Proceedings from the U.S. Environmental Protection Agency (EPA)
Coliphage Experts Workshop March 1-2, 2016
822-R-17-003
EPA Office of Water
Office of Science and Technology
Health and Ecological Criteria Division
July 25, 2017
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Disclaimer
The information in this document was funded by the U.S. Environmental Protection Agency under
Contract EP-C-11-005 and C_EPC12045_93_0_RCI and was subjected to Agency review and
approved for publication. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use. Furthermore, this document is a summary of the views
of the individual workshop participants; approval for publication does not signify that the contents
reflect the views of the Agency, and no official endorsement should be inferred.
Acknowledgments
The U.S. Environmental Protection Agency would like to thank the speakers and others who
participated in the Coliphage Experts Workshop. Their contributions to the workshop and
dedication to produce these proceedings are greatly appreciated.
Workshop Participants and Presenters include: Nicholas Ashbolt (University of Alberta), William
Burkhardt (U.S. Food and Drug Administration), Kevin Calci (U.S. Food and Drug Administration),
Jack Colford (University of California, Berkeley), Sorina Eftim (ICF), John Griffith (Southern
California Coastal Water Research Project), Vincent Hill (Centers for Disease Control and
Prevention), Juan Jofre (University of Barcelona, Spain), Naoko Munakata (Sanitation Districts of
LA County), Sharon Nappier (U.S. Environmental Protection Agency), Rachel Noble (University of
North Carolina at Chapel Hill), Joan Rose (Michigan State University), Mark Sobsey (University of
North Carolina at Chapel Hill), Jeff Soller (Soller Environmental), Timothy Wade (U.S.
Environmental Protection Agency)
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Table of Contents
Acronyms iv
Foreword vi
Executive Summary vii
Introduction 1
Purpose of the Workshop 1
Workshop Design 1
Background 2
Welcome and Introductions 2
Topic 1: Need for Viral Indicator 5
Topic 2: Coliphage as a Predictor of Gastrointestinal Illness 8
Topic 3: Coliphage as an Indicator of Wastewater Treatment Performance 11
Topic 4: F-Specific Coliphage versus Somatic Coliphage 14
Topic 5: Systematic Literature Review of Viral Densities 17
Future Research 19
Closing Statements 20
References 22
Appendix A. Workshop Agenda and Participant List A-l
Appendix B. Expert's Written Responses to Charge Questions B-l
Figures and Tables
Figure 1. Conceptual model for recreational exposures 3
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Acronyms
AS activated sludge
AOR adjusted odds ratio
AWQC ambient water quality criteria
BAV beach action value
C Celsius
CA California
CDC Centers for Disease Control and Prevention
CFU colony forming unit
cm centimeters
CPE cytopathic effects
CT contact time
CWA Clean Water Act
DNA deoxyribonucleic acid
ds double-stranded
EPA Environmental Protection Agency
EU European Union
FDA Food and Drug Administration
FIB fecal indicator bacteria
FIO fecal indicator organism
FRNA F-specific RNA coliphage
g gram
Gl gastrointestinal
GM geometric mean
GPD gallons per day
HAB harmful algal bloom
HECD Health and Ecological Criteria Division
HRC high rate clarification
ICR Information Collection Rule
ISO International Organization for Standardization
ISSC Interstate Shellfish Sanitation Conference
IWA International Water Association
L liter
MCRT mean cell resident time
mg milligram
MGD million gallons per day
mL milliliter
mm millimeter
MPN most probable number
MS male specific
MSC male-specific coliphage (same as F-specific coliphage)
MST microbial source tracking
m-TEC membrane thermotolerant E. coli agar
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MWRDGC
Metropolitan Water Reclamation District of Greater Chicago
NEEAR
National Epidemiological and Environmental Assessment of Recreational
Water Study
NOAEL
no observed adverse effects level
NPDES
National Pollutant Discharge Elimination System
PCR
polymerase chain reaction
PFU
plaque forming unit
POTW
publicly owned treatment works
QMRA
quantitative microbial risk assessment
qPCR
quantitative polymerase chain reaction
RCT
randomized controlled trial
RNA
ribonucleic acid
RT-PCR
reverse transcriptase polymerase chain reaction
RWQC
recreational water quality criteria
SC
somatic coliphage
SCCWRP
Southern California Coastal Water Research Project
ss
single stranded
TMDL
total maximum daily load
U.S.
United States
UV
ultraviolet light
WERF
Water Environment Research Foundation
WRP
water reclamation plant
WWTP
wastewater treatment plant
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Foreword
The goal of the Coliphage Experts Workshop was to obtain input on science questions related to
coliphage from experts in the fields of environmental microbiology, microbial risk assessment, and
environmental epidemiology to inform coliphage criteria development. The goal of the workshop
was not to reach consensus, rather, it was designed to be a critical thinking and information
gathering exercise. Therefore, the workshop proceedings below provide a record of the workshop
presentations, discussions, and primary outcomes, but do not contain official Agency
recommendations.
This document was peer reviewed in accordance with EPA's Peer Review Handbook (EPA/100/B-
15/001).
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Executive Summary
The United States (U.S.) Environmental Protection Agency (EPA) Office of Science and Technology
convened a workshop, hereafter referred to as the Coliphage Experts Workshop, in March, 2016
where twelve invited technical experts in the fields of environmental microbiology, microbial risk
assessment, and recreational water epidemiology met with Agency staff to engage on how best to
protect public health from human viral pathogens that can be found in water that contains fecal
contamination. The EPA is developing Clean Water Act (CWA) §304(a) Recreational Water Quality
Criteria (RWQC) for coliphage, a viral indicator, to ensure public health is protected from water
sources influenced by fecal contamination, particularly wastewater. In this Workshop EPA sought
scientific insights in five topic areas: 1) the need for an enteric viral indicator; 2) coliphages as a
predictor of gastrointestinal illness; 3) coliphages as an indicator of wastewater treatment
performance; 4) evaluation of F-specific (also known as "male-specific") and somatic coliphages;
and 5) systematic review of enteric viral densities. Participants were also asked to identify future
research needs. These proceedings report the findings of the Coliphage Experts Workshop, as
described below.
Topic 1 Need for Viral Indicator
EPA previously published a Review of Coliphages as Possible Indicators of Fecal Contamination for
Ambient Water Quality (hereafter referred to as EPA's Coliphage Literature Review). The workshop
participants commented on EPA's conclusion that the research literature supports that human
enteric viruses are an important cause of illnesses associated with ambient recreational water
exposures. Overall, it was noted that data are well described in the literature regarding viral
illnesses and occurrence in recreational waters, viral-associated outbreaks, epidemiological
studies, and quantitative microbial risk assessments (QMRA). The participants also identified
important advantages and disadvantages when using coliphages for assessing viral fecal
contamination in ambient waters compared to traditional fecal indicator bacteria (FIB).
Topic 2 Coliphages as a Predictor of Gastrointestinal Illness (Gl)
Workshop participants commented on the strength of the association between coliphage and
human health illness in epidemiological studies conducted in ambient recreational waters. The
experts agreed that available evidence is suggestive that coliphage may be a useful indicator of Gl
illness, particularly at sites impacted by human fecal contamination. The participants also
commented on specific characteristics that influence the association between coliphage and
human health illness, principally the source and intensity of fecal contamination. The panel
suggested that future epidemiological studies should include coliphages as a measured indicator
and that investigators should collect larger water samples so that coliphages are more easily
quantified. The participants also discussed whether specific conditions exist under which
traditional FIB do not adequately protect public health. The participants noted that coliphage
deserve further consideration, especially for situations with sporadic or predominately human-
impacted fecal sources.
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Topic 3 Coliphage as an Indicator of Wastewater Treatment Performance
Workshop participants also commented on EPA's Coliphage Literature Review conclusion that
human pathogenic viruses can enter surface waters via wastewater treatment effluent. Experts
stated that viruses can enter surface waters via wastewater treatment plant (WWTP) effluent, and
noted that treatment configurations exist where viruses are reduced to levels below the sensitivity
of the assays utilized. Episodic loading and periods where WWTPs exceed design flows are key
causes of viral surface water contamination; these are influenced by wet weather, storm events,
snow melt, and hydraulic influencers (inflow and infiltration).
Participants summarized the most important reasons coliphages might be useful indicators (or
models) of the behavior of human enteric viruses in wastewater treatment and disinfection
processes, and commented on EPA's Coliphage Literature Review conclusion that monitoring for
coliphages would be more useful than enterococci and Escherichia coli (E. coli) in predicting
removal of human viral pathogens during wastewater treatment.
Topic 4 Evaluation of F-specific and Somatic Coliphages
Workshop participants commented on important advantages and disadvantages of using the two
types of coliphages as 1) predictors of human health illness in recreational waters and 2) indicators
of wastewater treatment performance. Experts' opinions varied on whether somatic coliphages, F-
specific coliphages, or both would be better for the various applications. Participants also
discussed whether specific attributes of the two coliphage types or site conditions (e.g., fecal
source) influence the usefulness of the indicator or would favor the use of one type of coliphage.
Finally, the participants briefly discussed research conducted in other countries that are currently
investigating or assessing the use of coliphages for various purposes. Specifically, academic
research on the use of coliphages as indicators of water quality has been conducted in Singapore,
Australia, Canada, Argentina, Columbia, Brazil, South Africa, Japan, South Korea, New Zealand,
Tunisia, and the European Union (EU). The participants noted that regulatory authorities in
different parts of the world, including Australia, are beginning to consider coliphages as indicators
of water quality, noting they are traditionally used in the shellfish industry.
Topic 5 Systematic Literature Review of Viral Densities
EPA is planning to use a quantitative microbial risk-based approach to derive RWQC for coliphages.
The risk methodology relies on densities of key viral pathogens and coliphages in raw wastewater.
EPA conducted systematic literature reviews to understand and document these viral densities.
The participants commented on the risk assessment approach, the information collected to date,
and what additional data might be considered. Overall, there was support for how the analysis was
structured and conducted. Individual experts provided search databases suggestions and
considerations regarding the bootstrap and risk assessment approaches. Experts supported the
study inclusion criteria EPA used and recommended that future data from the United States and
other countries should be subject to the same inclusion criteria as the data currently in the
analysis.
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Future Research
Recommendations to address data gaps were also captured from the discussion during the two-
day workshop. At the end of the workshop, participants were asked to classify the various research
projects as long-term or short-term and high, medium, or low priority. Topic 6 lists all short and
long-term research projects discussed.
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Introduction
The U.S. EPA's Office of Science and Technology convened a workshop, Coliphage Experts
Workshop, at the EPA Potomac Yard Office in Arlington, Virginia on March 1-2, 2016. Twelve
invited technical experts in the fields of environmental microbiology, microbial risk assessment,
and recreational water epidemiology met with Agency staff over two days.
Purpose of the Workshop
The purpose of the Coliphage Experts Workshop was to engage internationally recognized experts
on how best to protect public health from viral contamination of water given currently available
information. EPA organized the workshop into five topic areas, with 16 total charge questions. EPA
views this workshop as part of an ongoing commitment to protect public health through
enhancement of CWA 304(a) RWQC.
Specific goals of the workshop included:
• Obtain input on science questions from experts in the fields of environmental microbiology,
microbial risk assessment, and environmental/recreational water epidemiology.
• Gather scientific insight to determine the best coliphage type (F-specific or somatic) for use
in CWA 304(a) criteria.
• Define conditions where coliphages might be most useful for preventing illnesses and
identifying impaired waters.
• Identify research needs that can be completed in the short-term (3 to 5 years).
