broth solution was also spread on a fresh m-FC agar plate to serve as a positive control, and a fresh unused m-FC agar plate was used as a negative control.

All Colilert tubes and agar plates collected from the BioStage impactor were incubated at 44oC for 24 hours. After 24 hours, both the agar plates and Colilert test tubes were taken out of the incubator. For the agar plates, positive and negative controls were recorded as showing colonies or no colonies respectively.

The impaction method of sampling may cause lots of bacteria colonies to grow close or on top of each other and this may affect the number of bacterial colonies counted (Macher, 1989; ACGIH, 1999). After counting the number of colonies on the agar plates for each trial, the colony counts were corrected using the correction tables for samplers with 400 holes (Macher, 1989; ACGIH, 1999).The bioaerosol concentration in Colony Forming Units (CFU) per m3 for each experimental condition for each toilet type was calculated using this formula:

( ) Bioaerosol concentration (CFU/m3) = …..… ( ) x Eq. 3.1 𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 𝐶𝐶𝐶𝐶𝐶𝐶 1000 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉 𝑜𝑜𝑜𝑜 𝐴𝐴𝐴𝐴𝐴𝐴 𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 m3 The volume of air sampled is determined using the average flow rate after the BioLite pump was calibrated for the Biostage impactor, and the sampling time for each trial. Since the calibration was done in litres, a conversion factor of 1000 L/m3 must be applied to give the bioaerosol concentration in CFU per m3 of sampled air. Appendix shows all the sample calculations to determine the bioaerosol concentrations in this study.

After 24 hours, all Colilert tubes were taken out of the incubator and a 6-watt, 365-nm UV light was used to look for fluorescence in the tubes in a dark environment as shown in figure 3.9 below. The interpretation of results for the Colilert tests shown in Appendix.

Figure 3.9. Determining fluorescence in Colilert test tubes using UV light.

3.5 Statistical Analysis Line, distribution and strip plots were used to observe the distribution of bioaerosol concentrations across the experimental conditions and particle concentrations across the experimental conditions and bin sizes for the two toilet types.

A t-test was performed to determine the statistical differences between bioaerosol and particle concentrations at an experimental condition for the test gravity toilet and at the same condition for the test vacuum toilet. Paired t-tests (significance level 5%) were conducted for each toilet type, within pre and post seeding, pre and post flush, lid open and lid closed and first and subsequent flush experimental conditions This was done to determine the statistical differences between the bioaerosol concentrations for two chosen conditions and the particle concentrations for the same chosen conditions for each of the toilet types. Paired t-tests were also done to determine the significant differences between the concentrations of the particle sizes for two chosen conditions for the gravity, and then the vacuum toilet. For the testing of the hypothesis of this study, separate t- tests were performed using the average bioaerosol and particle concentrations for the flush conditions which were closest to what happens when an individual uses a toilet (C, D, H and I) but between the two toilet types. This was to determine the significant differences between bioaerosol concentrations in a chosen experimental condition for the gravity toilet and bioaerosol concentrations in the same condition for the vacuum. The same was done for the particle concentrations measured in a chosen experimental condition for the gravity toilet and the same condition for the vacuum toilet. The generated p-values were then compared to the desired significance level of the t-test (p=0.05). Any observed differences were considered as significant when the p-value was less than 0.05. Based on this, the null hypothesis of this study that, gravity toilets produce toilet plume which contains bioaerosols, but vacuum toilets do not, was rejected or not rejected. 24

CHAPTER 4 - RESULTS AND DISCUSSION

4.1 Results 4.1.1 Bioaerosol concentrations. 4.1.1.1 Gravity toilet For the gravity toilet, the agar plates retrieved from the impactor showed growth after the incubation period, for only experimental conditions which included flushing the toilet with the lid open with no disinfection prior to flushing (Appendix). As shown in figure 4.1, experimental condition C, which mimicked the flushing of loose faecal matter during an incidence of diarrhea, produced the highest bioaerosol concentration of 58.976 CFU/m3. This was followed by experimental condition D (35.386 CFU/m3), which is a representation of what happens when an individual flushes a toilet for the second time after the first flush to get rid of residual waste in the toilet. Experimental condition H, representing the flushing of faecal matter with solid consistency, had the third highest bioaerosol concentration (23.59 CFU/m3). Experimental Conditions, E, I and J followed with the same bioaerosol concentration of 11.795 CFU/m3. In experimental conditions where there was either no flushing done, or flushing was done with the lid closed or flushing was done after disinfection (A, B, F, G, K and L), the agar plates showed no growth of E. coli; hence, the colony counts were zero. The resulting bioaerosol concentration for these conditions were also zero when the equation (Eq. 3.1) for calculating the bioaerosol concentration was applied.

Figure 4.1. Bioaerosol concentrations for the gravity toilet across all experimental conditions.

4.1.1.2 Vacuum toilet. For the vacuum toilet, all agar plates retrieved from the impactor for all the experimental conditions showed no growth after the 24-hour incubation period (Appendix). The bioaerosol sampler was placed at the same position during sampling in the gravity toilet. In the case of the vacuum toilet however, the position of the sampler was varied across different distances from the center of the toilet bowl in replicates 2 and 3 to make the trials as random as possible when it was observed that no colony counts were being recorded for the vacuum toilet. Despite this, the bioaerosol concentrations remained zero for the three replicates in all the experimental conditions for the vacuum toilet. Figure 4.2 is a graph which illustrates this result.

Figure 4.2. Bioaerosol concentrations for the vacuum toilet across all experimental conditions.

Determination of the significant differences between bioaerosol concentrations across experimental conditions for the gravity toilet and that of the vacuum toilet gave a p-value of zero due to the zero bioaerosol concentrations for the vacuum toilet. The zero bioaerosol concentration values obtained for the vacuum toilet implies a variance of zero. This means that the bioaerosol concentration obtained from the vacuum toilet assumes a constant value of 0, making it deterministic. Thus, a t- test was not necessary.

4.1.2 Particle Concentrations 4.1.2.1 Gravity toilet. For the gravity toilet, 75-85% of all the particles generated in each experimental condition were of bin size 0.3 µm or less in all the three minutes of sampling (Appendix). Except for experimental conditions B and E which produced particle concentration for the 10 µm bin size in the first minute, zero particle concentration was recorded for bin size 10 µm in all experimental conditions during the 3 minutes of sampling.

In experimental conditions where there was no flushing, the average particle concentration did not significantly increase over time. For experimental conditions which involved flushing, the particle concentrations increased immediately, for all three minutes. Of all the flush conditions, experimental condition C recorded the highest total particle concentration and the highest particle concentrations for the 0.3 µm and 0.5 µm bin sizes (figure 4.3). The condition which recorded the next highest particles for these two bin sizes was experimental condition D.

