EPA/600/R-18/221 | July 2018
www.epa.gov/homelarid-security-research
United States
Environmental Protection
Agency
oEPA
Exposure Pathways to High-Consequence
Pathogens in the Wastewater Collection
and Treatment Systems
COLLECTION
TREATMENT
TRANSIT
Office of Research and Development
-------
EPA/600/R-18/221
July 2018
Exposure Pathways to High-Consequence
Pathogens in the Wastewater Collection
and Treatment Systems
by
Sandip Chattopadhyay, Ph.D.
Sarah Taft, Ph.D.
Threat and Consequence Assessment Division
National Homeland Security Research Center
Cincinnati, OH 45268
Contract No. EP-C-14-001 to ICF under Work Assignment 40
U.S. Environmental Protection Agency Project Officer
Office of Research and Development
Homeland Security Research Program
-------
Disclaimer
The U.S. Environmental Protection Agency (EPA) through its Office of Research and
Development funded and managed the research described here under Contract No. EP-C-14-001
to ICF under Work Assignment 40. It has been subjected to the Agency's review and has been
approved for publication. Note that approval does not signify that the contents necessarily reflect
the views of the Agency. Any mention of trade names, products, or services does not imply an
endorsement by the U.S. Government or EPA. The EPA does not endorse any commercial
products, services, or enterprises.
Questions concerning this document or its application should be addressed to:
Sandip Chattopadhyay, Ph.D., M.B.A.
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
26 W. Martin Luther King Drive, MS NG16
Cincinnati, OH 45268
Phone: 513-569-7549
Fax: 513-487-2555
E-mail: chattopadhyay.sandip@epa.gov
-------
Table of Contents
Table of Contents iii
Acronyms vii
Executive Summary ix
1 Introduction 1
2 Problem Formulation 3
3 Potential Disease Transmission During Wastewater Collection and Treatment 5
4 Fate and Transport of Pathogens During Wastewater Collection and Treatment 8
4.1 Collection 8
4.2 Pathogens in Wastewater During Transport and Treatment 12
4.3 Formation of Bioaerosols 16
4.3.1 Background 16
4.3.2 Bioaerosol Generation During the Toilet Flush 20
4.3.3 Bioaerosol Generation During Wastewater Treatment 26
5 Challenges to Performance of Quantitative Microbial Exposure Assessment 34
6 Overview of Screening Process 36
6.1 Conceptual Exposure Model for Exposure in the Wastewater System 36
6.2 Elements of Screening Process 39
6.2.1 Pathogen Disease Transmission Characteristics 42
6.2.2 Pathogen Potential to Persist in Wastewater or Deposited Droplet 43
6.2.3 Pathogen Potential to Form Viable Bioaerosols 43
6.2.4 Bridging Pathogen-specific Data Gaps for Fate and Transport 44
6.3 Selection of Emerging Pathogens for Two Case Study Evaluations 49
7 Case Study: Ebola Virus 50
7.1 Does Pathogen Exhibit Identified Disease Transmission Characteristics? 50
7.1.1 Pathogen Shedding in Feces, Urine, or Vomit 50
7.1.2 Disease Transmission and Associated Exposure Doses by Route of
Exposure 52
7.1.3 Is Ebola Virus a High-Consequence Pathogen? 53
7.2 Does the Pathogen Persist in Wastewater or Deposited Droplet? 53
7.2.1 W astewater Persi stence 53
7.2.2 Deposited Droplet Persistence 59
7.3 Does Pathogen Form Viable Bioaerosols from a Toilet Flush or the Wastewater
Treatment Process? 59
7.4 Conclusion: Could Ebola Virus Form Viable Exposure Pathways in the
Wastewater System? 61
8 Case Study: Bacillus anthracis Spores 63
8.1 Does Pathogen Exhibit Identified Disease Transmission Characteristics? 63
8.1.1 Direct Entry via Decontamination Wastewater 63
8.1.2 Disease Transmission and Associated Exposure Doses by Route of
Exposure 63
8.1.3 Is the Spore Form of Bacillus anthracis a High-Consequence
Pathogen? 64
8.1 Does the Pathogen Persist in Wastewater or a Deposited Droplet? 66
8.1.1 Wastewater Persistence 66
-------
8.1.2 Deposited Droplet Persistence 66
8.2 Does Pathogen Form Viable Bioaerosols from the Wastewater Treatment
Process? 67
8.3 Conclusion: Could the Spore Form of Bacillus anthracis Form Viable Exposure
Pathways in the Wastewater System? 67
9 Data Gaps and Suggested Research to Further Refine Screening Process 69
10 Glossary 71
11 References 73
-------
List of Tables
Table 4-1. Characteristics with Potential to Lead to a Higher or Lower Dilution Factor 14
Table 4-2. Available Data for Bioaerosol Concentration of Microorganisms from Toilet Flush. 23
Table 4-3. Range of Reported Bacterial Bioaerosol Concentrations from Identified Wastewater
Treatment Processes and Maintenance Activities 32
Table 4-4. Range of Reported Viral Bioaerosol Concentrations from Identified Wastewater
Treatment Processes 33
Table 4-5. Estimated Bioaerosol Concentration Range for Bacterial or Viral Wastewater
Pathogens with Inhaled Doses for 10 Minutes, 60 Minutes, and 8 Hours 34
Table 6-1. Assumptions Incorporated in Screening Process 41
Table 6-2. Terminology, Definitions, and Examples for Process to Bridge Data Gaps 46
Table 7-1. Summary of Ebola Disease Transmission Characteristics 54
Table 7-2. Potential Surrogates and Benchmark Indicators for Persistence of Ebola Virus in
Wastewater 55
Table 8-1. Summary of Bacillus anthracis Disease Transmission Characteristics 65
-------
List of Figures
Figure 4-1. Overview of exposure assessment pathways for wastewater collection and treatment.
10
Figure 4-2. Toilet flush showing aerosol and particle deposition 21
Figure 6-1. Conceptual exposure model for pathogen exposure during wastewater collection and
treatment 37
Figure 6-2. Flow chart for screening process for a high-consequence pathogen (HCP) 40
Figure 7-1. Published data describing time for a 2-log reduction of Ebola virus in wastewater and
water 56
Figure 7-2. Summary of Ebola virus exposure pathways and screening process outputs 62
Figure 8-1. Summary of Bacillus anthracis spore exposure pathways and screening process
outputs 68
-------
Acronyms
cfu
colony-forming unit(s)
EBOV
Ebola virus(es)
ELISA
enzyme linked immunosorbent assay
EPA
U.S. Environmental Protection Agency
EVD
Ebola virus disease
HCP
high-consequence pathogen(s)
HIV
human immunodeficiency virus
MHV
murine hepatitis virus
MPN
most probable number
NACWA
National Association of Clean Water Agencies
NHP
nonhuman primate(s)
PCR
polymerase chain reaction
pfu
plaque-forming unit(s)
qPCR
quantitative polymerase chain reaction
RNA
ribonucleic acid
rRT-PCR
real-time reverse-transcription polymerase chain reaction
RT-PCR
reverse-transcription polymerase chain reaction
SARS
severe acute respiratory syndrome
SSO
sanitary sewer overflow
TCIDso
50% tissue culture infective dose
UV-APS
ultraviolet-aerodynamic particle sizer
VEP
viable exposure pathway(s)
WWTP
wastewater treatment plant(s)
-------
Acknowledgements
The following individuals and organizations have been acknowledged for their contributions
towards the development and/or review of this document.
United States Environmental Protection Agency (EPA), Office of Research and Development
(ORD), National Homeland Security Research Center (NHSRC)
Sandip Chattopadhyay, Ph.D., M.B.A. (Principal Investigator)
Sarah Taft, Ph.D.
Eric Rhodes, Ph.D.
EPA Office of Research and Development, National Exposure Research Laboratory
Shannon M. Griffin
EPA Office of Research and Development, Office of Wastewater Management
Robert K. Bastian, M.S.
Phil Zahreddine, M.S. Env. Eng.
Occupational Safety & Health Administration (OSHA), Office of Emergency Management and
Preparedness
Chris Brown
Marti Sinclair (CSRA) is acknowledged for technical editing; and quality assurance reviewer
Eletha Brady-Roberts (ORD, NHSRC) is acknowledged for contributions to this report.
-------
Executive Summary
The recent development of disease outbreaks in the United States and throughout the world has
heightened concerns regarding the potential for exposure to emerging pathogens during
wastewater collection and treatment. Emerging pathogens may enter wastewater systems from
pathogen shedding in human waste, introduction of decontamination wastewater, illicit activity,
or surface water runoff following a wide-area biological incident. Emerging pathogens may
exhibit fate and transport characteristics that provide for atypical transmission pathways or
higher exposure concentrations than natural transmission sources (e.g., human-to-human
transmission, fomite contamination from infected individuals). Given the significant health threat
posed by some emerging pathogens (e.g., Ebola virus [EBOV], severe acute respiratory
syndrome [SARS]), exposure to emerging pathogens in a wastewater system could result in
potentially serious health outcomes. As a result, there is a need to evaluate potential exposure
and disease transmission from wastewater systems.
There are significant data gaps in understanding emerging pathogens that may limit or preclude
the performance of a quantitative exposure assessment. Research on fate and transport of
pathogens that enter the wastewater system focuses on bacteria and enteric viruses, though
quantitative data describing persistence and fate for estimation of bioaerosol generation and
concentration are scarce. As a result, the estimation of bioaerosol concentration generated by an
individual wastewater collection or treatment process exhibits very high uncertainty for bacterial
and enteric viruses. Published data that quantitatively describe the fate and transport behavior
(e.g., persistence, formation of viable aerosols) of enveloped viruses or bacterial spores in a
wastewater system are also scarce. Collectively, these data gaps significantly limit the current
capability to perform a quantitative exposure assessment for emerging pathogens in the
wastewater system.
This report describes a conceptual exposure model based upon a review of the relevant literature
for fate and transport elements for pathogens when present in wastewater systems. A screening
process is then presented that evaluates emerging pathogens for the presence of two
characteristics: (1) the potential to exhibit high-consequence disease transmission characteristics
in wastewater systems, and (2) the potential to exhibit viable exposure pathways (VEP)1 for
human receptors who have contact with wastewater systems. The screening process is developed
for use with pathogens with varying levels of available data. However, the screening process is
designed to be usable with pathogens with limited data, and for the screening process to
incorporate quantitative data when it is available. Furthermore, this report assists users on the
selection and use of data on surrogate microorganisms in decision-making for elements of the
screening process. Lastly, case studies for the EBOV and the spore form of Bacillus anthracis
are then presented to illustrate use of the screening process to evaluate pathogens and the
presence of VEP.
The EBOV is determined to be a high-consequence pathogen (HCP) with potentially severe or
lethal disease transmission from all routes of exposure identified in the conceptual exposure
model: inhalation, incidental ingestion, dermal contact, and ocular (including conjunctival) or
1 A VEP is a complete exposure pathway for a microorganism that includes routes of exposure along with
documented disease transmission potential.
-------
oral mucous membrane contact (Koonin et al., 2015; Fischer and Wohl, 2016; NASEMSO,
2018; Public Health England, 2018). There is high uncertainty in the determination that viable
infectious pathogens are shed in the feces. However, there is the potential for exposure from the
toilet flush from released bioaerosols or the splash of the toilet contents. One study was
identified for the toilet flush that utilized the enveloped Phi6 bacteriophage, but reported no
detection of bioaerosol over a 20-minute period (Lin and Marr, 2017). There is increasing
evidence that enveloped viruses can survive in wastewater, primarily supported by the recent Ye
et al. (2016) study documenting persistence exceeding one day for enveloped viruses in raw
wastewater. Given the rapid transit time between wastewater collection and arrival at the
wastewater treatment plant (WWTP), the hypothesized persistence of EBOV could allow for
potential exposure to individuals prior to WWTP entry (e.g., combined sewer overflow, sanitary
sewer overflow [SSO]), or during wastewater treatment. Data reviewed in the report indicate that
under certain conditions, EBOV in wastewater deposited on surfaces (before or after drying)
could persist. Additionally, the EBOV has been shown to form viable bioaerosols when
aerosolized from a nebulizer in a protective fluid medium, such as tissue culture fluid. However,
there are no bioaerosol data for enveloped viruses that originate from WWTP processes. The
model predicted that EBOV has the potential to exhibit the defining characteristics of an HCP
and to result in VEP for all exposure pathways identified in the wastewater system.
The spore form of B. anthracis is also determined to exhibit HCP characteristics in a wastewater
system. Disease transmission is documented to occur for all routes of exposure identified in the
conceptual exposure model: inhalation, incidental ingestion, dermal contact, and ocular or oral
mucous membrane contact. There is low uncertainty in the identified routes of exposure
associated with disease transmission. The case study presented in the report evaluated the
introduction of B. anthracis spore-containing wastewater as part of the management process for
wastewater generated from decontamination activities. Therefore, an assessment of the potential
for shedding of viable B. anthracis spores by individuals infected with anthrax was not
performed. With the exception of the toilet flush, all potentially complete exposure pathways
identified in the conceptual exposure model for wastewater systems are VEP for B. anthracis
spores. Thus, the spore form of B. anthracis is found to exhibit behavior of an HCP and result in
a VEP in the wastewater system for all pathways evaluated.
The primary benefit of the screening process is a systematic approach to evaluate disease
transmission and potential exposure to pathogens in the wastewater system. The successful case
study evaluations for EBOV and the form of B. anthracis demonstrate the overall proof of
concept for pathogens with a range of disease transmission characteristics, differences in fate and
transport characteristics, and variability in the amount of available data for the assessment. The
screening process was also demonstrated to be resource-efficient for those pathogens for which
sufficient data were available to easily determine either disease transmission and/or fate and
transport determinations.
The review of available literature and development of the screening process highlighted data
gaps that could be bridged with further research to increase the reliability of the screening
process evaluations. Five key areas were identified for further research:
-------
Development of analytical techniques for counting enveloped viruses in the wastewater
medium and bioaerosol form with known levels of recovery, which would support
quantitative microbial exposure assessment,
Development of data sets to better understand the driving mechanisms and quantitative
relationship between culture-based and molecular-based approaches for biological groups
in matrices of interest (e.g., human feces, wastewater, and bioaerosols) with the goal of
ultimate development of viability corrected measures,
Development of data sets that describe the type and magnitude of exposure relative to the
range of potential technologies used in each treatment unit processes (e.g., primary,
secondary, sludge management),
Evaluation of aggregate exposure of individual WWTP workers based on contact with
multiple unit processes during typically defined job descriptions, and
Performance of studies for persistence and other measures in environmental conditions
(e.g., high relative humidity, winter temperature conditions) typical for WWTP in indoor
and outdoor settings for a variety of regions and weather conditions to generate data
suitable for assessment across the United States.
-------
1 Introduction
Interest in the potential for disease transmission during wastewater management has been
heightened over the past decade by the potential for emerging pathogens to pose novel hazards
when introduced into wastewater management systems. Concern regarding the potential for
human exposure to pathogens during wastewater management has been heightened by recent
disease events. Some recent disease events have included emerging pathogens (e.g., Ebola virus
[EBOV], pandemic influenza), about which less information on disease transmission is available.
Recent disease events include the care of Ebola virus disease (EVD) patients by United States
(U.S.) hospitals during the recent African epidemic (Bibby et al., 2015a), pandemic preparedness
activities after appearance of the H5N1 influenza virus in Asia (World Health Organization,
2007), and the severe acute respiratory syndrome (SARS) outbreak in 2002 (Yu et al., 2014). As
noted by Wigginton et al. (2015), "Should a major virus pandemic occur, wastewater and
drinking water treatment industries would be under increased scrutiny for serving as a potential
means of transmission" So, these recent disease events have exposed the need for better
information on the potential for disease transmission to occur through environmental exposure
routes, including the wastewater exposure pathway.
There are a number of ways that pathogens can enter the wastewater management system. For
pathogens that remain viable when shed in bodily fluids, human disease outbreaks can introduce
emerging pathogens into wastewater systems via the collection of wastewater from residential
toilets. Emerging pathogens could also enter wastewater treatment systems from illicit activity or
through capture of surface-water runoff following a wide-area biological incident.
Decontamination wastewater is another way pathogens can make it into the wastewater
management system. Decontamination wastewater is defined as wastewater generated during
decontamination activities, such as remediation of building interiors after release of biological
materials (e.g., anthrax spores). For this report, decontamination wastewater does not include
infectious materials generated from medical treatment in or outside a medical treatment facility.
In the aftermath of the release of biological materials, the decontamination wastewater could be
released to the wastewater treatment system and enter the WWTP (wastewater treatment plant).
Decontamination wastewater and surface water runoff carrying pathogens could enter the
wastewater collection system through sanitary or combined sewer-sanitary systems (NACWA,
2005). Decontamination wastewater is assumed to be pre-treated prior to discharge to the
collection system or prior to direct addition at the WWTP. However, there is uncertainty in the
actual loadings of residual pathogens as well as concern for potential exposure to WWTP
workers or others who may contact pathogens in the wastewater system.
Emerging pathogens can exhibit fate and transport characteristics in the wastewater system that
provide for novel exposure pathways relative to pathways associated with natural disease
transmission. Bioaerosol generation during the wastewater treatment process provide for atypical
transmission pathways or generate higher exposure concentrations than those produced by
infected individuals in natural transmission environments. As a result, pathogens that are not
typically transmitted from human-to-human via inhalation exposure in a natural environment
could be transmitted via inhalation exposure in the built environment (Roy and Milton, 2004).
In the Woolhouse and Gaunt (2007) systematic literature review of emerging human pathogens
reported between 1980 to 2005, approximately 66% of emerging pathogens were viruses and
-------
91% of these viruses were enveloped. Fungal species represented the next highest percentage of
emerging pathogens at 15% (Woolhouse and Gaunt, 2007). To date, the study of viral pathogens
in wastewater systems has focused on nonenveloped enteric viruses because they have been
generally considered to exhibit significantly better survival in the aqueous environment than
enveloped viruses (Wigginton et al., 2015). The high representation of viruses for recently
identified emerging pathogens, coupled with the observation that viruses may be less effectively
removed by wastewater treatment processes than bacteria (Dias et al., 2015), could indicate an
increased potential of exposure or higher exposure levels throughout the wastewater treatment
process. Few studies have addressed the persistence of viable fungal or bacterial spores in the
wastewater environment. As a result, there are significant uncertainties associated with
infectivity, persistence, and the ultimate fate of enveloped and nonenveloped viruses (Wigginton
et al., 2015), of the spore form of bacteria, and of other groups of nonbacterial microorganisms
(e.g., fungal microsporidia) that are likely to include emerging pathogens.
Data needed to predict the presence, persistence, and fate of emerging pathogens when a human
disease outbreak introduce such pathogens into a typical wastewater management system are
scarce. Data need to meet challenges presented by the introduction of emerging pathogens into
the wastewater management system from collection of decontamination wastewater are similarly
scarce. Studies performed to evaluate the hazard posed by wastewater pathogens have often
relied on epidemiological tools or serological analyses to conduct their assessments (e.g., Khuder
et al. [1998]). Alternatively, however, bacterial pathogens and nonenveloped viruses in
wastewater are generally amenable to environmental recovery and laboratory analysis using
available techniques, and exposure data can readily be developed. On the other hand, however,
the overrepresentation of enveloped viruses in the list of likely emerging pathogens challenges
the assumption that data can easily be generated to estimate exposure. Current culture-based
analytical capabilities exhibit limitations for the enumeration of viable (i.e., infective) enveloped
viruses in wastewater, with difficulties associated with cell culture techniques and virus
extraction methods identified as potential causes (Wigginton et al., 2015). This raises questions
whether available analytical capabilities can be rapidly deployed to quantitatively evaluate
exposure, especially when these pathogens are aerosolized or present in complex media (e.g.,
feces, wastewater).
An additional impediment to gathering analytical data for many emerging pathogens is stringent
requirements that limit the laboratories and personnel that can perform studies relative to typical
wastewater pathogens (Wigginton et al., 2015). For example, work with live EBOV for culture-
based analysis requires the highest laboratory biosafety level and is generally restricted to
specialized research laboratories or governmental agencies with high performance costs
(Broadhurst et al., 2016). In contrast, more common wastewater pathogens from bacterial or
nonenveloped virus groups can be analyzed in many laboratories at reasonable cost without
highly specialized facilities or protective equipment. The lack of quantitative data and
appropriate models for emerging pathogens significantly limit technical capabilities to perform
an exposure assessment for the wastewater system. The difficulty in obtaining new data for many
emerging pathogens drives the current need to assess potential wastewater system exposure using
qualitative approaches that leverage available quantitative data.
In this report, we examine the potential for a viable exposure pathway (VEP), i.e., a complete
exposure pathway for a microorganism that includes routes of exposure with documented disease
-------
transmission potential. An exposure pathway has five parts: a source of contamination, an
environmental media and transport mechanism, a point of exposure, a route of exposure, and a
receptor population (USEPA 2012). "When all five parts are present, the exposure pathway is
characterized as "complete ", that is, capable of contributing to human health risks'' (USEPA
2012). For a VEP, in the case of a microbial contaminant, the contaminant must not only be
capable of reaching the receptor, it must also retain its infectivity upon reaching the receptor.
This report presents a screening process to evaluate emerging pathogens for the presence of two
traits: (1) the potential to exhibit high-consequence disease transmission characteristics in the
wastewater system, and (2) the potential to exhibit viable exposure pathways (VEP) for human
receptors who may have contact with the wastewater system. Receptors include individuals who
use and then flush the toilet, and WWTP workers or others who may contact wastewater during
collection or treatment processes. Case studies for the EBOV and spore form of Bacillus
anthracis are then presented to illustrate use of the screening process to evaluate pathogens for
high-consequence pathogen (HCP) disease transmission characteristics and the presence of VEP.
A Glossary (Section 10) is also included to define exposure assessment and disease transmission
terms used in the report.
2 Problem Formulation
A screening process is presented to evaluate potentially complete exposure pathways resulting
from the introduction of emerging pathogens into a wastewater system. In the development of
the screening process, no primary data are gathered and the project relies on secondary data for
the analysis. Given the limited availability of data, the screening process outputs likely exhibit
high levels of uncertainty. The wastewater system in this study includes (1) the toilet as the
collection point for human bodily waste or some other introduction point for pathogen-
containing wastewater from other sources (e.g., decontamination wastewater), (2) the collection
system that transports wastewater from households to the WWTP, (3) wastewater treatment
processes, and (4) locations where maintenance activities are performed. The purposes of the
screening process are: (1) to identify distinguishing characteristics of an HCP in the context of
wastewater collection and treatment processes, and (2) to evaluate the presence of a VEP for
identified HCP in a wastewater system. The literature on emerging pathogens typically exhibits
significant data gaps that may limit or preclude quantitative exposure assessment. Accordingly,
the screening process is qualitative, but quantitative data are incorporated when available. The
output is a determination of the presence of a VEP for an identified HCP when present in the
wastewater system. The risk of disease transmission or severity of illness is not determined.
The screening process evaluates exposure pathways for emerging pathogens that are introduced
to wastewater from bodily fluids (i.e., defined as feces, urine, vomit) shed by infected individuals
or from other means of entry into the system. Other means of direct entry to the system can
include management of decontamination wastewater, illicit activity, and surface runoff after a
wide-area biological incident. Decontamination wastewater is assumed to have undergone agent-
specific pre-treatment (e.g., bleach addition to wastewater containing B. anthracis spores), but
wastewater added to the system may have residual low levels of biological contamination.
However, pathogens that enter the wastewater system via the toilet will not be assumed to have
had any pretreatment (e.g., chemical introduction, increased retention time), nor will pathogens
-------
that enter the system through entry into sewer system from nonpoint surface runoff or infiltration
of lines.
Exposure pathways are identified for three human receptors: (1) individuals that shed viable
pathogens into the toilet and are then exposed to these pathogens during the flush of the toilet,
(2) individuals that contact wastewater containing viable pathogens during the collection and
treatment process, and (3) individuals that contact untreated wastewater containing pathogens
during a spill or release of wastewater from the collection system. For toilet usage, the exposure
assessment begins with the determination of potential receptor exposure pathways resulting from
the flush of the toilet. However, receptor exposure from the introduction of decontamination
wastewater or other means of entry to the wastewater system are not assessed. Exposure to
pathogens from decontamination wastewater is evaluated only after the pathogens enter the
wastewater system during maintenance or treatment. Once in the wastewater system, exposure is
evaluated relative to potential contact with wastewater during a spill or release of wastewater
from the collection system (e.g., sanitary sewer overflow [SSO], combined sewer overflow, or
sewer main break), treatment in WWTP processes, and general maintenance activities (e.g.,
spray cleaning) for treatment units.
Given the lack of fate and transport data for emerging pathogens necessary to rigorously quantify
exposures from emerging pathogens in wastewater systems, this screening process is designed to
allow for a qualitative evaluation of available data to estimate the potential for exposure and
disease transmission for receptors. As published data were available, they were incorporated into
the screening model but data have not been consistently generated to address the diversity of
potential treatment processes or possible configurations of wastewater treatment systems
throughout the United States. The boundaries of the assessment were drawn to focus effort on
assessing direct exposures from wastewater collection and treatment. As a result, exposure to
treated wastewater effluent or biosolids is not evaluated, nor it the potential for the presence or
generation of reservoirs (e.g., biofilms) that may extend of otherwise alter the character of the
initial exposure scenario. Raw sewage sludge could be produced by the waste water treatment
system if this material is managed off-site at a centralized processing or disposal facility. Class B
sewage sludge (biosolids) generated by wastewater treatment system could be handled with land
application. Class B material is treated to significantly reduce pathogen content and relies on
natural die-off to control residual levels in the soil. This management scenario raises questions
for waste that had been contaminated with a viral pathogen. However, those questions are
beyond the scope of this assessment. The potential for exposure via land application of biosolids
to farmland, forested areas, land reclamation sites, or other sites are not evaluated here. Exposure
is not evaluated from residential sewage overflows, or contact with receiving surface water
bodies either prior to treatment (e.g., combined sewer overflow) or after treatment. Since the
evaluation is focused on the human receptor, exposure to ecological receptors is not considered.
Exposure of receptors is evaluated for wastewater bioaerosol, bulk or splashed wastewater, and
wastewater that is deposited on surfaces from bioaerosol particles, droplets, or splashed
wastewater (Chattopadhyay et al., 2017). For the toilet flush, surfaces are defined to include the
toilet tank, flush handle, toilet lid, and sink or vanity surfaces. For the wastewater treatment
processes, surfaces can include piping, table tops, floors, or other horizontal and vertical surfaces
that individuals may contact them with their hands. Surfaces can be composed of materials that
are porous or nonporous, but most are anticipated to be nonporous surfaces. Exposure to
-------
bioaerosol and to bulk or splashed wastewater are assessed for the following potential routes of
exposure: inhalation, incidental ingestion, dermal, and potential contact with mucous membranes
(e.g., ocular, oral). The identified routes of exposure from complete exposure pathways are then
considered relative to the routes of exposure documented to be associated with disease
transmission.
This screening process is designed to answer the following questions:
• What are the potentially complete exposure pathways for HCP during and after
introduction to the wastewater system?
• Which disease transmission characteristics are associated with the potential for a
pathogen to exhibit HCP activity in the wastewater system?
• How can an HCP be screened for the potential presence of VEP in the wastewater
system?
3 Potential Disease Transmission During Wastewater Collection and
Treatment
For pathogens not directly introduced into the wastewater system, disease transmission may
result from viable pathogens that are shed in bodily fluids and remain infectious until exposure.
Potential linkages between pathogens in wastewater and disease transmission were identified in
the early 1900s, with the first published report dating back to 1907 (Johnson et al., 2013a).