Workshop Design
The workshop was designed to provide an opportunity to share and listen to ideas, not to reach
consensus on any particular topic. All relevant discussion, including conflicting opinions, are
included in this document. Experts provided individual views and were not asked for
recommendations or agreement. Experts represented a spectrum of perspectives from academia,
EPA Office of Research and Development, other federal agencies (Centers for Disease Control and
Prevention [CDC] and the Food and Drug Administration [FDA]), and the wastewater industry.
Each expert participant was assigned the role of "Topic Lead" for one agenda topic. Prior to the
workshop, Topic Leads were asked to prepare written responses to the charge questions for their
topic. At the workshop, each Topic Lead provided a 15-minute oral summary of their responses to
the charge questions. Two to four Topic Leads were assigned to each workshop topic. Following
the oral presentations, the whole expert group discussed the topics and associated charge
questions.
The remaining sections of this workshop proceedings document are organized parallel to the
workshop agenda (Appendix A). For each topic, the charge questions are presented, followed by a
topic summary, and highlights from the group discussion.
Peer reviewer comments were incorporated into these proceedings to provide additional expert
views. Several more recently published studies were added, as suggested by peer reviewers.
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Background
Welcome and Introductions
Elizabeth Behl, Director of the Health and Ecological Criteria Division (HECD) in EPA's Office of
Water, provided opening remarks describing the purpose and importance of the workshop and
welcoming and thanking the participants. She explained EPA's role in the development of RWQC is
to provide national criteria that are scientifically defensible and protective of designated uses (e.g.
primary contact recreational use) so that states may adopt the criteria into their Water Quality
Standards to protect their waters under the CWA. States may also adopt other scientifically
defensible criteria into their standards, which are final after approval by EPA. In 2012, EPA
published RWQC that maintained the FIB water quality levels from the 1986 Ambient Water
Quality Criteria (AWQC). Although the 2012 RWQC included supplemental tools, such as
Enterococcus measured by quantitative polymerase chain reaction (qPCR) and the Beach Action
Value (BAV), many stakeholders expressed a need for further tools for protection of recreational
waters and to take advantage of the latest science. This workshop is a key milestone in the effort
to address the need for viral indicators to enhance the protection of people recreating in those
waters.
Dr. Sharon Nappier (HECD) presented a background perspective to help frame the workshop
discussions.
The CWA establishes the basic structure for state water quality standards, including regulation of
pollutant discharge into the waters of the United States. CWA 304(a) RWQC are recommendations
intended to be used by states, territories, and authorized tribes adopting water quality standards
to protect the designated use of primary contact recreation. RWQC are used for different purposes
including:
1. Preventing illness by preventing fecal contamination and pathogens from entering surface
waters through point source permits (National Pollutant Discharge Elimination System
[NPDES] permits).
2. Identifying impaired waters through CWA 303(d) Listing and restoring impaired waters by
developing Total Maximum Daily Loads (TMDLs).
3. Enabling states to identify potentially hazardous conditions to beachgoers by the issuance
of beach notifications.
Figure 1 shows a conceptual model for recreational exposures of fecally associated pathogens.
Dominant elements (or elements with the most available information for consideration in RWQC
development) are captured by fully enclosed boxes. Elements with less information which are
more difficult to quantify are captured in boxes with dashed lines. Fecally associated pathogens
can enter surface waters via point and non-point sources. Humans can be exposed to pathogens
via fresh and marine waters; sand exposure has also been linked to Gl illness. The dominant
exposure route is ingestion, though inhalation and dermal exposures can also occur. Receptors are
both children and adults. The predominant endpoint is Gl illness, but some pathogens are
associated with other health endpoints.
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Figure 1. Conceptual model for recreational exposures
EPA's most recent RWQC, published in 2012, recommends two FIB: enterococci (for marine and
freshwater) and E. coli (for freshwater). The magnitude, duration (30-day), and frequency are
specified for both indicator types and for two different illness rates (32 and 36 National
Epidemiological and Environmental Assessment of Recreational Water Study [NEEAR] Gl illness per
1,000 recreators). The 2012 RWQC also include supplemental tools, such as a qPCR method for
same-day notification and BAV for precautionary notification.
Application of the RWQC can both prevent illnesses and identify waters that need improved water
quality. For example, the use of FIB has led to the targeting and control of bacterial pathogens in
wastewater discharges. Historically, bacterial pathogens caused the most serious disease
outbreaks (e.g., cholera and typhoid), and wastewater treatment improvements and discharge
permits based on FIB effectively control such bacterial pathogens. More recently, quantitative
microbial risk assessment (QMRA), epidemiology, and microbial water quality studies indicate that
viruses may be a more significant cause of swimming-associated illnesses in human-impacted
waters. For example, U.S. outbreak surveillance data collected by CDC points to noroviruses as
being the leading viral pathogen responsible for untreated recreational water outbreaks (with
noroviruses responsible for ~17% of untreated recreational outbreaks between 2003 and 2012
[http://www.cdc.gov/healthywater/surveillance/recreational/2011-2012-figures.html]). Current
wastewater treatment processes, however, do not specifically target enteric virus
removal/inactivation. Thus, viruses can enter surface waters from both treated and untreated
human sources.
Cultural FIB are effective at predicting bacterial impairments of water quality, but epidemiological
studies indicate they may not always predict all types of illnesses, such as those caused by viruses
(e.g., norovirus and adenovirus). For example, several epidemiological studies suggest high illness
rates occurring at EPA's recommended water quality levels (Marion et al., 2010; Lamparelli et al.,
2015). Additionally, other epidemiological studies found statistically significant relationships
between Gl illness and the viral indicator coliphage (Lee et al., 1997; Colford et al., 2005, 2007;
Wiedenmann et al., 2006; Wade et al., 2010; Griffith et al., 2016).
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Additional advantages of using coliphage as an indicator of recreational water quality include: they
are of fecal origin and thus highly concentrated in sewage; they are physically similar to human
enteric viruses of concern (ribonucleic acid [RNA] coliphages are more like norovirus and somatic
coliphages are more like adenoviruses); and they have similar persistence patterns to human
enteric viruses of concern during treatment and in response to environmental conditions (e.g., no
appreciable re-growth in ambient waters). In summary coliphages are useful models for fate and
transport of human enteric viruses. Further, coliphages are non-pathogenic and are easy to
measure compared to human pathogenic viruses, which have methodological constraints (e.g.,
length of time to obtain results after samples are taken and need to concentrate multiple liters of
water). An additional advantage is that methods are available to easily measure culturable viruses,
rather than only nucleic acid targets.
Coliphage has been recommended for use as a viral indicator by EPA and FDA. EPA's Ground Water
Rule recommended coliphage to detect and quantify viruses in groundwater in addition to E. coli
and enterococci. The Interstate Shellfish Sanitation Conference (ISSC) and FDA have used F-specific
coliphage for shellfish bed opening decisions, after closure resulting from contamination from
wastewater discharge (FDA, 2009). Also, the National Water Research Institute and the Water
Research Foundation recommend use of viruses and viral indicators to measure disinfection of
treated wastewater to support water reuse.
Availability of a coliphage-based RWQC could enable states to enhance public health protection
from viruses in vulnerable locations by facilitating development of discharge permits to prevent
viruses entering recreational or source drinking waters and by identifying impaired waters or
potentially hazardous conditions at beaches.
In April 2015, EPA published Review of Coliphages as Possible Indicators of Fecal Contamination for
Ambient Water Quality (hereafter referred to as EPA's Coliphage Literature Review), which
included the following overarching conclusions:
• Methods — Coliphage methods are available for water quality monitoring. EPA is in the
process of validating methods in ambient waters and wastewaters.
• Epidemiological Studies — Five of the eight relevant epidemiological studies report a
statistically significant relationship between coliphage and Gl illness levels.
• Occurrence in the Environment — Coliphages are not always significantly correlated with
the presence of human viruses in environmental waters (because of source and fate
differences) but are better correlated with pathogens than traditional FIB, which are the
only currently recommended recreational criteria.
• Environmental Fate — Coliphages are generally good surrogates for the behavior of human
enteric viruses. Behaviors between coliphages and human enteric viruses with
temperature, sunlight, pH, salinity, environmental degradation, and inorganic/organic
matter are similar.
• Wastewater Treatment — F-specific and somatic coliphages are more conservative
indicators of viral pathogen removal overall than traditional FIB in wastewater treated by
most disinfectants.
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Topic 1: Need for Viral Indicator
In EPA's Coliphage Literature Review, EPA concluded that coliphages likely are better indicators of
viruses in fecal contamination, compared to currently recommended FIB (i.e., enterococci and
E. coli). Topic 1 addressed the overall need for a viral indicator as evidenced in epidemiological,
microbial risk assessment, outbreak, and microbial water quality studies. The following charge
questions were provided to the Topic Lead Experts. These experts each provided a 15-minute
presentation based on their submitted written responses to the charge questions (Appendix B).
Charge Questions:
1. Comment on EPA's conclusion that the literature (including epidemiological, risk
assessment, outbreak, and microbiological) supports that viruses are an important
cause of illnesses associated with exposure to ambient recreational waters.
2. Comment on EPA's conclusion that the literature supports that coliphages can be used
as an indicator of human viral fecal contamination.
3. What are the most important advantages and disadvantages of using coliphage for
assessing human viral fecal contamination compared to traditional FIB in ambient
waters?
Topic 1 Overview
Ample evidence exists documenting that human enteric viruses are the leading cause of illnesses
associated with exposure to ambient recreational waters, as was reviewed in EPA's Coliphage
Literature Review. Supportive data are well described in the recreational water literature on viral
occurrence, outbreaks, epidemiological studies, and QMRA. However, varied information exists on
the importance of enteric viruses in different types of human fecal sources of contamination (i.e.,
wastewater, septic). Depending on the source of fecal contamination, coliphages may address the
viral etiologies documented in epidemiological studies.
The individual experts agreed that coliphage would be useful as an additional indicator of fecal
contamination for CWA 304(a) RWQC purposes. However, clarity is needed on the most suitable
coliphage type (F-specific, somatic, or both). Additionally, epidemiological studies vary regarding
the association between coliphages and illnesses. Some important factors that influence coliphage
relationships in epidemiological studies might include geography, temperature, time, study design,
fecal source, and other key variables. Further investigation could reveal which coliphage types are
associated with specific fecal sources.
Important advantages and disadvantages of using coliphage for assessing human viral
contamination in ambient waters compared to traditional FIB were identified. Key advantages
include: 1) coliphages have physical, chemical, and functional characteristics that are similar to
human pathogenic viruses, and thus mimic or model human pathogenic viruses; 2) coliphages and
human viruses are both consistently present in large municipal sewage systems (>1 million gallons
per day [MGD]); 3) coliphages have been shown to be useful in evaluating individual wastewater
treatment processes, disinfection efficacy, and shellfish harvesting waters; and 4) coliphage
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enumeration methods are developed, inexpensive, and could be incorporated into easy-to-use
commercial kits. Rapid methods (<8 hours) are available, but have not undergone multilaboratory
validation in wastewater. Noted disadvantages, particularly for F-specific coliphage, include that
excretion of coliphage by individuals is variable and inconsistent. The smaller the treatment
system, the lower the probability of having coliphage present. A peer-reviewer noted that because
viruses are more variable and dilute in the environment, concentration steps may need to be
added to the current enumeration methods, so that they are more readily detectable in ambient
waters. Additionally, coliphages are very diverse, consequently designing molecular methods that
capture the full diversity of coliphages potentially of interest is difficult. Note, a discrepancy exists
between analytical methods measuring infectiousness and those measuring the presence of viral
nucleic acids. However, this disadvantage is related to how we apply methods for making risk and
management decisions and is not an inherent disadvantage. A major advantage of coliphage is that
culturable methods measure infectious virus particles, allowing for the prediction of removal of
human viral pathogens during wastewater treatment.