A strip plot showing particle concentrations across time in the experimental conditions for the gravity toilet (figure 4.4) showed that, the highest particle concentrations were recorded within the first two minutes of sampling for all the flush conditions. Another strip plot, showing the particle concentrations in the bin sizes across time (figure 4.5) showed that, in all the 3 minutes of sampling, particles of bin sizes 0.3 µm and 0.5 µm had the highest concentrations for all experimental conditions. Based on this result, paired t-tests were conducted between pre-flush and post flush conditions appendix to detect significant differences between the 0.3 µm and 0.5 µm particle concentrations within each condition. For the 0.3 µm bin size, a significant difference was observed between experimental condition D and E (p= 0.0016) and for the 0.5 µm bin size, significant differences were observed between experimental conditions B and G (p=0.005) and D and E (p=0.003).

The distribution of particle concentration (figure 4.6) for the gravity toilet showed that the particle concentration data was positively skewed to the right. No significant difference was observed when paired t tests were between the experimental conditions using the average particle concentrations appendix.

Figure 4.3. Particle concentrations across the bin sizes for the gravity toilet.

Figure 4.4. Particle concentrations across time in experimental conditions for the gravity toilet.

Figure 4.5. Particle concentrations in bin sizes across time for the gravity toilet.

Figure 4.6. The distribution of particle concentrations for the gravity toilet. 29

4.1.2.2 Vacuum toilet. For the vacuum toilet, 70-81% of all the particles generated in each experimental condition were of bin size 0.3 µm or less in all the three minutes of sampling (Appendix). Except for experimental conditions D and G which produced particle concentrations for the 10 µm bin size in the first minute, zero particle concentration was recorded for bin size 10 µm in all experimental conditions during the 3 minutes of sampling.

The results particle concentration results for the vacuum toilet share some similar trends with the results reported for the gravity toilet. As in the case of the gravity toilet, in experimental conditions where there was no flushing, the average particle concentration did not significantly increase over time for the vacuum toilet. Again, like the gravity toilet, the particle concentrations increased immediately in all minutes for experimental conditions for the vacuum toilet which involved flushing. Also, of all the flush conditions, experimental condition C recorded the highest total particle concentration and the highest particle concentrations for the 0.3 µm and 0.5 µm bin sizes (figure 4.7). However, instead of experimental condition D as in the results of the gravity toilet, figure 4.7 shows that, the condition which recorded the next highest particles for these two bin sizes for the vacuum toilet was experimental condition H.

A strip plot showing particle concentrations across time in the experimental conditions for the vacuum toilet (figure 4.8) showed that, the highest particle concentrations were recorded within the first two minutes of sampling for all the flush conditions. Another strip plot, showing the particle concentrations in the bin sizes across time (figure 4.9) showed that, in all the 3 minutes of sampling, particles of bin sizes 0.3 µm and 0.5 µm had the highest concentrations for all experimental conditions for the vacuum toilet. Based on this result, paired t-tests were conducted between pre-flush and post flush conditions appendix to detect significant differences between the 0.3 µm and 0.5 µm particle concentrations within each condition. For the 0.3 µm bin size, significant differences were observed between experimental conditions B and G (p= 0.042), B and C (p=0.004), C and D (p=0.003), C and F (p=0.008), H and K (p=0.004) and C and H (p=0.006). For the 0.5 µm bin size, significant differences were observed between experimental conditions B and C (p=0.009), C and D (p=0.004), C and F (p=0.006), H and K (p=0.005) and C and H (p=0.02). The distribution of particle concentration (figure 4.10) for the vacuum toilet showed that the particle concentration data was positively skewed to the right. No significant difference was observed when paired t-tests were conducted between the experimental conditions using the average particle concentrations appendix.

Because the bioaerosol concentration data for the gravity and vacuum toilet are deterministic and required no hypothesis testing, t-tests were performed using particle concentrations for the flush conditions which were closest to what happens when an individual uses a toilet (C, D, H and I)

30 between the two toilet types (Appendix D). The results showed no significant difference between gravity toilet particle concentration data and vacuum toilet particle concentration data at the 5% significance level. Particle concentration data for the gravity toilet versus particle concentration data for the vacuum toilet generated p-values of 0.498, 0.138, 0.600 and 0.087 for experimental conditions C, D, H and I respectively.

Figure 4.7. Particle concentrations across the bin sizes for the vacuum toilet.

Figure 4.8. Particle concentrations across time in experimental conditions for the vacuum toilet.

Figure 4.9. Particle concentrations in bin sizes across time for the vacuum toilet.

Figure 4.10. The distribution of particle concentrations for the vacuum toilet.

4.1.3 Colilert Test Results 4.1.3.1 Gravity toilet. For the test gravity toilet, all surfaces tested in experimental conditions A and L (no seeding with E- coli and disinfection prior to flushing) tested negative for E-coli or total coliforms. In experimental conditions B, G, H and I, only the insides of the toilet bowl and the water in the toilet bowl tested positive for E-coli only, the rest of the surfaces in those conditions were negative for E-coli or total coliforms. Experimental conditions C, D tested positive for E-coli only at the top of the toilet seat cover, underside of the toilet seat cover, insides of toilet bowl and the water in the toilet bowl. At these conditions, visible splashes were observed on the toilet seat cover and the BioStage sampler which was positioned on the seat (figure 4.11). Splashes were also noticed on the floor (figure 4.12) in these two conditions. However, the results proved negative when the splashes on the floor were picked up with cotton swab sticks. The rest of the tested locations for these conditions were negative for E-coli or total coliforms. Only the insides of the toilet bowl tested positive for E-coli in experimental conditions E and J, the rest of the locations in that condition tested negative for E-coli or total coliforms. Even though visible splashes were not observed on the toilet seat covers, conditions F and K tested positive for E-coli at the top of the toilet seat cover, underside of the toilet seat cover, insides

33 of toilet bowl and the water in the toilet bowl just like conditions C and D. However, F and K had closed lids and the toilet lids also tested positive for E-coli on the lid in this condition. The rest of the surfaces in experimental conditions F and K tested negative for E-coli or total coliforms.

Figure 4.11. Visible splashes (circled) from the gravity toilet on the BioStage sampler (left) and the toilet seat cover (right).

Figure 4.12. Visible splashes (circled) from the gravity toilet on floor.

4.1.3.1 Vacuum toilet. For the test vacuum toilet, all surfaces tested in experimental conditions A and L tested negative for E-coli or total coliforms. In experimental conditions B, C, D, E, G, H, I and J only the insides of the toilet bowl and the water in the toilet bowl tested positive for E-coli only, the rest of the surfaces in those conditions were negative for E-coli or total coliforms. For the vacuum toilet flush conditions above, the toilet lid was open, however, no visible splashes were seen on the top of the toilet seat as in the case of conditions C and D for the gravity toilet. One interesting observation that was made for

34 the vacuum toilet was the fact that, the top of the toilet seat cover and the toilet lid showed visible splashes when the toilet was closed and flushed (figure 4.13). Hence, conditions F and K tested positive for E-coli at the top of the toilet seat cover, underside of the toilet seat cover, insides of toilet bowl, the water in the toilet bowl and toilet lid. The rest of the surfaces in experimental conditions F and K tested negative for E-coli or total coliforms.