Research interest in occupational exposure to wastewater peaked in the 1970s and 1980s after the
published descriptions of sewage workers' syndrome by Clark et al. (1977) and Rylander et al.
(1976). During this time, the primary transmission hazard posed to workers during wastewater
treatment was identified as oral exposure to enteric viruses through incidental ingestion via
contaminated hands (U.S. Environmental Protection Agency, 1980). It is commonly accepted
that fecal-oral pathogens, primarily from bacterial and nonenveloped viral biological groups,
have the potential for disease transmission from incidental ingestion of feces-contaminated
wastewater.
Traditionally, disease transmission of respiratory viruses was assumed to be driven by: (1)
person-to-person contact with bioaerosols generated from an infected individual who was
shedding virus (e.g., cough, sneeze, exhalation) or (2) fomites contaminated from bioaerosols or
large droplets from the infected individual (Weber and Stilianakis, 2008). Respiratory viruses
were not considered to be transmissible from water sources (e.g., wastewater, drinking water)
(Weber and Stilianakis, 2008). During the 1980s, there were few pathogens that were both
known to initiate infection in the lungs and frequently occur in wastewater (U.S. Environmental
Protection Agency, 1980). It was viewed as an anomaly if an enteric pathogen was "uniquely
infectious by the aerosol route", with the noted exception of the respiratory bacterium
Mycobacterium tuberculosis (U.S. Environmental Protection Agency, 1980). There were also no
available analytical methods to quantitate enveloped viruses in wastewater. As a result,
wastewater treatment disease transmission studies from that time did not usually consider
respiratory pathogens.
However, there were preliminary indications in the literature prior to the 1970s that disease
transmission from wastewater collection and treatment may not be limited to fecal-oral
-------
pathogens and the oral route of exposure. Darlow and Bale (1959) hypothesized that aerosols
generated by the toilet flush could provide for disease transmission from fecal-oral pathogens
that could produce infection via the respiratory tract (e.g., poliovirus) or pathogen-containing
particles that were incidentally ingested after inhalation of bioaerosols. Darlow and Bale (1959)
also noted the potential hazard posed by sewage treatment sources by processes that resulted in
turbulent movement of sewage and may generate aerosols. Consistent with U.S. Environmental
Protection Agency (1980) assertion, Darlow and Bale (1959) also identified the potential for
fecal-associated transmission pathways for inhalation exposure to the respiratory pathogen, M.
tuberculosis.
Independent of the Darlow and Bale (1959) paper, Slote (1976) developed a conceptual model to
describe the potential linkage between the shedding of identified enveloped and nonenveloped
viruses in human feces and urine, the confirmed presence of these viruses in wastewater, and the
pathogen-specific potential to transmit disease via oral, nasal, or inhalation exposure. Through
this process, Slote (1976) hypothesized the potential for exposure and disease transmission from
a broad variety of viruses (e.g., infectious hepatitis, smallpox) present in the wastewater and
routes of exposure (e.g., ingestion, inhalation) known to be present during wastewater treatment.
The hypothesis of disease transmission from wastewater or sewage containing respiratory viruses
as described by Darlow and Bale (1959) and Slote (1976) gained credibility from disease
transmission studies during the 2003 SARS epidemic in Hong Kong. The SARS outbreak was
fueled by bioaerosol generation during the collection and transport of sewage that allowed for
distant disease transmission (Roy and Milton, 2004; Yu et al., 2014). The movement of SARS-
contaminated sewage through the floor drains generated high concentrations of aerosolized virus
that remained virulent and of sufficient dose to cause infection after airborne travel a
considerable distance from the original source (Roy and Milton, 2004).
Interestingly, the SARS outbreak advanced the understanding of conditions for airborne
transmission of pathogens that seemingly lack this form of transmission in the natural
environment or human-to-human transmission. Using the SARS virus and other respiratory
pathogens as examples, Roy and Milton (2004) conceptualized aerosol disease transmission by a
range of descriptors that describe potential fluidity of disease transmission for pathogens with
varying levels of dependence on respiratory pathways (i.e., obligate, preferential, or
opportunistic). Each type of airborne transmission shares the common element that the pathogen
exhibits a reasonable probability of initiation of infection from aerosol inhalation exposure
through a small dose in the lung (Roy and Milton, 2004).
Pathogens characterized as obligate respiratory pathogens are transmitted solely via respiratory
exposure to aerosols (e.g., tuberculosis) (Roy et al., 2010). Preferential2 and opportunistic3)
respiratory pathogens can be transmitted through both respiratory and non-respiratory exposure,
2 Diseases with preferentially airborne transmission are caused by agents that can naturally initiate infection through
multiple routes but are predominantly transmitted by aerosols deposited in distal airways (airways less than 2 mm in
diameter and are comprised of both membranous bronchioles and gas exchange ducts).
3 Diseases with opportunistically airborne transmission are infections that naturally cause disease through other
routes but that can also initiate infection through the distal lung and may use fine-particle aerosols as an efficient
means of propagating in favorable environments.
-------
with the potential for differing disease presentation and severity based on the type of exposure
(Roy et al., 2010). No process is agreed upon to evaluate whether an individual pathogen may
exhibit preferential or opportunistic transmission. However, approaches are described to
determine the potential for transmission from bioaerosols that could facilitate the identification
of these potential respiratory pathogens. For example, pathogens that exhibit replication in the
lungs for at least one stage in their life cycle may also exhibit the potential for transmission via
bioaerosols (Tang et al., 2006).
In the assessment of wastewater disease transmission, emerging pathogens may pose an
opportunistic disease transmission hazard in the built environment or in association with specific
human activities. For example, "unorthodox transmission patterns" in the built environment may
exist when sources that generate concentrated aerosols are combined with an agent that exhibits a
high probability of respiratory infection (Roy and Milton, 2004). This combination produces
conditions for the presence of novel exposure sources and/or routes of exposure that allows for
disease transmission to differ from natural transmission patterns (Roy and Milton, 2004). Unique
transmission patterns can arise when pathogen exposure from novel exposure pathways is greater
than natural transmission pathways. This condition can result from two potential causes: (1)
generation of bioaerosols that exceed natural levels generated by infected individuals and (2)
production of bioaerosols that optimize production of respirable particle sizes relative to natural
sources. Pathogen concentrations in bioaerosols that are generated from infected individuals have
a biological ceiling based on degree of pathogenicity of a microorganism (i.e., how easily it can
invade a host and the severity of the disease it can cause) and the ceiling can easily be exceeded
in aerosols artificially generated under optimized environmental conditions (such as hot tub,
showering, flushing) (Roy et al., 2010; Chattopadhyay et al. 2017). Dependent on the
technology4 or the built environment5, artificially produced bioaerosols may also exhibit particle
size distributions not constrained by the size distribution of particles expelled from the human
respiratory system or released from natural environmental reservoirs.
The Legionella spp. bacterium provides an example of disease transmission facilitated by built
environment conditions that lead to higher bioaerosol concentrations and a more optimized
particle size for inhalation than natural environmental sources. Legionella spp. is a naturally
occurring bacterium in surface water bodies that is rarely associated with transmission from
these natural environments. However, hot tubs, spas, shower, and hot water heaters provide
optimal conditions for bacterial multiplication and for release mechanisms for human exposure.
Breiman (1996) characterized the hazard posed by Legionella spp. exposure as a function of
conditions of the built environment (e.g., high water temperatures in hot water heater that could
allow for extensive bacterial growth) and the presence of technology (e.g., showerhead) to
produce aerosol particle sizes that are ideal for disease transmission via inhalation. In another
example of disease transmission facilitated by the built environment, novel disease pathways
associated with sewage collection practices in a large apartment complex contributed to the
SARS outbreak (Roy and Milton, 2004; Yu et al., 2014). The collection and movement of human
4 Collison nebulizer, atomizer, bubbling generator, liquid sparging aerosolizer or other technologies.
5 Major sources in the built environment include plumbing sources (e.g., showers, faucets, and toilets), cooling
towers, respiratory devices (e.g., humidifiers, vaporizers, and nebulizers), swimming pools (including spas/hot tubs
and whirlpools), steam-producing appliances, and ornamental fountains.
-------
waste aerosolized the respiratory virus and facilitated distant airborne transmission. In addition
to structures in the built environment, human activities that increase the potential for
aerosolization of emerging pathogens may also facilitate opportunistic disease transmission.
Though human-to-human airborne transmission of EBOV from aerosols of small droplets or
droplet nuclei is thought to be unlikely from the natural epidemiology of the disease, disease
transmission may be facilitated by generation of aerosolized body fluids containing the virus
(Judson et al., 2015). Aerosol generating procedures in the medical setting, including intubation
or manual ventilation, may generate large droplets or aerosols from bodily fluids or respiratory
secretions that are associated with disease transmission potential (Judson et al., 2015). The
conditions associated with the nonhuman primate (NHP) outbreak of EBOV-Reston that was
hypothesized to result from mechanical aerosolization of EBOV generated during cage cleaning
and other activities provides support for this mechanism (Judson et al., 2015). In the context of
wastewater treatment and collection, it could also be assumed that the movement of the
wastewater or movement of air through wastewater could result in aerosolization of the EBOV if
present in wastewater. Given the lack of epidemiological evidence for human-to-human
respiratory exposure of aerosol or droplets released from the human respiratory tract as a likely
means of transmission of human EBOV infection (Judson et al., 2015), EBOV could be
considered an opportunistic respiratory pathogen in this context.
4 Fate and Transport of Pathogens During Wastewater Collection and
Treatment
The identification of potential exposure pathways from the wastewater system to human
receptors requires knowledge of initial pathogen loading and the anticipated fate and transport of
pathogens as they move with the wastewater through the wastewater system from collection to
treatment. The following sections will describe fate and transport characteristics of pathogens in
the wastewater systems as the pathogens move through wastewater collection, transport, and
treatment. Fate and transport characteristics considered include the initial loading in bodily fluids
and resulting wastewater concentration, viability of pathogens in wastewater, adhesion to
solids/organics that may affect exposure, and the potential for phase shifts (e.g., aerosolization of
the wastewater medium to the air medium) from wastewater.
4.1 Collection
The residential toilet is the first location for collection of wastewater as well as the first
generation point for potential receptor exposure to wastewater pathogens (Figure 4-1). Many
infectious diseases result in the shedding of viable pathogens in urine, feces, or vomit from
infected individuals (Johnson et al., 2013a). The system-wide loading of pathogens from the
collection system is determined by the total number of infected individuals (i.e., ill and
convalescent) who shed viable pathogens combined with the daily volume or mass of waste and
an associated pathogen concentration for the waste.
Fecal pathogen loadings for common fecal-oral pathogens (e.g., norovirus) can be as high as 108
to 109 genomic copies per gram of feces and at least 106 genomic copies per milliliter of vomit
(Johnson et al., 2013a). Pathogen numbers reported per gram of feces for typical indicator fecal
pathogens (e.g., bacteria, nonenveloped viruses) associated with fecal-oral transmission
-------
pathways have also been reported (e.g., Campylobacter spp. [106 number/gram feces], Vibrio
cholerae [105 number/gram feces], and enteroviruses [106 number/gram feces]) (World Health
Organization, 2017).
Numerous respiratory viruses have been reported in feces, including respiratory syncytial virus,
SARS coronavirus, adenovirus, and bocavirus (Arena et al., 2012). For enveloped respiratory
viruses like human influenza (both seasonal and pandemic forms), molecular measurements
(e.g., viral ribonucleic acid [RNA] identification) are the most frequently reported (Minodier et
al., 2015). The isolation of viable virus from feces is less frequent (Minodier et al., 2015). As a
result, quantitative data that describe viable pathogen numbers in bodily fluids, especially feces,
for many respiratory pathogens are scarce and/or highly uncertain.
-------
r
Routes of
Exposure for
Toilet Use
Inhalation
Incidental
Ingestion
Contact with
Ocular or
Oral
Mucous
Membranes
No Exposure
Assessment for
Introduction of
Decontamination
Wastewater, Illicit
Activity, Surface
Runoff
Contact with
Wastewater during
Maintenance and
Treatment
Evaluated
2^ /\
Routes of
Exposure for
Wastewater
Maintenance
Activities or
Combined/Sanitary
Sewer Overflows
Inhalation
Incidental
Ingestion
Contact with
Ocular or
Oral Mucous
Membranes
Routes of
Exposure for
Wastewater
Treatment
Inhalation
Incidental
Ingestion
Contact with
Ocular or
Oral Mucous
Membranes
wastewater
w,
wastewater
COLLECTION
-------
The total load of pathogens collected in the toilet from an individual can be affected by pathogen-
specific and intra-individual variability in disease presentation. For example, pathogen contributions
by an infected individual may exhibit atypically high pathogen waste concentrations (i.e., "super-
spreaders" who shed disproportionately high levels of pathogens relative to the average or even
upper-bound levels), increased volumes of generated waste per individual, and the possibility of an
extended duration of shedding after resolution of clinical illness. In addition to increased loading
levels per unit mass or volume of waste, diarrhea can significantly increase the waste volume per
day for some pathogens. For example, waste generation of up to 9 L of liquid waste per day is
reported for individuals with EVD (Lowe et al., 2014). Chughtai et al. (2016) also reported extended
shedding of EBOV RNA in urine after resolution of clinical disease symptoms for up to 30 days
after the clinical illness was identified. Phenotypic variation in viral shedding among EBOV strains
is hypothesized based on observations of the 2013-2016 EVD outbreak where patients exhibited
prolonged disease progression and more frequent diarrhea (Vetter et al., 2017).
Once pathogen-containing bodily fluids are added to the toilet, pathogens may partition to surfaces
or other solid elements in the toilet. Prior to the flush, pathogens that enter the toilet may be
adsorbed to solid fecal or vomit material; absorbed within the solid fecal or vomit material;
contained unbound in liquids (e.g., urine, vomit, or water); associated with particles in the toilet
bowl; or adsorbed to the material lining the bowl. The bowl surface and contents (i.e., clean water,
feces, urine, vomit) have a very short time period (i.e., minutes) for potential partitioning between
solid and liquid states. Titcombe Lee et al. (2016) evaluated short-term partitioning (i.e., 5 to 10
minutes) of the nonenveloped MS-2 and the enveloped Phi6 bacteriophages between water, diarrhea
surrogates (i.e., synthetic sludge, anaerobically digested sludge), and fabricated bowl surfaces (i.e.,
porcelain, concrete, polyvinyl chloride, polypropylene). After spiking the sludge with 108 plaque-
forming units (pfu) of bacteriophage and additional water to mimic diarrhea, the liquid and solid
fractions were then generated via centrifugation after 5 to 10 minutes (Titcombe Lee et al., 2016).
The time duration allowed for sorption in this study is generally consistent with a slightly extended
partitioning time prior to a toilet flush. Adsorption between unsterilized sludge, water, and a range of
material surfaces was found to be generally low over short time periods with at least 94% of MS-2
and Phi6 as measured by quantitative polymerase chain reaction (qPCR) remaining in the liquid
fraction across all replicates (Titcombe Lee et al., 2016). The viral load in water is then available for
aerosolization from the toilet water during the flush (Titcombe Lee et al., 2016).
The flushing of the toilet may contribute to exposure from aerosolization of toilet contents, including
potential pathogens, and deposition of splashed toilet water or aerosol particles on surfaces that may
act as fomites (Johnson et al., 2013a). Pathogens or pathogen-containing materials that sorb to the
sidewall of the toilet may generate bioaerosols over subsequent flushes even absent the introduction
of additional pathogen sources to the toilet (Gerba et al., 1975; Barker and Jones, 2005; Johnson et
al., 2013a). In contrast to the limited direct partitioning to toilet bowl surfaces reported by Titcombe
Lee et al. (2016), biofilms present on toilet surfaces are suggested to easily capture and then release
pathogens during subsequent flushes (Johnson et al., 2013a). Receptors who may be exposed to
pathogens released from the toilet flush include infected individuals that use and flush the toilet
and/or contact fomites as well as individuals that may be exposed to successive flushes and/or
contact with pre-existing or newly deposited fomites.
-------
4.2 Pathogens in Wastewater During Transport and Treatment
After pathogens are introduced to wastewater during collection via the toilet, they are transported
with wastewater to the WWTP through the collection system. The concentration of pathogens in
wastewater during transport is of potential concern to individuals who may contact wastewater
during transit (e.g., via maintenance activities, combined sewer overflow) or during the early stages
of treatment when pathogens are present at their highest levels.
There is evidence that even relatively small numbers of individuals can contribute to detectable
pathogens in wastewater, with considerable mixing of introduced pathogens noted during release and
catch studies of poliovirus in collection systems (Hovi et al., 2001). As an example, it was estimated
that poliovirus excretion by 70 people in a population of 700,000 could be detected downstream in
wastewater based on capture of poliovirus spiked in the wastewater system. (Hovi et al., 2001).
Researchers developing polio surveillance programs reported that the shedding of live poliovirus at
average levels of 50% tissue culture infective dose (TCID50) equal to 1.3 x 105 per gram of feces
was associated with detectable peak measurements of approximately 102 pfu per liter of sewage
(Lodder et al., 2012). It is relevant to note that the average poliovirus fecal levels (i.e., 105 order of
magnitude) reported in the Hovi et al. (2001) study are within the general levels described in Section
4.1 for fecal-oral pathogens of bacterial or nonenveloped origin. Poliovirus is hypothesized to
provide a conservative estimate for persistence in wastewater because of its demonstrated ability to
survive in feces and wastewater. For similar loading levels, pathogen characteristics and wastewater
collection systems; the peak measurement value of 102 pfu per liter of sewage could be considered as
a ceiling concentration for hardy, nonreplicative pathogens. In this context, hardy is defined to
describe pathogens with known persistence in wastewater, with classic examples being enteric
bacteria or viruses.
The wastewater concentration of a pathogen during transport is a function of several parameters:
pathogen loading of the waste, wastewater volume over which the pathogen will be diluted (i.e.,
system size, expected wastewater volume), location within the collection network and type of
equipment used to transport wastewater, and persistence of the pathogen in wastewater relative to
the expected time between collection and the time of interest. One approach to estimate the potential
wastewater concentration after pathogen entrance to the wastewater system is the development of a
dilution factor. A dilution factor describes the quantitative relationship between the concentration of
the pathogen in feces (or other bodily wastes) and concentration as diluted by wastewater. As noted
earlier, quantitative data describing the viable fecal pathogen loading per gram of feces are scarce for
the primary biological group of enveloped pathogens that are most often associated with emerging
pathogens. However, fecal concentration data are available for some common fecal indicator
microorganisms or fecal-oral pathogens and these data can inform the development of estimated
ranges for emerging pathogen wastewater concentrations. For example, published ranges of
pathogen concentrations in feces and associated wastewater concentrations are reported for several
commonly recognized fecal-oral pathogen groups (e.g., World Health Organization [2017], Feachem
et al. [1983]).
The U.S. Environmental Protection Agency (1980) estimated a dilution factor range of 1,000 to
10,000 for the relationship between the feces concentration of normal intestinal flora relative to the
expected wastewater concentration of these flora prior to WWTP entry. The U.S. Environmental
Protection Agency (1980) dilution factor predicted that a fecal concentration of 108 pathogens per
gram of feces when present in 1% to 10% of the population would result in 105 to 107 pathogens per
-------
liter of sewage (U.S. Environmental Protection Agency, 1980). However, no further information on
the generation of the U.S. Environmental Protection Agency (1980) dilution factor range was
identified.
Table 4-1 summarizes disease transmission, pathogen, wastewater system, and service population
characteristics that may contribute to a higher or lower dilution factor value in an individual
wastewater system. For example, pathogen-specific data provide insight into relevant fate
characteristics (e.g., persistence time outside the host in wastewater) that can be used with specified
system-specific environmental conditions (e.g., seasonal temperature, variance in pH) to identify a
potential dilution factor. Data describing these characteristics are expected to be unavailable or
highly uncertain for most emerging pathogens. However, a modeling approach could also be
considered to estimate pathogen concentration during transport and treatment.
When considering the application of a generic dilution factor to estimate wastewater concentration
of a pathogen, it should be noted that dilution factors implicitly incorporate the characteristics that
are identified in Table 4-1. When determining the appropriateness of the published generic factors
for application to emerging pathogens, comparability among the characteristics (e.g., persistence in
wastewater, percentage of population excreting pathogen) of the emerging pathogen relative to the
pathogen represented by the range should be evaluated to avoid potential over- or underestimation of
wastewater concentration. Similarly, comparability among the system characteristics should also be
explicitly considered.
Given the complex interactions between the characteristics that can affect the dilution factor, the
identification of quantitative thresholds for characteristic values associated with pathogen detection
cannot be reliably determined. For example, there are no general guidelines that can be identified
with regard to the values individual characteristics most likely to be associated with receptor
exposure (e.g., loading of pathogen in waste). As a result, relative descriptors (e.g., higher versus
lower percentage of infected individuals) were used in Table 4-1 to provide general direction on how
individual elements may affect the value of the dilution factor (i.e., potential tradeoffs) with other
elements remaining constant unless specifically identified.
-------
Dilution factors do not describe the distribution of pathogens within the wastewater system.
However, predictions regarding the partitioning of pathogens within wastewater and the potential for
subsequent media shifts (e.g., aerosolization from water medium to air medium) can be made to
inform identification of potential routes of exposure (Chattopadhyay et al., 2017). Adsorption to
solids may also affect aerosolization and the resulting bioaerosol concentration (Hejkal et al., 1981;
Chattopadhyay et al., 2002). One key mobilization determination is whether pathogens remain
unbound (i.e., dispersed or aggregated and not adhered to any particle, surface, or other organic
matter) in wastewater or are associated with particles (Chattopadhyay and Puis, 1999). For unbound
pathogens in wastewater, there is the potential for sequestration by existing wastewater particles,
system piping, or biofilms associated with the piping or treatment processes. Wastewater contains an
abundance of solid particles available for binding. Most virus aggregates and viruses adsorbed to
solids are associated with submicron-sized particles, with almost three-quarters of particles reported
to be 0.3 |im or smaller in size (Hejkal et al., 1981). Surface properties (e.g., surface charge,
hydrophobicity) of a microorganism and surface properties of solids, and wastewater pH are
common factors associated with the efficacy of common wastewater treatment elements including
coagulation, disinfection, environmental transport, and adhesion to surfaces (White et al., 2012).
However, the vast majority of studies cited by White et al. (2012) report data for bacteria (spores and
vegetative form) or the protozoa Cryptosporidium spp. Studies are needed to evaluate potential
surface charge relationships between viruses, especially enveloped viruses, and common treatment
processes within the relevant pH range of wastewater. Pathogens that strongly sorb to solids in
wastewater are more likely to be captured by the settling process. For example, sewage sludge is the
ultimate fate of particle-associated viruses and can function as an integrator of viruses introduced to
wastewater (Bibby and Peccia, 2013). Worker exposure from some downstream treatment processes
(e.g., secondary treatment technologies) may be reduced, but exposures from the sludge treatment
processes may increase. As result, potential exposures to wastewater workers are not removed, but
may be shifted to other processes in the treatment plant.
Fecal matter, urine, and most domestic sewage exhibit a neutral range of pH (Sobsey and Meschke,
2003; Rose et al., 2015). However, wastewater typically exhibits fluctuating pH conditions due to
ongoing wastewater additions to the system, but generally stays within the neutral range. Viruses are
generally described as exhibiting an isoelectric point (i.e., the pH value at which the zeta potential is
approximately zero indicating no net electrical charge of the substrate) below the neutral pH range
observed for wastewater, though some strain-specific variability in virus isoelectric points is also
described (Sobsey and Meschke, 2003). In the neutral pH of wastewater, the virus is hypothesized to
be positively charged and therefore attracted to the solids in wastewater that typically exhibit a
negative charge.
However, the composition and surface properties of enveloped viruses are different from bacteria
and nonenveloped viruses. Enveloped viruses have multiple structures emanating from their
envelope and each structure can have a unique isoelectric point. In contrast, the isoelectric point of
nonenveloped viruses is principally determined by the functional groups of the coat protein (Michen
and Graule, 2010). The individual isoelectric points presented by an enveloped virus may be
alternately higher or lower than the wastewater pH at any given point in time. As a result, it may not
be useful to conceptualize surface charge for enveloped viruses in the same manner as nonenveloped
viruses and bacteria. Enveloped viruses may have a constantly shifting electric net charge as well as
the potential for individual structures to sorb to solids with varying levels of intensity. The varying
-------
changes in net charge and potential for differing levels of sorption strength for enveloped viruses
complicate the use of general rules regarding their predicted net charge relative to wastewater.
4.3 Formation of Bioaerosols
4.3.1 Background
Aerosols are generally defined as small solid or liquid particles that are suspended in air. In this
report, the term particle is inclusive of liquid or solid forms of aerosol particles. However, the term
droplet is commonly reported in various forms in disease transmission research to describe the size,
potential fate, or likely exposure characteristics of liquid particles. In contrast to the restriction
imposed in some texts that droplets originate only from the human respiratory tract, the term droplet
is used in this report without any limitation on its source. One use of the term droplet maintained in
the report is to describe the phases of liquid particle fate after initial release of the bioaerosol (i.e.,
droplet to droplet nuclei). Consistent with some published descriptions of bioaerosols (e.g., Johnson
et al. [2013a]), the terms droplet and droplet nuclei will be maintained to differentiate between the
particle that is released immediately after aerosolization (i.e., droplet) and the particle that remains
after water evaporation (i.e., droplet nuclei). The second use is the term large droplet to describe
liquid droplets released to the air that are sufficiently large (e.g., approximately 10 to 20|im or
greater in size) that they will exhibit shorter time durations of suspension in air and therefore have
less propensity to travel distances when airborne. However, there is no generally agreed upon size
classification to distinguish between droplet sizes. For example, Weber and Stilianakis (2008)
proposed a 10|im size (as measured post-evaporation after droplet nuclei formation) cutoff between
post-evaporation size of droplet nuclei versus large droplets. Judson et al. (2015) indicated a cutoff
of 20|im size between large and small droplets, and characterized droplet nuclei as less than 20|im in
size.
When aerosols contain microbiological organisms, they are termed bioaerosols. The generation of
bioaerosols containing microbial pathogens during toilet use (Darlow and Bale, 1959; Barker and
Jones, 2005; Johnson et al., 2013b) and during treatment of wastewater (U.S. Environmental
Protection Agency, 1980, 1981; Bauer et al., 2002; Wen et al., 2009) is well documented. Bioaerosol
particles can be in the form of individual microorganisms (i.e., bacterial cells, spores, or viruses),
aggregates composed of multiple microorganisms, or a combination of microorganisms with other
nonmicrobiological materials (Morawska, 2006). As a result, bioaerosol particle size can be
considerably larger than an individual microorganism.
The generation of a viable (infective) bioaerosol is a two-part process: (1) the release of airborne
particles containing microorganisms through a mechanism known to aerosolize pathogens from an
aqueous medium (e.g., bubble bust mechanism), and (2) bioaerosol survival in the air environment
upon release (Thomas et al., 2011). The infectivity of bioaerosols for both bacteria and viruses is
significantly affected by aerosolization technique and environmental conditions (Thomas et al.,
2011; Turgeon et al., 2014). A number of environmental conditions affect the viability of viral
bioaerosols including temperature, relative humidity, ultraviolet light, and the medium of the aerosol
(e.g., mucus from sneeze or cough that aerosolized the virus) (Turgeon et al., 2014). Thomas et al.
(2011) identified a similar list for bacteria, though additional factors were included (e.g., particle
size, oxidative shock).