Topic 1 Group Discussion
Below are additional items discussed by the entire panel of experts that are related to Topic l's
charge questions.
Sediments: The experts indicated sand and sediments support the accumulation of microbes that
affect water quality. FIB accumulate and grow in the environment and can have high
concentrations in sediment and sand. Coliphages need a high density bacterial host for replication
and thus, do not readily regrow in the environment. The panel discussed how the data for
coliphage in sediment were mixed. One expert indicated that coliphage survive longer in
sediments than in water (as do FIB and pathogens). However the fact that coliphage do not easily
regrow in the environment, as compared to traditional FIB, is a benefit to the use of coliphage,
though this has not been studied extensively.
Coliphage types: One expert suggested it would be easiest to consider both coliphage groups and
measure them simultaneously, using the bacterial host CB390, which is infected by both somatic
and F-specific coliphage. In addition, they noted, deciphering which plaques are somatic coliphage
and which are F-specific coliphage is easy because the somatic coliphage plaques are large and the
F-specific plaques are small. Coliphage types have been confirmed by picking plaques on the
bacterial host CB390. A peer-reviewer indicated that the E. coli strains used in typical assays are
optimized for detection of coliphage associated with sewage, and that environmental coliphage
may more efficiently replicate in E. coli isolated from the environment (Reyes and Jiang, 2010).
Variability: Experts discussed that coliphage and FIB have similar levels of variability in ambient
waters and future research could further evaluate variability of coliphages compared to FIB. One
expert questioned the distance from the fecal source at which one should measure the variability.
Several experts agreed that more than one indicator should be employed, and determining which
conditions are best for the different indicators is important. For example, F-specific coliphage
seems to be more common in some geographic locations, but less common in others. In studies in
Spain and Florida with warm ambient waters, F-specific coliphages are not found. Thus, F-specific
coliphages might not be useful indicators for warmer waters.
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Septic, municipal, and groundwater sources: Experts noted that F-specific coliphages are
consistently found in municipal sewage, but not necessarily in individual septic tanks because not
all individuals excrete F-specific coliphage. For example, a CDC study reported approximately 50%
of tested septic tanks were positive for F-specific coliphages. However, once the tank was positive
for F-specific coliphages, it tended to stay positive. One expert noted their concern about viruses
from WWTPs traveling to shellfish growing areas. They agreed that, because the carrier rate of F-
specific coliphages among individuals in a population is low, it is not a good indicator for sewage
coming from a single house septic system or a non-point source, such as a boat. One exception is
septic systems for coastal rental properties, which mimic larger municipal systems because of
multiple users. Sources contaminating shellfish waters often include overboard discharges or one
septic system; and in these cases, F-specific coliphage may not be effective indicators. One expert
emphasized that F-specific coliphages are most effective when large municipal sewage sources
impact the overlying shellfish waters. It was indicated that because traditional FIB are inactivated
by chlorine disinfection, a widely used disinfectant which has little or no effect on viruses,
coliphage are needed to adequately protect public health, especially in some waters.
Another expert pointed out that floods and rise in sea level may enhance surface and groundwater
connections in some locations and the rise in sea level could impact many coastal states. This
connection is already manifested in many areas and leads to septic system overruns. Another
expert indicated that with septic systems, viruses including coliphages travel more efficiently and
more extensively from on-site systems. On average, attenuation of viruses and coliphages in
groundwater downgradient from a subsurface source will be less than for FIB. One expert
suggested that we should be designing experiments to ask if there are sources specifically related
to the different somatic coliphages and to determine which taxonomic groups of coliphages are
present or predominantly associated with humans. Another expert noted source tracking
strategies are needed. For example, when low-flow contamination was present, septic systems
were shown to link to the human marker; and when it rains, run-off markers (i.e., human and cow)
were found.
A peer-reviewer indicated that there are increased levels of enteric viruses during outbreaks and
there can be seasonal differences in occurrence. For example, virus levels will increase during
increased illness incidences among a population (Sinclair et al., 2009). In the case of norovirus,
incidence of infection increases during the winter in temperate climates. It is unknown if coliphage
would be expected to increase along with increases in the incidence of enteric virus infections in a
community.
Experts further discussed sewage-contaminated groundwater as another source of fecal
contamination. One expert discussed the Avalon, California (CA) beach epidemiological study site
conducted by the Southern California Coastal Water Research Project (SCCWRP). At this site, the
sewage infrastructure is faulty and pipes are corroded because salt water is used to flush toilets. In
this instance, it was noted that viral indicators are the best for protecting people from sources that
are not always obvious and where FIB are not detected. For example, at Avalon, viruses were
detected in the water, in the absence of FIB, and there was a health risk, indicating that coliphage
would be useful in this situation.
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Topic 2: Coliphage as a Predictor of Gastrointestinal Illness
The second topic discussed was coliphage as a predictor of Gl illness. In EPA's Coliphage Literature
Review, eight epidemiological studies were reviewed. Five of the eight studies found a statistically
significant relationship between coliphage and illness. Four studies found a relationship between
F-specific coliphages and Gl illness. One additional study found an association between Gl illness
and somatic coliphage and suggested a no observed adverse effects level (NOAEL) of 10 plaque
forming units (PFU) per 100 milliliters (mL). Topic 2 Experts were asked to reflect on the strength
of association identified in epidemiological studies along with conditions under which coliphage
may better predict illness than FIB.
Charge Questions:
1. Comment on the overall strength of the association between coliphage and human health
illness in epidemiological studies conducted in ambient recreational waters.
2. Are there specific characteristics that influence the association between coliphage and
human health illness (i.e., source of contamination, salinity)?
3. Are there specific conditions under which traditional FIB are not adequate to protect
public health (i.e., Lamparelli et a!., 2015; Marion et a!., 2010) and if so, comment on the
potential for coliphages to be useful in those situations?
Topic 2 Overview
Workshop participants commented on the strength of the association between coliphage and
human health illness in epidemiological studies conducted in ambient recreational waters. The
experts agreed that available evidence is suggestive, but inconsistent. The inconsistency may be
due in part to the wide range of methods, sites, study designs, and measurement methods. A main
concern noted was the frequency of studies that did not detect coliphage or had few detects. This
poses a major issue for establishing exposure response associations or establishing threshold
values. However, some studies do provide evidence that coliphage may be a useful indicator of Gl
illness under conditions where human sources of fecal contamination are likely. Future
epidemiological studies should specifically include coliphage as a measured indicator.
The participants also noted that as with traditional FIB, various factors influence epidemiological
relationships. In particular, the source and intensity of the fecal contamination are important.
Specifically, a peer-reviewer noted most of the positive associations between coliphage and
illnesses have been found at beach areas impacted by sewage pollution. Studies that collected
larger water samples detected coliphage more frequently. Additionally, the type of disinfection (or
lack of disinfection) used at the contamination source could influence the association between
coliphage and illness (e.g., see Wiedenmann et al., 2006).
The participants discussed whether specific conditions exist under which traditional FIB do not
adequately protect public health and if coliphages are potentially useful in those situations.
Culturable coliphage deserve further consideration for situations with sporadic or predominately
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human sources where FIB are not a strong indicator or for determining how soon a beach can be
reopened after a contamination event.
Topic 2 Group Discussion
Below are additional items discussed by the group that are related to Topic 2's charge questions.
Viral illnesses: The experts discussed whether viruses are causing illnesses in epidemiological
studies. In addition to the studies presented by the topic leaders (Appendix B), one expert
mentioned a study of bathing beaches impacted by stormwater in Sydney Harbor that supported a
viral etiology. In this study, norovirus was isolated both from children and the stormwater drain
(Ferson et al., 1993). Experts agreed that future epidemiological studies need to collect pathogen
data, as well indicator data. One expert mentioned EPA's epidemiological study conducted at
Washington Park beach and that salivary immunoassays were included, which will provide
pathogen exposure information (to date the work has not been published).
Additionally, experts discussed overall health outcomes associated with recreational water
exposure. Several experts agreed that the respiratory infections, such as those associated with
adenovirus, are important, in addition to the Gl illnesses. Epidemiological studies by Fleisher et al.
(2006, 2010) found the illness burden was high for respiratory infections. One expert questioned if
an indicator specific for the respiratory health endpoint is needed. Another expert noted that
adenovirus may persist longer than traditional FIB in the environment and respiratory infections
could be observed in the absence of Gl illness. It was noted that in recreational water
epidemiological studies, if participants are asked about Gl symptoms, information on coughs and
related respiratory symptoms is also typically collected.
Accumulation of microorganisms in the environment. The group discussed the accumulation of
viruses and coliphages in the environment. It was noted that there is evidence that people are
being exposed to viruses accumulating in sand. However, evaluating differences in viral exposures
from recreational water versus sand is difficult to measure in epidemiological studies.
In addition to sand, it was noted that pathogenic viruses and coliphages might accumulate in algal
mats and possibly during harmful algal blooms (HABs). It was postulated that some of the illnesses
observed during algal blooms are not only associated with algal toxins, but may be caused by
pathogens accumulating during the blooms. Another expert agreed that some bacteria may
potentially accumulate and multiply in vegetative material, particularly when surface waters
receive effluent treated only by seasonal disinfection (rather than year-round disinfection).
Salinity. One expert asked if any conclusions can be drawn regarding the effects of salinity based
on available data. A peer-reviewer noted that there are reports on coliphage distribution along a
salinity gradient, but it is not clear that the pattern observed is due to source or due to die-off.
Another expert offered that epidemiology cannot address certain characteristics related to survival
of the indicator, such as salinity. One expert suggested going back to beaches where
epidemiological studies have been conducted previously. However, another expert cautioned that
many confounders exist when sampling for indicators at the same beach at a different time. For
example, the impacts from municipal sewage, bather population, or WWTP performance could be
different. A large amount of data on those parameters would need to be collected for comparisons
between years.
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Epidemiological study design attributes and future site needs: The group discussed various aspects
of epidemiological study designs and why the Wiedenmann et al. (2006) study was so successful at
detecting an association between Gl illness and coliphage. Wiedenmann et al. (2006) included six
locations and more than 2,000 individuals, including children as young as four years of age. The
fecal sources impacting the site were untreated human point sources, which are rich with
indicators, and the study participants had variation in exposure to different levels of indicators.
Individual experts noted the randomized controlled trial (RCT) study design has benefits when
well-designed.
Additionally, sanitary surveys and water quality monitoring are important for choosing a beach site
for an epidemiological study. A certain percentage of samples should be positive for the indicators
of interest. Additional monitoring could be conducted at WWTPs to determine the loading coupled
with calculations of dilution at the beach. Monitoring the sites for inclusion in possible
epidemiological studies could be done in 12 months. The precipitation over the previous year,
including overall drought or wet weather conditions, should be recorded. The larger patterns can
also influence study results, beyond just the daily measures. Experts noted that animal
contributions, in particular birds, are confounding factors that need to be considered in
epidemiological studies.
Several experts suggested having a national mobile application for enrolling individuals to self-
report health after going to a beach and concurrently mobilizing a national team to do a water
quality assessment. Another expert indicated an epidemiological study of California surfers did use
a mobile application for collecting data from surfers, which may be useful in future studies (Arnold
et al., 2017).
Sufficient information: The group discussed whether the epidemiological studies supported moving
forward with coliphage criteria. One expert said there is not enough information to discard the
idea of developing coliphage criteria. Another noted an epidemiological dose-response
relationship for developing a guideline value for predicting risk is not available. However, it was
noted at the workshop that EPA is considering a risk assessment-based approach to deriving the
criterion value. Another expert suggested the group consider what was sufficient for decision
making previously and not be biased by the abundance of information currently available on FIB.