Figure 4.1.3 Visible splashes on the seat and lid when the lid of the vacuum toilet was closed, and the toilet was flushed.

4.2 Discussion The results of this study are significant because, no previous, published work has done a comparative study on the bioaerosol and particle concentrations between a vacuum and gravity toilet, using the international standards and recommendations for sampling indoor air (ACGIH, 1999; Environment Agency, 2018; ASTM International, 2019).

Bioaerosol concentration data for the gravity and vacuum toilet suggest that, the vacuum toilet did not produce any significant concentrations of aerosolized E. coli to be captured by the impactor. This is similar to the unpublished tests results (Marshall, 2012) of the literature reviewed work which compared a vacuum toilet to three flush toilet types and detected no E. coli counts on the agar plates positioned on the vacuum toilet seats, floor and walls.

The main difference between a gravity toilet and a vacuum toilet is the flushing mechanism. The flushing mechanism of the gravity toilet has been confirmed to cause the agitation of colloidal suspension of water and faecal matter resulting in the release of tiny droplets and splashes which may entrain microbes from faecal matter (Jessen, 1955; Raabe, 1968; Gerba, Wallis and Melnick, 1975; Johnson et al., 2013; Verani, Bigazzi and Carducci, 2014). The vacuum toilet on the other hand does not make use of water to transport faecal waste from the toilet bowl through the sewage pipes as in the case of the gravity toilet. Rather, the vacuum toilet makes use of air suction pressure to transport faecal matter and the water that gently flows from the sides to fill up the vacuum toilet bowl only serves as a rinse liquid (Oldfelt and Söderström, 1987).

This may explain why zero bioaerosol concentrations were recorded despite how randomly the sampling was done for the vacuum toilet. The deterministic nature of the bioaerosol data between the gravity and vacuum toilet suggests that, a gravity toilet will produce bioaerosols via visible splashes or overspray when faecal matter of loose or solid consistency is flushed with the lid open, but a vacuum toilet would not produce bioaerosols or visible splashes under the same conditions. Studies have suggested that, bioaerosols produced from flushing mechanisms similar to that of the gravity toilet, tend to contaminate surfaces in the washroom which are closest to the toilet, especially the toilet (Barker and Jones, 2005a). This was confirmed by the results of the Colilert test done on the gravity toilet for conditions C, D, H and I (conditions closest to what happens when an individual uses a toilet). If the vacuum toilet tested in this study indeed produced bioaerosols, then the toilet seat especially should have been contaminated with E. coli but the Colilert tests performed on the vacuum toilet for the same conditions (C, D, H and I) on the gravity toilet in which the toilet seat was contaminated during flushing proved otherwise.

The only time the vacuum toilet seat was contaminated, were in experimental conditions (F and K) when the lid was closed, and the toilet was flushed. The inside of the raised lid was covered with lots of heavily contaminated water droplets which run unto the toilet seat (figure 4.13) thus, contaminating the seat with bacteria. For the same experimental conditions on the gravity toilet, this observation was not made, even though the toilet seat was contaminated. The vacuum toilet system works using differential air pressures such that, the pressure in the sewer pipes is kept negative (below atmospheric pressure) and the pressure in the toilet bowl when the lid is open, is the same as the atmospheric pressure in the washroom (Oldfelt and Söderström, 1987). When the flush button is pressed, air (atmospheric pressure in the toilet bowl with the lid open) is sucked into the system to transport the faecal matter through the sewer pipes to the waste containment system. The observations made for the vacuum toilet in conditions F and K suggest that, closing the vacuum toilet

36 lid disrupted the required differential air pressure between the toilet bowl and the sewer pipes for the vacuum toilet system to function properly.

The reduction in bioaerosol concentrations as the gravity toilet was seeded to mimic waste of solid consistency (toilet tissue paper and E. coli mixture) confirm the findings of a previous study which suggests that flushing loose faecal waste may generate higher bioaerosol concentrations (Newsom, 1972; Knowlton et al., 2018). This also the deduction that the presence of anal cleansing paper, the proportion of loose faecal matter and the consistency of faecal matter may reduce the turbulent movement of the toilet water during flush and limit the generation of bioaerosol and particle concentrations (Knowlton, 2017).

Compared to other studies (Barker and Jones, 2005; Best, Sandoe and Wilcox, 2012; Knowlton et al., 2018), the bioaerosol concentrations recorded for the gravity toilet across all conditions in this study are relatively low due to the low collection efficiency of E. coli. The low collection efficiency of E. coli is due to desiccation of the organism (Li et al., 1999). For high collection efficiencies, some studies have used bacteria like Salmonella sp. which are potentially dangerous and can resist desiccation (Newsom, 1972). A study performed on the death mechanisms of airborne E.coli suggests that, the survival of bacteria during aerosolization is complexly related to the relative humidity of the atmosphere, and between relative humidity values of 75% - 85%, the survival of E. coli can decrease from 36% up to 8% (Benbough, 1967). Benbough also suggested that, the death rate of E. coli aerosolized in air is faster than in a gaseous medium like Nitrogen. This study did not measure the relative humidity in either the gravity or vacuum toilet cubicles before sampling but assumes that, the aerosolization of pathogens took place in air.

Another reason for the low bioaerosol concentrations in the gravity toilet is the relatively low concentration of E. coli that was seeded into the toilet. Extremely high concentrations of 105–109 Shigella, (Thomson, 1955), 104–108 Salmonella (Thomson, 1955) and 108–109 norovirus (Atmar et al., 2008) per gram of stool and at least 106 norovirus per milliliter of vomit (Caul, 1994) may be found in the faeces and vomit of infected persons (Johnson, Lynch, et al., 2013). When a contaminated toilet is flushed, a fraction of the aerosols produced is expected to contain these pathogens (Raabe, 1968). Most studies which have measured high bioaerosol concentrations for gravity flush toilets did not state the concentration of the bacteria that was seeded to get high bioaerosol concentrations. Despite the low concentrations of E. coli, there is a potential for Escherichia coli O157 to cause disease ay an infectious dose of 10 CFU (Willshaw et al., 1994).

The low dosage of E. coli seeded into the toilet is also confirmed by the results of the gravity toilet Colilert tests which suggests that the seeded bacteria was relatively low to produce bioaerosols or heavily contaminated splashes (Newsom, 1972) at the various locations in the toilet which were 37 checked for the presence of bacteria. This explains why the visible splashes which were noticed on the floor during flush conditions C and D, tested negative for E. coli when Colilert tests were performed.