-------
Bioaerosol formation follows known mechanisms of aerosol generation. The bubble-burst
mechanism is generated by the movement of air through a volume of water resulting in the bursting
of a bubble through the air-water interface and release of the bioaerosol particle to the air. The
bubble-burst mechanism is a relevant aerosolization mechanism for droplet nuclei formation during
the toilet flush (Johnson et al., 2013a) and wastewater treatment processes (Sanchez-Monedero et al.,
2008). Types of the bubble-burst mechanism can be differentiated based on the size of the bubble
moving through water; the film droplet is initiated by bubbles from submicron to 20 |im sizes
whereas the jet droplet is formed by bubbles approximately an order of magnitude larger in size
(Johnson et al., 2013a). The jet droplet mechanism is thought to be active in the aerosols formed in
surface water bodies (i.e., sea surf) (Baylor et al., 1977a; Baylor et al., 1977b) and may have general
applicability to wastewater processes with large volumes of water and wave-like movement (e.g.,
clarifying tanks).
When evaluating the applicability of earlier bioaerosol studies to wastewater treatment, Fernando
and Fedorak (2005) noted that many studies were based on observations of a single bubble as it
moved upward through water to the surface. However, aeration systems in wastewater treatment
processes utilize an ongoing flow of air bubbles allowing for the potential combination of air
bubbles prior to reaching the water surface (Fernando and Fedorak, 2005). As a result, Fernando and
Fedorak (2005) hypothesized that aerosol formation in wastewater aeration systems that
simultaneously inject multiple bubbles is more likely to result from film droplets rather than jet
droplets, given the lack of jet droplet formation from the larger combined bubbles. The size of the
bubble is also associated with the number of film droplets, with coarse bubble aeration (i.e., larger
sized bubbles) leading to a larger number of film droplets and a greater number of bioaerosolized
microorganisms generated than fine aeration (Fernando and Fedorak, 2005).
One unique aspect of bioaerosols formed by the jet or film droplet processes is the potential for the
concentration of microorganisms in the bioaerosol relative to the source water. The released
bioaerosol is composed of bacterial or viral content distributed throughout the volume of the liquid
as well as the bacteria and viruses that preferentially concentrate at the air-water interface (Slote,
1976; Blanchard and Syzdek, 1982). The presence of concentrated microorganisms at the interface is
reported for enveloped hydrophobic viruses, nonenveloped hydrophilic viruses, vegetative bacteria,
and spores (Baylor et al., 1977a; Hejkal et al., 1980; Falkinham III, 2003; Sobsey and Meschke,
2003). The ratio of bioaerosol concentration relative to source water exhibits a greater than 1,000-
fold increase in numbers of viable mycobacteria (Falkinham III, 2003), a range of zero increase to
approximately 100-fold increase in concentration for a variety of bacteria (Hejkal et al., 1980), a
four-fold increase in concentration for bacterial spores (Hejkal et al., 1980), and a 100- to 200-fold
or more increase for nonenveloped viruses (Baylor et al., 1977a). Concentration in the individual
drops is size-dependent, with smaller drops exhibiting higher concentrations than larger drops
because the smaller drops are derived from a higher relative amount of water drawn from the
enriched surface layer (Hejkal et al., 1980). However, there is a hypothesized limit of bubble size
below which the pathogen concentration cannot continue to increase due to accompanying
reductions in the relative volume of water captured from the concentrated surface layer (Hejkal et
al., 1980).
The hydrophobicity of the microorganism surface is an important factor in aerosolization of
microorganisms from water (Falkinham III, 2003). Consistent with the observation on the role of
hydrophobicity in promoting aerosolization, Johnson et al. (2013b) reported that the highly
-------
hydrophobic bacterium, Mycobacterium tuberculosis, could be aerosolized from a relatively minor
disruption of the liquid surface when contained in water. Similarly, the outer exosporium of Bacillus
spores is also very hydrophobic, with noted differences in the level of hydrophobicity among
individual species (Greenberg et al., 2010). Additional factors may also affect aerosolization
potential even within the same bacterial species. For example, Blanchard and Syzdek (1982) report
differences in bioaerosol formation based on the age of bacterial cells in Serratia marcescens.
The process of aerosolization can impart significant stress on the associated microorganism from the
addition of air-to-water interface surfaces, heat, or the breakage of chemical bonds (Hatch and
Wolochow, 1969). The hardiness of the outer membrane is an important determinant of the potential
for a microorganism to withstand aerosolization stress and maintain viability. Bacteria exhibit
susceptibility to shear stress, with the cellular outer membrane noted as a site of significant damage
during aerosolization studies using Escherichia coli (Thomas et al., 2011). Damage may be
expressed in the form of cell death or loss of culturability, as is reported for a group of Gram-
negative bacteria (i.e., S. marcescens, Klebsiellaplanticola, Cytophaga allerginae) (Heidelberg et
al., 1997).
Viruses exhibit significant variation in response to the stress from the aerosolization process and
subsequent sampling stress from recovery and enumeration (Turgeon et al., 2014). Similar to the
description of shear stress impacts on bacterial viability after aerosolization, enveloped viruses are
hypothesized to lose infectivity from conditions that promote virus accumulation on the surface of
aerosol droplets when accompanied by sufficient surface forces to burst the outer viral membrane
(Weber and Stilianakis, 2008). The magnitude of the stress is dependent on the viral species or
subtype. For example, the Phi6 bacteriophage exhibited losses of greater than 99.8% after
aerosolization, and sampling stress assays confirmed that damage to the viral envelope was
significant (Gendron et al., 2010). However, the MS-2 bacteriophage was reported to have losses of
approximately 80% in the same study reported by Gendron et al. (2010). The MS-2 bacteriophage is
reported to exhibit significant resistance to the stresses of aerosolization and sampling, as evidenced
by the same order of magnitude recovery by qPCR and plaque-based assays (Turgeon et al., 2014).
However, small differences in physical structure of enveloped viruses identified at the subtype level
for influenza viruses may result in variable losses in infectivity after aerosolization (Verreault et al.,
2015).
The evaluation of sampling stress must incorporate consideration of the specific mechanism of
aerosolization. A key component in the evaluation of potential surrogate data for viable aerosol
generation is that a similar mechanism for aerosolization is used to generate the surrogate data
relative to the mechanism likely in place for the wastewater system environment. For bacteria, the
physiology of the aerosol can be strongly associated with the aerosolization process (Thomas et al.,
2011). This linkage is also true for viruses (Verreault et al., 2015), especially enveloped viruses that
by the nature of their envelope may exhibit similar outcomes from shear stress as bacteria. As a
result, identification of aerosolization ratios or resistance to aerosolization should be considered to
be specific to the aerosolization mechanism and pathogen.
Most of the laboratory-based assessments of aerosolization of bacteria and viruses use a nebulizer to
produce the aerosol. While nebulizers are not used in wastewater treatment processes, the
atomization process in a nebulizer is similar to the bubble-burst mechanism that is generated by the
rapid movement of air through a volume of water. However, the relative aerosolization stress
imparted from a nebulizer is unknown when compared to the aerosolization stress from the toilet
-------
flush or wastewater treatment processes that exhibit a bubble-burst mechanism. It is known that
nebulizers exhibit a high shear stress (National Research Council, 2006), but published data to
compare these two mechanisms have not been identified. If the bubble-burst mechanism imparted
more stress, bacterial bioaerosol data measured from an actual wastewater treatment process may be
more representative of viable bioaerosol generation than viral data developed using a nebulizer. The
differential impacts of aerosolization stress may be even more pronounced if the nebulizer tests were
performed under conditions of low or moderate relative humidity.
Though few studies are performed specifically for viruses, the medium from which the pathogen is
aerosolized is also important to the generation of a viable bioaerosol (Turgeon et al., 2014). No
published data describe aerosolization of EBOV or other enveloped viruses from the wastewater
medium, which is known to contain high levels of solids and proteins that may provide protection
during the process. However, Verreault et al. (2008) described the Ijaz et al. (1985) study that
reported an 80% infectious virus recovery at 24 hours under conditions of mid-range relative
humidity when the nonenveloped rotavirus was aerosolized from a fecal matter spray. Verreault et
al. (2008) hypothesized that the organic matter and compounds present in fecal matter may serve to
protect aerosolized microorganisms from desiccation and other environmental stressors.
The two most important environmental factors associated with inactivation of viral bioaerosols are
relative humidity and temperature (Piercy et al., 2010). The working definition for relative humidity
used in Yang and Marr (2012) will be used for this report: "ratio of the actual water pressure to the
saturation vapor pressure of ambient air" (Yang and Marr, 2012). Though there are exceptions, it is
generally accepted that enveloped viruses exhibit greater persistence at lower relative humidity
levels, and nonenveloped viruses demonstrate greater stability at higher relative humidity levels
(Verreault et al., 2008; Yang and Marr, 2012). Generally agreed upon values for higher or lower
relative humidity levels are not available to assist in the placement of experimental scenarios into
higher or lower relative humidity levels. However, Yang and Marr (2012) distinguished lower from
higher humidity based on whether relative humidity levels were lower or higher than 50% and that
definition is useful for the evaluations likely to be conducted in wastewater systems.
Relative humidity affects aerosol viability when the aerosolized particles lose water to equilibrate
with airborne water levels, causing both a concentration of non-volatile components in the particle
and shrinkage in particle size (Hatch and Wolochow, 1969). The equilibration happens rapidly after
the aerosol particles are released, and the final particle size is dependent on the relative humidity
(Verreault et al., 2008). In high relative humidity conditions, aerosol particles exhibit a high air-to-
water interface that can lead to a greater inactivation of hydrophobic viruses (e.g., enveloped
viruses) (Trouwborst et al., 1974). This inactivation could be sufficiently rapid after aerosolization
under conditions of high relative humidity that it may preclude the measurement or detection of
viable bioaerosols of enveloped viruses. For example, the enveloped Phi6 bacteriophage
immediately lost infectivity when aerosolized at temperatures of 30°C and 80% relative humidity
(Verreault et al., 2015). Given that many WWTP have high (percentage unspecified by cited author)
relative humidity (Masclaux et al., 2014), the environmental conditions at most plants may reduce
the potential for enveloped viruses to generate viable bioaerosols.
To evaluate reasons for lessened persistence by enveloped viruses at high relative humidity levels,
Yang and Marr (2012) hypothesized that viruses could be separated by virtue of their pH
requirements for cellular fusion with the host cell during disease. As an example, viruses that require
low pH to enter host cells (e.g., avian influenza, SARS) are less stable at 50% to 90% relative
-------
humidity levels (Yang and Marr, 2012). Results from recent aerosolization studies conducted with
the Phi6 bacteriophage are also consistent with the generalization that enveloped viruses exhibit
lesser resistance at higher relative humidity levels (Verreault et al., 2015). Interestingly, the
commonly identified enveloped viral surrogate Phi6 bacteriophage is in the same host cellular fusion
group identified by Yang and Marr (2012) as several emerging pathogens of likely interest for the
screening process (e.g., SARS, avian influenza). As a result, the Phi6 bacteriophage and many
emerging pathogens may exhibit similar behavior in the relative humidity conditions of a WWTP.
In contrast, enveloped viruses that require a neutral pH for cellular fusion exhibit greater stability at
50% to 90% relative humidity (e.g., Rous sarcoma virus, bovine rhinotracheitis virus) (Yang and
Marr, 2012). Viruses that do not have specific pH requirements for fusion due to multiple pathways
of cellular entry may not exhibit sensitivity to relative humidity (e.g., vaccinia virus, causative agent
of smallpox) (Yang and Marr, 2012). While further data are needed to fully describe the relationship
between relative humidity, aerosol viability, and virus type; the Yang and Marr (2012) hypothesis
provides a mechanistic basis to identify surrogates predictive of target pathogen bioaerosol
generation based on an identified relationship between viral type, relative humidity, and aerosol
persistence. It also helps to explain why enveloped viruses of interest may exhibit lower aerosol
stability within the range of relative humidity levels likely to be found at some WWTP. When
selecting surrogate organisms to evaluate the generation of viable bioaerosols of enveloped viruses,
the suitability may vary based on ambient temperatures and relative humidity. As a result,
environment-specific surrogate selections should be identified, with the assumption that they may
not be broadly applicable to all environmental settings.
4.3.2 Bioaerosol Generation During the Toilet Flush
Darlow and Bale (1959) provided one of the earliest reports that the toilet flush could generate
bioaerosols of pathogens contained in waste. The flushing of a toilet produces a bioaerosol with a
range of particle sizes (Figure 4-2). Lin and Marr (2017) reported a particle size distribution for a
toilet flush with one L of anaerobically digested sludge in the toilet bowl. A scanning mobility
particle sizer was used for particles 14-700 nm and an aerodynamic particle size spectrometer for
particles 0.5-20 |im, and the results merged to generate a distribution (Lin and Marr, 2017). Based
on the measurement of two commercial autoflush mechanism toilets, a total particle number of 1.7 to
2.6 million per flush and the total volume of aerosols generated in the range of 10 9 to 10 8 mL were
reported (Lin and Marr, 2017). The distribution exhibited multiple peaks over the particle size range
of approximately 10 nm to 1,000 nm (i.e., 10"2 |im to 1 |im). However, a description of the size
distribution across a particle size range (e.g., 1 |im to 20 |im, large droplets) of greatest interest for
the screening assessment was not quantitatively described by Lin and Marr (2017).
-------
Large particles
da>10 (jm
Inhalable large particles
10 < da<100 pm
Aerosol respirable particles
da<10 (jm
Figure 4-2. Toilet flush showing aerosol and particle deposition.
Particle size is a key descriptor of the bioaerosol generated during toilet usage because the size of an
aerosolized particle predicts its fate and potential exposure pathways of receptor exposure. As a
result, the distribution of particle sizes is an important summary statistic that describes the relative
proportion of particle sizes in the bioaerosol and thereby informs the determination of the mixture of
potential exposure pathways present. The bioaerosols generated by the toilet flush are generally
smaller than the threshold for respirable particle size (diameter 10 urn) with the greater portion of
the mean particle sizes reported to be 5 jam or less in size based on an evaluation of commonly cited
studies describing toilet bioaerosol particle sizes (Darlow and Bale, 1959; O'Toole et al., 2009;
Johnson et al., 2013a). In one of the first published studies of bioaerosol particle size from the toilet
flush, Darlow and Bale (1959) reported that 87% of the aerosol was less than 4 |im in size using
study data collected from impingers and pre-impingers. Darlow and Bale (1959) reported the mass
mean particle diameters at seat level for an open toilet lid as 2.33 ^im and a closed toilet lid as 1.99
jam. O'Toole et al. (2009) also reported particle number and size for 420 mm above toilet seat level
from water-efficient Australian toilets; the aerosol concentration was 44.9 aerosol particles/cm3 in
the size range of 0.2 to 1 jim, 1.7 aerosol particles/cnr' were in the size range of 2 to 3 (jm, and all
other measurements for ranges of particle sizes up to 20 um were below the detection limit of the
aerodynamic particle sizer.
Johnson et al. (2013a) evaluated the initial droplet size 15 seconds post-flush and the resulting
droplet nuclei for two modern toilets with tanks (i.e., high efficiency, pressure-assisted high
efficiency) and one without a tank (i.e., flushometer) after seeding with fluorescent microspheres of
varying sizes (i.e., 0.25, 0.5, 1.0, and 1.9 jam). Assessment of larger-sized aerosolized particles was
-------
not part of the study design; these particles were not measured and time was permitted to allow them
to settle out prior to measurement of other droplet sizes. Johnson et al. (2013a) reported that aerosol
generation did not appear to be proportional to pre-flush particle concentration when comparing
varying toilet types with comparable volumes of bowl and flushing water. Key findings by Johnson
et al. (2013a) included that aerosolized particle numbers demonstrated an increase relative to
increasing flush energy, aerosol generation was not proportional to the initial particle loading in the
toilet, and droplet formation likely occurs through two mechanisms (i.e., bubble burst for the droplet
nuclei and splashing for large droplets). Particles greater than 5 |im in diameter reached their
maximum counts during 15 to 20 seconds post-flush and began to decline after 60 seconds (Johnson
et al., 2013a). Mean droplet nuclei generation rates, with generation rate defined as droplet count per
liter flushed, ranged from approximately 2,100 (high efficiency, high-volume flush) to 25,663
(flushometer). When using fluorescent particles to measure droplet nuclei generation rates, the mean
droplet nuclei generation rates (i.e., droplet nuclei produced per 100 million fluorescent particles
present pre-flush) were reported by trial to range from 0.072 to 0.256 (Johnson et al., 2013a). These
data, in conjunction with known flush energy values by toilet type, were used to support the
hypothesis that flush energy and aerosol production were associated (Johnson et al., 2013a).
Considerable variability was present in individual results when comparing the averages generated by
toilet type (Johnson et al., 2013a).
Differences in particle size distribution between open and closed toilet lids are reported. For
example, Darlow and Bale (1959) reported that a closed toilet lid preferentially removed larger-sized
S. marcescens particles and resulted in a reduced overall bioaerosol concentration. However, a
higher proportion of remaining particles emitted were sufficiently small to remain airborne for
potential inhalation (Darlow and Bale, 1959). Best et al. (2012) reported that the closed toilet lid
resulted in an approximately 10-fold reduction in airborne Clostridium difficile, and also noted that a
reduced load of aerosolized microorganisms was still being transported through gaps between the
toilet lid and the bowl. However, few data sets evaluating the impact of lid placement on subsequent
aerosolization allow for comparisons across toilet designs and biological groups. As a result, it is
difficult to distinguish the potential impact of differences in toilet design (e.g., toilet design, lid
placement) versus the innate propensity to form viable bioaerosols exhibited by the different
biological groups in the studies.
The literature search identified bioaerosol measurement data from the toilet flush for nonenveloped
viruses (Barker and Jones, 2005), enveloped viruses (Lin and Marr, 2017), and bacteria (Darlow and
Bale, 1959; Barker and Jones, 2005) (Table 4-2). No data describing bacterial or viral bioaerosol
concentrations from vomit or simulated vomit when present during the toilet flush were identified.
Additional studies (Gerba et al., 1975; Wallis et al., 1985; Best et al., 2012) describe water
concentration data and measurements of captured microorganisms (i.e., enteric poliovirus, MS-2
phage, bacteria, C. difficile) after flushing, but these studies do not provide air concentration data.
However, these studies describe important phenomena (e.g., release during successive flushes)
associated with bioaerosol formation during the toilet flush that will be reviewed later in this section.
Study designs varied in the means of introduction of microorganisms to the toilet bowl for
measurement of bioaerosols in the studies summarized in Table 4-2, as well as additional identified
studies that did not report a bioaerosol concentration measurement (Gerba et al., 1975; Wallis et al.,
1985; Best et al., 2012). Pathogens were introduced into the toilet by direct addition to the toilet in
-------
Table 4-2 summarizes the studies that report bacterial and viral bioaerosol concentration for
commonly studied microorganisms and the associated study conditions. Table 4-2 identifies the
toilet type(s) that was evaluated, total pathogen number, method of introduction to the toilet bowl
water, and bioaerosol measurements. Reported bacterial bioaerosol concentrations ranged between
101 colony forming units (cfu)/m3 to 103 cfu/m3 for measurements between 20 to 30 cm above the
seat level and up to 30 cm in front of the toilet (Darlow and Bale, 1959; Barker and Jones, 2005).
One study reported bacterial bioaerosol concentrations at seat level of 321 organisms/m3 for
impinger measurements and 217 organisms/m3 for pre-impinger measurements (Darlow and Bale,
1959). The use of pre-impinger and impinger bioaerosol measurements by Darlow and Bale (1959)
allowed for the reporting of bioaerosol concentration by particle size ranges (i.e., greater than 4 |im
in particle size from the pre-impinger, less than 4 |im in particle size from the impinger). Barker and
Jones (2005) reported a viral bioaerosol concentration of 2,420 pfu/m3 for the MS-2 bacteriophage
after seeding agar to simulate feces, but no particle size data were captured. However, one study
reported nondetection of viral bioaerosol as measured by pfu after the flush of an automatic toilet
flush mechanism when a 1 L sludge sample was spiked to a concentration of 107 pfu/mL with MS-2
and Phi6, respectively (Lin and Marr, 2017). Lin and Marr (2017) provided particle size data from
the flush, but did not quantitatively report bioaerosol concentrations in common size ranges used in
exposure assessment.
The first apparent pattern exhibited in Table 4-2 is that bioaerosol concentrations appear to be
highest near seat level as would be expected. When bioaerosols were detected, little difference was
identified between the bacterial and viral bioaerosol concentrations. However, S. marcescens is a
hardy bacterium that exhibits a low decay constant when aerosolized (Darlow and Bale, 1959), and
the MS-2 bacteriophage is also recognized as being very resistant to aerosolization (Turgeon et al.,
2014). As a result, the potential for generation of viable bioaerosols may be anticipated to be fairly
similar between hardier bacteria (e.g., S. marcescens) and nonenveloped viruses (e.g., MS-2
bacteriophage).
Barker and Jones (2005) spiked agar to simulate pathogen-containing feces clinging to the toilet
bowl wall and reported first flush airborne particles at one-minute post-flush to contain 2,420 pfu/m3
for MS-2 bacteriophage and 1,370 cfu/m3 for S. marcescens. The Wallis et al. (1985) data directly
compared airborne attenuated poliovirus after release from stool collected from vaccinated infants to
airborne poliovirus after release from toilet bowl water to which the virus was directly added. Wallis
et al. (1985) reported that 65 pfu of poliovirus (measured as a capture and elution from filter) were
generated from a toilet flush after seeding the toilet with a feces concentration of 2.4 x 107 pfu and a
resulting water concentration of 2,040 pfu/mL. In another round of sampling, 6 pfu of poliovirus
(also measured as a capture and elution from filter) were generated from a toilet flush when 4.5 x
107 pfu poliovirus in feces were introduced and a resulting water concentration of 480 pfu/mL virus
was obtained (Wallis et al., 1985). For other sampling rounds with 107 total pfu in fecal samples, the
virus detected in the bowl water did not rise above 180 pfu/mL and no pfu were recovered from the
aerosol. In contrast, no aerosolized virus could be detected when virus was added directly to the
toilet bowl water until 3 x 108 pfu poliovirus levels were used (Wallis et al., 1985). The virus was
gently added and stirred in the toilet water (Wallis et al., 1985) and the reported airborne
measurement is reflective of the toilet flush only.
Best et al. (2012) seeded feces from elderly humans with the spore-forming bacterium C. difficile at
levels of 107 cfu/mL and measured aerosolization after the toilet flush. Spores are assumed to exhibit
-------
greater hardiness during aerosolization than vegetative bacteria and some viruses. As a result, the
spore biological group could represent a potential worst-case scenario for high levels of
aerosolization (Best et al., 2012). Best et al. (2012) did not measure aerosol concentration, but placed
a rotating plate portable sampler at various heights for tests with the toilet lid closed (i.e., toilet seat
height, 10 cm above the seat/handle height) and the toilet lid open (i.e., toilet seat height, 10 cm
above the seat/handle height, 25 cm above seat height). For the evaluation of aerosolization, a
standard wash-down toilet design commonly used in U.K. hospitals was examined. The highest
levels of C. difficile (35 cfu at seat height) were identified immediately after flushing with the lid
open (Best et al., 2012). Levels then exhibited an eight-fold reduction by 60 minutes and another
three-fold reduction by 90 minutes (Best et al., 2012). The highest counts in the first 30 minutes
were reported at seat-height, regardless of whether the lid was open or closed (Best et al., 2012).
When using a cling film over the toilet bowl to capture large droplets in the hospital ward setting, the
mean number of large droplets captured was 15 and 47 for the standard wash-down design and
rimless pan with raised seat toilets, respectively (Best et al., 2012). For the evaluation of droplet
formation, two toilet designs were evaluated: the standard wash-down toilet used in the
aerosolization assessment and a rimless pan toilet with a raised seat (Best et al., 2012). Both toilet
types are in common use in the United Kingdom (Best et al., 2012). Settle plate were also used to
measure the potential surface contamination levels resulting from the toilet flush. Settle plate results
were also compared for toilet flushes with the lid open (mean of 1 to 3 cfu/plate, except for the left-
hand side of toilet that had 0 cfu/plate) versus the lid closed when no droplets were reported (Best et
al., 2012). The direction of water flow during the water flush was hypothesized to cause the lack of
droplets reported on the left-hand side of the toilet (Best et al., 2012). For settle plates placed on the
floor during an open lid flush, C. difficile was recovered during a 90-minute period (Best et al.,
2012). However, there was no droplet recovery on settle plates when the toilet lid was closed (Best
etal., 2012).
Darlow and Bale (1959) seeded toilet water with S. marcescens and made measurements using
impingers at various heights from seat level (zero, one, and two feet) and slit samplers at seat level.
After the toilet flush, Darlow and Bale (1959) noted that the generated bioaerosol exhibited highest
concentrations in the immediate vicinity of the toilet seat but then became diluted over time from
losses of gravity and inactivation. For example, the reported bioaerosol concentrations were highest
at the toilet seat level during the time period from zero to two minutes post-flush, with a reported
impinger measurement of 11,329 cfu/ft3. The bioaerosol concentration then rapidly decreased from
seat level to 1,509 cfu/ft3 at one foot and 115 cfu/ft3 at two feet.
Using the Darlow and Bale (1959) bioaerosol data described above, Hines et al. (2014) reported an
emission factor ratio that could be used to estimate bioaerosol concentration after a toilet flush. The
emission factor value of 1.3E-6 (L/m3)is derived by the ratio of the bioaerosol concentration
(cfu/m3) over the bacterial pathogen water concentration (cfu/L) reported for the Darlow and Bale
(1959) data set. The emission factor value can be interpreted that the measured bioaerosol
concentration was approximately six orders of magnitude less than the initiating water concentration
for the Darlow and Bale (1959) data set. While the emission factor value should be considered to
have high uncertainty (Hines et al., 2014), it does provide a general indication that relatively high
levels of bacterial contamination may need to be present in the toilet for generation of a detectable
bacterial bioaerosol.
-------
There is also evidence that pathogens added to the toilet bowl may not be fully removed during the
first flush. Darlow and Bale (1959) first noted that there were not proportionate reductions in
bioaerosol concentrations after a reduction in the inoculum introduced to the toilet bowl. For
example, a ten-fold reduction in total inoculum added to the bowl (ranging from approximately 109
to 1012) only resulted in an approximately one-quarter decrease in bioaerosol concentration (Darlow
and Bale, 1959). The lack of proportionality was highlighted for small inocula (e.g., bowl residues)
which contributed to subsequent sporadically high bioaerosol concentrations in successive flushes.
Pathogens may become attached to the sidewalls and provide an ongoing source over multiple
flushes (Gerba et al., 1975; Barker and Jones, 2005; Johnson et al., 2013a). Viruses may be more
difficult to remove than bacteria from the toilet bowl (Gerba et al., 1975), and it has been suggested
that biofilms on the surface of the bowl may contribute to viral persistence in the toilet (Johnson et
al., 2013a).
It is unclear how bioaerosol generation may differ based on whether pathogens are introduced
directly to the toilet bowl water as might occur in urine or are associated with solid or semi-solid
material. With the exception of one of the Wallis et al. (1985) data sets and the Darlow and Bale
(1959) data, contaminated feces or a simulant were added to clean toilet water in the reported
studies. However, the use of agar or other carriers may affect the aerosolization potential relative to
actual feces and the resulting air concentration (O'Toole et al., 2009). Wallis et al. (1985)
hypothesized that the hydrophobic nature of the added fecal materials may enhance aerosolization of
the included microorganisms given the relationship identified between the increased aerosolization
noted for increased levels of hydrophobicity. The added materials may also increase turbulence
during flushing and thereby potentially increase aerosolization (Wallis et al., 1985). Barker and
Jones (2005) performed a non-statistical comparison of the bacterial bioaerosol concentration when
an equivalent loading of bacteria was seeded directly to the bowl water or when bacteria were placed
in agar on the sidewall to mimic contamination from diarrhea, but no apparent differences were
identified (Barker and Jones, 2005).