One expert recalled that some of the first epidemiological studies were conducted by EPA in the
1970s. At that time, EPA looked at the best candidate indicators and the study authors (Cabelli et
al., 1982) concluded that enterococci was best. At the time, it was recommended to EPA to
evaluate coliphage in the future. This expert proposed that if the goal is to advance knowledge and
understanding, a variety of phages should be studied in the future.
A peer-reviewer pointed out that direct relationships between the incidence of illness and
coliphage may never be possible because of the limitations of any epidemiological study. However,
this does not preclude their eventual use as a measure of recreational water quality as more
reflective of the risk of viral infection, as compared to traditional bacterial indicators.
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Topic 3: Coliphage as an Indicator of Wastewater Treatment Performance
Topic 3 Experts evaluated coliphage as an indicator of wastewater treatment performance. In
EPA's Coliphage Literature Review, EPA summarized indicator attributes and treatment removal
efficiencies of FIB, coliphages, and human enteric viruses. EPA concluded that coliphages are likely
a better indicator of viruses across wastewater treatment, compared to the currently
recommended FIB (i.e., enterococci and E. coli).
Charge Questions:
1. Comment on EPA's conclusion that human pathogenic viruses are entering surface waters
via wastewater treatment effluent.
2. Summarize the most important reasons that coliphages might be useful models or
indicators of the behavior of enteric viruses in wastewater treatment and disinfection
processes.
3. Comment on EPA's conclusion that monitoring for coliphages would be more useful than
enterococci and E. coli in predicting human viral pathogens in wastewater treatment
effluent.
Topic 3 Overview
The experts agreed that human pathogenic viruses are entering surface waters via wastewater
treatment effluent. Treatment configurations exist, however, where pathogens are at levels below
detection. In addition, the national variability and national distribution of pathogens coming out of
WWTPs is not known. Episodic loading and when WWTPs exceed design flows are important
events, which are influenced by wet weather, storm events, snow melt, and hydraulic influences.
The most important reasons that coliphages might be useful models or indicators of the behavior
of human enteric viruses in wastewater treatment and disinfection processes include: 1)
coliphages are more similar to human enteric viruses than FIB, and 2) coliphages are consistently
present in municipal sewage, thus providing a baseline for looking at log reductions by different
treatment processes under varied conditions; and 3) the literature suggests that coliphage and
human viruses have similar reductions during wastewater treatment, however not all disinfection
processes and treatment configurations have been evaluated. For example, there is some evidence
that coliphage log reductions reflect human virus log reductions during wastewater treatment
better than enterococci, particularly for chloramines, free chlorine, and ultraviolet light (UV)
treatment. For ozonation, coliphage and viral pathogens are inactivated at lower doses, compared
to enterococci. However, not all coliphage react the same to treatments. It was additionally noted
that F-specific coliphage have been demonstrated to work well for assessing shellfish safety in
waters impacted by wastewater treatment. In particular, they perform much better than
traditional FIB as treatment indicators when WWTPs are operating at flow capacities above their
design level. In these cases, FIB may be within permit limits, but F-specific coliphage increase
steadily as flow gets higher and higher above design capacity.
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Topic 3 Group Discussion
Below are additional items discussed by the group that are related to Topic 3's charge questions.
Non-culturable viruses: Experts discussed the fact that some pathogens are culturable in cells,
allowing for the evaluation of infectivity, while others, such as norovirus, are not readily
culturable. When assessments are limited to evaluating culturable viral assays, only a small portion
of the pathogens are quantified. For example, in the case of shellfish, one expert noted that
evaluating only infectious viruses fails to capture norovirus, but it is known that many of the
illnesses are caused by noroviruses. Recently norovirus has been cultured, but the method has not
been developed into a quantitative assay for either clinical or environmental samples. One expert
noted that FDA compared reduction of viable coliphage to numbers of norovirus detected with
molecular tools. Norovirus RNA signals are found in effluent and reductions across treatment
correlate with coliphage, but information on the infectivity of norovirus in the effluent is lacking.
However, one expert noted norovirus could be useful for direct pathogen measurement and an
index of other fecalborne pathogens.
Disinfection: Regarding UV treatment, UV inhibits the replication function of viral RNA, but leaves
viral capsids intact. In theory, if some RNA viruses are inactivated by UV, then others should react
similarly because the target is the same. One expert discussed his studies on viral, protozoan, and
bacterial pathogens in UV and chlorine treated effluent. If oxidized tertiary effluent is treated with
UV and chlorine, E. coli, enterococci, coliphages, and Clostridium perfringens are not detectable
(per 100 mL); however molecular analyses detected adenovirus and norovirus nucleic acids, and
Giardia and Cryptosporidium were detected microscopically as immunofluorescent (oo)cysts.
Unfortunately, it is unclear if the molecular and immunofluorescent microscopy assay results
represent viable organisms. Experts discussed several ongoing projects that include reoviruses,
secondary treatment, and UV disinfection.
Another expert noted qPCR signals are very good at evaluating the activated sludge step and the
physical processes related to removal of viruses from wastewater. Disinfection needs to be
evaluated separately, however, because the molecular signal can persist when the pathogen is
inactivated (versus physically removed). Disinfection, in particular, has not been thoroughly
studied, and disinfection processes vary in their effectiveness against inactivating different
pathogen classes.
Laboratory/small-pilotstudies: The group discussed full-scale versus laboratory bench-model
systems as data sources. One expert discussed the challenge in obtaining data from full-scale
plants. Several experts noted that in the short-term, laboratory, bench-top data, and small pilot
studies can be conducted cheaply and quickly, and can compare different coliphages, other
viruses, culture, and qPCR methods against various unit treatment processes. Experts described
the utility of previous bench-scale studies on MS2 coliphage and hepatitis A virus treated with
chlorine and monochloromine. Several experts countered that seeded and indigenous viruses
show different persistent patterns to treatment, and it can be difficult to translate the bench-scale
information conducted on limited strains to full-scale operations (Gerba et al., 2015). Limitations
were noted regarding imitation of microbial clumping when seeding samples. One expert
expressed the importance of normalizing the metrics (such as descriptions of flow rate and facility
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treatment capacity) used across treatment plants so it is easier to link unit treatments to
standardized log viral reductions.
Similar reductions of coliphages and human viruses: Experts agreed that reductions of coliphages
overall are more similar to pathogenic virus reductions than bacteria across treatment processes,
and viruses are in general more resistant to disinfection than bacteria. However, there are
disinfectants where the reverse is true (i.e., ozone).
The group discussed where coliphage have demonstrated to be useful. One expert offered that
although FIB can identify catastrophic failures of treatment processes, F-specific coliphage can also
identify more subtle upsets with treatment processes. For example, FDA's shellfish data indicate
that sometimes when WWTPs meet all fecal coliform discharge permits, culturable F-specific
coliphage can still be detected. If human viral pathogen densities occur at up to 109 particles per
liter in raw sewage, and the WWTP facility provides only 2 to 4 viral log reductions, then norovirus
can still occur in effluent (infectious and non-infectious).
Overall, the group agreed that if forward progress is to be made, pathogen indicators representing
viruses and protozoa shold be considered in future criteria recommendations. Specifically,
coliphages and C. perfringens spores would be useful for addressing the persistence of these
microbial groups.
Continuing advancements: A peer-reviewer pointed out that changes in wastewater treatment are
occurring. Particularly in the Western United States, denitrification and enhanced phosphorus
removal is becoming more common as wastewater treatment plants upgrade. These upgrades
have resulted in enhanced removal of human enteric viruses over conventional activated sludge
and more effective disinfection with chlorine (Schmitz, et al., 2016). Data are not yet available to
determine whether coliphages behave in a similar manner. Separately, the composition of raw
wastewater is also changing as low-flush toilets, water efficient washing machines, and use of cold
water rather than hot water for washing changes the dilution and survival of pathogens in sewage
(Gerba et al., 2017).
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Topic 4: F-Specific Coliphage versus Somatic Coliphage
Topic 4 Experts were tasked with looking at the different types of coliphages and providing an
assessment of whether one type is more useful than the other. EPA's Coliphage Literature Review
provides background information relevant to the use of both F-specific and somatic coliphages, as
indicators of viral fecal contamination.
Charge Questions:
1. What are the most important advantages and disadvantages of using these two types of
coliphages as (a) predictors of human health illness in recreational waters; (b) indicators
of wastewater treatment performance?
2. Are there specific attributes of these coliphages or conditions (i.e., source) that influence
the usefulness of the indicator, or would favor the use of one type of coliphage over the
other?
3. EPA is aware that other countries have considered coliphages for various purposes.
Please provide summaries and commentaries for any of these efforts you deem helpful.
Topic 4 Overview
The two types of coliphages (F-specific and somatic) were compared as (a) predictors of human
health illness in recreational waters and (b) indicators of wastewater treatment performance.
Experts noted that the application drives the preference, and both somatic and F-specific have
advantages. Among experts, opinions ranged on whether somatic, F-specific coliphage, or both
would be better for the various applications. Experts agreed that bacterial host selection for
coliphage assays is important, and it was noted that a single available cell host can capture both
coliphage types.
Epidemiological evidence is suggestive of relationships between both groups of coliphages and Gl
illness. For measuring log reductions in wastewater treatment, some individual experts indicated
somatic coliphages are consistently more numerous in wastewater and thus provide the most
dynamic range in log reduction by wastewater treatment. Somatic coliphages, as a group, also
have the most diverse viron types to represent the broad range of human enteric viruses. Under
some conditions, they persist longer in the environment, and thus may provide a more useful
conservative surrogate role compared to F-specific coliphages. However, others noted that F-
specific RNA coliphages are present in sufficiently high densities (approximately 106/liter) in raw
sewage and they behave more similarly to the RNA human viruses of concern. Additionally, there
are situations where F-specific coliphages have been seen to outnumber somatic coliphage, such
as reclaimed water with high UV treatment, clay sediments, and groundwater from an alluvial
gravel aquifers.
The experts agreed that there is diversity within both coliphage groups, and data are sparse on
how the full diversity of these coliphage groups behave during wastewater treatment.
Unfortunately, while many published studies provide information on either somatic or F-specific
coliphages, most studies do not include both types. Beyond occurrence information, there are
even fewer studies that investigate environmental factors or viral attributes that could be different
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for somatic coliphages compared to F-specific coliphages. From a methodological perspective,
somatic coliphages are easier to count in plate assays because the plaques can be more easily
visualized by their larger size and clarity.
The participants briefly discussed other countries where coliphages have been studied for various
purposes including The Netherlands (Havelaar et al., 1986), Singapore (Liang et al., 2015; Vergara
et al., 2015), Australia (Keegan et al., 2012; Charles et al., 2009), Canada (Payment et al., 1988),
Argentina (Lucena et al., 2003), Columbia (Lucena et al., 2003; Venegas et al., 2015), Israel (Alcalde
et al., 2003; Armon et al., 1995), South Africa (Grabow et al., 1986, 2001; Momba et al., 2009),
Japan (Hata et al., 2013), New Zealand (Wolf et al., 2008), Tunisia (Yahya et al., 2015), China (Fu et
al., 2010) and the EU (Contreras-Coll et al., 2002; Lucena et al., 2003; Ibarluzea et al., 2007; Araujo
et al., 1997; Arraj et al., 2005, Blanch et al., 2004).
Internationally, bacteriophages have been included in guidelines affecting water reclamation in
Australia (Queensland Government, 2005) and biosolids applied in agriculture in Australia and
Colombia (Western Australia Government, 2012; Republica de Colombia, 2014). Additionally, two
drafts of regulatory documents regarding drinking and reclaimed water including coliphages are
being circulated for discussion in the European Union.
Topic 4 Group Discussion
Below are additional items discussed by the group that are related to Topic 4's charge questions.