In this study, apart from the insides of the toilet bowl and the toilet bowl water, the locations closest to the gravity toilet (toilet lid, top of toilet seat cover and underside of toilet sea covert) were the only contaminated surfaces for all flush conditions with either the lid open or closed. In other studies, the flush handle, shelf, wall behind toilet, and toilet paper dispenser were contaminated after flushing a gravity-flush (Sassi et al., 2018). The visible splashes on the toilet seats in the flush conditions for the gravity toilet may confirm the findings from Barker and Jones (2005) that, the toilet seat may have the highest counts of bacteria because it is the surface which is closest to the toilet bowl Baker and Jones (2005), also suggested that droplets from flushing the toilet can contaminate surfaces within a range of 1m from the toilet bowl. This study could not completely confirm that suggestion since the shelf, flush button, sink tap and door handles were all within a range of 1m from the toilet bowl but were not contaminated. One reason may be due to the fact that, the toilet used in Baker and Jones’ study had different characteristics (flush energy and water usage) to the gravity toilet used in this study. In their study, the toilet was described as a ‘domestic toilet with a cistern reservoir of 12-litre flush’

Another study which detected a high concentration of bacteria at the insides of the door handles and sink taps (Mendes and Lynch, 1976) suggested that, the door handle and sink tap are usually touched by individuals who use the washroom (before and after washing the hands) and the presence of bacteria on these surfaces are more due to the fact that, moisture from the hands aid bacterial survival, rather than aerosolized bacteria.

For the gravity toilet, subsequent flushes may continue to produce bioaerosols because, bacteria residues may remain in the toilet bowl water and the insides of the bowl after the first flush. Residual bacteria adhering to the insides of the toilet bowl can be detached by the agitation of the bowl water during flush, and subsequently released into the air. This incidence has been confirmed by other studies which also seeded toilets with bacteria (Darlow and Bale, 1959; Newsom, 197;, Barker and Jones, 2005a; Best, Sandoe and Wilcox, 2012; Knowlton et al., 2018).The bioaerosol concentrations and the Colilert test results for the gravity toilet in this study show that, reduction in the concentration of bowl water contamination by subsequent flushes produced a proportionate decrease in bioaerosol concentrations. Other studies have however confirmed that, the reduction in bowl water contamination is not necessarily directly proportional to generated bioaerosol concentrations (Darlow and Bale, 1959; Scott and Bloomfield, 1985; Barker and Jones, 2005a).

In this study, the issue of residual bacteria was confirmed for both the gravity and vacuum toilet by the Colilert tests done for the experimental conditions which involved flushing for the two toilet types. For both the gravity and vacuum toilet, Sodium Hypochlorite bleach was able to get rid of all residual bacteria from the toilet bowl water, insides of toilet bowl and other contaminated surfaces within the toilet cubicle. This confirms the suggestion by Scott and Bloomfield (1985) that, disinfection drastically reduce bacterial contamination.

Flushing with the lid closed contaminates the lid for both vacuum and gravity toilets and this supports the reasoning that the toilet lid may be a source of bacteria and not desirable to touch (Knowlton, 2017).

Both the gravity and vacuum toilet had the distribution of their average particle concentration positively skewed to the right stipulating that; the particles of smaller sizes were higher in concentration than the particles of larger sizes. This study found the 0.3 µm bin size to have the highest particle concentrations (108 particles per m3) across all conditions for both the gravity and vacuum toilet. The results of this study for the gravity toilet, is similar to a previous study which measured particle concentrations for a 4/6-litre dual flush gravity toilet in a healthcare setting (Knowlton, 2017), and found 0.3 µm particles dominating the bin sizes with a concentration as high as 107 particles per m3.

This study did not determine the composition of the particle concentrations or the proportion of particles which are bioaerosols for either the gravity toilet or the vacuum toilet. However, a large proportion of the particles are within the 0.3 µm bin size, then there is a possibility that a proportion of these particles could be inhaled or swallowed, increasing the risk of diseases (Barker and Jones, 2005a).

Flushing the toilet without seeding with the mixture of toilet tissue paper and E. coli and with the lid open, yielded the highest particle concentrations for both the gravity and vacuum toilet. This suggests again that, for a gravity-flush toilet, flushing loose faecal waste may lead to high particle concentrations (Knowlton et al., 2018) and the presence of tissue paper and the consistency and thickness of faecal waste in the toilet bowl may reduce the generation of particles. According to Johnson et al., particle concentrations measured may result from water droplets from flushing effect or aerosolized microbes. The particles generated in this study may be due to human movement before or during the particle sampling. However, the results of this study indicate that, particle concentrations increased immediately after flush. This may be a source of evidence for the gravity toilet that, flushing may lead to the aerosolization of pathogens which may increase concentration of particles in the air in the washroom where the gravity toilet is located.

With the relatively low concentration of bioaerosols determined from the gravity toilet, it can be deduced that either the particle concentrations were made up of majority of aerosolized water droplets because, the flushing energy was quite high to the extent that droplets could be seen on the floor for the conditions that yielded the highest particle concentrations. Another reason could be that, the particle concentrations were made up of non-viable or dead aerosolised E. coli particles which were suspended in the air. Microbes and water droplets are components of the particles generated when a gravity toilet is flushed (Johnson et al., 2013; Knowlton, 2017).

Significant differences could not be determined between the particle concentrations for the pre-flush and post-flush conditions for both the gravity and vacuum toilet. However paired t tests of the same pre flush and post flush conditions, showed significant difference in particles in the smaller size bins (0.3 µm and 0.5 µm) for both the gravity and the vacuum toilet. This indicates that for a gravity toilet, flushing may produce particles with sizes less than 0.5 µm.

Even though the particle concentration results for the gravity and the vacuum toilet share similar trends, the same conclusions cannot be drawn for the two toilet types. This is based on the same argument used to explain the results of the vacuum toilet bioaerosol concentrations that, the flushing mechanism of the vacuum toilet does not lead to the aerosolization of pathogens in the toilet bowl. There is limited information on vacuum toilets and particle generation.

In vacuum engineering and applied physics, a vacuum is defined as any space in which the pressure is lower than atmospheric pressure (Harris, 1989) and not necessarily the absence of particles. There is no complete vacuum; every vacuum is hypothesized to be made up of appearing and disappearing ghost particles which may interfere with real particles and contradict the contribution of real particles to any space (Sakharov, 1991). The vacuum toilet used in this study was connected to a vacuum pump of 40% vacuum. Ultra-high vacuum which can achieve up to 80% vacuum is known to contain about 2.5 x 1013 particles /m3 (Festo, n.d.). A phenomenon known as photoelectric emission, is known to cause isolated particles in vacuum to attain a positive charge (Sickafoose et al., 2000). According to charge Sickafoose et al., this positive charge exerts an electric force that causes the particle to be in a direction against gravity (by floating). Because bacteria had been flushed through the sewer pipes, it cannot be totally overruled that the vacuum sewer pipes were contaminated with bacteria. This could also mean that, particle in the vacuum sewer pipes may have been made up of bacteria in the sewer pipes and the ghost particles mentioned by Sakharov (1991). With this theory and the relatively low vacuum percentage used for the vacuum toilet pump, this study suggests that, the vacuum that existed in the sewer pipes contained bacteria particles which floated (against gravity) into the sampling area anytime the flush button pressed, opening the discharge valve connecting the

40 sewer pipe to the toilet bowl. This could explain why the particle concentrations in the vacuum toilet increased immediately after flush.