4.3.3 Bioaerosol Generation During Wastewater Treatment
4.3.3.1 Measurement-based Bioaerosol Data
Sanchez-Monedero et al. (2008) described aerodynamic diameter particle sizes for WWTP
bioaerosols as ranging from less than 1 |im to 100 |im in size. However, the expected statistical
distribution of the data was not described. In general, respirable or smaller size particles are most
frequently reported. Laitinen et al. (1994) reported aerodynamic diameter sizes less than 4.7 |im for
88% of detected bacterial bioaerosols in WWTP. Bauer et al. (2002) reported that 99.99% of
particles were less than 6.12 |im and 99.9% were less than 2 |im for bacterial and fungal bioaerosols,
but did not describe a statistical distribution associated with the reported particle distributions. Bauer
et al. (2002) noted that these results were not consistent with the particle sizes reported by Brandi et
al. (2000 ) where 60% to 80% of bacterial bioaerosol particles were greater in size than 2.1 |im.
Advances in instrumentation capabilities provide greater precision in measurement of particle size
while also allowing for simultaneous determination of viability for bioaerosol particles. Viable
bioaerosol concentrations (using an ultraviolet-aerodynamic particle sizer [UV-APS]) were reported
for multiple wastewater treatment process locations in a single plant, with the highest viable particle
number greater than 2 |im in size (6,533 particles/m3) identified in the sludge thickening basin (Li et
al., 2016). Lower particle numbers, from largest to smallest, were reported in the biological reaction
-------
basin, office building, screen room, effluent outlet, and downwind plant outdoor boundary (Li et al.,
2016). For particles 2 |im or smaller in size, the highest number of viable particles were found at the
biological reaction basin (1,300 to 3,867 particles/m3) and the office building (1,133 to 3,667
particles/m3) (Li et al., 2016). The potential contribution of bacteria from office workers was
identified as the likely reason for the larger proportion of smaller-sized particles in the office
building (Li et al., 2016). For most sampling sites, fluorescent peaks of viable particles were
identified in the 3 to 4 |im range leading Li et al. (2016) to hypothesize that the particles were
reflective of aggregates of fungal or bacterial material. However, Li et al. (2016) did not report
measurements that described the distance between the treatment processes and sample collection.
The selected analytical methods used to measure bioaerosol concentrations may affect the level of
magnitude of reported concentrations. When analyses are selected that measure a large number of
bacterial families (e.g., heterotrophic plate counts, mesophilic bacteria), bacterial bioaerosol counts
reach their highest levels. Medema et al. (2004) reported heterotrophic bacteria plate count measures
as high as approximately 106 microorganisms/m3 from raw sewage intake screens and near tricking
filters. When using mesophilic group bacterial measurements, Sanchez-Monedero et al. (2008)
identified a bioaerosol concentration of approximately 1000 cfu/m3 from pretreatment processes, and
Fracchia et al. (2006) reported an equivalent order of magnitude measurement (3,370 cfu/m3) from
the pretreatment grit chamber. Similarly, measurements of the Gram-negative bacteria group were
also within the 103 cfu/m3 order of magnitude in the pretreatment area (i.e., sewage inflow, including
primary screening and the grit collection tank) (Fracchia et al., 2006). In the context of potential
emerging pathogens that may be introduced to a wastewater system, individual pathogen species
may exhibit loadings that are considerably less than those described by analyses that measure
multiple species or families of microorganisms.
The literature is inconsistent on whether higher capacity WWTP are associated with higher
bioaerosol levels. Masclaux et al. (2014) reported no statistically significant differences between
plant processing capacity and adenovirus bioaerosol concentration across a range of 31 WWTP in
Switzerland that receive household waste (with the highest size category serving 50,000 inhabitants
or more) and use an activated sludge treatment process. In contrast, Heinonen-Tanski et al. (2009)
reported that higher levels of bacterial bioaerosols were identified in pretreatment areas of large- and
medium-sized plants relative to smaller plants because the larger plants must operate their
pretreatment systems longer to accommodate larger volumes of wastewater. In the Heinonen-Tanski
et al. (2009) evaluation, Helsinki, Finland was the largest size plant with an influent volume of up to
267,000 m3/day and total culturable bacterial bioaerosol levels of 31.1 x io3 cfu/m3. In contrast,
Siilinjarvi, Finland was the smallest size plant with an influent volume of up to 200 m3/day influent
volume and reported total culturable bacterial bioaerosol levels of 4.8 x io3 cfu/m3. Brandi et al.
(2000) also described higher levels of bioaerosol generation in larger versus smaller plants.
Generation of the highest bacterial bioaerosol levels is associated with wastewater treatment
processes that have rapid movement, mechanical agitation, or forced aeration of wastewater (Pascual
et al., 2003; Sanchez-Monedero et al., 2008). Though exceptions are identified for some sludge
management processes, bacterial bioaerosol concentrations tend to decrease as the wastewater moves
through the treatment process and bacterial loads progressively decrease (Fracchia et al., 2006). The
higher levels of bioaerosols associated with sludge management are generated from more aggressive
processing of materials (e.g., high turbulence, movement through rotating parts) that can result in a
higher bioaerosol concentration even when a lower wastewater concentration is present. High levels
-------
of bioaerosols may also be generated during maintenance activities due to the use of high-pressure
water to clean screens or other treatment equipment (Heinonen-Tanski et al., 2009). Bacterial
bioaerosol generation may also be elevated during high pressure cleaning activities, with maximum
total bacterial levels identified at 104 cfu/m3 and total coliforms reported at 103 cfu/m3 (Haas et al.,
2010). Bacterial bioaerosols in the range of 101 and 102 cfu/m3 for maintenance activities (e.g.,
screens, cleaning of sludge centrifuge) were reported for fecal coliforms and enterococci,
respectively (Heinonen-Tanski et al., 2009). Medema et al. (2004) also reported heterotrophic plate
count measurements of approximately 104 5 microorganisms/m3 for belt filter press cleaning
activities.
Bauer et al. (2002) hypothesized that microorganisms were primarily transported from water to air
during aeration processes in wastewater treatment. As noted earlier by Blanchard and Syzdek (1982)
and Slote (1976), aerosol generation by the forcing of air bubbles upward through water allows for
the potential concentration of bioaerosol microorganisms relative to wastewater source
concentration. The raw sewage entry point and associated pretreatment processes are commonly
identified as generating high levels of bioaerosols (Brandi et al., 2000; Fracchia et al., 2006; Karra
and Katsivela, 2007; Heinonen-Tanski et al., 2009). Given the potential for use of covered clarifiers
at the primary stage, this stage may also exhibit greater variability than other treatment stages when
considering the presence or absence of covers that limit release of aerosols (Pascual et al., 2003).
Other contributors to variability in bioaerosol generation in primary treatment may include exposed
surface area, indoor versus outdoor placement of equipment, environmental conditions, settings of
treatment equipment (e.g., clarifier rake arm speed).
The type of aeration process used for sludge management is a key determinant in the level of
measured bioaerosol (Fracchia et al., 2006). Heinonen-Tanski et al. (2009) reported a positive
correlation between the number of bioaerosolized microorganisms and the rate of water aeration.
Fracchia et al. (2006) determined that mechanical aeration of sludge was associated with higher
levels of bioaerosols than submerged microbubble systems or fixed film reactors (e.g., Brandi et al.
[2000]; Bauer et al. [2002]; Fernando and Fedorak [2005]). Consistent with Sanchez-Monedero et al.
(2008), Heinonen-Tanski et al. (2009) reported that aeration performed with a brush aerator or an air
stripping aerator produced greater bioaerosol concentrations than a diffused aerator. Depending on
the type of bacterial analysis performed, reported values ranged from single bacterial group (i.e.,
enterococci) measures of approximately 10 cfu/m3 (Heinonen-Tanski et al., 2009) to a heterotrophic
plate count reflective of multiple bacterial families of up to 106 microorganisms/m3 (Medema et al.,
2004).
When comparing bacterial bioaerosol concentrations at a WWTP that was converted from a coarse
bubble aeration to a fine bubble aeration process, a significant decrease in bioaerosol concentration
was identified where reported levels were similar to a background location (Fernando and Fedorak,
2005). In studies comparing bioaerosol concentrations across types of aeration, the use of diffused or
fine bubble systems was also found to generate lower bioaerosol concentrations (Heinonen-Tanski et
al., 2009) due to a less vigorous forced aeration of the water. In one of the highest mean mesophilic
bacterial measurements identified for sludge management, Bauer et al. (2002) reported a
concentration of 1.7 x io4 cfu/m3 in an activated sludge system that used paddle mixers to stir the
tank continuously.
In comparison with the available bacterial bioaerosol data, few published studies describe viral
bioaerosol generation from WWTP processes (Masclaux et al., 2014). Masclaux et al. (2014)
-------
evaluated bioaerosols of identified nonenveloped viruses (i.e., adenovirus, norovirus) and the
enveloped virus (i.e., hepatitis E) using qPCR techniques. Samples were collected from 39 WWTP at
various treatment stages, including raw wastewater inflow and bubbling aeration basins. Adenovirus
was found frequently (84%) in 124 samples and aerosol concentrations were as high as 22.76 x 105
viral particles/m3 (Masclaux et al., 2014). Hepatitis E was not detected, but low wastewater
concentrations were identified as the reason for lack of detection in the aerosols (Masclaux et al.,
2014). Heinonen-Tanski et al. (2009) reported somatic and f-specific coliphage aerosol
concentrations, with the highest pretreatment geometric mean measurement reported for somatic
coliphage of 137.8 pfu/m3 (raw wastewater pumping) and f-specific coliphage of 13.4 pfu/m3
(aerated fine screen grit removal). Medema et al. (2004) reported average f-specific RNA-phage
bioaerosol concentrations as high as approximately 104 to 105 microorganisms/m3 at the intake
screen for raw sewage and trickling filter process locations. Carducci et al. (2000) reported viral
bioaerosol (of unspecified virus) in various pretreatment locations at an activated sludge plant as
ranging from 4.51 x 10"4 most probable number (MPN)/L to 3.92 x 10"3 MPN/L. Slightly higher
values were reported for an anaerobic sludge plant that ranged from 20.2 x 10"3 MPN/L to 17.2 x
10"3 MPN/L (Carducci et al., 2000).
Some WWTP bioaerosol concentration studies identified operational control measures to reduce the
level of bioaerosol production from identified WWTP processes. Deployment of casing or additional
covering of processes associated with generation of high levels of bioaerosols has been identified to
reduce bioaerosol exposure levels of workers (Fernando and Fedorak, 2005; Heinonen-Tanski et al.,
2009). Actions taken to reduce formation of odors by outdoor WWTP (i.e., covering outdoor grit
tanks and primary settling tanks) are associated with measured reductions of bioaerosols (Fernando
and Fedorak, 2005). Based on evaluations to determine emissions of bioaerosols relative to unit
volumes of wastewater, Bauer et al. (2002) recommended that reductions in the surface area of the
aeration tank could reduce the flux of bioaerosol emissions. In an approach designed to effectively
limit the surface area of bulk wastewater exposed to air, Hung et al. (2010) reported E. coli
bioaerosol reductions between 50% and 100% when polystyrene balls with diameters sizes of 1.9 to
4.7 cm were placed at the water surface of a laboratory-scale tank.
Few studies that reported bioaerosol concentration included measurement of wastewater
concentrations, either at sewage entry to the plant or at various stages of treatment. However, two
studies (Bauer et al., 2002; Karra and Katsivela, 2007) measured wastewater and bioaerosol at an
individual treatment stage and then calculated emission factor values for bioaerosol generation from
wastewater. Bauer et al. (2002) developed an aerosolization ratio to describe the order of magnitude
difference between the mesophilic bacterial concentration of wastewater and bioaerosol that was
generated. After converting to a common air and treated water volume of 1 m3, a nine to 11 order of
magnitude difference between wastewater and bioaerosol concentration was identified at a fixed film
reactor WWTP and the aeration tank of an activated sludge WWTP (Bauer et al., 2002). A five to
seven order of magnitude difference was reported for fungi using the same process and sampling
locations (Bauer et al., 2002). With the assumption that the aerosolization ratio is linear, the ratio can
be interpreted to indicate that fairly high levels of mesophilic bacteria must be present in wastewater
(e.g., 109cfu or greater) before measurement of mesophilic bacteria results in detectable levels.
Karra and Katsivela (2007) also evaluated relationships between bacterial group wastewater
concentrations (i.e., heterotrophs, total coliforms, fecal coliforms, enterococci) and found that the
measured bacterial group wastewater concentrations were at least 108 times greater than the
bioaerosol concentration (e.g., total coliforms data of 1.6 xio4 cfu/mL compared to 127 cfu/m3).
-------
Medema et al. (2004) generated emission factors (i.e., ratio of the concentration in the air over the
concentration in the water) using data from five WWTP plants in the Netherlands for identified
treatment and maintenance processes. Water and bioaerosol samples were collected for heterotrophic
plant count, coliform, f-specific RNA phage, and sulfite-reducing Clostridium spore analyses
(Medema et al., 2004). Emission factor values were calculated for processes from which there were
detections in both the wastewater and air media (Medema et al., 2004). As a result, emission factors
for some biological groups, such as the viral f-specific RNA phage, are available for fewer processes
than analyses with more frequent detections in both media (e.g., heterotrophic plant count data). The
reported emission factors range across all evaluated processes for the four groups of microorganisms
evaluated was approximately 10"4 to 10"10 (Medema et al., 2004). This range can be interpreted as an
approximate four to 10 orders of magnitude difference between the bioaerosol concentration relative
to the wastewater concentration. The highest emission factor values for an individual process were
often derived for sulfite-reducing Clostridium spores (e.g., belt filter press operation, belt filter
maintenance, and screening of raw wastewater). For other processes (e.g., aeration tank mixer
operation, sludge screw pump, and the diffused aeration tank), the spore-based emission factor was
less consistently identified as the highest emission factor value relative to other biological groups for
a given process. Calculated emission factors for cleaning and operation of sludge dewatering belt
filter presses were associated with higher values (ranging from 10"4 to 10"8) for all biological groups,
with cleaning activities for the belt filter press associated with the highest emission factor range (i.e.,
approximate value of 10"4 to 10"6) across all biological groups. The operation of the belt filter press
was associated with a calculated emission factor range of approximately 10"6 and 10"8 across
multiple WWTP. Emission factors were calculated for the f-specific RNA phage for aeration tanks
(i.e., approximately 10"8 to 10"10), press filtrate collar (e.g., approximately 10"8), belt filter press (i.e.,
10"6), and belt filter press maintenance (i.e., 10"7) (Medema et al., 2004). Lower emission factor
values for all biological groups were determined for other processes including aeration tanks,
covered primary clarifiers, diffused aeration tanks, and discharge sludge screw pumps (i.e., 10"8 to
10"10) (Medema et al., 2004). The published ratio values may not be directly comparable across
studies because of different approaches used to calculate the emission factors (e.g., adjustment by
Bauer et al. (2002) to the units of 1 m3 treated wastewater). However, the ratios do indicate that
some bacterial pathogens may need to be present at relatively high levels in wastewater to generate
detectable levels of pathogens in bioaerosols.
4.3.3.2 Modeled Bioaerosol Data
Models have been developed to estimate bacterial or viral bioaerosol concentrations. In the Monte
Carlo simulation developed to estimate EBOV risks to sewer workers, Haas et al. (2017) fit
published data for mesophilic heterotrophic bacteria to a beta distribution (parameters: alpha =
2.3281 Logio, beta = 1.96512 Logio, range of-11.46 to -5.88 Logio) for the generation of a
wastewater and bioaerosol partition coefficient (pathogens per m3 sewer headspace/pathogens per m3
wastewater). Mesophilic heterotrophic bacteria were selected based on the assumption that the
hydrophobicities of the bacteria and EBOV would be equivalent when bacteria were reported in
units of cfu and the EBOV reported in RNA copies (Haas et al., 2017). For data sets that lacked a
reported wastewater concentration associated with the bioaerosol concentration, Haas et al. (2017)
assumed a range of bacterial concentration of 1010 to 1012 cfu/m3, based on wastewater data
identified in Hung et al. (2010).
-------
Pascual et al. (2003) developed a global linear model to predict bacterial bioaerosol concentration
based on daily inflow, wind speed, treatment stage, and bacterial type parameters. Karra and
Katsivela (2007) compared predictions from the Pascual et al. (2003) model with sampling data and
reported a difference of one to two orders of magnitude between measured values and model
predictions. Given the variability and complexity of wastewater treatment systems, this level of
agreement may be reasonable for a general model.
4.3.3.3 Summary of Bioaerosol Data and Potential Levels of Inhalation Doses
Table 4-3 identifies reported high and low measured bacterial bioaerosol concentrations (arithmetic
or geometric mean) by WWTP process based on a review of published data (Brandi et al., 2000;
Carducci et al., 2000; Bauer et al., 2002; Medema et al., 2004; Fernando and Fedorak, 2005;
Fracchia et al., 2006; Karra and Katsivela, 2007; Sanchez-Monedero et al., 2008; Heinonen-Tanski
et al., 2009; Haas et al., 2010). No values derived from modeling of WWTP bacterial bioaerosol
concentrations were included in the identified ranges in Table 4-3. The low values represent the
lowest quantified detection identified in the reviewed data. Data were reported for bacterial
bioaerosol concentrations associated with raw wastewater inflow (Medema et al., 2004; Fracchia et
al., 2006; Heinonen-Tanski et al., 2009), pretreatment or primary treatment (Carducci et al., 2000;
Fernando and Fedorak, 2005; Fracchia et al., 2006; Karra and Katsivela, 2007; Sanchez-Monedero et
al., 2008; Heinonen-Tanski et al., 2009), tertiary treatment (Sanchez-Monedero et al., 2008), sludge
management (Carducci et al., 2000; Bauer et al., 2002; Medema et al., 2004; Sanchez-Monedero et
al., 2008), and maintenance activities (Heinonen-Tanski et al., 2009; Haas et al., 2010). No data
were identified for secondary treatment from these sources.
Consistent with the reviewed literature, mean bacterial bioaerosol concentrations exhibited multiple
orders of magnitude difference within and across wastewater system activities (Table 4-3). The
overall range in mean bioaerosol concentration across all WWTP processes was approximately 101
to 106 cfu/m3 (Table 4-3). Differences between the identified high and low bioaerosol concentration
values in Table 4-3 for an individual wastewater process may be reflective of variable wastewater
microorganism concentrations and differing processes for a given treatment stage, environmental
conditions (e.g., indoor versus outdoor temperature), and choice of analysis with regard to the
biological group being sampled.
The range of reported bioaerosol concentrations for an individual treatment stage often incorporates
the influence of different treatment processes on resulting bacterial bioaerosol concentrations. For
example, Fernando and Fedorak (2005) described significant differences in the potential bioaerosol
generation between fine versus coarse bubble aeration in sludge management processes. Lower
bioaerosol generation was reported in fine bubble aeration relative to mechanical aeration of sludge
or course bubble aeration (Fernando and Fedorak, 2005).
-------
recognize that there may also be issues associated with comparability across commonly used
molecular measurements. For example, there are no standardized assays in place for EBOV that
would facilitate quantitative comparisons in reported viral loads generated using real-time reverse-
transcription polymerase chain reaction (rRT-PCR) measurements (Vetter et al., 2017).
The difference in measurement results obtained by molecular-based approaches and viability-based
approaches relative to the actual pathogen number is not well understood for current wastewater
pathogens. Given the complexity of the wastewater medium and the diverse range of environmental
conditions associated with wastewater systems, interactions between the medium and environment
(i.e., medium-environment interactions) may have the potential to significantly affect culturability in
culture-based methods and/or introduce interferences or inhibitions into molecular-based methods.
In the wastewater medium, variability can be present in pathogen type and loading, general
chemistry (e.g., pH), and the presence and type of solid particles. The environment in which the
wastewater is present can exhibit variability in temperature, relative humidity, air flow, wastewater
plant operations, and other characteristics that may affect measurements from wastewater, the
surrounding air, or surfaces. Because of this variability, interactions between the medium and
environment may not affect culture- and molecular-based methods in a consistent manner within or
across wastewater system settings. This will likely preclude the identification of a single adjustment
factor that may be universally applied to relate molecular-based method results to culture-based
results for all treatment systems and media. A greater understanding of conditions that increase or
decrease the likelihood of culturability relative to molecular presence must be present before
generalizations can be reliably made.
The second key data gap is the lack of basic fate and transport data for pathogens and associated
biological groups of interest in the wastewater system. Research on fate and transport of pathogens
that enter the wastewater system focuses on bacteria and enteric viruses, though quantitative data
describing persistence and fate for estimation of bioaerosol generation and concentration are scarce.
The scarcity of data for enveloped viruses results from the significant analytical difficulty associated
with the recovery and enumeration of enveloped viruses in the wastewater, especially when derived
from fecal sources (Wigginton et al., 2015). While there are some recent advances in the
development of virus recovery methods (e.g., Ye et al. [2016]) for wastewater resulting from work
initiated after the 2014-2015 EVD outbreak, the current body of available data is still insufficient to
perform a quantitative exposure assessment. Given that the vast majority of emerging pathogens
over the past 25 years are enveloped viruses, there is a mismatch between desired quantitative data
and the technical capability to rapidly generate needed data sets. As a result, the estimation of
bioaerosol concentration generated from a wastewater collection or treatment process exhibits very
high uncertainty for bacterial and enteric viruses. Of greatest importance, published data that
quantitatively describe the basic fate and transport behavior of enveloped viruses or bacterial spores
in a wastewater system are not identified.
The lack of data on viable pathogen in waste - along with the identified analytical challenges for
downstream fate and transport measurements for pathogens in all relevant exposure media in the
wastewater system - significantly limit the ability to perform a quantitative microbial exposure
assessment. So, lacking data, any comprehensive analysis must necessarily incorporate assumptions
regarding fate and transport and regarding potential exposure. As a result, a qualitative screening
process is necessary to provide a robust evaluation that can incorporate quantitative data and
necessary assumptions.
-------
6 Overview of Screening Process
A qualitative screening process is presented to identify pathogens with the potential to pose a serious
human health threat if individuals were to be exposed to them from a wastewater system. The
screening process will address the following two questions:
• Does the pathogen have the potential to exhibit HCP disease transmission characteristics in a
wastewater system?
• Is the HCP likely to generate YEP for individuals that contact the pathogen in the wastewater
system?
Given the recognized difficulty of performance of a quantitative microbial exposure assessment for
emerging pathogens, the assessment is designed with pathogens and leverages available quantitative
data to inform the process.
The following subsections describe the conceptual exposure model for pathogen exposure from the
wastewater system (Section 6.1) and the screening process (Section 6.2). A framework is provided to
describe data used to bridge identified data gaps for key fate and transport characteristics (Section
6.2.4). Relevant considerations for fate and transport of pathogens in wastewater are also identified
(Section 6.2.4). Case studies are then presented to demonstrate the use of the screening process for
the EBOV (Section 7) and spore form of B. anthracis (Section 8).
6.1 Conceptual Exposure Model for Exposure in the Wastewater System
A conceptual exposure model is presented to describe pathogen fate, transport, and exposure
pathways for pathogens present in the wastewater system (Figure 6-1). Complete exposure pathways
are defined to include a source or source-releasing mechanism, transport if present, exposure
medium, receptor, and route(s) of exposure. Sources for introduction of pathogens that enter the
wastewater system are: (1) infected individuals that shed viable pathogens in bodily wastes (i.e.,
defined as urine, feces, vomit) that are collected in the toilet and (2) intentionally generated
pathogens that enter the system from illicit activity, management of decontamination of wastewater
generated during a biological agent incident, and surface runoff from contaminated surfaces into the
wastewater collection system. Surface runoff with pathogens can be introduced after a wide-area
biological agent incident followed by rainfall that carries pathogens into a combined sewer system or
from infiltration of sewer lines. The receptors of interest are the residential individuals who use and
flush the toilet and the WWTP worker that may contact wastewater treatment processes during
potential maintenance activities. Exposure from the toilet can occur in residential, medical, or other
facilities. There are also opportunities for exposure to the general public and WWTP workers from
uncontained releases of wastewater (e.g., sewer main break).
Pathogens can be released from activities that are defined to include collection of wastewater (i.e.,
toilet flush for residential exposure), maintenance or overflow events during wastewater transit, and
during wastewater treatment processes (i.e., pretreatment, primary treatment, secondary treatment,
tertiary treatment, and sludge management). Source-releasing mechanisms include the splash or
aerosolization of toilet bowl contents resulting from water movement during the toilet flush,
wastewater treatment processes, overflow events, maintenance activities, and equipment
malfunctions.
-------
Source
Source-Releasing
Mechanism
Routes of
Exposure
Toilet flush
Re-aerosolization
Large particles
Deposition
Water
flow
Contact
with
hands
Contact
with
hands
Water flow
Splash
and Drench
Deposited
on Surface
Air
Feces / Urine /
Vomit Added
to Residential
Toilet
Inhalation
Aerosol and
Droplet
Generation
Wastewater
Collection
Decontamination
Wastewater
Illicit Activity
Surface Runoff
Maintenance
Event in
Collection System
Overflow Event
Contact with
Ocular or Oral
Mucous
Membranes
and Skin
Incidental
Ingestion
Treatment Process
Pre-treatment /
Primary Treatment
Secondary T reatment
Tertiary Treatment
Sludge Management
Figure 6-1. Conceptual exposure model for pathogen exposure during wastewater collection and treatment.
-------
Reported particle size measurements of toilet flush and wastewater treatment bioaerosols
indicate the potential for a wide range of particle sizes. Particle size is important to assessing the
fate and exposure potential of bioaerosol particles. Most mean or measured particle size peaks
are reported to be less than 5 |im, but reported distributions include a size range inclusive of
large droplet-sized particles (Sections 4.3.2 and 4.3.3). Though there is not a universally
accepted nomenclature for particle sizes, the following particle size categories have been
identified: respirable particles (i.e., aerodynamic diameter < 10 |im), inhalable particles (i.e.,
aerodynamic diameter between 10 |im and 100 |im), and large droplets6 (i.e., aerodynamic
diameter >100 |im) (Weber and Stilianakis, 2008). Respirable-sized particles are primarily
associated with respiratory transmission (also termed airborne transmission) (Weber and
Stilianakis, 2008) for pathogens that exhibit obligate, preferential, or opportunistic respiratory
transmission as defined by Roy and Milton (2004).
Inhalable particles are generally associated with disease transmission through inhalation, particle
contact with mucous membranes, or subsequent contact with fomites (i.e., contaminated surfaces
where particles land), whereas large droplets are associated with disease transmission through
particle contact with mucous membranes or fomites (Weber and Stilianakis, 2008). However, all
particle sizes may ultimately contribute to biological contamination of surfaces to which
receptors may have exposure if the microorganism remains viable for the duration of the particle
settling time through receptor exposure.
The distinguishing characteristics of the large droplet relative to respirable or inhalable particle is
the relatively larger size of the droplet and an increased likelihood of rapid settling on a surface
(Siegel et al., 2007). It is important to acknowledge that the settling time for a large droplet can
be relatively short in stagnant air. For example, a 10|im particle has a settling time of 491
seconds over a 1.5 m height (Weber and Stilianakis, 2008). The presence of air currents in the
location of particle release may facilitate longer airborne suspension times, especially in
enclosed building spaces (Fernstrom and Goldblatt, 2013). As a result, there can be potentially
short airborne transport distances (e.g., up to 10 feet for human respiratory droplets), though it is
noted that pathogen-specific or environmental conditions (e.g., temperature, relative humidity,
dispersed or aggregated states of pathogens) can be important determinants of the transport
distance (Siegel et al., 2007).