Training and ease of method: The group discussed the importance of focusing on laboratory
training. WWTP operators are concerned about adopting coliphage methods because they prefer
the simplicity of Colilert and Enterolert. If coliphage analysis can be done with pre-packaged
methods, like the Easyphage kit (from Scientific Methods), then fewer objections exist. Most
commercial systems are currently too complicated. One expert indicated that companies do not
develop pre-packaged kits until the methods are needed for regulatory monitoring. The individual
experts agreed that kits provide quality control. Of importance is that the level of precision is
meaningful for the level of decision making needed. Simple and complete kits can equal more
success in multi-laboratory studies. Another expert noted that many laboratories test both
recreational and shellfish waters. This expert recommended using videos and other modern
outlets to provide training and convey the process practically to reduce variability. This expert felt
that there were lessons learned from how the qPCR method was originally presented. Because
researchers focused on problems and extensive protocols, the method was not accessible to the
broader community. For new methods, simplicity and training are important. One expert noted
that somatic coliphages are easier for training because F-specific coliphage have smaller plaques.
The group agreed about the value and importance of training and implementation of a consistent
program.
Lessons learned from shellfish programs: The expert group agreed that lessons learned from the
shellfish program would be useful for EPA's evaluation of coliphage in recreational waters. The
shellfish community took 30 years to transition from Most Probable Number (MPN) to a direct
membrane Thermotolerant E. coli Agar (m-TEC) method, demonstrating that transitions can be
slow. The U.S. shellfish program includes regional laboratory training where laboratory technicians
practice the method. One expert noted that the EU shellfish program found a strong correlation
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between the occurrence of F-specific coliphages in shellfish and noroviruses. Some EU researchers
recommended F-specific coliphage for shellfish water monitoring, but the idea was not
implemented because it would have downgraded many waters.
Personal sampling devices and composite samples: The group discussed composite samples and
personal sampling devices in epidemiological studies. One expert noted that taking samples at
different times during the day is difficult logistically, thus integrated composite samples are
preferred. Researchers have not yet developed user-friendly, tamper-proof personal samplers,
thus there is an opportunity for engineers to design samplers that can be put on individuals to
better represent exposure. Another expert indicated that the California Surfer Study (Arnold et al.,
2017) intended to provide personal sampling devices to the surfers enrolled in the epidemiological
study, but a small enough device was not available. Another expert added that the situation is
complex, and added that tracer dye studies should be conducted to allow for placement of
sentinel monitoring stations close to the fecal sources.
Environmental replication: There was discussion of the possibility of replication of coliphage in the
environment. The individual experts agreed that phages would not appreciably replicate in the
environment because they need a concentrated bacterial host population for replication (Jofre,
2009). However, a peer-reviewer expert noted microbial ecology overall is very complicated and
that coliphage replication is possible. One expert posed the question of whether one could
distinguish release of phage from bursting cells versus actual phage replication. One expert
thought there might be a way to look at packaging versus presence of replicons. Another expert
thought that growth curves could differentiate between bursting cells and replication, but was not
aware that anyone has sewage-related data. Another expert suggested looking beyond sewage at
other places of potential replication such as Cladophora mats. Another expert indicated that he
published a paper on indigenous phage survival in sewage, looking at survival in seawater and the
effects of sunlight and whether coliphages can grow in shellfish. This expert observed that
coliphage could grow in shellfish spiked with the E. coli host (Famp) at elevated temperatures,
such that the shellfish were essentially incubators. However, this expert indicated that levels of
hosts in wastewater are orders of magnitude higher than anything that is growing on Cladophora
mats, thus replication in mats would be unlikely. A peer-reviewer indicated that storm drains could
be another potential place of coliphage replication. During Southern California's dry, summer
season, storm drains can accumulate very high concentrations of E. coli and can reach
temperatures needed for coliphage replication.
Somatic and F-specific coliphages: One expert suggested that we know less about somatic than F-
specific coliphages, but more hosts are possible for somatic coliphages. It was noted that the F-
specific coliphage methods perform more homogenously than the somatic coliphage methods.
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Topic 5: Systematic Literature Review of Viral Densities
EPA has developed ambient water quality criteria using risk-based approaches for chemicals. EPA
is considering using a quantitative microbial risk-based approach to derive recreational criteria for
coliphage. The risk methodology relies on identification of densities of key viral pathogens and
coliphages in raw wastewater. EPA has conducted systematic literature reviews to understand and
document these viral densities. In this session, approaches EPA is considering for the risk
assessment, the systematic literature review, and the non-parametric method for building
distributions of virus occurrence in wastewater influent were presented (Appendix B). An updated
version of this research was published after the workshop (Eftim et al., 2017).
Charge Questions:
1. Comment on the information collected on viral densities to date.
2. Are there any additional data that should be considered?
Topic 5 Overview
There was overall support for how the systematic literature review and analysis was structured
and conducted. Experts recommended that any future data from the United States and other
countries should meet the same inclusion criteria as the data currently in EPA's analysis.
Topic 5 Group Discussion
Below are additional items discussed by the group that are related to Topic 5's charge questions.
Systematic Review: Transparency regarding the criteria used for data inclusion is very important
because it addresses the quality of the data and the importance of access to raw data. Both the
criteria used to screen the papers and methodology used for bootstrapping, would provide the
public with valuable information The bootstrapping methodology could be used for other types of
datasets, like the Chicago River study (MWRDGC, 2008; Asian et al., 2011) with culture and qPCR
data.
Data suggestions: Experts offered several suggestions on data inclusion, such as the use of: 1) the
Embase database for literature searches; 2) publication date for use as a proxy for study quality; 3)
publications other than those written in English; 4) and non-peer reviewed reports, such as those
from sanitation districts. However, they recommended that unpublished data would need to pass
the study inclusion criteria.
Bootstrgp gnglysis suggestions: Experts offered several comments on the bootstrap analysis of
data, such as 1) possible error may be introduced by normalizing small volumes to liters; 2) a
threshold for minimum sample size for the bootstrapping approach should be employed; and 3)
significant figures should be adjusted in the presentation.
Criterion risk gssessment gpprogch suggestions: Experts offered several comments on the risk
assessment approach presented. One expert noted inclusion of culture-based reovirus because
infectious reoviruses pass through wastewater treatment. Reovirus is a good index of virus
behavior during treatment because they do not cause illnesses, but are excreted in human feces.
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One expert noted that culture data provide greater certainty when making health risk estimates,
compared to molecular data, because culture date provide information on infectivity. If the goal is
to predict human health risks, then methods that indicate infectivity are useful. Additionally, log
reductions from molecular data are less reliable through treatment. For example, log reductions
based on molecular data are less than the log reductions of culturable coliphages. However,
another expert indicated that FDA did a meta-analysis that looked at efficacy of WWTPs to reduce
total coliphage and genome copies of norovirus. He acknowledged that genome copies were very
beneficial in identifying reductions of viruses during wastewater treatment. The numbers were
variable based on sampling location and season, but they found that WWTP provided a 2 to 3 log
reduction of genome copies (Pouillot et al., 2015).
Ultimately, it was noted that the difficulty of using molecular methods, like qPCR, to estimate log
reductions is not relevant to the risk assessment approach presented because distributions of
pathogens were modeled for influent only, which is before treatment process are applied. One
researcher indicated that a study in Sydney, Australia looked at over 1,000 samples of effluent
from primary and tertiary treated water. In that data set, presence/absence of adenoviruses,
enteroviruses and reoviruses by cell culture were compared, indicating reoviruses as generally the
most resistance infectious human enteric viruses in environmental waters (Ashbolt et al., 1993), as
also reported in a recent review (Betancourt and Gerba, 2016). Furthermore, adenoviruses have
been shown to be more prevalent than noroviruses in bathing waters (Wyn-Jones et al., 2011).
While there appears to be no constant ratio of total viruses (qPCR) to cell culture infectious viruses
(Asian et al., 2011), in groundwaters the most stable to least stable appears to be: coliphage
PhiX174 (0.5 d_1) > adenovirus 2 > coliphage PRD1 > poliovirus 3 > coxsackie virus B1 (0.13 d_1),
whereas the order for qPCR results was: norovirus genogroup II > adenovirus > norovirus
genogroup I > enterovirus (Charles et al., 2009).
Coliphage methods are culture-based and should be collected and used for the analysis. Experts
suggested that EPA should proceed with the presented risk assessment approach to support
criteria and noted that QMRA has been used in Australia for standards. The QMRA methodology is
a good pathway forward on how to evaluate human enteric viruses as agents of global concern,
such as norovirus.
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Future Research
Sixteen research ideas were captured during the two-day meeting. The workshop organizers then
asked the expert participants to indicate whether these research ideas were long-term or short-
term and high, medium, or low priority. This was not a consensus activity, rather experts
individually prioritized projects. At the conclusion of the workshop, the results of th exercise were
collated by workshop organizers to determine the order of preferred priority for both the short-
term and longer-term projects.
Short-term projects in order of preferred priority:
• Evaluate many indicators and pathogens using small model (bench-scale) studies to
understand treatment efficacy for a variety of processes.
• Model persistence of coliphage related to many different factors.
• Compare variability of coliphage to traditional FIB, including temporal and spatial
differences depending on source and distance from source.
• For epidemiological studies, although Gl illness is the most plausible endpoint, the health
endpoints with the highest burden of health impacts should also be considered (such as
respiratory illness).
• Evaluate existing data to identify specific pathogens causing swimming-associated illnesses.
• Study coliphage diversity from different household sewage sources using metagenomics
and a variety of host cells.
• Collect more data on FIB and coliphage in wastewater effluent.
Longer-term projects in order of preferred priority:
• Develop guidance on standard epidemiology methods so studies can be more easily
compared. Also encourage researchers to make data publicly available for meta-analyses.
• Support groups who are interested in conducting epidemiological studies.
• Determine which coliphage are associated with animal and which with humans for
microbial source tracking purposes.
• Determine if coliphage replicate in algal mats.
• Investigate what is happening with coliphage in sewage, including differences in survival
and whether phage are replicating or increasing because cells burst.
• Conduct a community analysis of bacteriophage in algal mats, biofilms, and other natural
environments that influence source waters.
• Support building better maps for sanitary surveys, including locations of fecal sources.
• Support development of phone applications for use in epidemiological studies, so citizens
can self-report.
• Monitor beaches for coliphage to help identify sites for future epidemiological studies
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Closing Statements
Following discussion of all topics and charge questions, all experts were asked to state what they
thought were the most relevant messages from the workshop. The participants' individual
comments are below:
• The time has come to move away from using FIB to determine the efficacy of WWTPs.
Although there are data gaps associated with coliphage, a viral indicator should be used.
The old science is based on typhoid and not on the greatest virus of concern today—
norovirus.
• In examining the difficulties with the available epidemiological data, it is important to
ensure coliphage are useful in the context of TMDLs as a process indicator of log
reductions. As long as samples are collected close to the source of fecal contamination,
confidence can be placed in using coliphage.
• Norovirus is a major burden of disease that needs to be recognized and addressed,
however possible. Coliphage can be a useful tool and provides a better model or surrogate
for norovirus than FIB.
• Development of simple, user-friendly detection methods that can be readily implemented
is important for the implementation of coliphage criteria. It is also important to understand
coliphage types and densities in raw sewage and effluent (see: Sobsey et al., 2014). Near-
term studies doing parallel detection of coliphages by both infectivity (culture) and
molecular methods in the same samples would provide valuable information on their ratios
in untreated and treated wastewaters and in ambient recreational waters. Doing the same
for culturable human enteric viruses at the same time would provide even better
information for greater insights into the potential usefulness of molecular methods to
provide information predictive of infectivity and human health risk.