Although relative humidity was not measured in this study, it could have been a source of error for both the gravity and vacuum toilet. If the relative humidity was high during the time of conducting the particle sampling trials, there would be more water droplets present in the air within the sampling area, and this could reduce the rate of evaporating, allowing the water droplets to stay for a longer time (Hinds, 1999). The reason why for both the gravity and vacuum toilet, significant differences in particle concentrations could not be determined after conducting the paired t-tests between pre and post-flush conditions can be attributed to the fact that particle sampling was done at different relative humidity values for each trial condition.

When the relative humidity is low (less than 100%), pure water droplets tend to evaporate and soluble nuclei like bacteria can grow in this unsaturated air conditions by a condition termed heterogeneous nucleation (Hinds, 1999). Gram negative bacteria such as the E. coli used in this study, may be a source of soluble bacteria and can grow up to a diameter of 20 µm (Bauer et al., 2003; Möhler et al., 2008). 20 µm particles are relatively large and would settle quickly compared to smaller particles. This indicates that, under unsaturated low relative conditions, it may be impossible to observe larger particles due to their larger settling times. This explains why there were no particle concentrations recorded for the 10 µm bin size across all the conditions for both the gravity and the vacuum toilet. According to Johnson et al. (2013), particles which are larger than 5 µm attain maximum particle concentration within the first 30 seconds of flush. Considering the one-minute sampling intervals at which the particle sampling was done, the time after flush was relatively long for the detection of 10 µm particles across most of the experimental conditions for both the gravity and vacuum toilet.

Particle concentrations may be made up of aerosolized microbes, background dust particles and water droplets from the flushing effects of toilets (Johnson et al., 2013; Knowlton, 2017) . This statement indicates that, particle concentration may be a function of bioaerosols produced when a gravity toilet is flushed. The reverse of this can be said based on the arguments raised for the vacuum toilet that, particle concentration is not a function of bioaerosols when a vacuum toilet is flushed. With this established, conclusions on the null hypothesis of was made using the results of the t-tests performed on the particle concentration data for the flush conditions closest to what happens when an individual uses the gravity versus the vacuum toilet (see table 4.1). The results of the hypothesis testing for this study, were supported by the Colilert tests performed on the gravity and vacuum toilet for the experimental conditions under question.

Table 4.1. Results for hypothesis tests conducted using the particle concentrations for the chosen experimental conditions.

Experimental Condition Label p-value Remarks Conclusion

Toilet bowl seeded with At the 5% significance level, is no Accept null 450ml of diluted E. coli significant difference between the hypothesis. broth solution, flushed gravity and the vacuum toilet when and with lid open. either of the toilets is seeded with C 0.498 450ml of diluted E. coli broth solution and flushed with the lid open. The probability that the observed results are due to random chance is high.

Immediate 2nd flush with At the 5% significance level, is no Accept null lid still open right after 1st significant difference between the hypothesis. flush in Experimental gravity and the vacuum toilet when Condition C, toilet not either of the toilets is subsequently

seeded with E. coli. D 0.138 flushed after condition C, with the lid open.

The probability that the observed results are due to random chance is high.

Toilet bowl seeded with a At the 5% significance level, is no Accept null mixture of toilet tissue significant difference between the hypothesis. paper and 450ml of gravity and the vacuum toilet when diluted E. coli broth either of the toilets is seeded with a solution, flushed and with mixture of toilet tissue paper and H 0.600 lid open. 450ml of diluted E. coli broth solution and flushed with the lid open. The probability that the observed results are due to random chance is high.

Immediate 2nd flush with At the 5% significance level, is no Accept null lid still open right after significant difference between the hypothesis. first flush in Experimental gravity and the vacuum toilet when Condition H, toilet not either of the toilets is subsequently seeded with E. coli and flushed after condition H, with the tissue paper mixture. I 0.087 lid open.

The probability that the observed results are due to random chance is high.

CHAPTER 5 – CONCLUSIONS

5.1 Main Conclusions This research was aimed at comparing vacuum toilets and gravity toilets in terms of bioaerosol production under various flushing and pre-flushing conditions as stated in Table 3.1. Based on the qualitative and quantitative analysis of the results obtained from the various experiments, it can be concluded that, gravity toilets produce bioaerosols via visible splashes or overspray when faecal matter of loose or solid consistency is flushed with the lid open, but a vacuum toilet would not produce bioaerosols or visible splashes under the same conditions. This phenomenon experienced in the gravity toilet is responsible for contaminating surfaces in the washroom which are closest to the toilet bowl, especially the toilet seat. The study further concludes that, the toilet seat may have the highest counts of bacteria for the gravity toilet because, it is the surface which is closest to the toilet bowl.

Flushing the vacuum toilet with the lid open does not contaminate the toilet seat, as in the case of the gravity toilet. The only time the vacuum toilet seat will be contaminated is when the lid of the vacuum toilet is closed. This is attributable to the fact that, closing the lid disrupted the required differential air pressure between the toilet bowl and the sewer pipes for the vacuum toilet system to function properly. Thus, vacuum toilets should not be used with lids.

The study further reveals that, contamination of surfaces such as flush handles and doorknobs may not be due to bioaerosols but as a result of people’s contaminated hands from touching the contaminated surfaces within the toilet like the seat and lid, or from touching faecal matter whilst cleaning their anus.

For the gravity toilet, subsequent flushes may continue to produce bioaerosols because, bacteria residues may remain in the toilet bowl water and the insides of the bowl after the first flush and second flushes in a row. It was observed that, subsequent flushes in this study corresponded to a proportionate decrease in bioaerosol concentrations.

The study further reveals that flushing a gravity toilet with tissue paper or faecal material of solid consistency and thickness in the toilet bowl may reduce the generation of particles and bioaerosols for the toilet in a washroom.

For both the gravity and vacuum toilet, Sodium Hypochlorite bleach was able to get rid of all residual bacteria from the toilet bowl water, insides of toilet bowl and other contaminated surfaces within the toilet cubicle. This confirms the suggestion by Scott and Bloomfield (1985) that, disinfection is effective at drastically reducing bacterial contamination.

This could also mean that, particle in the vacuum sewer pipes may have been made up of bacteria in the sewer pipes and the ghost particles mentioned by Sakharov (1991). With this theory and the relatively low vacuum percentage (40%) used for the vacuum toilet pump, this study suggests that, the vacuum that existed in the sewer pipes contained bacteria particles which floated (against gravity) into the sampling area anytime the flush button was pressed, opening the discharge valve connecting the sewer pipe to the toilet bowl. This could explain why the particle concentrations in the vacuum toilet increased immediately after flush.

The results of this study are significant because, no previous, published work has investigated bioaerosol and particle concentrations for a vacuum toilet, or done a comparative study on the bioaerosol and particle concentrations between a vacuum and gravity toilet, using the international standards and recommendations for sampling indoor air (ACGIH, 1999; Environment Agency, 2018; ASTM International, 2019).