Based on the identified potential exposure pathways associated with the source-releasing
mechanisms of bioaerosol generation and wastewater splash identified in Sections 4.3.2 and
4.3.3, the potential routes of exposure include: inhalation of bioaerosols (including possible
incidental ingestion of larger-sized inhaled particles); ocular, oral, and dermal exposure from
contact with wastewater (e.g., splash or drenching); and ocular, oral, and dermal exposure from
contamination of hands after touching fomites. The conceptual exposure model indicates that a
complete exposure pathway to pathogens in the wastewater system will be present when at least
one of the following conditions is met: (1) pathogens persist in wastewater until exposure to
wastewater or surfaces contaminated by splashed wastewater, and (2) pathogens are shed in
bodily waste and form viable bioaerosols from a toilet flush or remain viable in wastewater
6 The term droplet is referencing toilet bowl content- or wastewater-derived particles and is not defined to include
respiratory droplets generated from the respiratory tract of an infected individual.
-------
through viable bioaerosol formation that results in inhalation exposure or contact with pathogen
from deposited particles on a surface.
6.2 Elements of Screening Process
The conceptual exposure model identifies the potentially complete exposure pathways for
pathogens that are introduced to the wastewater system. The screening process then leverages the
knowledge gained from the conceptual exposure model to determine if emerging pathogens have
the disease transmission potential to be HCP and to exhibit VEP in the wastewater system. To
conduct this assessment, pathogen-specific disease transmission potential (Section 6.2.1) is
evaluated relative to pathogen-specific fate and transport characteristics (Sections 6.2.2 and
6.2.3) to determine the presence of complete exposure pathways in the wastewater system that
may transmit disease. The fate and transport evaluation in the screening process is based on the
conceptual exposure model finding that the presence of a complete exposure pathway requires at
least one of the following pathogen fate and transport conditions: (1) the persistence of pathogen
in wastewater or surfaces until exposure or (2) formation of viable bioaerosol from the toilet
flush or wastewater system processes.
Figure 6-2 provides a flow chart to identify the sequence of screening questions and answers that
are associated with determination of the presence or absence of VEP(s) for the wastewater
system. The screening questions were generated from known or suspected modes of disease
transmission for wastewater pathogens (Section 3) and the potential exposure pathways indicated
in the conceptual exposure model (Section 6.1). The first question determines whether the
pathogen exhibits characteristics that could be associated with disease transmission in a
wastewater system. If disease transmission is not documented to occur from any of the routes of
exposure identified in the conceptual exposure model (Figure 6-1), there can be no VEP present.
Once an emerging pathogen is determined to be an HCP, the process then evaluates for the
presence of complete exposure pathways associated with routes of exposure that exhibit
confirmed disease transmission. The determination of a complete exposure pathways is based on
the fate and transport characteristics assumed for the emerging pathogen.
There are several assumptions that were made to develop the screening process from the
conceptual exposure model. Table 6-1 identifies the assumptions, the basis for each assumption,
affected exposure pathways, and the uncertainty associated with the assumption. For example,
the assumption that viable pathogens are shed in bodily fluids based on the molecular
confirmation of the presence of detected generic material or sequences was selected as a
conservative option to address the uncertainty associated with this parameter.
-------
Does Potential
HCP Exhibit al
Least One
ld»ntilie
-------
6.2.2 Pathogen Potential to Persist in Wastewater or Deposited Droplet
For a complete pathway of exposure for pathogens in wastewater, the pathogen must persist in
wastewater until the time of exposure, until contact with the wastewater (in the form of bulk
wastewater or splashes), or until contact with aerosols, droplets, or deposited wastewater
particles on surfaces. The time between collection of pathogen-containing waste and exposure of
the receptor varies and depends on where in the collection and treatment systems that the point of
contact is located. The flush of the toilet is the only event where the timing from introduction to
the collection system is known as it can be assumed to occur immediately after pathogen-
containing waste enters the toilet. After the flush of the toilet, additional contact events for the
public and WWTP workers may vary unpredictably in timing. The public and workers may be
exposed to untreated wastewater (e.g., collection or pumping station repair, sewer main break),
and workers may also be exposed to partially treated wastewater at later points in the wastewater
system.
Wastewater typically exhibits a short transit time to the WWTP that can range from a minimum
of minutes (e.g., one to 10) (Ort et al., 2010) to approximately 24 hours or less (U.S.
Environmental Protection Agency, 1980). The actual transit time is dependent on the layout of
the collection system (U.S. Environmental Protection Agency, 1980) and placement of the toilet
within the overall system. As a result, there may be significant variability in the timing of
exposure accompanied by uncertainty in the exposure duration for an individual receptor. Since
the screening process is designed to evaluate potential exposures throughout the collection and
treatment process, the screening process assumes that an HCP does not require extended
persistence to provide a potential for exposure.
Given these conditions, the following pathogen characteristic is necessary for an HCP to exhibit
persistence in wastewater or persistence when deposited on surfaces from a wastewater splash or
bioaerosol:
• Data that report persistence in wastewater or after surface deposition for a minimal
period of time (e.g., five minutes).
6.2.3 Pathogen Potential to Form Viable Bioaerosols
The formation of a viable bioaerosol is necessary for a complete exposure pathway for inhalation
of HCP from the toilet flush or wastewater treatment. For complete inhalation pathways in these
scenarios, the pathogen must persist until the time of initial aerosolization and then form a viable
bioaerosol. Formation of a viable bioaerosol is also a source-releasing mechanism by which
wastewater can be deposited on surfaces for subsequent contact with hands and/or inoculation of
oral or ocular mucous membranes.
Viable bioaerosols are defined by the presence of airborne pathogens that survive the
aerosolization process and retain sufficient infectivity to allow for detection and measurement.
The persistence time of the viable bioaerosol is not emphasized as a determinant of inhalation
exposure because there is the potential for WWTP worker or toilet user contact with bioaerosol
immediately after generation. The formation of a viable bioaerosol is a function of compatibility
between the physical structure of the microorganism, the mechanism generating the aerosol, and
the release environment (e.g., temperature, relative humidity). The review of published
bioaerosol data in Section 4.3 documents that bioaerosols from bacterial or viral biological
-------
groups can be generated by toilet and wastewater system processes. As a result, the evaluation of
the potential to form viable bioaerosols focuses on pathogen-specific aspects related to the
aerosolization mechanisms (e.g., bubble-burst mechanism with jet droplet formation) associated
with wastewater systems.
Three simplifying assumptions have been made in the development of the screening process. The
first assumption is that viable pathogens shed in bodily waste retain viability until at least the
time of the toilet bowl flush. A second assumption is that detection and measurement of
aerosolized pathogens is sufficient to document their potential for exposure; the screening
process does not assume any decay or inactivation of pathogens after aerosolization. By
definition, an HCP allows for the potential of disease transmission from pathogen exposure of
short exposure durations. Therefore, the screening process has assumed that viability sufficient to
allow measurement of bioaerosol formation identifies appropriate conditions for potential
inhalation exposure. The third assumption is that biological group-level data may be used to
describe the propensity of emerging pathogens to form viable bioaerosols from identified
aerosolization mechanisms when emerging pathogen data are unavailable. A specific biological
group taxonomy is not recommended for use in the screening process, instead the characteristics
of the target pathogen most relevant to formation of viable bioaerosols should be considered.
Examples of possible categories and rationale include mycobacteria (i.e., to reflect highly
hydrophobic vegetative bacteria), Gram-negative bacteria (i.e., to reflect higher levels of
hydrophobicity relative to Gram-positive bacteria), and enveloped viruses (i.e., to reflect higher
levels of hydrophobicity and potential susceptibility to shear stress exposure to the envelope).
The rationale for the evaluation of biological groups is that similarities in physical-chemical
structure, by which the biological groups are identified, may be associated with similarities in
likelihood to form viable bioaerosols. As described in Section 4.3, data describing aerosolization
are scarce for many potential target pathogens, especially enveloped viruses.
The following characteristic is necessary for an HCP to be considered to form viable bioaerosols
from the toilet flush or wastewater system:
• Measured bioaerosol data for the emerging pathogen or potential surrogate from
wastewater or a similar medium with an aerosolization mechanism consistent with a
toilet flush (e.g., bubble-burst mechanism) or wastewater treatment (e.g., aeration-
associated bubble-burst mechanism).
6.2.4 Bridging Pathogen-specific Data Gaps for Fate and Transport
Based on the conceptual exposure model presented in Section 6.1, key fate and transport data to
estimate human exposure in the wastewater system are: (1) persistence in bulk wastewater,
deposited wastewater aerosol particles, or wastewater splash deposited on surfaces present in the
wastewater system (e.g., nonporous bathroom counter, nonporous work surfaces with potential
for hand contact), and (2) propensity to produce viable bioaerosols during the collection or
treatment process. However, these data gaps limit the ability to perform a quantitative exposure
assessment for many potential emerging pathogens in the wastewater system (Section 5) and
may also contribute to difficulties in performance of the screening process. To maximize utility
-------
of the screening process for data-poor pathogens, a framework using customized terminology is
provided to identify and transparently describe the surrogate data used to fill identified data gaps.
The evaluation of surrogate data through the screening process should consider two areas: (1)
characteristics of the microorganism that may affect fate and transport relative to the target
pathogen, and (2) medium and environmental characteristics of test conditions relative to
conditions in the wastewater system. The evaluation assumes that key inferences on data
suitability can be derived from an evaluation of the type of microorganism (e.g., enveloped
versus nonenveloped virus, single- versus double-stranded RNA) and test conditions (e.g., raw
iversus sterilized wastewater, temperature of wastewater).
The screening process utilizes the Sinclair et al. (2012) identification of surrogate selection
considerations when evaluating environmental pathogen fate and transport, with some
modification to better reflect the wastewater system. The term 'target pathogen' is retained as it
was originally described in Sinclair et al. (2012). As shown in Table 6-1, the term "target
pathogen" is used here to mean a pathogen of interest for which the ability to perform screen
process or exposure assessment is limited by a lack of data regarding the fate and transport of the
pathogen. The concept of environmental attributes is maintained as originally described in
Sinclair et al. (2012), i.e., the common parameters of the environment which the pathogen
inhabits or the engineered or natural system under study. However, the environmental attributes
concept is further developed to accommodate anticipated data gaps for pathogens in the
wastewater system. Three additional terms are defined for use in the screening process:
surrogate, benchmark indicator, and benchmark conditions (Table 6-1).
-------
The terminology is designed to convey relevant differences between the microorganism(s) and
test condition(s) associated with available data relative to the wastewater system conditions
where it will be applied. As a result, the terminology describes the microorganism and
environmental conditions relative to their likelihood to over- or underestimate the fate and
transport characteristic. The categorization of surrogate data should also aid in clearly describing
the rationale for hypothesized differences in fate and transport relative to the target pathogen.
The term surrogate7 is generally defined as data that are used to fill an identified data gap during
defined environmental conditions. For the screening process, a surrogate is defined as a
microorganism with similar biological, physical, and chemical features that allow measurements
derived from the microorganism to be directly applied in place of the missing target pathogen
data under similar environmental conditions. Surrogates can be determined by matching the
biological attributes relevant to fate and transport for both the surrogate microorganism and
target pathogen, with the accompanying determination that there are also no known mechanisms
by which the fate and transport characteristics may differ. Surrogate selection should focus on
estimates that describe exposure for a limited number of days for wastewater contact or
immediately after aerosolization for the toilet flush and wastewater contact. Relevant data sets do
not need to estimate the longest possible persistence time or describe kinetics of persistence. The
screening model incorporates the assumption that data only need to reliably describe persistence
for the limited duration of time between wastewater collection and completion of treatment time.
The general approach of surrogate selection described by Sinclair et al. (2012) should be used in
the screening process. Surrogates should be identified based on an evaluation of the biological
attributes of the target pathogen that are relevant for fate and transport in the relevant
environment (i.e., wastewater system) (Sinclair et al., 2012). Relevant biological attributes
include genetics and taxonomy, fundamental morphology, hydrophobicity and isoelectric point,
and preparation of organisms (Sinclair et al., 2012). However, genetic and taxonomic elements
should not be overly relied upon to the exclusion of actual biological, physical, or chemical
features that may have greater utility (Sinclair et al., 2012). The selection of surrogates for
emerging pathogens may be associated with higher levels of uncertainty from both lack of
biological group data for likely emerging pathogens (e.g., enveloped viruses) as well as general
fate and transport data gaps for microorganisms in the wastewater system environment. As a
result, surrogates selected for emerging pathogens may provide estimates that may differ from
the target pathogen by multiple orders of magnitude.
Similar to the concept of benchmarking described by Sinclair et al. (2012), two additional terms
are defined for use in the screening process. The first term, benchmark indicator, is defined as a
microorganism that will provide an overestimate of the fate and transport characteristic of
interest and therefore generate a more conservative estimate of exposure relative to the target
pathogen. The term surrogate is reserved for only those microorganisms for which the data are
directly applicable based on biological similarity to the target pathogen. It is not intended for use
for microorganisms that are known to be likely to overestimate or to underestimate the fate and
transport characteristic of interest. As an example, nonenveloped enteric viruses are
acknowledged to exhibit greater resistance to the stresses of aerosolization than enveloped
viruses. Specifically, Casanova and Weaver (2015) hypothesize that the presence of a double-
7 The term indicator or simulant is often used in the wastewater treatment community to describe a biological agent
that predicts the fate and behavior of an identified pathogen.
-------
stranded DNA or RNA virus enveloped virus (e.g., Phi6 bacteriophage) may be more stable in
water and exhibit longer persistence than a single-stranded enveloped virus (e.g., EBOV, SARS,
avian influenza). This hypothesis is supported by Decrey et al. (2016) who reported potentially
lower resistance to stressors exhibited by single-stranded RNA viruses in human waste when
compared to double-stranded RNA viruses and single- or double-stranded DNA viruses.
However, Decrey et al. (2016) also reported observations from stored human waste that did not
have the addition of gray or flush water more typical of wastewater. The Phi6 bacteriophage,
identified as a conservative surrogate for enveloped viruses by Casanova and Weaver (2015),
would be considered a benchmark indicator in the screening process. In contrast, the single-
stranded enveloped virus murine hepatitis virus (MHV) evaluated in the persistence studies
reported by Ye et al. (2016) could be considered a surrogate for enveloped viruses in the
screening process with other elements of comparison remaining consistent.
The second term, benchmark condition, describes a test environment (i.e., combination of testing
medium and environmental conditions) that provides a more challenging situation for the target
pathogen relative to the identified fate and transport characteristic than the wastewater system
environment in which the data will be applied. Sinclair et al. (2012) identified relevant
environmental conditions of natural or engineered systems to include pH, temperature, relative
humidity, ultraviolet, organic matter, nutrients, air or water currents, biofilm, and turbidity. For
wastewater systems, additional relevant conditions could include treatment processes in place
(e.g., specific wastewater treatment operation and associated mechanism of aerosolization) or
other aspects of the built environment and the time duration over which the surrogate data were
derived. The latter reflects the relatively finite time duration between the introduction of the
pathogen into the wastewater system and completion of its flow through the treatment process.
Overall, the environment in which the surrogate data are applied should be appropriately
consistent with the conditions from which the surrogate data were generated.
The microbial background is also an important environmental characteristic of the wastewater
system. While there are many established methods to concentrate, recover, and analyze bacteria
and nonenveloped viruses in wastewater (e.g., enteric viruses including polioviruses,
enteroviruses), these methods are not suitable for enveloped viruses due to incompatibility of the
methods with maintenance of the viral envelope (Ye et al., 2016). As a result, much of the
available data for enveloped viruses is developed using sterilized or pasteurized wastewater to
reduce the impact of microbial background on the analysis. Alternatively, nonenveloped viruses
are used in place of enveloped viruses because the hardier nonenveloped viruses remain viable
during use of standard analytical methods. Sterile or pasteurized wastewater is commonly
assumed to facilitate longer persistence times than raw wastewater for enveloped pathogens such
as EBOV (Bibby et al., 2015b). In this context, sterile or pasteurized wastewater would be
considered a benchmark condition because it should provide a conservative estimate of
persistence (i.e., likely to overestimate).
When evaluating the use of potential benchmark conditions, care must be taken to document the
rationale for identifying the presence of a conservative fate and transport environment. It is also
important to recognize that there may be conflicting claims regarding the identification of
conditions that facilitate target pathogen persistence. For example, Casanova and Weaver (2015)
reported a comparison of study results indicating that enveloped viruses should exhibit greater
persistence in sterile water relative to wastewater. However, the time to achieve a ten-fold
-------
reduction for the enveloped human immunodeficiency virus (HIV) was reported to be longer for
primary and secondary wastewater effluents relative to sterile water (12 versus 6 hours,
respectively) at the same temperature (25°C) and generally similar pH conditions (ranges of 5 to
7.5 and 6.5 to 7) (Casson et al., 1992; Moore, 1993). Moore (1993) hypothesized that increased
survival in wastewater is associated with suspended solids and other organic loadings in
wastewater for enteric viruses, and that the presence of increased survival may also be true for
the enveloped HIV. The potential protective role for viral aggregation with particles was also
noted by Bibby et al. (2015b) for the enveloped EBOV. Given the limited and sometimes
conflicting data available for emerging pathogens, the identified basis for the determination of
benchmark conditions should also note potential uncertainties.
6.3 Selection of Emerging Pathogens for Two Case Study Evaluations
Two emerging pathogens were selected for case studies using the screening process. The first
pathogen, EBOV, was selected because it is an emerging pathogen of significant current interest
that is hypothesized to enter wastewater systems in bodily waste. The EBOV is representative of
an enveloped virus for which there are significant data gaps for fate and transport measurements
that are necessary to assess exposure. It is likely that there is the potential for differential
persistence in the exposure media in wastewater systems. For example, Ebola may persist in
wastewater throughout the period of wastewater collection and treatment, but may have limited
persistence after aerosolization. Though the EBOV is hypothesized to aerosolize from
mechanical means, but transmissible aerosols are not thought to be generated from human
respiratory system for person-to-person transmission (Vetter et al., 2017). The EBOV is
recognized for low-dose disease transmission and lethality from multiple routes of exposure,
including viral contact of infectious droplets with ocular and oral mucous membranes. Concerns
regarding EBOV exposure from wastewater were heightened during the most recent outbreak
when U.S. citizens were brought to U.S. hospitals for treatment and recovery. Given these
concerns, the EBOV was selected for a case study to apply the screening process.
The second pathogen, the spore form of B. anthracis, was selected because of its high lethality
from systemic illness, exceptional persistence in a variety of environments, and a potential entry
point into the WWTP if the facility were used to manage decontamination wastewater. Exposure
to B. anthracis spores from inhalation, ingestion, and dermal contact with open wounds can
result in lethal systemic anthrax illness (Inglesby et al., 2002). Inhalation anthrax poses the
greatest concern for potential disease transmission due to illness from low-doses (i.e., value of
less than 105 CFU) combined with a high degree of lethality (i.e., case fatality rate of 45% during
the 2001 anthrax letter) despite appropriate medical treatment (U.S. Environmental Protection
Agency, 2016). Given the recognized hardiness of the B. anthracis spore under harsh
environmental conditions, there is concern that spores could remain viable at sufficient levels in
wastewater and during potential aerosolization processes to provide for disease transmission.
Given these concerns, the spore form of B. anthracis was selected as a case study to evaluate
using the screening process.
-------
7 Case Study: Ebola Virus
The EBOV is in the hemorrhagic fever virus family (Filoviridae) and has five known species:
Zaire, Bundibugyo, Sudan, Reston, and Tai Forest (previously termed Cote d'lvoire) (Vetter et
al., 2017). Human pathogenicity of the species varies from no appearance of pathogenicity (e.g.,
EBOV- Reston) (Bausch, 2011) with asymptomatic infection (Olejnik et al., 2017) to case
fatality ratios of approximately 50% in EBOV-Sudan (Borio et al., 2002) and up to 90% in
EBOV-Zaire (Borio et al., 2002; Bausch, 2011). During the 2013-2016 African EVD outbreak,
greater than 28,500 cases and over 11,000 deaths were reported (based on data gathered up to
March 2016) (Vetter et al., 2017).
7.1 Does Pathogen Exhibit Identified Disease Transmission Characteristics?
To determine that the EBOV may exhibit HCP characteristics in a wastewater system, all disease
transmission features identified below must be present:
• Shedding of viable pathogen in feces, urine, or vomit associated with infection in the
human or relevant animal model,
• Disease transmission in the human or relevant animal model for at least one of the
following routes of exposure identified in the conceptual exposure model: inhalation of
bioaerosol, dermal contact, incidental ingestion, ocular or other mucous membrane
contact, and
• Severe or lethal illness resulting from the types of exposures generated during wastewater
collection and treatment and identified in the conceptual exposure model (Figure 6-1).
7.1.1 Pathogen Shedding in Feces, Urine, or Vomit
There is uncertainty in the available data regarding the shedding of viable infectious EBOV in
feces, urine, or vomit by infected individuals. Vetter et al. (2017) reviewed the literature
describing viral shedding and associated transmission of EBOV since its discovery in 1976
through the beginning of June 2016. Feces are identified as a "major source of infection during
acute disease" (Vetter et al., 2017). Vetter et al. (2017) reports that no culture-positive EBOV are
reported in feces despite numerous positives for viral RNA. Vetter et al. (2017) cites known
challenges in isolation and culture of virus from feces as one potential explanation for the lack of
positive culture data. However, culture-positive results for EBOV are reported in urine (Vetter et
al., 2017). One attempt to culture virus from human vomit is identified in the literature, and no
recovery of virus is reported (Bausch, 2011; Vetter et al., 2017).
Another literature review was performed by Brainard et al. (2016) to assess data describing
filovirus (specifically, EBOV and Marburg virus) presence and persistence in bodily fluids.
Brainard et al. (2016) also reported the scarcity of culture-positive confirmation of viable,
infectious EBOV in feces or urine samples and noted significant data gaps for all bodily fluids,
except for saliva and blood. Brainard et al. (2016) identified five EBOV studies meeting their
literature review requirements that measured virus in feces, with only two studies identified that
reported culture data. In one study using molecular methods, Brainard et al. (2016) reported
maximum viral loadings for feces from two patients in the range of 105 to 105 5 genomic
copies/mL waste (Wolf et al., 2014; Schibler et al., 2015). Interestingly, the 105 to 1055
-------
copies/mL feces loadings described in the human were within an order of magnitude of the
reported mean fecal swab results from five EBOV-infected non-human primate (NHP) at the
time of euthanasia as reported by Prescott et al. (2015).
Citing a personal communication from Hunter (a co-author of the Brainard et al. [2016] study),
the World Health Organization (2015) reported that shedding of viable EBOV occurred with low
frequency in feces and urine. Filovirus RNA was detected in 8.4% of urine and 20.8% of stool
samples when PCR or other molecular methods were used. From that same data set, World
Health Organization (2015) reported that culture-based methods confirmed viable virus in 2.3%
of urine samples and 0% of stool samples. As a result, the World Health Organization (2015)
reported in May 2015 that "most of the faecal matter at Ebola care facilities did not contain any
Ebola virus." However, Brainard et al. (2016) reported "too few samples" to draw strong
inferences from the available data. For example, only one patient was evaluated with both culture
and reverse-transcription polymerase chain reaction (RT-PCR) testing, but with no positive
results for either method (Brainard et al., 2016).
Schuit et al. (2016) evaluated recovery of infectious EBOV from feces as part of a larger
evaluation of viral persistence in bodily fluid matrices on specific surfaces. Interestingly,
infectious virus was not recoverable from wet or dried feces from any of the evaluated surfaces
immediately after introduction, leading Schuit et al. (2016) to hypothesize that feces could have a
virucidal action for EBOV. It is also possible that the feces matrix may decrease the sensitivity
of the microtitration assay (Schuit et al., 2016), but the exact cause is the lack of viable EBOV
and recovery is unknown. Additionally, differences in persistence may also be associated with
differences in stool type. The primary type of stool typically exhibited in EVD infection is loose,
extremely watery and cholera-like (Schuit et al., 2016). However, the Schuit et al. (2016) study
used stool from healthy volunteers
Haas et al. (2017) generated a distribution for human EBOV bodily waste concentration as part
of a Monte Carlo simulation to estimate WWTP worker exposure. Using the Towner et al.
(2004) report of an approximately 4 Logio differential in paired human blood samples when
measuring both EBOV (Sudan) RNA copies versus culture-based pfu measurements, Haas et al.
(2017) applied a 3 to 4 Logio reduction to the range of viral RNA copies reported in the literature
(2.8 to 7.2 Logio viral RNA copies) to calculate a viremia concentration of viable virus particles.
These data were pooled across bodily fluids (i.e., identified as sweat, urine, feces) and fit to a
logistic distribution (parameters: mean [or location] of 4.38 Logio, scale of 0.61 Logio). Support
for the use of the 3 to 4 Logio reduction was noted based on consistency in the literature for
reported comparisons of RNA viral copies to pfu in humans and NHP as well as pfu and TCID50
in NHP and pigs for unspecified bodily fluids and EBOV strains (Haas et al., 2017). However,
the Vetter et al. (2017) review of EBOV data in bodily fluids cautions that the lack of
standardized RT-PCR EBOV assays poses a challenge to comparison across assays (Vetter et al.,
2017). This caveat may also be applicable to assumptions regarding similar performance of
assays across bodily fluids (e.g., blood, feces).
In Bayesian belief network model generated to evaluate the risk of EBOV exposure for
wastewater workers, Zabinski et al. (2017) also modeled the viral concentration of patient liquid
waste. In this system, the initial EBOV concentration from an individual patient in liquid waste
was assumed to range from 103 to 107 particles/mL for those with severe illness. Based on a
review of published literature of hospital days when waste production of an individual patient
-------
exceeded 1 L per day, severe illness was assumed to exhibit a prior probability of 33% (Zabinski
et al., 2017). The prior probability term in the model reflects the base state of information that is
assumed for the parameter before additional information is included in the assessment. Patients
with a waste volume less than 1 L were assigned to have nonsevere illness and an initial virus
concentration of up to 103 particles/mL (Zabinski et al., 2017). Two potential corrections were
then identified to this value: (1) a hemorrhagic correction to reflect the model's assumption that
the sole source of EBOV in feces was from patients that exhibited clinically determined
gastrointestinal hemorrhage, and (2) a PCR correction for particle viability to distinguish
between "active" and "inactive" viral particles (Zabinski et al., 2017). The PCR correction was
based on data reported for poliovirus and ranged from 0 to 0.1, with bin-specific probabilities
assigned for discrete portions of the range in the model (Zabinski et al., 2017). The level of
uncertainty associated with a PCR correction based on poliovirus is unknown as well as the
assumption that gastrointestinal hemorrhage is the only source of EBOV in bodily waste.
The lack of culture-based data to document shedding of infectious virus is a major uncertainty in
the evaluation of potential exposure to EBOV from wastewater systems. However, there are
acknowledged analytical difficulties in recovering infectious enveloped viruses from the feces
matrix that significantly impede the reliable determination of viable virus. For the screening
process, the conservative assessment decision is made that the presence of EBOV RNA genomic
copies is indicative of the potential presence of shedding of infectious virus particles in feces and
urine. Given the presence of one single negative study assessing the presence of viable virus in
vomit, the screening process will assume infectious virus to be present in vomit due to the high
uncertainty in this model element also.
7.1.2 Disease Transmission and Associated Exposure Doses by Route of Exposure
Judson et al. (2015) reviewed the current scientific consensus on the likelihood of EBOV
transmission from contact with bodily fluids. The available epidemiology and experimental data
indicate that it is "very likely" that contact between EBOV-contaminated bodily fluids and
mucous membranes or broken skin may result in disease transmission, citing data that sharing
needles or handing infected or deceased individuals are high risk factors for disease transmission
(Judson et al., 2015). However, data that report disease transmission associated with toilet use or
wastewater systems are not identified in Judson et al. (2015).