• A global problem is the management of human and animal excreta. The problem grows as
populations increase. It is attached to every ecosystem service. Risk frameworks and
discussions of wastewater treatment in a One Water multi-barrier approach for health are
important. The CWA does not specify risk-based and evidence-based assessments, but
these are important tools for identifying how to best manage wastewater treatment in the
future. Excreta management has not moved forward, but wastewater treatment has. The
questions that need to be answered are: how much wastewater treatment is needed for
virus removal in the "One Water Framework" and how can coliphage inform the decision?
• There needs to be an Alternate Test Procedure process for coliphages, so it can be
published as an EPA standard method. When characterizing coliphages, microbial source
tracing should be used to identify the source(s), especially when no human sewage is
identified (by HF183 or HumM2 microbial source tracking [MST] markers), as the health risk
may be substantially less.
• Coliphage is very valuable as an additional tool for managing water quality. In dry areas, as
for example Spain, water is recycled and it is important to have water quality indicators
Coliphage Experts Workshop
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that address multiple uses, including coliphages. With warmer climate, water reuse will be
more frequent.
• I am glad to see EPA moving towards measuring viruses, even if it is only coliphage. The
strength of coliphage in epidemiology studies is not strong, possibly due to method-related
issues. Additional studies will be needed to determine the efficacy of coliphage as a general
indicator of recreational water quality, especially at non-wastewater impacted beaches.
• I am optimistic about where this is going, but I wonder if we have really addressed the
conditions where coliphage are better than FIB. I think there are situations where that is
possible, but we need to be clear about that moving forward. Epidemiology will not provide
a lot of information and only supplemental information. We will need to consider how
genomic copies can be used in risk assessments
• More research is needed to fully evaluate whether coliphage would be better indicators
than FIB. Changes in indicator organisms may require publicly owned treatment works
(POTWs) to make substantial infrastructure investments, so we should make sure that
these changes also provide substantial improvements over existing indicators for human
health outcomes, and that their selection is well-supported enough to be stable for
years/decades (not part of a revolving door of indicators that would create a moving target
for regulatory compliance)
• This is a well-run workshop and the charge questions highlighted important health issues.
The basic epidemiology paradigm is exposure and outcome. Measurement of personal and
fixed exposures, coupled with the ability to capture variability, will be useful in future
epidemiological studies. Additionally, identification of the pathogens that cause
recreational water illnesses is key.
• Viral and bacterial pathogens by nature differ broadly in pathogen traits and modes of
infectivity, so it is reasonable to conclude that a viral indicator may be of interest.
Coliphage are an interesting and viable option for multiple reasons as an addition to
current criteria, but direct viral pathogen detection, especially via digital polymerase chain
reaction (PCR) is gaining ground rapidly and ought to be included in this process.
Coliphage Experts Workshop
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Appendix A. Workshop Agenda and Participant List
U.S. Environmental Protection Agency
Coliphage Experts Workshop
March 1-2, 2016 I One Potomac Yard, South Bldg, Room S4370/80
2777 S. Crystal Drive, Arlington, VA 22202
Agenda
Participant List:
Name
Affiliation
Coliphage Experts
Nicholas Ashbo It
U niversity of Al berta
William Burkhardt
U.S. Food and Drug Administration
Kevin Calci
U.S. Food and Drug Administration
JackColford
University of California, Berkeley
Sorina Eftim
ICF
John Griffith
Southern California Coastal Water Research Project
Vincent Hill
Centers for Disease Control and Prevention
Juan Jofre
University of Barcelona, Spain
Naoko Munakata
Sanitation Districts of Las Angeles County
Rachel Noble
University of North Carolina
Joan Rose
Michigan State University
MarkSobsey
University of North Carolina
Jeff Soller
Soller Environmental
Timothy Wade
U.S. Environmental Protection Agency
Disclaimer: This meeting is not a federal advisory committee, and EPA will not be seeking consensus or
recommendations. The Coliphage Experts Workshop is an information gathering exercise and individual
opinions of the experts will be captured.
Coliphage Experts Workshop
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Approx. Timing
Draft Agenda Item
Goal of Agenda Item
Day 1: March 1, 2016
8:15-8:30 AM
Meet in Lobby for Escort
Participants meet in lobby; EPA escort to
meeting room required. Check-in and receive
nametags and meeting materials.
Welcome and Introduction
8:45-9:00 AM
Welcome and Introductions
Elizabeth Behl (EPA)
Open workshop and introduce S. Nappier.
Introduce experts.
9:00-9:30 AM
Introduction on RWQC Efforts
Sharon Nappier (EPA)
Provide historical context and present
Coliphage related efforts today to date.
9:30-9:45 AM
Overview of Workshop
Sharon Nappier
leff Soller
Clarify understanding of scope, objectives,
outputs, agenda, and schedule. Introduce the
facilitator.
9:45-10:00 AM
Break
Topic 1: Need for Viral Indicators
Charge Questions:
In April 2015, EPA published Review of Coliphages as Possible Indicators of Fecal Contamination for
Ambient Water Quality (hereafter referred to as EPA's Coliphage Literature Review). In this review, EPA
concluded that coliphages are likely better indicators of viruses in fecal contamination, compared to
currently recommended fecal indicator bacteria (FIB) (i.e., enterococci and E. coli).
1. Comment on EPA's conclusion that the literature (including epidemiological, risk assessment, outbreak,
and microbiological) supports that viruses are an important cause of illnesses associated with exposure to
ambient recreational waters.
2. Comment on EPA's conclusion that the literature supports that coliphages can be used as an indicator
of viral fecal contamination.
3. What are the most important advantages and disadvantages of using coliphage for assessing viral fecal
contamination compared to traditional FIB in ambient waters?
10:00 -10:45 AM
Background on Topic
lohn Griffith
Vincent Hill
Mark Sobsey
Provide information and individual responses
to the topic charge questions.
10:45-11:30 AM
Discuss Charge Questions
Facilitator: Jeff Soller
Discuss charge questions for this topic.
11:30-11:45 AM
Discussion Summary
11:45-1:15 PM
Lunch on your own
Coliphage Experts Workshop
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Topic 2: Coliphage as a Predictor of Gastrointestinal Illness
Charge Questions:
In EPA's Coliphage Literature Review, eight epidemiological studies were reviewed. Four of the eight
studies found a statistically significant relationship between F-specific coliphages and gastrointestinal (Gl)
illness. One additional study found a statistically significant association between Gl illness and somatic
coliphage and suggested a no observed adverse effects level (NOAEL) of 10 plaque forming units (PFU) per
100 milliliters (mL).
1. Comment on the overall strength of the association between coliphage and human health illness in
epidemiological studies conducted in ambient recreational waters.
2. Are there specific characteristics that influence the association between coliphage and human health
illness (i.e., source of contamination, salinity)?
3. Are there specific conditions under which traditional FIB are not adequate to protect public health (i.e.,
Lamparelli et al., 2015a; Marion et al., 2010b) and if so, comment on the potential for coliphages to be
useful in those situations?
aLamparelli, C.C., Pogreba-Brown, K., Verhougstraete, M., Zanoli Sato, M.I.Z., de Castro Bruni, A., Wade,
T.J., Eisenberg, J.N.S. 2015. Are fecal indicator bacteria appropriate measures of recreational water risks in
the tropics: A cohort study of beach goers in Brazil? Water Research 87: 59-68.
b Marion, J.W., Lee, J., Lemeshow, S., Buckley, T.J. 2010. Association of gastrointestinal illness and
recreational water exposure at an inland U.S. beach. Water Research 44(16): 4796-4804.
1:15-2:00 PM
Background on Topic
Tim Wade
lack Colford
Provide information and individual responses
to the topic charge questions.
2:00-2:45 PM
Discuss Charge Questions
Facilitator: Jeff Soller
Discuss charge questions for this topic.
2:45 - 3:00 PM
Discussion Summary
3:00-3:45 PM
Break
Topic 3: Coliphage as an Indicator of Wastewater Treatment Performance
Charge Questions:
In EPA's Coliphage Literature Review, EPA summarized indicator attributes and treatment removal
efficiencies of FIB, coliphages, and enteric viruses. EPA concluded that coliphages are likely a better
indicator of viruses across wastewater treatment, compared to the currently recommended FIB (i.e.,
enterococci and E. coli).
1. Comment on EPA's conclusion that human pathogenic viruses are entering surface waters via
wastewater treatment effluent.
2. Summarize the most important reasons that coliphages might be useful models or indicators of the
behavior of enteric viruses in wastewater treatment and disinfection processes.
Coliphage Experts Workshop
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3. Comment on EPA's conclusion that monitoring for coliphages would be more useful than enterococci
and E. coli in predicting human viral pathogens in wastewater treatment effluent.
3:15-4:00 PM
Background on Topic
Bill Burkhardtand Kevin Calci Naoko
Munakata
loan Rose
Provide information and individual responses
to the topic charge questions.
4:00 - 4:45 PM
Discuss Charge Questions
Facilitator: Jeff Soller
Discuss charge questions for this topic.
4:45 - 5:00 PM
Discussion Summary
5:00 PM
Adjourn Day 1
6:00 PM
Group Dinner (Optional)
Approx. Timing
Draft Agenda Item
Goal of Agenda Item
Day 2: March 2,2016
8:00 AM
Meet in Lobby for Escort
Participants meet in lobby; EPA escort to
meeting room required.
Welcome and Introduction
8:15-8:30 AM
Announcements
Sharon Nappier
Audrey Ichida
Jeff Soller
Logistical announcements
Topic 4: F-Specific Coliphage vs Somatic Coliphage
Charge Questions:
EPA's Coliphage Literature Review provides background information relevant to the use of both F-specific
and somatic coliphages, as indicators of viral fecal contamination. EPA is considering these two possible
viral indicators for use in future recreational water quality criteria (RWQC).
1. What are the most important advantages and disadvantages of using these two types of coliphages as
(a) predictors of human health illness in recreational waters; (b) indicators of wastewater treatment
performance?
2. Are there specific attributes of these coliphages or conditions (i.e., source) that influence the usefulness
of the indicator, or would favor the use of one type of coliphage over the other?
3. EPA is aware that other countries have considered coliphages for various purposes. Please provide
summaries and commentaries for any of these efforts you deem helpful.
8:30-9:15 AM
Background on Topic
Rachel Noble
Juan Jofre
Nick Ashbolt
Provide information and individual
responses to the topic charge questions.
Coliphage Experts Workshop
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Approx. Timing
Draft Agenda Item
Goal of Agenda Item
9:15-10:00 AM
Discuss Charge Questions
Facilitator: Jeff Soller
Discuss charge questions for this topic.
10:00-10:15 AM
Discussion Summary
10:15-10:30 AM
Break
Topic 5: Systematic Literature Review of Viral Densities
Charge Questions:
EPA has developed ambient water quality criteria using risk-based approaches for chemicals. EPA is
considering using a quantitative microbial risk-based approach to derive RWQC for coliphage. The risk
methodology relies on densities of key viral pathogens and coliphages in raw wastewater. EPA has
conducted systematic literature reviews to understand and document these viral densities. The relevant
information collected to date will be reviewed and summarized.
1. Comment on the information collected to date.
2. Are there any additional data that should be considered?
10:30-11:00 AM
Background on Topic
Sorina Eftim
Provide additional information to facilitate
the topic's discussion.
11:00-11:45 AM
Discuss Charge Questions
Facilitator: Jeff Soller
Discuss charge questions for this topic.
11:45 -12:00 PM
Discussion Summary
12:00-1:15 PM
Lunch on your own
Future Research
Charge Questions:
EPA is in the process of developing future RWQC for Coliphage. EPA anticipates a draft publication will be
available in 2017.
1. What are the key uncertainties regarding the development of future RWQC for Coliphage?
2. Please describe research that could be completed in a relatively short time period to address these key
uncertainties, which would support the development of future RWQC for Coliphage?