5.2 Limitations of the study and Recommendations for Future Work One of the limitations of this study is the small number of replicates conducted per experimental condition. Only three replicates may not be able to generate sufficient statistical power for the analysis of the data collected during the study. Variables such as minor errors in starting and turning off the samplers whilst flushing the toilets simultaneously, may have affected the measured bioaerosol and particle concentrations for both the gravity and vacuum toilet.

Another limitation of this study is that, the experiments were conducted without measuring relative humidity and temperature. These are conditions which greatly affect the survival of bioaerosols and the distribution of particles in an indoor setting. The absence of relative humidity data limited the interpretation of the data in this study.

To better understand the implications of the results of this study, future work should address the composition of the particle concentrations or the proportion of particles which are bioaerosols for either the gravity toilet or the vacuum toilet. Also, relative humidity and temperature measurements should be incorporated into further studies. 45

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Appendix A - EXPERIMENTAL SETUP A1: Schematics of Water supply

DRAIN TO SEWER 1. Gravity toilet cubicle COLD WATER SUPPLY

2. Vacuum drainage toilet cubicle 3. Vacuum pump (located in a separate plant room)

VACUUM DRAINAGE GRAVITY TOILET TOILET TOILET MODEL Jets Vacuum Armitage Shanks Model: Jade Model: Contour 21 Plus Mount type Wall hang Floor standing Toilet Flush Volume 0,8 litre/flush 6 / 4 litre/flush Cold water supply 15dia pipe 15 dia pie Min. 2 bar - Max. 4 bar min. 1 bar Vacuum system pipework 50dia pipe with N/A 165 litres (or 100m of 50dia) Vacuum pressure at pump 40% N/a

Gravity toilet components (to be ordered): 1x S0437 (WC HD) Contour 21+ 70cm projection back to wall rimless WC pan with horizontal outlet and anti-microbial glaze 1x S3624(67) Conceala 2 cistern 6/4 litre dual flush valve top inlet and internal overflow, alternative height plastic flushbends 1 x Set dual flush button and WC PAN fixing brackets or frame/bolts

Optional WC Seat - Contour 21+ slim slow close seat and cover with top fix hinges 1 x S0670(01)

Appendix : Sample Calculations for Determining the Bioaerosol Concentrations in CFU/m3. A1: Determining the volume of air sampled. The BioLite Pump was calibrated with a rotameter to sample air at a flow rate of 28.3 litres per minute. The calibration was done three times and the following values were obtained. 28.6, 27.8, 28.4.

. . . Average flow rate = =28.26 L/min 28 6+27 8+28 4 3 Bioaerosol sampling was done for 3 minutes for every experimental condition and trial.

Hence, the total volume of air sampled = 28.26L/minute x 3 minutes = 84.78 Litres

i. Correcting colony counts

Colony counts were correcte Cc is the estimated amount of colonies impacted on each plate obtained from tables correcting for 400 holes (ACGIH, 1999; Macher, 1989) .

Appendix B: Table of Values for Bioaerosol Concentrations Measured Across All Experimental Conditions for the Two Toilet Types B1: Bioaerosol Concentrations for the Test Gravity Toilet Across All the Experimental Conditions

Colony counts from Averag incubated agar e plates (CFU) Colony

Count Correcte Labe Bioaerosol Experimental Condition rounde d Colony l concentration Trial Trial Trial d to the Counts (CFU/m3) 1 2 3 nearest whole number Toilet used as installed in new state, not seeded with E. coli, A 0 0 0 0 0 0 Not flushed and with lid open. Toilet bowl seeded with 450ml of diluted E. coli broth solution, not B 0 0 0 0 0 0 flushed and with lid open. Toilet bowl seeded with 450ml of diluted E. coli broth solution, C 4 7 5 5 5 58.976 flushed and with lid open. Immediate 2nd flush with lid still open right after 1st flush in D 2 3 3 3 3 35.386 Experimental Condition C, toilet not seeded with E. coli. Immediate 3rd flush with lid still open right after 2nd flush in E 1 2 0 1 1 11.795 Experimental Condition D, toilet not seeded with E. coli. Toilet bowl seeded with 450ml of diluted E. coli broth solution, F 0 0 0 0 0 0 flushed and with lid closed

Toilet bowl seeded with a mixture of toilet tissue paper and 450ml of G 0 0 0 0 0 0 diluted E. coli broth solution, not flushed and with lid open. Toilet bowl seeded with a mixture of toilet tissue paper and 450ml of H 2 2 1 2 2 23.590 diluted E. coli broth solution, flushed and with lid open. Immediate 2nd flush with lid still open right after first flush in Experimental Condition H, toilet not seeded with E. coli and tissue I 0 1 2 1 1 11.795 paper mixture. Immediate 3rd flush with lid still open right after 2nd flush in Experimental Condition I, toilet not seeded with E. coli and tissue J 0 0 2 1 1 11.795 paper mixture. Toilet bowl seeded with a mixture of toilet tissue paper and 450ml of K 0 1 0 0 0 0 diluted E. coli broth solution, flushed and with lid closed. Toilet disinfected and cleaned with Sodium Hypochlorite bleach and L 0 0 0 0 0 0 flushed twice with lid open.

B2: Bioaerosol Concentrations for the Test Vacuum Toilet Across All the Experimental Conditions

Colony counts from Averag incubated agar Correcte Bioaerosol Labe e Experimental Condition plates (CFU) d Colony concentration l Colony Trial Trial Trial Counts (CFU/m3) Count 1 2 3 Toilet used as installed in new state, not seeded with E. coli, A 0 0 0 0 0 0 Not flushed and with lid open.

Toilet bowl seeded with 450ml of diluted E. coli broth solution, not B 0 0 0 0 0 0 flushed and with lid open. Toilet bowl seeded with 450ml of diluted E. coli broth solution, C 0 0 0 0 0 0 flushed and with lid open. Immediate 2nd flush with lid still open right after 1st flush in D 0 0 0 0 0 0 Experimental Condition C, toilet not seeded with E. coli. Immediate 3rd flush with lid still open right after 2nd flush in E 0 0 0 0 0 0 Experimental Condition D, toilet not seeded with E. coli. Toilet bowl seeded with 450ml of diluted E. coli broth solution, F 0 0 0 0 0 0 flushed and with lid closed Toilet bowl seeded with a mixture of toilet tissue paper and 450ml of G 0 0 0 0 0 0 diluted E. coli broth solution, not flushed and with lid open. Toilet bowl seeded with a mixture of toilet tissue paper and 450ml of H 0 0 0 0 0 0 diluted E. coli broth solution, flushed and with lid open. Immediate 2nd flush with lid still open right after first flush in Experimental Condition H, toilet not seeded with E. coli and tissue I 0 0 0 0 0 0 paper mixture. Immediate 3rd flush with lid still open right after 2nd flush in Experimental Condition I, toilet not seeded with E. coli and tissue J 0 0 0 0 0 0 paper mixture. Toilet bowl seeded with a mixture of toilet tissue paper and 450ml of K 0 0 0 0 0 0 diluted E. coli broth solution, flushed and with lid closed. Toilet disinfected and cleaned with Sodium Hypochlorite bleach and L 0 0 0 0 0 0 flushed twice with lid open.