Disease transmission resulting in lethal or severe EVD is confirmed for the following routes of
exposure and exposure levels for the NHP: inhalation when using a low-dose exposure level
(Johnson et al., 1995), ingestion or droplet contact with oropharynx when using a low-dose
exposure level or low-dose exposure medium (Jaax et al., 1996; Mire et al., 2016), and ocular or
conjunctival contact using a low-dose exposure level or low-dose exposure medium (Jaax et al.,
1996; Mire et al., 2016). Disease transmission from the contact of EBOV-contaminated bodily
fluids with broken skin is identified as a scientific consensus finding by Judson et al. (2015).
Given the potential for cuts or breaks in the skin of those that contact wastewater and
hypothesized disease transmission via introduction of EBOV through these skin areas, sufficient
evidence is present to identify dermal contact as a route of disease transmission for EVD in the
screening process.
-------
7.1.3 Is Ebola Virus a High-Consequence Pathogen?
Table 7-1 summarizes the results of the assessment of EBOV disease transmission characteristics
and details the determination that the EBOV is an HCP in the wastewater system. While there is
uncertainty with regard to human shedding of viable EBOV in feces, the pathogen is well
documented to transmit disease from routes of exposure associated with the toilet flush,
wastewater collection, wastewater treatment, releases of wastewater, and maintenance activities
(Table 7-1).
7.2 Does the Pathogen Persist in Wastewater or Deposited Droplet?
7.2.1 Wastewater Persistence
To answer this screening element affirmatively, a HCP must have data indicating that it has the
potential to persist in wastewater for a minimal period of time (e.g., five minutes). There are no
published data describing EBOV persistence in raw wastewater. However, studies that evaluate
the factors associated with persistence or inactivation of viruses in water or wastewater assist in
the identification of potentially relevant data sets to fill the data gap. The persistence of viruses
in water is multi-factorial, with dependencies identified for temperature, organic matter, and the
presence of other microorganisms (Gundy et al., 2008). The general factors identified for viral
persistence in water are also assumed to be relevant for EBOV in wastewater.
A recent regression model generated by Brainard et al. (2017) to describe viral persistence in
wastewater also identifies relevant characteristics for categorization of potential surrogates,
benchmark indicators, and benchmark condition determinations. The following characteristics
were identified as predictive of viral persistence in wastewater: DNA or RNA structure, presence
of an envelope, primarily transmission as fecal-oral pathogen, temperature, and relative level of
waste contamination (i.e., low, medium, high) (Brainard et al., 2017). Brainard et al. (2017)
defined a low level of contamination as media with no fecal or urine content and a high level of
contamination as media with unclear or unknown levels of fecal content or greater than or equal
to 10% fecal material. All other media that did not fit into the first two categories as a high level
of contamination, except for media that was diluted to less than or equal to 1% which would
result in placement in the medium category (Brainard et al., 2017).
Table 7-2 summarizes potential surrogates and benchmark indicators with potential relevance for
the persistence of EBOV in wastewater reported in the literature. The literature identifies the
enveloped Phi6 bacteriophage as a potential wastewater persistence surrogate for the EBOV
(Bibby et al., 2015a; Bibby et al., 2015b; Casanova and Weaver, 2015; World Health
Organization, 2015), avian influenza (Adcock et al., 2009), and the enveloped virus biological
group (Casanova and Weaver, 2015). Bibby et al. (2015a) identified a number of potential
surrogates for EBOV based on biological similarity, though some microorganisms lack published
persistence data (e.g., carrot mottle virus, tobacco mosaic virus). Ye et al. (2016) identified a
broader range of enveloped viruses for evaluation of fate and transport characteristics that
included the Phi6 bacteriophage and MHV.
-------
• wale', <2 ox
• W«MOQ*Cto25*C
• >25'C
¦ R1S"C
~ Sterile w Paiteuraed Wastewater,
iS*C
Ebola +
Benchmark
Condition
Surrogate
in Raw
Wastewater
~
~
a
a
Surrogate +
Benchmark
Condition
Benchmark
Indicator
Benchmark
Indicator +
Benchmark
Condition
Surrogate - Microorganism with sufficiently similar biological, physical, or chemical characteristics to
allow for direct application of data, not hypothesized to over- or underestimate persistence
Benchmark Indicator-Microorganism likely to provide a conservative estimate of persistence
Benchmark Condition - Test conditions likely to provide a conservative estimate of persistence
Figure 7-1. Published data describing time for a 2-log reduction of Ebola virus in
wastewater and water.
The published data for EBOV persistence are reflective of a variety of potential benchmark
conditions, including pasteurized or sterilized wastewater and cold water temperatures. Figure
7-1 identifies the temperature of the wastewater or water for an individual data set using blue to
indicate temperatures less than 20°C, green to indicate temperatures between 20°C to 25°C, and
red to indicate temperatures greater than 25°C. The longest reported persistence data were
identified for EBOV surrogate or benchmark indicators at temperatures less than 20°C
combined with other benchmark conditions (e.g., water medium) (Adcock et al., 2009;
Casanova et al., 2009) (Figure 7-1). Accordingly, it is reasonable to consider persistence studies
performed using water as the test medium and cooler temperatures as benchmark conditions
when evaluating data for the screening process. In contrast, studies that utilize warmer
temperatures regardless of the medium are likely to exhibit reduced persistence relative to
-------
moderate or cooler temperature conditions. Decreased temperatures have been suggested to be
protective for the enveloped virus influenza A due increased stability and lipid ordering of the
envelope (Weber and Stilianakis, 2008). The same mechanism may be active for other
enveloped viruses.
In the only data set reporting EBOV persistence in wastewater, Bibby et al. (2015b) spiked
EBOV (Guinea Makona) into sterilized untreated wastewater at two titers (106 and 102
TCIDso/mL) to evaluate persistence over an eight-day period under environmental conditions of
20°C and 40% relative humidity. Results indicated a rapid decrease of approximately 99%
within the first day of the test, but EBOV was still detected through day eight for the 106
TCIDso/mL concentration. The 102TCIDso/mL spike was not detectable after day one. Bibby et
al. (2015b) reported two time values for a 90% reduction of sterile wastewater seeded with 106
EBOV TCIDso/mL held at 20°C: 2.1 days or 6.6 days depending on the modeling assumption
approach used to generate the 90% reduction value (i.e., inclusion or exclusion of the time zero
concentration value).
Sterilized or pasteurized wastewater is hypothesized to provide a conservative estimate of viral
persistence for EBOV (Bibby et al., 2015b; Casanova and Weaver, 2015) and is evaluated as a
benchmark condition for the EBOV assessment. However, Section 6.2.4 identifies conflicting
evidence regarding whether sterilized or pasteurized wastewater will consistently provide a
conservative estimate of persistence in the wastewater medium. In their analysis, Bibby et al.
(2015b) noted that there is uncertainty in whether the mechanism of loss for the viruses was
inactivation, viral particle aggregation, or adsorption to other wastewater particles that may
have promoted the apparent loss of virus in their data set. When noting the increased persistence
of the enveloped HIV in sterile versus primary and secondary effluents to sterile water, Moore
(1993) hypothesized that increased survival in wastewater is associated with suspended solids
and other organic loadings in wastewater for enteric viruses, and that the same mechanism
could be present for the enveloped HIV.
The water medium is also hypothesized to promote viral persistence relative to raw, pasteurized,
or sterile wastewater. However, there are conflicting data for some enveloped pathogens (e.g.,
HIV) (e.g., Casson et al. (1992) persistence data for water versus wastewater). The potential
protective role provided by the particles in wastewater has previously been hypothesized for
EBOV (Bibby et al., 2015b). However, available data generally support increased persistence
for water relative to wastewater for the enveloped virus surrogate MHV (Casanova et al., 2009)
and the benchmark indicator Phi6 bacteriophage (Adcock et al., 20091) with approximately 17
to 22 days reported for a 2-log reduction at temperatures of 27 °C and 28 °C, respectively. In
contrast, Fischer et al. (2015) developed a regression equation to describe EBOV persistence
using study data from water at 27°C and estimated a 2-log reduction time of 2.19 days. Fischer
et al. (2015) reported EBOV persistence in deionized water for the same strain as the Bibby et
al. (2015b) dataset (EBOV Guinea Makona-WPGC07). The estimate for a 90% decrease in
EBOV at a similar temperature was sufficiently close to the value reported by the Bibby et al.
(2015b) study using sterilized wastewater, with detectable virus found up to day six at 21°C
from a starting titer between 104 and 105 TCID50 /mL. However, the persistence in water was
half as long at 27°C (three days) compared to 21°C (six days) (Fischer et al., 2015). Given these
data, data generated from EBOV in water could be categorized as data reflective of a target
pathogen in benchmark conditions.
-------
Several biological characteristics are associated with water or wastewater persistence that can
be used to distinguish a surrogate from a benchmark indicator. Using the hypothesis that
double-stranded RNA or DNA viruses are hardier than single-stranded viruses, the Phi6
bacteriophage virus would be considered to be a benchmark indicator due to the presence of
double-stranded RNA. Persistence data for the Phi6 bacteriophage are currently available for
water (Adcock et al., 2009), pasteurized wastewater (Casanova and Weaver, 2015; Ye et al.,
2016), and unpasteurized wastewater (Ye et al., 2016). Using Phi6 bacteriophage persistence
data for pasteurized wastewater, enveloped viruses would be expected to exhibit a 6 to 7 Logio
inactivation rate over three to seven days in wastewater (Casanova and Weaver, 2015).
The MHV, a single-stranded RNA virus, could be identified as a surrogate for EBOV. Due to
the increased vulnerability of single-stranded viruses, the persistence values of MHV in
wastewater are similar to single-stranded emerging pathogens (e.g., EBOV, SARS, avian
influenza). Ye et al. (2016) reported persistence data in the form of time for a 90% reduction for
the single-stranded enveloped MHV in 25°C pasteurized and raw wastewater of 19 hours and 13
hours, respectively. At 10°C, the time for a 90% reduction in pasteurized and raw wastewater
increased to 149 hours and 36 hours, respectively (Ye et al., 2016).
As noted by Arduino (2015), the HIV provides the historical example of an enveloped single-
stranded RNA virus shed in bodily waste that also prompted concerns for exposure from
wastewater. As such, the HIV may represent an appropriate surrogate for the EBOV (Arduino,
2015). Ansari et al. (1992) first reported the presence of HIV genetic material in raw
wastewater, but did not test for infectivity. In a later study that evaluated primary and secondary
wastewater treatment effluent, Palmer et al. (1995) first reported the presence of proviral and
viral HIV-1 nucleic acids in wastewater, with positives identified for two unique locations.
However, no infectious virus was detected (Palmer et al., 1995). To ensure the methods used
were capable of recovery of viable HIV, Palmer et al. (1995) also seeded wastewater with HIV
to demonstrate recovery of viable virus from wastewater. The HIV continues to share an
important data gap with the emerging pathogen EBOV as there is uncertainty regarding the
presence of infectious virus in feces. Studies have described the presence of HIV genomic
material in feces and urine (Chakrabarti et al., 2009), but few studies have evaluated the number
of infectious virus in feces and urine.
The screening process determination is that the EBOV meets the conditions for persistence in
wastewater for the screening process given the documented presence of persistence beyond the
five-minute time duration. Given the lack of data for EBOV persistence in wastewater,
identified benchmark indicator and benchmark condition data provide sufficient evidence that
the persistence time condition is met. The Bibby et al. (2015b and 2017) data set clearly
indicates persistence in the timeframe of days for EBOV in the benchmark condition of
sterilized wastewater. The Ye et al. (2016) data set describes persistence of MHV, a potential
surrogate of EBOV, in raw wastewater that is indicative of persistence on the order of hours to
days before a 90% reduction at both high and low temperatures.
-------
7.2.2 Deposited Droplet Persistence
For the screening process, pathogen persistence when deposited on surfaces requires data that
document pathogen persistence after deposition on a surface for a minimal period of time (e.g.,
five minutes). Study data were not identified for wastewater persistence of enveloped viruses on
surfaces that originated from either splash of wastewater or deposited aerosolized wastewater.
However, EBOV persistence data for deposition on surfaces is reported for varying
combinations of viral medium (e.g., tissue culture, blood) and surface type (e.g., plastic,
stainless steel).
A key consideration for the assessment of pathogen persistence is uncertainty regarding the
identification of benchmark conditions for aerosolization media other than wastewater. It can be
assumed that tissue culture fluid provides a favorable medium for persistence on surfaces after
deposition. Piercy et al. (2010) evaluated the persistence of EBOV after it was diluted in either
guinea pig sera or tissue culture media and then dried on polyvinyl chloride plastic, stainless
steel, and glass substrates. No EBOV could be recovered for evaluations performed at room
temperature and 55% relative humidity (Piercy et al., 2010). However, low-temperature
conditions were found to promote persistence leading to extended viral survival that lasted
multiple weeks for some surfaces (Piercy et al., 2010). For example, samples that were dried on
glass substrates were recovered at 26 and 50 days at 4°C (Piercy et al., 2010). Interestingly, no
virus was recovered from a metal surface at any time for tests performed at room temperature
and 4°C conditions (Piercy et al., 2010). Piercy et al. (2010) noted that several hemorrhagic
viruses have also demonstrated decreased persistence on metal surfaces (e.g., 90% decrease in
less than two hours). In contrast, tests performed with deposited droplets from another
enveloped single-stranded RNA virus (influenza A [H1N1]) reported persistence of greater than
24 hours at 22°C and 50 to 60% relative humidity on stainless steel substrates when using a
phosphate-buffered saline medium (Noyce et al., 2007).
It is unknown whether the lack of recovery from stainless steel surfaces is unique to EBOV and
the hemorrhagic fever viruses or whether the suspension medium is also affecting deposited
particle persistence. Enhanced inactivation on metal surfaces may be an important finding given
the typical substrates that could be associated with receptor contact in a WWTP. In contrast,
persistence at room temperature (temperature unspecified) was less than two days when the
virus was applied to glass or plastic surfaces. Poliquin et al. (2016) reported that hospital
bedrails, which were not visibly soiled, and concrete floors were areas of EBOV RNA
persistence. Case reports in the United States also confirmed the presence of EBOV RNA in
various body fluids, including blood, urine, vomitus, feces, endotracheal secretions and semen
(Chughtai et al., 2016). For the screening process, it has been determined that EBOV can persist
when deposited on surfaces, especially during very low temperature conditions. However, there
may be conditions that do not favor survival (e.g., metal substrates, warm temperatures) that
should be considered as potential modifiers to the determination.
7.3 Does Pathogen Form Viable Bioaerosols from a Toilet Flush or the Wastewater
Treatment Process?
For the screening process, viable bioaerosols are defined by the presence of airborne pathogens
that survive the aerosolization process and retain sufficient infectivity to allow for detection and
measurement. Experimental data were not identified that described the formation of bioaerosols
-------
from EBOV-contaminated wastewater by any aerosolization mechanism. To determine that the
EBOV can form viable bioaerosols from a toilet flush or wastewater treatment process,
bioaerosol data must be identified for a surrogate or benchmark indicator that was generated
under relevant conditions (i.e., similar aerosolization mechanism, similar environmental
conditions, microorganism with similar susceptibility to aerosolization stress).
The bioaerosol data reviewed in Sections 4.3.2 and 4.3.3 describe the mechanistic potential for
aerosolization of EBOV from a toilet flush or wastewater treatment processes. One study was
identified that reported bioaerosol generation data from a toilet flush for an enveloped virus. Lin
and Marr (2017) reported the virus emission rates and aerosol emission volumes for 20 minutes
after a single flush of an automatic toilet containing anaerobically digested sludge spiked with
the nonenveloped MS-2 and enveloped Phi-6 virus, respectively. For each virus, no pfu was
detected after an individual flush by collection of aerosol in gelatin filter at a flow rate of 2 L for
20 minutes (Lin and Marr, 2017).
Persistence data for aerosolized EBOV generated from a nebulizer are available. Piercy et al.
(2010) reported persistence data for two EBOV strains (i.e., Zaire, Reston) that were aerosolized
from a tissue culture medium using a nebulizer to form small particle aerosols (i.e.,
predominantly 1 to 3 |im particles). When evaluating these data, it is important to recognize that
there are high levels of shear stress associated with use of the nebulizer to generate a bioaerosol
(National Research Council, 2006). It is possible that that bacteria or viruses could become
fragmented or lose viability during aerosolization (National Research Council, 2006) and may
generate persistence estimates that are biased low relative to other processes of aerosolization in
wastewater systems. An exponential decay curve was fitted to data from the 90-minute
observation period, with calculated half-lives identified for EBOV-Zaire and EBOV-Reston of
15 and 24 minutes, respectively (Piercy et al., 2010). The time for a 99% loss (i.e., 2-log loss) of
EBOV-Zaire and EBOV-Reston was 104 and 162 minutes, respectively (Piercy et al., 2010).
However, the relative humidity (50% to 55%) of the test conditions is in the lower range of
relative humidity levels identified by Yang and Marr (2012) for reduced stability of identified
enveloped virus bioaerosols, including EBOV. As a result, it is not known if EBOV
aerosolization in the potentially higher relative humidity levels of an indoor WWTP process
may exhibit reduced viability relative to the persistence described by Piercy et al. (2010).
Verreault et al. (2008) identified the Phi6 bacteriophage as a potential surrogate for aerosolized
small enveloped viruses. Using the terminology of the screening process, the Phi6
bacteriophage is most appropriately termed a benchmark indicator. Reasons for the selection of
aerosolized Phi6 bacteriophage to fill enveloped virus data gaps include the common biological
features of a viral envelope, small-size, and absence of a tail (unlike earlier proposed
bacteriophages) (Verreault et al., 2008). Phillpotts et al. (2010) also identified the Phi6
bacteriophage as an aerosolization surrogate for Venezuelan equine encephalitis virus for
generally similar reasons (i.e., size, surface structures, and lipid envelope). In an evaluation of
the effects of aerosolization and sampling on the endpoint of infectivity, the Phi6 bacteriophage
is also identified as an aerosolization surrogate for the enveloped influenza virus (Turgeon et al.,
2014). However, the Phi6 bacteriophage is double-stranded and may exhibit greater stability in
the wastewater medium than single-stranded viruses like EBOV (Casanova and Weaver, 2015).
For the screening process determination, available bioaerosol data for the benchmark indicator
Phi6 bacteriophage from Piercy et al. (2010) indicate that the microorganism can be aerosolized
-------
and remain sufficiently viable after aerosolization to allow for detection. It is uncertain whether
the nebulizer aerosolization mechanism provides for an increased or decreased potential for
formation of viable bioaerosols relative to the identified aerosolization mechanisms associated
with a toilet flush (e.g., bubble-burst mechanism) or wastewater treatment (e.g., aeration-
associated bubble-burst mechanism). It is also unknown how the medium used in Piercy et al.
(2010) impacts the potential for viable bioaerosol generation relative to the toilet bowl water or
wastewater media.
7.4 Conclusion: Could Ebola Virus Form Viable Exposure Pathways in the
Wastewater System?
The EBOV was determined to be an HCP with potential disease transmission from the
following routes of exposure: inhalation, incidental ingestion, dermal contact, and ocular or oral
mucous membrane contact (Figure 7-2). However, there is high uncertainty in the determination
that viable infectious pathogens are shed in the feces and other bodily fluids. The lack of
evidence for determination of viable pathogen in feces is a result of analytical challenges
associated with the medium as well as a lack of studies specifically designed to evaluate its
presence. There is increasing evidence that enveloped viruses such as EBOV can survive in
wastewater. For example, the Ye et al. (2016) study recently reported enveloped virus
persistence in excess of one day for enveloped viruses in raw wastewater. Given the rapid
transit time from wastewater collection to the treatment plant, this persistence time could allow
for potential exposure to receptors if exposure occurred prior to the plant (e.g., combined sewer
overflow) or during wastewater treatment. There are also sufficient data to indicate that
deposited wastewater on surfaces could persist to allow for worker exposure for specific
substrates and environmental conditions (e.g., low temperature with non-metal substrates).
Similarly, the EBOV can form viable bioaerosols when aerosolized from a nebulizer with a
potentially protective medium such as tissue culture fluid (Piercy et al., 2010). There are no
bioaerosol data for enveloped viruses that originate from WWTP processes. However, there is
one study that reported no aerosolization of the enveloped Phi-6 virus introduced to the toilet in
simulated sewage sludge (Lin and Marr, 2017). One major uncertainty is whether the stress of
aerosolization is greater under the conditions of the WWTP processes or the nebulizer. These
data would be necessary to provide certainty in the appropriate identification of benchmark
conditions. Figure 7-2 summarizes the potentially complete exposure pathways for EBOV in the
wastewater system and the outputs of the screening process. The figure is based on Figure 6-1,
but for EBOV, the residential toilet is a source of contamination whereas decontamination
wastewater, illicit activity, and surface runoff are not shown as potential sources. The final
determination is that the EBOV could exhibit behavior of an HCP and will result in a VEP in
the wastewater system.
-------
era
c
-!
C6
-4
K>
C/3
S
SS
-!
V3
W
cr
£.
5"
=r
c
W5
CP
X
¦c
o
an
C
-s
c&
¦C
as
s-
ss
v;
»
s
a
v:
rt
it
n
3
5'
TO
¦c
-s
o
o
n>
©
s
¦s
c
(i aa r
¦5s 01 If o
°» ^ Si.c
S 2 2; a
<5 gj.^ S
2 o i
3 :
o
5-
I*
g o o
ill
lr ^ 5^
§?()!
« og 3"
S'S.g'S
3 ii. in
U <5
11
2 o
a. H.
O U
3 ».
2 ^
3* 3*
3^ o m
»§ §¦
JSff
9"c §¦
- M
oS»
Sis.
O 2" CQ
w <§ =T
O ^ '
^ 3
I! II
° S j? :r
3 y! 2 p
2; | J a
5
§ 3 3
. ST W'
3 O
& 5 3
& 1 a
2L J» O
sM
1 3
8 a
> HI '
| "J M I
. 3 oi r*
I 3 1
t 3> § C
i J= 3
9 a
3 O •- W £&
5 ^ 3 5' S
2 O 3-5 5"
g a m 1 ^
ttw >
**' I -S
£i) U
§ 3
O 3
3 O
s l#S
o- 3 r> a.
^ 5 i a
1«| 3
Q. 3 m g
!S» O
? ~
iA -<
*&l-a
® Q. o w
III
cr o q»
c w 'j,
" ®s
9- 3 ^
S u ^
3 <¦> a
5' = |
ais
2 1" a.
1 5 I
51 5 "
8 »B
.?3
s) u _
3 : 2
2. = £
ffl W
S "0
fi) 0)
OS J
o "¦ o
n 1 IQ
ST = ®
2. a. 3
¦¦° a "o
CD ®
1 |
w W
O 2 ^ ^
2 2. w c
11 su I
2 3 a 8.8
o ;r w ? ^
3 ^ 3- ^
'^¦o
5 5 3 a
8 a |l
}!|!
5 2 S.S-
•?? S *
3' J g' 5.
2:2 S< n
O » O w
" -g 5 3
" O
S.3
' E. S
3 J
¦ J ^
? S"» «
;• w -
O . X x
? 3 «
§ s a
a.'S 3
v s®
Q- S
0 o
31 ^
1 £
3-1
i ?
tn =
J 2, J
s S
~ I
® 3
- ~n
O < (B
3] 9 °
H ff 1. J
S^aC
CD q. n)
J _ D)
o m ^
5(D
2- 3
C/) -. 0)
*< 3 2
cn o
(/) —I C/) T) "0 H
p » » 2, 3 2
Q. 9- " 3 ? S
P o-v? "> 3
2h«! h | ®
3 S HS ® I
fl) B) —I Q) TJ
0 g S 3^3
S ® a ® 8
1 a 3 = ®
a 3 8
1-3
(DOW
^ "D O
»f Js
ws o o a
5 ? » O |
-------
8 Case Study: Bacillus anthracis Spores
Bacillus anthracis spores could enter the wastewater system if a WWTP is used to manage
decontamination wastewater after a biological agent incident. For the evaluation of exposure,
only the spore form is considered in the case study because the spore form has historically been
of greatest human health concern due to its persistence in indoor or outdoor environments,
demonstrated lethality if infection results from human inhalation exposure, and prior use in
biological terrorism (U.S. Environmental Protection Agency, 2016). The screening process
scenario does not assess how potential exposure may result from sporulation and the production
of vegetative bacteria during wastewater collection and treatment.
8.1 Does Pathogen Exhibit Identified Disease Transmission Characteristics?
8.1.1 Direct Entry via Decontamination Wastewater
For the B. anthracis spore case study, it is assumed that the spores enter the wastewater system
directly (e.g., introduction of decontamination wastewater). In contrast to bodily fluids that
enter the wastewater system through collection in the toilet, it is assumed that pretreatment of
decontamination wastewater is performed prior to discharge to the collection system and/or
WWTP. Pre-treatment is consistent with the management of decontamination wastewater from
previous anthrax incidents (e.g., use of sodium hypochlorite solution to pre-treat
decontamination wastewater generated during the 2001 anthrax letter attacks) (NACWA, 2005).
Despite treatment, it is possible that residual infective spores may remain after pretreatment,
providing the potential for exposure to human receptors during wastewater transit and
treatment.
8.1.2 Disease Transmission and Associated Exposure Doses by Route of Exposure
The four types of anthrax illness are generally differentiated based on the route of exposure
associated with initiation of infection: inhalation anthrax from inhalation exposure,
gastrointestinal or oro-pharyngeal anthrax from oral exposure, cutaneous anthrax from dermal
exposure, and infection anthrax from injection of drugs contaminated by B. anthracis spores
(U.S. Environmental Protection Agency, 2016). The spores of B. anthracis can transmit disease
for all routes of exposure identified in the conceptual exposure model presented in Figure 6-1.
Human and animal model data support the potential for disease transmission and lethality of B.
anthracis from the following routes of exposure: inhalation, incidental ingestion, dermal, and a
special form of dermal exposure associated with the eye identified as periocular. Disease
transmission and subsequent lethality from inhalation exposure is described for low-dose
exposures for the NHP animal model (Lever et al., 2008) and the human (Inglesby et al., 2002).
The U.S. Environmental Protection Agency (2016) reviewed animal model and human data for
gastrointestinal anthrax from ingestion exposures. Animal model data indicate that relatively
high exposure doses are necessary to transmit B. anthracis from ingestion and dose levels may
exceed over 108 cfu for some animal models (i.e., rabbit, NHP) (U.S. Environmental Protection
Agency, 2016). However, the presence of an immunocompromised state in the human receptor
may generate susceptibility to anthrax infection U.S. Environmental Protection Agency, 2016.
One case of gastrointestinal anthrax resulted from exposure to aerosolized spores from the use
of a contaminated drum, but the exact route of exposure remains unknown (e.g., ingestion or
inhalation and subsequent mucociliary transport of spores to the gastrointestinal tract) (U.S.
-------
Centers for Disease Control and Prevention, 2010). Though B. anthracis data are not available
for the wastewater collection or treatment setting, gastrointestinal anthrax in an occupational
mill environment has been reported with the hypothesis that hand-to-mouth contact with spore-
contaminated materials is a potential route of exposure that could lead to disease transmission
(MacDonald, 1942). Accordingly, these data are sufficient for the screening process
determination that incidental ingestion of spores is a potential exposure pathway that could
result in disease transmission.