1:15-2:00 PM
Discussion of future research
Facilitator: Jeff Soller
Discuss research that could be completed
in the next 1 to 2 years. A list of future
research needs will be tracked during all
topic discussions and revisited during this
session.
2:00 - 3:00 PM
Concluding Statements and Final Remarks
Jeff Soller
Sharon Nappier
Discuss conclusions from the workshop and
next steps. Revisit take-home messages from
each topic.
3:00 PM
Adjourn Day 2
Coliphage Experts Workshop
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Appendix B. Expert's Written Responses to Charge Questions
This appendix includes the written responses to the charge questions that the Experts submitted
to EPA prior to the workshop. The Expert's responses are presented without editorial
modifications or proof reading.
Topic 1: Need for Viral Indicators
Topic Leads:
• John Griffith
• Vincent Hill
• MarkSobsey
Charge Questions:
In April 2015, EPA published Review of Coliphages as Possible Indicators of Fecal Contamination for
Ambient Water Quality (hereafter referred to as EPA's Coliphage Review). In this review, EPA
concluded that coliphages are likely better indicators of viruses in fecal contamination, compared
to currently recommended fecal indicator bacteria (FIB) (i.e., enterococci and E. coli).
1. Comment on EPA's conclusion that the literature (including epidemiological, risk assessment,
outbreak, and microbiological) supports that viruses are an important cause of illnesses
associated with exposure to ambient recreational waters.
2. Comment on EPA's conclusion that the literature supports that coliphages can be used as an
indicator of viral fecal contamination.
3. What are the most important advantages and disadvantages of using coliphage for assessing
viral fecal contamination compared to traditional FIB in ambient waters?
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Response: John Griffith
Charge Question 1: Overall, the literature supports that viruses are an important cause of Gl illness
from exposure to recreational waters. However, despite a great deal of effort, it has been difficult
to establish a direct correlative relationship between viruses and illness in epidemiological studies.
This like due to the fact that human pathogenic viruses are inherently difficult to measure in
environmental waters and epidemiological studies must as a practical matter, rely on a fixed time
and sampling point for water quality assessment, while swimmers and bathers move freely about.
Thus, the exposure to viruses at the point where water samples are collected for viral analysis are
only a rough proxy for the actual exposure of an individual swimmer.
The most compelling evidence for viruses as the etiological agent of Gl illness from contact with
recreational water comes from the modeling exercise conducted by Soller et al. (2015), which
determined that the onset of Gl illness observed in the NEEAR study most closely resembled that
of norovirus infection. While not based on actual measurements, this work is supported by
empirical data from a study conducted at Surfrider Beach in Malibu, CA, in which the authors
concluded that the onset of diarrhea in swimmers most closely resembled that expected of
norovirus infection (Arnold et al., 2013).
While it is plausible that norovirus is the likely cause of Gl illness in the above-mentioned studies,
the lack of direct correlative evidence supporting these assertions in troubling. Without such
evidence, it is difficult to ascertain how much Gl illness to attribute to norovirus or to know
whether or not there are other as yet unknown viruses that are at least in part responsible.
Norovirus was unknown until fairly recently and only became detectable with the advent of
molecular measurement methods. It seems equally plausible that there may be additional viral
agents capable of producing similar symptoms that have yet to be characterized by virologists, but
may play a role in the Gl illnesses observed in the swimmers in these studies. Further
epidemiological studies which utilize advanced detection technologies, such as digital droplet PCR,
capable of enumerating norovirus at the low concentrations found in environmental waters are
needed to answer this question.
Charge Question 2: Overall, the literature supports the conclusion that coliphages may be used as
an indicator of viral fecal contamination. Although the studies cited had disparate designs,
differences in the type of exposure (e.g. swimming vs. rafting) and differed as to whether water
exposure was to fresh or marine water, coliphages were positively correlated with illness in more
than half of the studies cited.
While coliphage were often correlated with illness in swimmers, an unresolved issue is how to
choose which coliphages to measure and which measurement method to use. The studies cited in
EPA's review target a variety of different types of coliphage using an equally large number of
methods. Without additional research this issue cannot be resolved. One of the hallmarks of a
reliable fecal indicator organism (FIO) is that the measurement methods produce equivalent
results. The literature as well as personal experience show that different coliphage measurement
methods often produce different results in terms of their observed relationship to water contact
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health risk. These issues will need to be resolved and measurement methods standardized before
coliphage can be considered a reliable water quality indicator.
Charge Question 3: Perhaps the biggest advantage of coliphage as an water quality indicator
versus traditional FIB is that they are more likely to mimic the fate and transport mechanisms of
human enteric viruses in the environment than are bacteria. This is important because viruses, not
bacteria, are posited to cause >90% of waterborne illnesses (Soller et al., 2015).
Coliphage have two main attributes that make them a superior water quality indicator over
traditional FIB. The first is that they are not as easily removed by typical wastewater treatment
regimens as FIB, making them a more viable indicator of the presence of "disinfected" wastewater
plumes. This is because everything about traditional wastewater treatment facilities is geared
toward removing and inactivating FIB in order to bring concentrations in the final effluent, with
dilution, into compliance with regulations. Unfortunately, this treatment regimen does not remove
or inactive enteric viruses or coliphage with similar efficiency. To understand why this is so, one
only need look to the fact that bacteria and viruses are very different in terms of their biology.
Bacteria are living cells that must maintain cellular integrity to survive. They expend energy for
growth, locomotion and to maintain homeostasis under often hostile environmental conditions
and are easily inactivated or killed by UV light, oxidation and other common water treatment
practices. In contrast, viruses, including coliphage, are defined as bordering on the living and non-
living. Like all viruses, coliphage require a specific host for reproduction, are non-motile and are
not as easily inactivated by common wastewater treatment processes as are FIB. Thus it is that the
relative concentrations of active viruses compared to FIB in wastewater effluent are often higher
in treated wastewater than in the untreated water entering the plant. These attributes potentially
make coliphage a much better indicator for human enteric viruses in wastewater than are FIB.
The second advantage of coliphage over FIB is that it is more likely to share the same fate and
transport as human enteric viruses, both in terms of dispersion in the environment and decay
characteristic in sunlight. This is especially important when viruses may be discharged into water
bodies with groundwater contaminated by leaking sanitary infrastructure. For example, in a
epidemiology study conducted at Doheny State beach, FIB concentrations were only correlated
with Gl illness on the few days when sewage contaminated water was flowing from San Juan
Creek. Despite this, water contact continued to be correlated with Gl illness throughout the study
period, even though levels of FIB were exceedingly low (Griffith et al., 2016). Further, coliphage
was positively associated with the presence of human adenovirus and had a stronger relationship
to Gl illness than did Enterococcus under high-risk conditions. A retrospective investigation at
Doheny State Beach revealed a degraded sanitary collection system which was hydrologically
connected to the beach.
Despite a stronger relationship to Gl illness in some studies, coliphage do have some
disadvantages compared to FIB. On disadvantage is that it is more expensive and labor intensive to
measure coliphage than traditional FIB. A second disadvantage is that no one coliphage type or
measurement method has yet distinguished itself as superior to all others in epidemiology or
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laboratory studies. Finally, there is recent evidence in the literature to suggest that coliphage may
be able to replicate in the environment in warmer freshwater environs (Ravva and Sarreal, 2016).
While this should not exclude its use in marine and cold water bodies, it does call into question the
conventional wisdom that coliphage cannot reproduce in the environment.
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Response: Vincent Hill
Charge Question 1: I agree with EPA's general conclusion that there is sufficient scientific and
epidemiological evidence that viruses are an important cause of illnesses associated with exposure
to ambient recreational waters. However, the evidence base for this conclusion is more robust
with respect to gastrointestinal illness than respiratory, skin or other illnesses. U.S. outbreak
surveillance data points to noroviruses as being the leading viral pathogen responsible for
untreated recreational water outbreaks (with noroviruses responsible for ~17% of untreated
recreational outbreaks between 2003 and 2012
[http://www.cdc.gov/healthywater/surveillance/recreational/2011-2012-figures.html]), although
recreational water-associated disease outbreaks in the U.S. have also been reportedly caused by
adenoviruses and hepatitis A virus. While relatively few risk assessments have focused on viruses
in recreational waters, several have reported higher than tolerable risk values for general viral
infection and adenovirus in particular (in Lake Michigan; Wong et al., 2009), hepatitis A virus (in
South Africa; Venter et al., 2007), but some (e.g., Kundu et al., 2013, van Heerden et al., 2005)
have found no elevated risk (for adenovirus in both studies).
With respect to the eight epidemiological studies discussed in the EPA review, the conclusions that
can be drawn from these studies is mixed. Three of the eight studies provide no statistically
significant support for the association of coliphages with increased risk of disease after
recreational water bathing. Of the other five studies, only two provide strong support for such
associations (Wiedenmann et al., 2006 for somatic phages and Griffith et al., 2016 for F-specific
phages). Three studies provide some support, but each of these with important caveats. First, the
study by Lee et al. (1997) should be reconsidered by EPA as to whether it has sufficient scientific
standing to provide guidance for EPA's decision-making process. One issue with using Lee et al.
(1997) as a reference is that the manuscript was published in Water Science & Technology from an
International Water Association (IWA) Health-Related Water Microbiology conference proceedings
and was not subject to formal peer review. EPA epidemiologists and statisticians should review
this manuscript, preferably by providing comments and questions for the study authors to answer,
to obtain additional information regarding study methods (e.g., the statistical methods are
described in one sentence) In addition, while Lee et al. (1997) appears to provide solid support for
the correlation between F-specific RNA (FRNA) coliphages and gastrointestinal illness, the authors
used Salmonella Typhimurium WG49 apparently without pretreating the water samples with S.
Typhimurium WG45 to removal somatic Salmonella phages (as was recommended by EPA's Stetler
and Williams, 1996 and Handzel et al., 1993). Thus, Lee et al. (1997) used a non-standard
methodology (by current standards) that appears to have resulted in yielding data for total
coliphages. In the second of the three studies that provide some support for coliphages as disease
risk indicators, Wade et al. (2010) provides suggestive evidence, but no statistically significant
associations were found for coliphages (as they were for enterococci by qPCR). The reason given
for this was an insufficient number of coliphage detections. However, Wade et al. (2010) did
report that the adjusted odds ratio (AOR) of Gl was significantly higher on days when F-specific
phages were detected, so this finding provides some support, but no support for establishing a
NOAEL (as was reported in the Wiedenmann et al., 2006 study). Similarly, while the Colford et al.
studies (2005; 2007) reported statistically significant associations between the levels of F-specific
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coliphages in marine beach water and several categories of reported illness, but the authors
cautioned that the conclusions should not be strongly interpreted because the statistical
association were based on relatively few F-specific coliphage detections (max concentration of 1
MPN/100 mL and detections in only 11% of samples). I am afraid that this (relative infrequency of
detection vs. FIB) is going to be a recurring theme for coliphages as reliable indicators of fecal
contamination and risk of illness.
Charge Question 2: My experience with coliphages during research on animal waste systems,
domestic sewage systems, decentralized wastewater systems, and fecally impacted receiving
water is that coliphages are useful as indicators of treatment system efficacy for virus removal and
inactivation, but coliphages may not be reliable indicators of fecal contamination in ambient
waters unless these waters receive wastewater (including stormwater) loadings from large
population centers and (in the case of F-specific phages) large-volume samples (~1 L) are tested.