Appendix C: Table of Values for Particle Concentrations Measured Across All Experimental Conditions for the Two Toilet Types C1: Particle Concentrations for the Test Gravity Toilet Across All the Experimental Conditions

Experimental Condition A: Toilet used as installed in new state, not seeded with E. coli, not flushed and with lid open.

3 Minute Bin Size(µm) Particles per m 1 0.3 26002470 0.5 5326502 1.0 2369611 3.0 571378 5.0 177032 10.0 0 2 0.3 32528620 0.5 6456890 1.0 2020141 3.0 396820 5.0 116254 10.0 0 3 0.3 43186930 0.5 10623320 1.0 3337809 3.0 660071 5.0 200707 10.0 0

Experimental Condition B: Toilet bowl seeded with 450ml of diluted E. coli broth solution, not flushed and with lid open.

3 Bin Size(µm) Particles per m 1 0.3 45936400 0.5 4482686 1.0 669258

3.0 177739 5.0 55124 10.0 353 2 0.3 46853000 0.5 4563251 1.0 688339 3.0 170671 5.0 50530 10.0 0 3 0.3 47193290 0.5 4517314 1.0 623675 3.0 158304 5.0 43463 10.0 0

Experimental Condition C: Toilet bowl seeded with 450ml of diluted E. coli broth solution, flushed and with lid open.

3 Minute Bin Size(µm) Particles per m 1 0.3 322543500 0.5 84350890 1.0 11814840 3.0 681979 5.0 104240 10.0 0 2 0.3 315191200 0.5 81343110 1.0 11242050 3.0 600707 5.0 89399 10.0 0 3 0.3 112605300 0.5 22677740

1.0 3240990 3.0 281625 5.0 74558 10.0 0

Experimental Condition D: Immediate 2nd flush with lid still open right after 1st flush in Experimental Condition C, toilet not seeded with E. coli.

3 Minute Bin Size(µm) Particles per m 1 0.3 276080900 0.5 68928270 1.0 9112721 3.0 554064 5.0 86219 10.0 0 2 0.3 272052700 0.5 69744880 1.0 9668198 3.0 621908 5.0 94700 10.0 0 3 0.3 129761500 0.5 28736750 1.0 3993286 3.0 332156 5.0 71025 10.0 0 Experimental Condition G: Toilet bowl seeded with a mixture of toilet tissue paper and 450ml of diluted E. coli broth solution, not flushed and with lid open.

3 Minute Bin Size(µm) Particles per m 1 0.3 49362900 0.5 13413080 1.0 3938869 3.0 757244 5.0 203180 61

10.0 0 2 0.3 74061140 0.5 13618380 1.0 2642050 3.0 448057 5.0 154064 10.0 353 3 0.3 83557950 0.5 16644880 1.0 2685866 3.0 239929 5.0 61131 10.0 0

Experimental Condition H: Toilet bowl seeded with a mixture of toilet tissue paper and 450ml of diluted E. coli broth solution, flushed and with lid open.

3 Minutes Bin Size(µm) Particles per m 1 0.3 156804200 0.5 37463600 1.0 5625442 3.0 359717 5.0 61484 10.0 0 2 0.3 146845200 0.5 33974560 1.0 5074205 3.0 360424 5.0 65724 10.0 0 3 0.3 142074200 0.5 33468550 1.0 5058537 3.0 434629

5.0 86926 10.0 0

Experimental Condition I: Immediate 2nd flush with lid still open right after first flush in Experimental Condition H, toilet not seeded with E. coli and tissue paper mixture.

3 Minute Bin Size(µm) Particles per m 1 0.3 196642000 0.5 46626150 1.0 6749470 3.0 443816 5.0 93640 10.0 0 2 0.3 187638200 0.5 45750180 1.0 6591166 3.0 413074 5.0 69965 10.0 0 3 0.3 174027900 0.5 40641340 1.0 5779152 3.0 351590 5.0 67491 10.0 0

Experimental Condition J: Immediate 3rd flush with lid still open right after 2nd flush in Experimental Condition I, toilet not seeded with E. coli and tissue paper mixture.

3 Minute Bin Size(µm) Particles per m 1 0.3 195219800 0.5 48154770 1.0 6675618 3.0 441696 5.0 81272

10.0 0 2 0.3 130407800 0.5 28462190 1.0 4133569 3.0 285866 5.0 61484 10.0 0 3 0.3 147728600 0.5 34895050 1.0 5233922 3.0 373498 5.0 65724 10.0 0

Experimental Condition E: Immediate 3rd flush with lid still open right after 2nd flush in Experimental Condition D, toilet not seeded with E. coli.

3 Minute Bin Size(µm) Particles per m 1 0.3 177223000 0.5 43667140 1.0 6366432 3.0 513074 5.0 100707 10.0 353 2 0.3 174043500 0.5 43131800 1.0 6270318 3.0 466078 5.0 89753 10.0 0 3 0.3 164415900 0.5 40562540 1.0 6196113 3.0 411307

5.0 71731 10.0 0

Experimental Condition F: Toilet bowl seeded with 450ml of diluted E. coli broth solution, flushed and with lid closed

3 Minute Bin Size(µm) Particles per m 1 0.3 81232510 0.5 21751590 1.0 3694700 3.0 407774 5.0 103534 10.0 0 2 0.3 82932160 0.5 18999290 1.0 3049117 3.0 216961 5.0 42756 10.0 0 3 0.3 64401770 0.5 14519790 1.0 2570672 3.0 209541 5.0 42049 10.0 0 C2: Particle Concentrations for the Test Vacuum Toilet Across All the Experimental Conditions

Experimental Condition B: Toilet bowl seeded with 450ml of diluted E. coli broth solution,NOT flushed and with lid open.

3 Minute Bin Size(µm) Particles per m 1 0.3 21454420 0.5 2337809 1.0 702474

3.0 101767 5.0 32509 10.0 0 2 Size Cumulative metre cube 0.3 22218380 0.5 2334629 1.0 715548 3.0 96113 5.0 23322 10.0 0 3 0.3 23849120 0.5 6059364 1.0 2636042 3.0 630742 5.0 171378 10.0 0

Experimental Condition C: Toilet bowl seeded with 450ml of diluted E. coli broth solution, flushed and with lid open.

3 Minute Bin Size(µm) Particles per m 1 0.3 184541700 0.5 45833570 1.0 6628622 3.0 423322 5.0 69258 10.0 0 2 0.3 161186900 0.5 38901050 1.0 5496113 3.0 395406 5.0 65371 10.0 0 3 0.3 139474900

0.5 32500350 1.0 4643816 3.0 356537 5.0 70318 10.0 0

Experimental Condition D: Immediate 2nd flush with lid still open right after first flush in Experimental Condition C, toilet not seeded with E. coli.