Cutaneous anthrax is the most common form of anthrax in natural settings (Inglesby et al.,
2002). Though cutaneous anthrax rarely progresses to fulminant systemic anthrax illness,
cutaneous anthrax is associated with reported lethality rates of 1% with antibiotic treatment and
10% to 20% without treatment (Beatty et al., 2003). Dermal exposure to B. anthracis spores can
result in cutaneous anthrax, though there is uncertainty about the necessity of breaks or
abrasions in the skin for disease transmission. For example, the seven-month old child who
developed cutaneous anthrax during the 2001 anthrax incident did not have any known cuts or
abrasions (Inglesby et al., 2002). Given the documented possibility for serious and potentially
lethal systemic anthrax from dermal exposures and the potential for receptors to have breaks or
cuts in the skin during exposure, dermal contact with B. anthracis spores is identified as a
means of disease transmission for lethal and serious anthrax illness.
Periocular cutaneous anthrax, also termed oculocutaneous anthrax, may result from cutaneous
anthrax infection of the skin in close proximity to the eyelid and/or periorbital area (David et al.,
2010; Gelaw and Asaminew, 2013). Anthrax infection of the eyelids can be complicated by
cicatricial ectropion, or a turning in of the eyelid toward the eye, with potential for subsequent
corneal scarring or impairment of vision despite appropriate treatment (Gelaw and Asaminew,
2013). Given the documented conditions for potential disease transmission and serious illness,
ocular exposure for the B. anthracis case study is defined more broadly than ocular mucous
membranes to include exposures to the overall ocular region that may contribute to severe or
lethal anthrax disease.
Disease transmission evidence from human incidence and animal studies indicates the potential
for severe or lethal illness for the following routes of exposure: inhalation, incidental ingestion,
contact with ocular or oral mucous membranes, and periocular dermal exposure. Dermal contact
through cuts in the skin may also result in disease transmission. The screening process
determination is that the potential for disease transmission is present for all routes of exposure
identified in the conceptual exposure model.
8.1.3 Is the Spore Form of Bacillus anthracis a High-Consequence Pathogen?
B. anthracis spores may act as an HCP in a wastewater system. The spore form of B. anthracis
is well documented to transmit disease for all routes of exposure associated with wastewater
systems after introduction of decontamination wastewater (Table 8-1).
-------
8.1 Does the Pathogen Persist in Wastewater or a Deposited Droplet?
8.1.1 Wastewater Persistence
To answer this screening question affirmatively, an HCP must have data indicating the potential
to persist in wastewater for a minimal period of time (e.g., five minutes). Bacillus anthracis
spores are known for their overall hardiness in a variety of environmental settings and
conditions. However, Sinclair et al. (2008) reviewed persistence data for B. anthracis spores
and noted a lack of current experimental data on spore persistence in water or wastewater, with
much of the experimental data collected in the late 1800s or early 1900s. One persistence study
performed in 1894 was identified by Sinclair et al. (2008) that reported persistence of B.
anthracis spores in sewage and distilled water to be 16 months and 20 months, respectively.
Minett (1950) reported that viable spore survival is longer in sterilized relative to unsterilized
water, but quantitative comparison data were not provided. Persistence data were also available
for a tube of spores placed in a sterile pond water medium and that were then set in a shaded
pond (Minett, 1950). No reduction of viable spores was identified over a two-year period
(Minett, 1950).
Though it may be possible to identify additional data from potential surrogate species of spores
similar to B. anthracis as noted by Sinclair et al. (2008), the available documentation is
sufficient for an affirmative determination of wastewater persistence for the duration identified
by the screening process of at least five minutes.
8.1.2 Deposited Droplet Persistence
For the screening process, the determination that the pathogen persists when deposited on
surfaces requires that data be available for persistence after deposition on a surface for a
minimal period of time (e.g., five minutes). Study data were not identified that described
wastewater persistence on surfaces for B. anthracis spores that were transported by either splash
of wastewater or deposited aerosolized wastewater.
Given the acknowledged persistence of B. anthracis spores in general and in water-based
settings, it is anticipated the spores should also exhibit sufficient persistence to allow for an
affirmative answer to the question for deposited droplets. Wood et al. (2015) reported
persistence times for B. anthracis spores deposited and dried on various surface types (e.g.,
glass, wood, unpainted concrete) and under differing conditions of ultraviolet light exposure.
Surface-dependent differences were reported in both initial recoveries and after ultraviolet light
exposure (Wood et al., 2015). The impact of ultraviolet light was also surface-dependent with
some surface types (e.g., concrete) thought to provide shelter from the light and promote
persistence (Wood et al., 2015). Decay was reported to be bi-phasic, with a first phase of more
rapid losses over the two days of exposure, than a slower phase with reduced loss rates (Wood
et al., 2015). However, rates of loss were sufficiently low that B. anthracis viable spores
remained over an extended period of time and persisted up to 56 days. The consideration of
ultraviolet light exposure by B. anthracis spores is of greatest relevance to WWTP with outdoor
equipment. For the screening process, it is therefore determined that B. anthracis can persist
when deposited on surfaces.
-------
8.2 Does Pathogen Form Viable Bioaerosols from the Wastewater Treatment Process?
For the screening process, viable bioaerosols are defined by the presence of airborne pathogens
that survive the aerosolization process and retain sufficient infectivity to allow for detection and
measurement. Data were not identified that described the formation of bioaerosols from
wastewater contaminated by B. anthracis spores for any of the relevant aerosolization
mechanisms.
To answer the screening question that B. anthracis spores can form viable bioaerosols from a
WWTP process affirmatively, bioaerosol data must be identified for a surrogate or benchmark
indicator that can be aerosolized for a wastewater treatment process. Since the spores were
introduced to the wastewater system from decontamination wastewater, the generation of
bioaerosols from the toilet flush is not considered in this evaluation.
The bioaerosol data reviewed in Section 4.3.3 indicate the mechanistic potential for
aerosolization of B. anthracis spores from wastewater treatment processes. Early studies
describing the jet drop mechanism referenced efforts that dropped B. subtilis spores in water
bodies to demonstrate bioaerosolization of spore forms (Baylor et al., 1977a). Given the
hydrophobic nature of the outer exosporium of Bacillus spores (Greenberg et al., 2010) and the
ease of bioaerosolization of hydrophobic microorganisms when in water (Falkinham III, 2003),
the B. anthracis spores could be aerosolized easily (Chattopadhyay et al, 2017). These same
microorganisms are also likely to exhibit limited sensitivity to shear stress and also remain
viable after aerosolization from the bubble-burst mechanism.
The nonenveloped viruses and bacterial data constitute benchmark indicator data that provide
for a conservative estimate of the potential for viable bioaerosols to be generated by B.
anthracis spores during wastewater treatment processes.
8.3 Conclusion: Could the Spore Form of Bacillus anthracis Form Viable Exposure
Pathways in the Wastewater System?
The spore form of B. anthracis is found to exhibit HCP characteristics in a wastewater system.
Disease transmission is documented to occur for all routes of exposure identified in the
conceptual exposure model: inhalation, incidental ingestion, dermal contact, and ocular or oral
mucous membrane contact. There is low uncertainty in the identified routes of exposure that
may contribute to disease transmission. The case study evaluated the introduction of B.
anthracis spores introduced to the system through the management of decontamination
wastewater. All potentially complete exposure pathways identified in the conceptual exposure
model for wastewater systems were VEP for B. anthracis spores. There is low uncertainty that
B. anthracis spores can persist in wastewater and deposited droplets for time durations relevant
to wastewater system exposure. Figure 8-1 summarizes screening process outputs. Figure 8-1
includes the conceptual exposure model, which is identical to Figure 6-1, with the exclusion of
the residential toilet as one of the sources. The final determination is that the spore form of B.
anthracis could exhibit the behavior of an HCP and will result in VEP in the wastewater system
for all identified pathways and associated routes of exposure.
-------
S Is
O C Pi f,
« 3 §¦§
2.5; So
s i
3 a
? a
"g a-§
J»o
& ~3
•Q <
=s. — <»
2! 3
5' o
cq a
m o
3 E. 3 a 5L
o- S{ | § a
I o ogo
® 0 § 3"
5? o* !J *9
w 3
d* o
^ 3 o
o £ (fl o r+
fi> § 3 ® ® -n
% ali ;I
511?sff
¦ s o £.s
-¦ 2. S" ®
3 5T ° Do
£. tu
§ a
° I b
sT ? ^
5: O 3
? 5'
O y 3
-*> <3 C?
^ 5- 3'
n?
SL o. <2
S" 5"
* §
s ? s-
w 2. w
0 ^ a
5 o-2
01 -r, ^
a Q.
O CD
5" S
Z 2
3 2.
3 w
Ills
Tj 5: 2- jl
3 f s |
8 •? § ^
a-3
sti
o®
c a ij r
3 2 73 o
9 3 o s
Ss3 =
3 o tr 3
IS II
?. 3 3 S
3 S 3
s>5«
§ a 5f 3
2 3- Q. 3.
|?!1
2. 3- O tu
O *5 3" £•
o S- 6i o
3 S 3 3
3.lli
i < o
CQ « 5*
-1" ?.
a ^!°
¦ s s -g
I i I
"* -i
tQ 3" 2. 3
O W (Q (Q
3 a i
3 ti tr
}• °
i|s.
ao»
Si>
g W o.
3 3a
d o
®
E p-
5 OT
oi = < a
§. » Si "
w C si o
O > (B 3
® a s sr
? < a 3
S -? ? 3'
a S:
o
3
f 2
co =
o o g
< —h O
£ _ Q)
o m =¦
—¦ < 3
§ 3 s
WHMUtlH
O. =h g 3 ?' 2
3 ^3 3
3 !K _i 0 ?.
0) g> ^ U n IP
¦s 3 n 3 C O
S ® a « §
m 3- 3 3. 3
3-3 8>
a
CD
-i
O
CD
o
(/)
0)
r^h
o'
T3
o
0
r+*
QJ
3
D
Q.
3 S
? a
I 3
I §
5 §
Ctt O
r« 3
0| r^.
O 3"
O
3 ?
g a-
s 2
S (it
3 SL
Si
a o
3
<" O S.
5 3=
-------
9 Data Gaps and Suggested Research to Further Refine Screening Process
The review of available literature and development of the screening process highlighted data
gaps that could be bridged with future research to increase the reliability of screening process
evaluations. Five key areas were identified for future research:
Development of analytical techniques for counting enveloped viruses in wastewater
and bioaerosols with known levels of recovery, which would support quantitative
microbial exposure assessment. Current technologies allow for infectious virus recovery from
liquid and aerosol media with live virus assay readouts and molecular methods (e.g. qPCR and
next-generation sequencing approaches) that analyze genetic material recovery in background
and spiked matrices. While these methods exist, they are typically used for qualitative or
relative assessments. As a result, the methods will need to be tested and validated using studies
exposing several surrogate agents to typical conditions and evaluating recovery. Methods
should incorporate testing in the presence of background material, both abiotic and biotic, to
ensure that recoveries are consistent with those expected in specific matrices and media.
Multiple surrogates should be evaluated to identify appropriate surrogates for diverse pathogens
and their recovery in each system. For example, multiple enveloped viruses may be used to
compare enveloped virus emerging pathogens known for increased stability (e.g., alphaviruses)
versus those with decreased stability (e.g., filoviruses). Surrogates may need to span multiple
virus families to appropriately determine quantitative microbial exposure. Data will define
surrogate agents with known recovery that can then be used for quantitative fate and transport
evaluations and microbial exposure assessment.
Development of data sets to better understand the driving mechanisms and
quantitative relationship between culture-based and molecular-based approaches for
biological groups in matrices of interest (e.g., human feces, wastewater, bioaerosols).
Currently, each culture-based approach must be linked with a specific molecular method, often
independent of other related methods. In addition, molecular methods are often specific to each
pathogen, and commercial-off-the shelf methods are often focused for biodefense or diagnostic
applications. With the large number of molecular and protein-based methods available for
pathogen detection (e.g., enzyme linked immunosorbent assay [ELISA], PCR,
immunofluorescence), the most appropriate in-use methods should be evaluated in direct
comparison between microorganisms spiked into WWTP matrices (e.g., feces, wastewater,
bioaerosols) and processed in parallel for generation of data to quantitatively compare culture-
based and molecular approaches. The ultimate goal would be the development of viability-
corrected measures suitable for use in the matrices of interest (e.g., Desneux et al. [2016],
developed for evaluation of animal manure).
Development of data sets that describe type and magnitude of exposure relative to
the use of toilet and the range of potential technologies used in each treatment unit
processes (e.g., primary, secondary treatment, sludge management). The results of the
literature review indicate the existence of significant data gaps for experimentally derived
exposure data associated with individual elements (e.g., toilet flush, treatment processes) in
wastewater systems. Data are not currently available for enveloped viruses, a biological group
that is identified as more likely to contribute to emerging pathogens than other biological
-------
groups. Exposure monitoring data should be developed to confirm the presence and potential
magnitude of hypothesized exposure pathways in the wastewater system.
Given the diversity of WWTP processes, exposure data should be developed that reflects the
breadth of treatment technologies in common use. Data should capture processes and generate
concurrent measurements for multiple media (e.g., wastewater, bioaerosol, deposited
wastewater on surfaces). Additionally, individual bioassessment data could also be captured
(e.g., wipe of hands, breathing zone air measurement) to better estimate exposures. Given the
inability to generate data for emerging pathogens of greatest current interest, surrogates should
be selected based on the most relevant fate and transport characteristics of the process of
interest.
Evaluate aggregate exposure of individual WWTP workers based on contact with
multiple unit processes during typically defined job descriptions. Data on typical job tasks,
work locations, and associated time durations for workers across the range of typical WWTP
sizes would add to the available data to quantify worker exposures to pathogens during the work
day. Ideally, a survey vehicle (i.e., methodology) could be designed to capture data describing
the size of the plant, treatment systems, region of county, seasonal variation, indoor/outdoor
environmental conditions (e.g., temperature, relative humidity), and process-specific location
exposure during the work day.
Performance of studies for persistence and other measures in environmental
conditions (e.g., high humidity, winter temperature conditions) typical for WWTP in
indoor and outdoor settings to generate data suitable for assessment across the United
States. Studies evaluating pathogen persistence in different environments may require an initial
evaluation of indoor and outdoor microbial load and environmental conditions. Pathogen- or
surrogate-spiked samples, in both liquid and aerosol forms, should be exposed to typical indoor
WWTP environments and exposures that WWTP workers encounter, including appropriate
background biotic material. Persistence data can be gathered in conjunction with live agent
recovery methods (e.g., culture-based) or molecular-based methods that are correlated with
infectious agent recovery. These data might enable quantitative risk assessment for WWTP
worker exposure to infectious aerosols and water for a wide variety of emerging pathogens.
-------
10 Glossary
bioaerosol: Bioaerosols are small solid or liquid particles that contain microbiological
organisms.
complete exposure pathway: An exposure pathway that includes a source of contamination, an
environmental media and transport mechanism, a point of exposure, a route of exposure, and a
receptor population.
conceptual exposure model: Model to describe the source, source-releasing mechanism,
exposure medium/media, and route(s) of exposure for all exposure pathways associated with the
wastewater exposure scenario.
droplet: Wastewater particle that is too large to be suspended in air for an extended period of
time or to be transported by air for distance (e.g., 5 to 10 meters) under still air conditions. The
term can be used to describe the phases of a released bioaerosol particle (i.e., droplet to droplet
nuclei after initial evaporation of water from the particle) or to distinguish between particles that
can be aerosolized versus large droplets that will stay airborne a short period of time (e.g.,
splash). In the context of the wastewater assessment, the droplets are released from the toilet
bowl contents or wastewater. In contrast to specific use of the terms droplet or droplet
transmission in human disease transmission studies, the term droplet does not imply that the
source is the human respiratory tract of an infected individual that then must land on mucous
membranes for disease transmission (e.g., see definition of droplet transmission in Siegel et al.
[2007]).
exposure: Contact of a microorganism with the outer boundary of a receptor and available for
absorption or intake. Exposure can be evaluated qualitatively (i.e., the presence/absence of a
complete exposure pathway for a specified route of exposure) or quantitatively (i.e., a measure
of the agent available at the outer boundary). Modified from U.S. Environmental Protection
Agency (2012).
exposure scenario: An exposure scenario is the set of conditions or assumptions about sources,
exposure pathways, amounts or concentrations of microorganisms, and the characteristics of the
exposed individual, population, or population that constitute one or more exposures. Source:
U.S. Environmental Protection Agency (2012).
exposure pathway: The course a microorganism (also termed biological agent) takes from a
source to an exposed receptor. Each complete exposure pathway includes a source or release
from a source, an exposure point (such as water, air, or a surface), an exposure route, and a
receptor. If the exposure point differs from the source, a transport/exposure medium (e.g., air) or
media (in cases of intermedia transfer) also is included. Modified from U.S. Environmental
Protection Agency (1989).
high-consequence pathogen: A pathogen that exhibits disease transmission potential in a
wastewater system as described by: (1) shedding of viable pathogen in feces, urine, or vomit
described in the human or relevant animal model or other means of entry to the wastewater
system, (2) documented disease transmission from acute exposure in the human or relevant
animal model for at least one of the following routes of exposure: inhalation of bioaerosol,
dermal contact, incidental ingestion, ocular or oral mucous membrane contact, and (3) severe or
-------
lethal illness documented to result from infection from routes of exposure associated with
disease transmission.
receptor: Humans who may have potential or actual exposure to microorganism. Source: U.S.
Environmental Protection Agency (2014).
route of exposure: Describes how the pathogen comes in contact with the vulnerable host
receptor cells that support intake and subsequent infection (e.g., inhalation, dermal contact,
oral). Source: U.S. Environmental Protection Agency (2014).
source: The entity (or entities) that supply microorganisms to an identified exposure pathway.
In the context of the wastewater assessment, potential sources include the infected individual
who sheds viable pathogen in defined bodily fluids (i.e., feces, urine, vomit) or intentionally
produced pathogens directly added to wastewater system. Modified from: U.S. Environmental
Protection Agency (2014).
viable exposure pathway: A complete exposure pathway for a microorganism that includes
routes of exposure with documented disease transmission potential.
wastewater system: The toilet, collection system, and wastewater treatment plant treatment
processes.
-------
11 References
Adcock, N. J., E. W. Rice, M. Sivaganesan, J. D. Brown, D. E. Stallknecht and D. E. Swayne
(2009). The Use of Bacteriophages of the Family Cystoviridae as Surrogates for H5N1
Highly Pathogenic Avian Influenza Viruses in Persistence and Inactivation Studies.
Journal of Environmental Science and Health, Part A 44(13): 1362-1366.
Ansari, S. A., S. R. Farrah and G. R. Chaudhry (1992). Presence of Human Immunodeficiency
Virus Nucleic Acids in Wastewater and Their Detection by Polymerase Chain Reaction.
Applied and Environmental Microbiology 58(12): 3984-3990.
Arduino, M. J. (2015). Ebola and Other Emerging Health Threats in Sewage and Wastewater:
Protecting Workers from Potential Exposures. The National Association of Clean Water
Agencies National Pretreatment and Pollution Prevention Workshop and Training Hyatt
Regency Greenville, Greenville South Carolina, May 12-15, 2015.
Arena, C., J. P. Amoros, V. Vaillant, K. Balay, R. Chikhi-Brachet, L. Varesi, J. Arrighi, T.
Blanchon, F. Carrat, T. Hanslik and A. Falchi (2012). Simultaneous Investigation of
Influenza and Enteric Viruses in the Stools of Adult Patients Consulting in General
Practice for Acute Diarrhea. Virology Journal 9: 116.
Barker, J. and M. V. Jones (2005). The Potential Spread of Infection Caused by Aerosol
Contamination of Surfaces after Flushing a Domestic Toilet. Journal of Applied
Microbiology 99(2): 339-347.
Bauer, H., M. Fuerhacker, F. Zibuschka, H. Schmid and H. Puxbaum (2002). Bacteria and
Fungi in Aerosols Generated by Two Different Types of Wastewater Treatment Plants.
Water Research 36(16): 3965-3970.
Bausch, D. G. (2011). Ebola Virus as a Foodborne Pathogen? Cause for Consideration, but Not
Panic. Journal of Infectious Diseases 204(2): 179-181.
Baylor, E. R., M. B. Baylor, D. C. Blanchard, L. D. Syzdek and C. Appel (1977a). Virus
Transfer from Surf to Wind. Science 198(4317): 575-580.
Baylor, E. R., V. Peters and M. B. Baylor (1977b). Water-to-Air Transfer of Virus. Science
197(4305): 763-764.
Beatty, M. E., D. A. Ashford, P. M. Griffin, R. V. Tauxe and J. Sobel (2003). Gastrointestinal
Anthrax: Review of the Literature. Archives of Internal Medicine 163(20): 2527-2531.
Best, E. L., J. A. T. Sandoe and M. H. Wilcox (2012). Potential for Aerosolization of
Clostridium difficile after Flushing Toilets: The Role of Toilet Lids in Reducing
Environmental Contamination Risk. Journal of Hospital Infection 80(1): 1-5.
Bibby, K., N. A. de Carvalho and K. Wigginton (2017). Research Needs for Wastewater
Handling in Virus Outbreak Response. Environmental Science & Technology 51: 2534-
2535.
Bibby, K., L. W. Casson, E. Stachler and C. N. Haas (2015a). Ebola Virus Persistence in the
Environment: State of the Knowledge and Research Needs. Environmental Science &
Technology Letters 2(1): 2-6.
Bibby, K., R. J. Fischer, L. W. Casson, E. Stachler, C. N. Haas and V. J. Munster (2015b).
Persistence of Ebola Virus in Sterilized Wastewater. Environmental Science &
Technology Letters 2(9): 245-249.
-------
Bibby, K. and J. Peccia (2013). Identification of Viral Pathogen Diversity in Sewage Sludge by
Metagenome Analysis. Environmental Science & Technology 47(4): 1945-1951.
Blanchard, D. C. and L. D. Syzdek (1982). Water-to-Air Transfer and Enrichment of Bacteria in
Drops from Bursting Bubbles. Applied and Environmental Microbiology 43(5): 1001-
1005.
Borio, L., T. Inglesby, C. J. Peters, A. L. Schmaljohn, J. M. Hughes, P. B. Jahrling, T. Ksiaszek,
K. M. Johnson, A. Meyerhoff, T. O'Toole, M. S. Ascher, J. Bartlett, J. G. Breman, E. M.
Eitzen Jr, M. Hamberg, J. Hauer, D. A. Henderson, R. T. Johnson, G. Kwik, M. Layton,
S. Lillibridge, G. J. Nabel, M. T. Osterholm, T. M. Perl, P. Russell, K. Tonat and For the
Working Group on Civilian Biodefense (2002). Hemorrhagic Fever Viruses: Medical
and Public Health Management. JAMA : Journal of the American Medical Association
287(18): 2391-2405.
Brainard, J., K. Pond, L. Hooper, K. Edmunds and P. Hunter (2016). Presence and Persistence
of Ebola or Marburg Virus in Patients and Survivors: A Rapid Systematic Review. PLoS
Neglected Tropical Diseases 10(2): e0004475.
Brainard, J., K. Pond and P. R. Hunter (2017). Censored Regression Modeling to Predict Virus
Inactivation in Wastewaters. Environmental Science & Technology 51(3): 1795-1801.
Brandi, G., M. Sisti and G. Amagliani (2000). Evaluation of the Environmental Impact of
Microbial Aerosols Generated by Wastewater Treatment Plants Utilizing Different
Aeration Systems. Journal of Applied Microbiology 88(5): 845-852.
Breiman, R. F. (1996). Impact of Technology on the Emergence of Infectious Diseases.
Epidemiologic Reviews 18(1): 4-9.
Broadhurst, M. J., T. J. G. Brooks and N. R. Pollock (2016). Diagnosis of Ebola Virus Disease:
Past, Present, and Future. Clinical Microbiology Reviews 29(4): 773-793.
Carducci, A., E. Tozzi, E. Rubulotta, B. Casini, L. Cantiani, E. Rovini, M. Muscillo and R.
Pacini (2000). Assessing Airborne Biological Hazard from Urban Wastewater
Treatment. Water Research 34(4): 1173-1178.
Casanova, L., W. A. Rutala, D. J. Weber and M. D. Sobsey (2009). Survival of Surrogate
Coronaviruses in Water. Water Research 43(7): 1893-1898.
Casanova, L. M. and S. R. Weaver (2015). Inactivation of an Enveloped Surrogate Virus in
Human Sewage. Environmental Science & Technology Letters 2(3): 76-78.
Casson, L. W., C. A. Sorber, R. H. Palmer, A. Enrico and P. Gupta (1992). HIV Survivability in
Wastewater. Water Environment Research 64(3): 213-215.
Chakrabarti, A. K., L. Caruso, M. Ding, C. Shen, W. Buchanan, P. Gupta, C. R. Rinaldo and Y.
Chen (2009). Detection of HIV-1 RNA/DNA and Cd4 mRNA in Feces and Urine from
Chronic HIV-1 Infected Subjects with and without Anti-Retroviral Therapy. AIDS
Research and Therapy 6: 20.
Chattopadhyay, S., and R. W. Puis. (1999) Adsorption of Bacteriophages on Clay Minerals.
Environmental Science & Technology 33(20): 3609-3614.
Chattopadhyay, D., S. Chattopadhyay, W. G. Lyon, and J. T. Wilson. (2002) Effect of
Surfactants on the Survival and Sorption of Viruses. Environmental Science &
Technology 36(19): 4017-4024.
-------
Chattopadhyay, S., S. D. Perkins, M. Shaw and T. L. Nichols. (2017). Evaluation of Exposure
to Brevundimonas diminuta and Pseudomonas aeruginosa During Showering. Journal of
Aerosol Science 114:77-93.
Chughtai, A. A., M. Barnes and C. R. Macintyre (2016). Persistence of Ebola Virus in Various
Body Fluids During Convalescence: Evidence and Implications for Disease
Transmission and Control. Epidemiology and Infection 144(8): 1652-1660.
Clark, C. S., A. B. Bjornson, G. M. Schiff, J. P. Phair, G. L. Van Meer and P. S. Gartside
(1977). Sewage Worker's Syndrome. Lancet 309(8019): 1009.
Darlow, H. M. and W. R. Bale (1959). Infective Hazards of Water-Closets. The Lancet 1(7084):
1196-1200.
David, S., J. Peter, R. Raju, P. Padmaja and P. Mohanraj (2010). Oculocutaneous Anthrax:
Detection and Treatment. Clinical Ophthalmology 4: 713-716.
Decrey, L., S. Kazama and T. Kohn (2016). Ammonia as an In-situ Sanitizer: Influence of Virus
Genome Type on Inactivation. Applied and Environmental Microbiology 82(16): 4909-
4920.
Desneux, J., A. Biscuit, S. Picard and A.-M. Pourcher (2016). Fate of Viable but Non-
Culturable Listeria Monocytogenes in Pig Manure Microcosms. Frontiers in
Microbiology 7: 245.
Dias, E., J. Ebdon and H. Taylor (2015). The Application of Removal Coefficients for Viruses
in Different Wastewater Treatment Processes Calculated Using Stochastic Modelling.
Water Science and Technology 71(9): 1382-1388.
Falkinham III, J. O. (2003). Mycobacterial Aerosol and Respiratory Disease. Emerging
Infectious Diseases 9(7): 763-767.
Feachem, R. G., D. J. Bradley, H. Garelick and D. D. Mara (1983). Sanitation and Disease:
Health Aspects of Excreta and Wastewater Management. Series: World Bank Studies in
Water Supply and Sanitation 3. New York: John Wiley & Sons.
Fernando, N. L. and P. M. Fedorak (2005). Changes at an Activated Sludge Sewage Treatment
Plant Alter the Numbers of Airborne Aerobic Microorganisms. Water Research 39(19):
4597-4608.