3 Minute Bin Size(µm) Particles per m 1 0.3 96004240 0.5 17854420 1.0 2693640 3.0 279152 5.0 78799 10.0 353 2 0.3 84472090 0.5 17846290 1.0 2808834 3.0 272438 5.0 65371 10.0 0 3 0.3 36232510 0.5 4940990 1.0 1129329 3.0 174912 5.0 48057 10.0 0

Experimental Condition F: Toilet bowl seeded with 450ml of diluted E. coli broth solution, flushed and with lid closed

3 Minute Bin Size(µm) Particles per m 1 0.3 60753000 0.5 5517314

1.0 666785 3.0 164311 5.0 48763 10.0 0 2 0.3 61824030 0.5 5746644 1.0 684099 3.0 162544 5.0 54417 10.0 0 3 0.3 61337100 0.5 5505300 1.0 680919 3.0 171378 5.0 53004 10.0 0

Experimental Condition G: Toilet bowl seeded with a mixture of toilet tissue paper and 450ml of diluted E. coli broth solution, not flushed and with lid open.

3 minute Bin Size(µm) Particles per m 1 0.3 11896470 0.5 3324028 1.0 1363251 3.0 277032 5.0 85512 10.0 353 2 0.3 11468900 0.5 3186926 1.0 1316254 3.0 284452 5.0 93640 10.0 0

Experimental Condition H: Toilet bowl seeded with a mixture of toilet tissue paper and 450ml of diluted E. coli broth solution, flushed and with lid open.

3 Minute Bin Size(µm) Particles per m 1 0.3 114902500 0.5 31418380 1.0 5249470 3.0 473498 5.0 104947 10.0 0 2 0.3 104221200 0.5 29019080 1.0 5226502 3.0 502120 5.0 108834 10.0 0 3 0.3 93329690 0.5 25845940 1.0 4797174 3.0 490813 5.0 109894 10.0 0

Experimental Condition K: Toilet bowl seeded with a mixture of toilet tissue paper and 450ml of diluted E. coli broth solution, flushed and with lid closed.

3 Minute Bin Size(µm) Particles per m 1 0.3 44986930 0.5 12768550 1.0 2830036 3.0 402827 5.0 106007 10.0 0 2 0.3 28922620

0.5 8019435 1.0 2044523 3.0 306714 5.0 89399 10.0 0

Experimental Condition I: Immediate 2nd flush with lid still open right after first flush in Experimental Condition H, toilet not seeded with E. coli and tissue paper mixture.

3 Minute Bin Size(µm) Particles per m 1 0.3 50057240 0.5 10519440 1.0 1945583 3.0 196113 5.0 50177 10.0 0 2 0.3 41966080 0.5 10798940 1.0 3190106 3.0 591166 5.0 146643 10.0 0 3 0.3 43313430 0.5 11017320 1.0 3287986 3.0 626502 5.0 161131 10.0 0

Appendix D: Results of Colilert Tests D1: Colilert Test Results for the Test Gravity Toilet Across All Experimental Conditions

Colilert Test Results Labe Flush Top of Under Water Bowl Floo Toilet Experimental Condition Door Sink l Butto Seat Seat Shelf in Toilet Sides r Bowl knob Tap n Cover Cover bowl Lid Toilet used as installed in new state, not seeded with E. coli, A Not flushed and with lid open. Toilet bowl seeded with 450ml of diluted E. coli broth solution, not flushed and with lid B open. Toilet bowl seeded with 450ml of diluted E. C coli broth solution, flushed and with lid open. Immediate 2nd flush with lid still open right after first flush in Experimental Condition C, D toilet not seeded with E. coli. Immediate 3rd flush with lid still open right after 2nd flush in Experimental Condition D, E toilet not seeded with E. coli. Toilet bowl seeded with 450ml of diluted E. coli broth solution, flushed and with lid F closed

Toilet bowl seeded with a mixture of toilet tissue paper and 450ml of diluted E. coli G broth solution, not flushed and with lid open. Toilet bowl seeded with a mixture of toilet tissue paper and 450ml of diluted E. coli H broth solution, flushed and with lid open. Immediate 2nd flush with lid still open right after 1st flush in Experimental Condition H, I toilet not seeded with E. coli and tissue paper mixture. Immediate 3rd flush with lid still open right after 2nd flush in Experimental Condition I, J toilet not seeded with E. coli and tissue paper mixture. Toilet bowl seeded with a mixture of toilet tissue paper and 450ml of diluted E. coli K broth solution, flushed and with lid closed. Toilet disinfected and cleaned with Sodium Hypochlorite bleach and flushed twice with L lid open.

D2: Colilert Test Results for the Test Vacuum Toilet Across All Experimental Conditions

Experimental Condition Colilert Test Results

Flush Top of Under Water Bowl Floo Toilet Labe Door Sink Butto Seat Seat Shelf in Toilet Sides r Bowl l knob Tap n Cover Cover bowl Lid Toilet used as installed in new state, not seeded with E. coli, A Not flushed and with lid open. Toilet bowl seeded with 450ml of diluted E. coli broth solution, not flushed and with lid B open. Toilet bowl seeded with 450ml of diluted E. C coli broth solution, flushed and with lid open. Immediate 2nd flush with lid still open right after first flush in Experimental Condition C, D toilet not seeded with E. coli. Immediate 3rd flush with lid still open right after 2nd flush in Experimental Condition D, E toilet not seeded with E. coli. Toilet bowl seeded with 450ml of diluted E. coli broth solution, flushed and with lid F closed Toilet bowl seeded with a mixture of toilet tissue paper and 450ml of diluted E. coli G broth solution, not flushed and with lid open.

Toilet bowl seeded with a mixture of toilet tissue paper and 450ml of diluted E. coli H broth solution, flushed and with lid open. Immediate 2nd flush with lid still open right after first flush in Experimental Condition H, I toilet not seeded with E. coli and tissue paper mixture. Immediate 3rd flush with lid still open right after 2nd flush in Experimental Condition I, J toilet not seeded with E. coli and tissue paper mixture. Toilet bowl seeded with a mixture of toilet tissue paper and 450ml of diluted E. coli K broth solution, flushed and with lid closed. Toilet disinfected and cleaned with Sodium Hypochlorite bleach and flushed twice with L lid open. D3: Colilert Test Results for the Already Existing Gravity and Vacuum Toilet in Use by Office Staff at the Testing Site

Top of Under Water in Insides Toilet Flush Doorkno Sink Toilet Type Seat Seat Shelf Toilet of Toilet Floor Bowl Button b Tap Cover Cover bowl Bowl Lid Gravity Toilet Vacuum Toilet

Appearance Colour Result

Less yellow than the comparator Negative for total coliforms and E. coli

Yellow equal to or greater than the comparator Positive for total coliforms Yellow and fluorescence equal to or greater Positive for E. coli than the comparator

Appendix E: Statistical Analysis Data