Fernstrom, A. and M. Goldblatt (2013). Aerobiology and Its Role in the Transmission of
Infectious Diseases. Journal of Pathogens 2013: 493960.
Fischer, R., S. Judson, K. Miazgowicz, T. Bushmaker, J. Prescott and V. J. Munster (2015).
Ebola Virus Stability on Surfaces and in Fluids in Simulated Outbreak Environments.
Emerging Infectious Disease 21(7): 1243-1246.
Fischer, W. A. and D. A. Wohl (2016). Responding to the Global Threat of High-Consequence
Pathogens: Protecting Health Care Workers and Caring for Patients. Ann. Am. Thoracic
Soc. 13(5): 584-585.
Fracchia, L., S. Pietronave, M. Rinaldi and M. Giovanna Martinotti (2006). Site-Related
Airborne Biological Hazard and Seasonal Variations in Two Wastewater Treatment
Plants. Water Research 40(10): 1985-1994.
Gelaw, Y. and T. Asaminew (2013). Periocular Cutaneous Anthrax in Jimma Zone, Southwest
Ethiopia: A Case Series. BMC Research Notes 6: 313.
-------
Gendron, L., D. Verreault, M. Veillette, S. Moineau and C. Duchaine (2010). Evaluation of
Filters for the Sampling and Quantification of RNA Phage Aerosols. Aerosol Science
and Technology 44(10): 893-901.
Gerba, C. P., C. Wallis and J. L. Melnick (1975). Microbiological Hazards of Household
Toilets: Droplet Production and the Fate of Residual Organisms. Applied Microbiology
30(2): 229-237.
Greenberg, D. L., J. D. Busch, P. Keim and D. M. Wagner (2010). Identifying Experimental
Surrogates for Bacillus anthracis Spores: A Review. Investigative Genetics 1: 4.
Gundy, P. M., C. P. Gerba and I. L. Pepper (2008). Survival of Coronaviruses in Water and
Wastewater. Food and Environmental Virology 1(1): 10-14.
Haas, C. N., T. Rycroft, K. Bibby and L. Casson (2017). Risks from Ebolavirus Discharge from
Hospitals to Sewer Workers. Water Environment Research 89(4): 357-368.
Haas, D., M. Unteregger, J. Habib, H. Galler, E. Marth and F. F. Reinthaler (2010). Exposure to
Bioaerosol from Sewage Systems. Water, Air, and Soil Pollution 207(1): 49-56.
Hatch, M. T. and H. Wolochow (1969). Bacterial Survival: Consequences of the Airborne State.
In: An Introduction to Experimental Aerobiology. R. L. Dimmick and A. B. Akers. (pp.
267-295). New York: John Wiley and Sons.
Heidelberg, J. F., M. Shahamat, M. Levin, I. Rahman, G. Stelma, C. Grim and R. R. Colwell
(1997). Effect of Aerosolization on Culturability and Viability of Gram-Negative
Bacteria. Applied and Environmental Microbiology 63(9): 3585-3588.
Heinonen-Tanski, H., T. Reponen and J. Koivunen (2009). Airborne Enteric Coliphages and
Bacteria in Sewage Treatment Plants. Water Research 43(9): 2558-2566.
Hejkal, T. W., P. A. LaRock and J. W. Winchester (1980). Water-to-Air Fractionalization of
Bacteria. Applied and Environmental Microbiology 39(2): 335-338.
Hejkal, T. W., F. M. Wellings, A. L. Lewis and P. A. LaRock (1981). Distribution of Viruses
Associated with Particles in Waste Water. Applied and Environmental Microbiology
41(3): 628-634.
Hines, S. A., D. J. Chappie, R. A. Lordo, B. D. Miller, R. J. Janke, H. A. Lindquist, K. R. Fox,
H. S. Ernst and S. C. Taft (2014). Assessment of Relative Potential for Legionella
Species or Surrogates Inhalation Exposure from Common Water Uses. Water Research
56: 203-213.
Hovi, T., M. Stenvik, H. Partanen and A. Kangas (2001). Poliovirus Surveillance by Examining
Sewage Specimens. Quantitative Recovery of Virus after Introduction into Sewerage at
Remote Upstream Location. Epidemiology and Infection 127(1): 101-106.
Hung, H. F., Y. M. Kuo, C. C. Chien and C. C. Chen (2010). Use of Floating Balls for
Reducing Bacterial Aerosol Emissions from Aeration in Wastewater Treatment
Processes. Journal of Hazardous Materials 175(1-3): 866-871.
Ijaz, M. K., A. H. Brunner, S. A. Sattar, R. C. Nair and C. M. Johnson-Lussenburg (1985).
Survival Characteristics of Airborne Human Coronavirus 229E. Journal of General
Virology 66 (Pt 12): 2743-2748.
Inglesby, T. V., T. O'Toole, D. A. Henderson, J. G. Bartlett, M. S. Ascher, E. Eitzen, A. M.
Friedlander, J. Gerberding, J. Hauer, J. Hughes, J. McDade, M. T. Osterholm, G. Parker,
-------
T. M. Perl, P. K. Russell, K. Tonat and for the Working Group on Civilian Biodefense
(2002). Anthrax as a Biological Weapon, 2002. Updated Recommendations for
Management. JAMA: Journal of the American Medical Association 287(17): 2236-
2252.
Jaax, N. K., K. J. Davis, T. J. Geisbert, P. Vogel, G. P. Jaax, M. Topper and P. B. Jahrling
(1996). Lethal Experimental Infection of Rhesus Monkeys with Ebola-Zaire (Mayinga)
Virus by the Oral and Conjunctival Route of Exposure. Archives of Pathology and
Laboratory Medicine 120(2): 140-155.
Johnson, D., R. Lynch, C. Marshall, K. Mead and D. Hirst (2013a). Aerosol Generation by
Modern Flush Toilets. Aerosol Science and Technology: the Journal of the American
Association for Aerosol Research 47(9): 1047-1057.
Johnson, D. L., K. R. Mead, R. A. Lynch and D. V. Hirst (2013b). Lifting the Lid on Toilet
Plume Aerosol: A Literature Review with Suggestions for Future Research. American
Journal of Infection Control 41(3): 254-258.
Johnson, E., N. Jaax, J. White and P. Jahrling (1995). Lethal Experimental Infections of Rhesus
Monkeys by Aerosolized Ebola Virus. International Journal of Experimental Pathology
76(4): 227-236.
Judson, S., J. Prescott and V. Munster (2015). Understanding Ebola Virus Transmission.
Viruses 7(2): 511-521.
Karra, S. and E. Katsivela (2007). Microorganisms in Bioaerosol Emissions from Wastewater
Treatment Plants During Summer at a Mediterranean Site. Water Research 41(6): 1355-
1365.
Khuder, S. A., T. Arthur, M. S. Bisesi and E. A. Schaub (1998). Prevalence of Infectious
Diseases and Associated Symptoms in Wastewater Treatment Workers. American
Journal of Industrial Medicine 33(6): 571-577.
Koonin L. M., D. J. Jamieson, J. A. Jernigan, C. A. Van Beneden, C. Kosmos, M. C. Harvey, H.
Pietz, J. Bertolli, J. F. Perz, C. G. Whitney, et al. (2015) Systems for rapidly detecting
and treating persons with Ebola virus disease: United States. Available online at
http://www.cdc.gov/mmwr/preview/mmwrhtml/mm6408a5.htm (accessed February 26,
2018).
Laitinen, S., J. Kangas, M. Kotimaa, J. Liesivuori, P. J. Martikainen, A. Nevalainen, R.
Sarantila and K. Husman (1994). Workers' Exposure to Airborne Bacteria and
Endotoxins at Industrial Wastewater Treatment Plants. American Industrial Hygiene
Association Journal 55(11): 1055-1060.
Lever, M. S., A. J. Stagg, M. Nelson, P. Pearce, D. J. Stevens, E. A. M. Scott, A. J. H. Simpson
and M. J. Fulop (2008). Experimental Respiratory Anthrax Infection in the Common
Marmoset (Callithrix jacchus). International Journal of Experimental Pathology 89(3):
171-179.
Li, J., L. Zhou, X. Zhang, C. Xu, L. Dong and M. Yao (2016). Bioaerosol Emissions and
Detection of Airborne Antibiotic Resistance Genes from a Wastewater Treatment Plant.
Atmospheric Environment 124, PartB: 404-412.
Lin, K. and L. C. Marr (2017). Aerosolization of Ebola Virus Surrogates in Wastewater
Systems. Environmental Science & Technology 51(5): 2669-2675.
-------
Lodder, W. J., A. M. Buisman, S. A. Rutjes, J. C. Heijne, P. F. Teunis and A. M. de Roda
Husman (2012). Feasibility of Quantitative Environmental Surveillance in Poliovirus
Eradication Strategies. Applied and Environmental Microbiology 78(11): 3800-3805.
Lowe, J. J., S. G. Gibbs, S. S. Schwedhelm, J. Nguyen and P. W. Smith (2014). Nebraska
Biocontainment Unit Perspective on Disposal of Ebola Medical Waste. American
Journal of Infection Control 42(12): 1256-1257.
MacDonald, W. D. (1942). Anthrax: Report of a Fatal Case Involving the Cutaneous and
Gastrointestinal Systems. The New England Journal of Medicine 226(24): 949-951.
Masclaux, F. G., P. Hotz, D. Gashi, D. Savova-Bianchi and A. Oppliger (2014). Assessment of
Airborne Virus Contamination in Wastewater Treatment Plants. Environmental
Research 133: 260-265.
Medema, G., B. Wullings, P. Roeleveld and D. van der Kooij (2004). Risk Assessment of
Legionella and Enteric Pathogens in Sewage Treatment Works. Water Science and
Technology: Water Supply 4(2): 125-132.
Michen, B. and T. Graule (2010). Isoelectric Point of Viruses. Journal of Applied Microbiology,
109(2): 388-397.
Minett, F. C. (1950). Sporulation and Viability of B. anthracis in Relation to Environmental
Temperature and Humidity. Journal of Comparative Pathology 60(3): 161-176.
Minodier, L., R. N. Charrel, P. E. Ceccaldi, S. van der Werf, T. Blanchon, T. Hanslik and A.
Falchi (2015). Prevalence of Gastrointestinal Symptoms in Patients with Influenza,
Clinical Significance, and Pathophysiology of Human Influenza Viruses in Faecal
Samples: What Do We Know? Virology Journal 12: 215. DOI 210.1186/sl2985-12015-
10448-12984
Mire, C. E., J. B. Geisbert, K. N. Agans, D. J. Deer, K. A. Fenton and T. W. Geisbert (2016).
Oral and Conjunctival Exposure of Nonhuman Primates to Low Doses of Ebola Makona
Virus. Journal of Infectious Diseases 214(Supplement 3): S263-S267.
Moore, B. E. (1993). Survival of Human Immunodeficiency Virus (HIV), HIV-infected
Lymphocytes, and Poliovirus in Water. Applied and Environmental Microbiology 59(5):
1437-1443.
Morawska, L. (2006). Droplet Fate in Indoor Environments, or Can We Prevent the Spread of
Infection? Indoor Air 16(5): 335-347.
NACWA (National Association of Clean Water Agencies). (2005). "Planning for
Decontamination Wastewater: A Guide for Utilities." Washington DC NACWA.
Available online at
http://www.ecoresourcegroup.com/pdf/Planning%20for%20Decontamination%20Waste
water0 o202005-10decon.pdf (accessed February 13, 2017).
NASEMSO (National Association of State EMS Officials). (2018). "Enhancing the Ability of
EMS to Transport Patients with Confirmed or Suspected Ebola and other High
Consequence Infectious Diseases. Available online at
https://www.nasemso.org/Councils/Health-Medical-
Preparedness/documents/N ASEMSO-HClD-Presentation.pdf (accessed February 26,
2018).
-------
National Research Council (2006). Chapter 3: Generation and Characterization of Aerosolized
Agents. Overcoming Challenges to Develop Countermeasures against Aerosolized
Bioterrorism Agents: Appropriate Use of Animal Models, (pp. 21-28). Washington DC:
National Academies Press.
Noyce, J. O., H. Michels and C. W. Keevil (2007). Inactivation of Influenza A Virus on Copper
Versus Stainless Steel Surfaces. Applied and Environmental Microbiology 73(8): 2748-
2750.
O'Toole, J., M. Keywood, M. Sinclair and K. Leder (2009). Risk in the Mist? Deriving Data to
Quantify Microbial Health Risks Associated with Aerosol Generation by Water-
Efficient Devices During Typical Domestic Water-Using Activities. Water Science and
Technology 60(11): 2913-2920.
Olejnik, J., A. Forero, L. R. Deflube, A. J. Hume, W. A. Manhart, A. Nishida, A. Marzi, M. G.
Katze, H. Ebihara, A. L. Rasmussen and E. Miihlberger (2017). Ebolaviruses Associated
with Differential Pathogenicity Induce Distinct Host Responses in Human Macrophages.
Journal of Virology 91(11): e00179-17.
Ort, C., M. G. Lawrence, J. Rieckermann and A. Joss (2010). Sampling for Pharmaceuticals and
Personal Care Products (PPCPs) and Illicit Drugs in Wastewater Systems: Are Your
Conclusions Valid? A Critical Review. Environmental Science & Technology 44(16):
6024-6035.
Palmer, C. J., M. H. Lee, G. F. Bonilla, B. J. Javier, E. B. Siwak and Y. L. Tsai (1995). Analysis
of Sewage Effluent for Human Immunodeficiency Virus (HIV) Using Infectivity Assay
and Reverse Transcriptase Polymerase Chain Reaction. Canadian Journal of
Microbiology 41(9): 809-815.
Pascual, L., S. Perez-Luz, M. A. Yanez, A. Santamaria, K. Gibert, M. Salgot, D. Apraiz and V.
Catalan (2003). Bioaerosol Emission from Wastewater Treatment Plants. Aerobiologia
19(3-4): 261-270.
Phillpotts, R. J., R. J. Thomas, R. J. Beedham, S. D. Piatt and C. A. Vale (2010). The
Cystovirus Phi6 as a Simulant for Venezuelan Equine Encephalitis Virus. Aerobiologia
26(4): 301-309.
Piercy, T. J., S. J. Smither, J. A. Steward, L. Eastaugh and M. S. Lever (2010). The Survival of
Filoviruses in Liquids, on Solid Substrates and in a Dynamic Aerosol. Journal of
Applied Microbiology 109(5): 1531-1539.
Poliquin PG, Vogt F, Kasztura M, A. Leaung, Y. Deschambault, R. Van den Bergh, C.
Dorion, P. Maes, A. Kamara, G. Kobinger, A. Sprecher and J. E. Strong (2016).
Environmental Contamination and Persistence of Ebola Virus RNA in an Ebola
Treatment Center. J. Infect. Dis. 214:145-52.
Prescott, J., T. Bushmaker, R. Fischer, K. Miazgowicz, S. Judson and V. J. Munster (2015).
Postmortem Stability of Ebola Virus. Emerging Infectious Diseases 21(5): 856-859.
Public Health England. (2018). Global high consequence infectious disease events. Emerging
Infections and Zoonoses Section. PHE Publications gateway number: 2017794.
Rose, C., A. Parker, B. Jefferson and E. Cartmell (2015). The Characterization of Feces and
Urine: A Review of the Literature to Inform Advanced Treatment Technology. Critical
Reviews in Environmental Science & Technology 45(17): 1827-1879.
-------
Roy, C. J. and D. K. Milton (2004). Airborne Transmission of Communicable Infection - The
Elusive Pathway. The New England Journal of Medicine 350(17): 1710-1712.
Roy, C. J., D. S. Reed and J. A. Hutt (2010). Aerobiology and Inhalation Exposure to Biological
Select Agents and Toxins. Veterinary Pathology 47(5): 779-789.
Rylander, R., K. Andersson, L. Belin, G. Berglund, R. Bergstrom, L. A. Hanson, M. Lundholm
and I. Mattsby (1976). Sewage Worker's Syndrome. Lancet 308(7983): 478-479.
Sanchez-Monedero, M. A., M. I. Aguilar, R. Fenoll and A. Roig (2008). Effect of the Aeration
System on the Levels of Airborne Microorganisms Generated at Wastewater Treatment
Plants. Water Research 42(14): 3739-3744.
Schibler, M., P. Vetter, P. Cherpillod, T. J. Petty, S. Cordey, G. Vieille, S. Yerly, C. A. Siegrist,
K. Samii, J. A. Dayer, M. Docquier, E. M. Zdobnov, A. J. Simpson, P. S. Rees, F. B.
Sarria, Y. Gasche, F. Chappuis, A. Iten, D. Pittet, J. Pugin and L. Kaiser (2015). Clinical
Features and Viral Kinetics in a Rapidly Cured Patient with Ebola Virus Disease: A
Case Report. The Lancet. Infectious Diseases 15(9): 1034-1040.
Schuit, M., D. M. Miller, M. S. Reddick-Elick, C. B. Wlazlowski, C. M. Filone, A. Herzog, L.
A. Coif, V. Wahl-Jensen, M. Hevey and J. W. Noah (2016). Differences in the
Comparative Stability of Ebola Virus Makona-C05 and Yambuku-Mayinga in Blood.
PLoS One 11(2): e0148476.
Siegel J. D., E. Rhinehart, M. Jackson, L. Chiarello and the Healthcare Infection Control
Practices Advisory Committee (2007). Guideline for Isolation Precautions: Preventing
Transmission of Infectious Agents in Healthcare Settings 2007. Available online from
https://www.cdc.gov/infectioncontrol/pdf/guidelines/isolation-guidelines.pdf (accessed
February 15, 2018).
Sinclair, R., S. A. Boone, D. Greenberg, P. Keim and C. P. Gerba (2008). Persistence of
Category A Select Agents in the Environment. Applied and Environmental
Microbiology 74(3): 555-563.
Sinclair, R. G., J. B. Rose, S. A. Hashsham, C. P. Gerba and C. N. Haas (2012). Criteria for
Selection of Surrogates Used to Study the Fate and Control of Pathogens in the
Environment. Applied and Environmental Microbiology 78(6): 1969-1977.
Slade, J. S., E. B. Pike, R. P. Eglin, J. S. Colbourne and J. B. Kurtz (1989). The Survival of
Human Immunodeficiency Virus in Water, Sewage and Sea Water. Water Science and
Technology 21(3): 55-59.
Slote, L. (1976). Viral Aerosols - a Potential Occupationally Related Health Threat in Aerated
Wastewater Treatment Systems. Journal of Environmental Health 38(5): 310-314.
Sobsey, M. D. and J. S. Meschke (2003). Virus Survival in the Environment with Special
Attention to Survival in Sewage Droplets and Other Environmental Media of Fecal or
Respiratory Origin. (Draft). World Health Organization International SARS
Symposium, Rome, Italy, August 21, 2003, available from
http://www.unc.edu/courses/2008spring/envr/421/001/WHQ VirusSurvivalReport 21A
ug2003.pdf (accessed November 2, 2017).
Tang, J. W., Y. Li, I. Eames, P. K. S. Chan and G. L. Ridgway (2006). Factors Involved in the
Aerosol Transmission of Infection and Control of Ventilation in Healthcare Premises.
Journal of Hospital Infection 64(2): 100-114.
-------
Thomas, R. J., D. Webber, R. Hopkins, A. Frost, T. Laws, P. N. Jayasekera and T. Atkins
(2011). The Cell Membrane as a Major Site of Damage During Aerosolization of
Escherichia coli. Applied and Environmental Microbiology 77(3): 920-925.
Titcombe Lee, M., A. Pruden and L. C. Marr (2016). Partitioning of Viruses in Wastewater
Systems and Potential for Aerosolization. Environmental Science & Technology Letters
3(5): 210-215.
Towner, J. S., P. E. Rollin, D. G. Bausch, A. Sanchez, S. M. Crary, M. Vincent, W. F. Lee, C.
F. Spiropoulou, T. G. Ksiazek, M. Lukwiya, F. Kaducu, R. Downing and S. T. Nichol
(2004). Rapid Diagnosis of Ebola Hemorrhagic Fever by Reverse Transcription-PCR in
an Outbreak Setting and Assessment of Patient Viral Load as a Predictor of Outcome.
Journal of Virology 78(8): 4330-4341.
Trouwborst, T., S. Kuyper, J. C. de Jong and A. D. Plantinga (1974). Inactivation of Some
Bacterial and Animal Viruses by Exposure to Liquid-Air Interfaces. Journal of General
Virology 24(1): 155-165.
Turgeon, N., M. J. Toulouse, B. Martel, S. Moineau and C. Duchaine (2014). Comparison of
Five Bacteriophages as Models for Viral Aerosol Studies. Applied and Environmental
Microbiology 80(14): 4242-4250.
U.S. Centers for Disease Control and Prevention (2010). Gastrointestinal Anthrax after an
Animal-Hide Drumming Event - New Hampshire and Massachusetts, 2009. MMWR.
Morbidity and Mortality Weekly Report 59(28): 872-877.
U.S. Environmental Protection Agency (1980). Wastewater Aerosols and Disease. Cincinnati,
OH: Health Effects Research Laboratory, U.S. Environmental Protection Agency. EPA
600/9-80/028.
U.S. Environmental Protection Agency (1981). Potential Health Effects from Viable Emissions
and Toxins Associated with Wastewater Treatment Plants and Land Application Sites.
Cincinnati, OH: Health Effects Research Laboratory, U.S. Environmental Protection
Agency. EPA 600/S1-81/006.
U.S. Environmental Protection Agency (1989). Risk Assessment Guidance for Superfund,
Volume I, Human Health Evaluation Manual (Part A) Interim Final. Washington, DC:
Office of Emergency and Remedial Response, U.S. Environmental Protection Agency.
Available from http://www.epa.gov/oswer/riskassessment/ragsa/index.htm (accessed
November 2, 2017). EPA/540/1-89/002.
U.S. Environmental Protection Agency (2011). Exposure Factors Handbook: 2011 Edition.
Washington, DC: Office of Research and Development, U.S. Environmental Protection
Agency. EPA/600/R-09/052F.
U.S. Environmental Protection Agency (2012). Microbial Risk Assessment Guideline:
Pathogenic Microorganisms with Focus on Food and Water. Interagency
Microbiological Risk Assessment Guideline Workgroup. EPA/100/J-12/001 and
USDA/F SIS/2012-001.
U.S. Environmental Protection Agency (2014). Microbiological Risk Assessment (MRA) Tools,
Methods, and Approaches for Water Media. Washington DC: Office of Science and
Technology, Office of Water, U.S. Environmental Protection Agency. EPA-820-R-14-
009.
-------
U.S. Environmental Protection Agency (2016). Review of Bacillus anthracis Dose-Response
Data for Human Health Risk Assessment. Cincinnati, OH: National Homeland Security
Center Threat and Consequence Assessment Division,, U.S. Environmental Protection
Agency. EPA/600/R-16/147.
Verreault, D., M. Marcoux-Voiselle, N. Turgeon, S. Moineau and C. Duchaine (2015).
Resistance of Aerosolized Bacterial Viruses to Relative Humidity and Temperature.
Applied and Environmental Microbiology 81(20): 7305-7311.
Verreault, D., S. Moineau and C. Duchaine (2008). Methods for Sampling of Airborne Viruses.
Microbiology and Molecular Biology Reviews 72(3): 413-444.
Vetter, P., W. A. Fischer, 2nd, M. Schibler, M. Jacobs, D. G. Bausch and L. Kaiser (2017).
Ebola Virus Shedding and Transmission: Review of Current Evidence The Journal of
Infectious Diseases 214 (Suppl 3): S177-S184.
Viau, E. and J. Peccia (2009). Survey of Wastewater Indicators and Human Pathogen Genomes
in Biosolids Produced by Class A and Class B Stabilization Treatments. Applied and
Environmental Microbiology 75(1): 164-174.
Wallis, C., J. L. Melnick, V. C. Rao and T. E. Sox (1985). Method for Detecting Viruses in
Aerosols. Applied and Environmental Microbiology 50(5): 1181-1186.
Weber, T. P. and N. I. Stilianakis (2008). Inactivation of Influenza A Viruses in the
Environment and Modes of Transmission: A Critical Review. Journal of Infection 57:
361-373.
Wen, Q., C. Tutuka, A. Keegan and B. Jin (2009). Fate of Pathogenic Microorganisms and
Indicators in Secondary Activated Sludge Wastewater Treatment Plants. Journal of
Environmental Management 90(3): 1442-1447.
White, C. P., J. Popovici, D. A. Lytle, N. J. Adcock and E. W. Rice (2012). Effect of pH on the
Electrophoretic Mobility of Spores of Bacillus anthracis and Its Surrogates in Aqueous
Solutions. Applied and Environmental Microbiology 78(23): 8470-8473.
Wigginton, K. R., Y. Ye and R. M. Ellenberg (2015). Emerging Investigators Series: The
Source and Fate of Pandemic Viruses in the Urban Water Cycle. Environmental
Science: Water Research & Technology 1(6): 735-746.
Wolf, T., G. Kann, S. Becker, C. Stephan, H.-R. Brodt, P. de Leuw, T. Griinewald, T. Vogl, V.
A. J. Kempf, O. T. Keppler and K. Zacharowski (2014). Severe Ebola Virus Disease
with Vascular Leakage and Multiorgan Failure: Treatment of a Patient in Intensive Care.
The Lancet 385(9976): 1428-1435.
Wood, J. P., K. M. Meyer, T. J. Kelly, Y. W. Choi, J. V. Rogers, K. B. Riggs and Z. J.
Willenberg (2015). Environmental Persistence of Bacillus anthracis and Bacillus subtilis
Spores. PLoS One 10(9): e0138083.
Woolhouse, M. and E. Gaunt (2007). Ecological Origins of Novel Human Pathogens. Critical
Reviews in Microbiology 33(4): 231-242.
World Health Organization (2007). Review of the Latest Available Evidence on Potential
Transmission of Avian Influenza (H5N1) through Water and Sewage and Ways to
Reduce the Risks to Human Health (Last Updated 10/10/2007). Geneva, Switzerland:
Water Sanitation and Health, World Health Organization. WHO/SDE/WSH/06,1 from
-------
http://www.who.int/water sanitation health/emerging/h5nlbackground.pdf (accessed
November 2, 2017).
World Health Organization (2015). Rapid Guidance on Decommissioning of Ebola Care
Facilities. World Health Organization WHO/EVD/Guidance/Strategy/15.1 from
http://vvvvvv.vvho.int/iris/handle/10665/160198#sthash.ivvHtOvDA.dpuf (accessed
November 2, 2017).
World Health Organization (2017). Guidelines for Drinking-Water Quality (Fourth Edition,
Incorporating the First Addendum). Geneva, Switzerland. ISBN 978-92-4-154995-0.
Yang, W. and L. C. Marr (2012). Mechanisms by Which Ambient Humidity May Affect
Viruses in Aerosols. Applied and Environmental Microbiology 78(19): 6781-6788.
Ye, Y., R. M. Ellenberg, K. E. Graham and K. R. Wigginton (2016). Survivability, Partitioning,
and Recovery of Enveloped Viruses in Untreated Municipal Wastewater. Environmental
& Science Technology 50(10): 5077-5085.
Yu, I. T., H. Qiu, L. A. Tse and T. W. Wong (2014). Severe Acute Respiratory Syndrome
Beyond Amoy Gardens: Completing the Incomplete Legacy. Clinical Infectious
Diseases 58(5): 683-686.
Zabinski, J. W., K. J. Pieper and J. M. Gibson (2017). A Bayesian Belief Network Model
Assessing the Risk to Wastewater Workers of Contracting Ebola Virus Disease During
an Outbreak. Risk Analysis Epub Ahead of Print doi: 10.1111/risa. 12827.
------- |