The coliphage detection frequency and concentration data from the EPA-reviewed epidemiological
studies also bear this out: somatic coliphages are typically present in surface waters at levels that
are ~ 4-10 times lower than E. coli (and also lower than enterococci), with concentrations of F-
specific coliphages being even lower. Somatic coliphages have the advantage that they can be
reliably detected in 100-mL sample volumes, but most studies indicate that reliable detection of F-
specific coliphages requires analysis of > 1 L, and even then the chances of detection may be
affected by human population size and person-person excretion variability. As part of a study we
recently published (Schneeberger et al., 2015), we analyzed two single-family home septic tanks 13
times over the course of 2 years for a suite of fecal indicators. We detected somatic coliphages
multiple times (5 of 13 samples) in one family's septic tank, but not in the other family's septic
tank (only 1 detection in 13 samples), and detected F-specific coliphages only once between the
two tanks (data not published). We also studied the presence of coliphages in larger scale
decentralized wastewater reuse systems. As the size of the system increased, the chance of
detecting coliphages (especially F-specific coliphages) in influent to the treatment systems
increased (see table below).
Site
ID
Facility Type
Scale of reuse
F-specific phage Detection
frequency (Geometric
Mean (GM) PFU/100 mL)
Somatic phage
Detection frequency
(GM PFU/100 mL)
A
Resort; golf course
community with two
hotels and small
commercial; 900
customers
Large-scale multi-
subdivision development;
-50,000 to 500,000
gallons per day (gpd) flow
depending upon season
4/4 (8700)
4/4 (4 x 104)
B
Resort; residential and
commercial resort
community; 475
customers
Large-scale subdivision &
commercial district;
~30,000 to 300,000 gpd
flow depending upon
season
4/4 (3 x 104)
4/4 (1 x 105)
C
Resort; retirement
community; small;
"residential" condo
Medium-scale condo
complex; ~5,000 to
50,000 gpd depending
upon season
3/3 (3500)
3/3 (1 x 105)
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D
Seasonal; high school
Small-scale:large flow
1/3
3/3 (6400)
and middle school
range due to school
(f. coli GM = 7 x 104/100
complex
schedule
mL)
E
Small cluster system
Small-scale : ~1,000 gpd
1/3
3/3 (3000)
serving 3 homes and a
design flow
(f. coli GM = 4 x 106)
business
F
High-rise building; 35-
Medium-scale on-site;
1/3
3/3 (2900)
story; > 250 units;
"sewer mining"; sewer
(f. coli GM = 1 x 106)
"residential"
backup; > 20,000 flow
G
High-rise family with
Medium-scale on-site;
0/4
2/4
commercial aspects;
"sewer mining"; sewer
(E. coli GM = 1 x 105)
>290 units
backup; > 20,000 flow
In a past outbreak investigation (O'Reilly et al., 2007) we measured somatic and F-specific
coliphages, along with FIB and a suite of pathogens, in ground water samples that were suspected
to be impacted by onsite wastewater systems and associated with the outbreak. We detected E.
coli in 6 of 13 ground water samples. We measured somatic coliphages in 3 of the 6 ground water
samples that were positive for E. coli (and in one ground water sample that was negative for E.
coli). We detected F-specific coliphages in only 1 of the 13 ground water samples (in a sample
containing E. coli at 118 colony forming units (CFU)/100 mL and somatic coliphages at 3 PFU/100
mL). We did not detect F-specific coliphages in a ground water sample found to contain E. coli at
420 CFU/100 mL, somatic coliphages at 92 PFU/100 mL, and which was positive for adenovirus and
enterovirus).
We have also observed the difficulty in detecting F-specific phages in an ongoing large-scale study
of irrigation ponds, where we have only detected F-specific coliphages in 4 of 110 irrigation pond
samples when we analyzed only 100 mL. When we analyzed 50-L water samples concentrated by
ultrafiltration, we detected F-specific coliphages in 23 of 110 irrigation pond samples
(corresponding to an approximate sample analysis volume of 15 L). Median E. coli for this set of
surface water samples was 12 CFU/100 mL (E. coli detected in 96 of 110 samples), so fecal
contamination of these ponds has been relatively low (and highly variable).
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Charge Question 3: One the primary advantages is that coliphages are viruses present in fecal
waste as part of normal gut microflora and they demonstrate similar physical properties and fate
and transport characteristics as enteric viral pathogens (e.g., based on waste treatment studies
showing them to be more resistant to treatment processes than FIB). They are not metabolically
active, like FIB are, and have thus far not been shown to replicate in the environment as has been
reported for E. coli (e.g., in sub-tropical climates) and enterococci (especially in conjunction with
plant matter in water systems). Aspects of coliphages vs. FIB that are important include:
Fecal Indicator
Advantages
Disadvantage
E. coli
Generally present in all warm-blooded
animals
Excreted at relatively high levels; relatively
higher concentrations in natural water vs.
other indicators
Narrowly-defined indicator (i.e., a bacterial
species) conducive to molecular detection
Standardized analytical methods for 100-mL
samples available in numerous formats
Easily inactivated by disinfection systems; faster
die-off in the environment
Cannot tolerate salt water
May replicate under warm, organic-rich
conditions
No rapid (< 8-h), quantitative viability method
available
Enterococci
Generally present in all warm-blooded
animals
Excreted at relatively high levels, though
lower levels than E. coli
Relatively narrowly-defined indicator (i.e.,
few dominant species) conducive to
molecular detection
Salt water tolerant
Standardized analytical methods for 100-mL
samples available in numerous formats
May be more readily inactivated by disinfection
systems; faster die-off in the environment
(however, they are more resistant to
inactivation than E. coli and may show similar
survival as coliphages.
Replicate under organic-rich conditions,
especially in presence of plant matter
No rapid, quantitative viability method available
Somatic
coliphages
Higher excretion frequency on population
basis than F-specific phages
Higher excretion levels and concentrations in
r and impacted water than F-specific phages
Lower inactivation rates than FIB in
treatment systems and environment
Rapid P/A method available
Not as readily detected as FIB in impacted
waters
No rapid, quantitative viability method available
Operational group comprised of multiple phage
families complicates some analyses (e.g.,
molecular)
F-specific
coliphages
Can provide animal versus human fecal
waste source information
Lower inactivation rates than FIB in
treatment systems and environment
Rapid P/A method available
Not excreted by all warm-blooded animals,
including humans
Excreted at lower levels than somatic phages
and FIB; likely need to assay at least 1 Lfor
chance of consistent detection in surface
waters
No rapid, quantitative viability method available
Operational group comprised of multiple phage
families complicates some analyses (e.g.,
molecular)
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Response: Mark Sobsey
Charge Question 1: There is ample but not rich evidence documenting that human enteric viruses
are important causes of illnesses associated with exposure to ambient recreational waters. Several
lines of evidence based on the available scientific literature support this position as was reviewed
in the EPA report. The three main types of evidence, specifically microbiological, epidemiological
and risk assessment-based, are available. There is well-documented, real-word evidence relevant
to exposure assessment and health effects assessment.
The exposure assessment evidence comes from the well-known and well-documented data on the
occurrence of enteric and respiratory viruses in the human population, their shedding from
infected human hosts and their presence in sewage and other human excreta such as sewer
overflows and sewage bypasses and on-site septic systems that impact ambient waters, including
those used for primary contact recreation. Virus shedding levels in feces, respiratory secretions
and other excreta are often in the millions to billions per gram or mL and many of them become
constituents of sewage and other sources of fecal wastes that may enter the ambient water
environment. It has been well-documented for many decades that human sewage contains a wide
range of human enteric, respiratory and other infectious viruses often at concentrations of
hundreds to thousands of infectious units per liter and sometimes higher. When measured by
molecular methods that quantify gene copies, the concentrations of these viruses are even higher.
Any human enteric or respiratory virus of health concern shed by members of a population will
almost certainly be present in sewage and related wastes and waste-impacted ambient waters.
Since the availability of mammalian cell culture systems to detect and quantify some of these
viruses, beginning in the 1950s, many of the viruses of health concern have been found and
quantified in sewage and in ambient surfaces waters impacted by sewage and other human waste
sources. Enteroviruses, reoviruses, rotaviruses and adenoviruses are examples of human enteric
viruses have been detected regularly in sewage, discharged sewage effluents and ambient waters
impacted by point and non-point human waste sources. Some of these viruses are documented
causes of waterborne outbreaks. With the development of nucleic acid based molecular methods,
primarily PCR and reverse transcriptase PCR (RT-PCR), in the 1990s it is now possible to detect and
quantify additional enteric, respiratory and other viruses of human health concern in sewage,
other human wastes and ambient waters impacted by such wastes that have difficult or so far
impossible to detect and quantify by infectivity in cell culture systems. These include noroviruses,
sappoviruses, astroviruses, and hepatitis A and E viruses, all of which are important because they
cause human infection and illness, including documented waterborne outbreaks by some of them.
All of these viruses have been detected in sewage and ambient waters.
There is credible epidemiological evidence from data of prospective cohort studies and
randomized controlled trials for excess human health effects in the form various illnesses or health
conditions consistent with possible and perhaps likely viral etiologies, including gastrointestinal,
respiratory, eye, and skin infections, among swimmers and others with recreational water
exposures. A variety of different human enteric and respiratory viruses can cause these kinds of
infections and illnesses. Unfortunately, few if any studies have collected clinical samples that
document the presence of human viruses or other evidence of recent viral infection such as
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immunological evidence, in recreational water bathers, except for a few waterborne outbreaks
reported to EPA and CDC in the United States. However, based in the signs and symptoms of illness
and other clinical findings, there is credible evidence to expect that at least some of the excess
illness and other adverse health effects occurring in recreational bathers compared to non-bathing
controls is caused by various viruses. The types of illnesses, adverse health effects and health
conditions reported by swimmers are consistent with possible and perhaps likely viral etiologies.
Other studies have addressed the risks of waterborne disease from recreational exposures to viral
pathogens using quantitative microbial risks assessment. Such studies have obtained data on virus
concentrations in recreational water and used human infectivity dose-response data from the
scientific literature to estimate the risks of viral infection and illness from recreational water
exposures. Such studies provide further evidence that excess risks of infection, illness or other
adverse health effects can be estimated based on measured concentrations of human viral
pathogens in recreational waters.
Charge Question 2: There is ample evidence from the literature supporting coliphages as viral
indicators of fecal contamination, based on them meeting the various criteria to be a suitable fecal
indicator, specifically for viral pathogens. First, coliphages ARE enteric viruses that are harbored by
human and other mammals and are shed fecally by these sources. They have physical,
biochemical, morphological and biological properties that are the same as or similar to human
enteric and respiratory viruses of health concern. Most people harbor and fecally shed coliphages
of one kind or another, although the carriage rates in individual humans and animals may be
somewhat variable. However, raw sewage and other fecal wastes from even small numbers of
people invariably contains coliphages at typical concentrations of 100s per mL. In addition, they
are applicable to and detectable in all types of water and wastewater and other relevant samples.
They are often documented to be present in feces, sewage and fecally contaminated ambient
waters when viral pathogens are present. In addition, their numbers are associated with amount
of fecal contamination, with higher concentrations in samples that are more fecally contaminated
based on other measurable properties. Coliphages outnumber viral pathogens in sewage and
other fecally contaminated samples, including ambient waters. Under most conditions, coliphages
do not "grow" in the environment or have non-fecal environmental sources. Although some
coliphages can replicate in host bacteria in aqueous media when concentrations of both are
sufficiently high, such conditions occur rarely if ever in most environmental samples, except
possibly fresh raw sewage containing susceptible host bacteria. On average coliphages survive and
persist as long as or greater than most human viral pathogens. However, their survival varies
among the different coliphages as does the survival of different human enteric viruses, including to
wastewater treatment processes. Coliphages are easily detected and quantified by simple
laboratory tests in a short time, making them a practical and affordable viral indicator of human
enteric viruses. Coliphages have defined characteristics that vary among the different coliphage
taxonomic groups but are constant and predictable with their different groups, as are the human
enteric viruses. Coliphages are also harmless to humans and other animals and therefore safe and
easy to culture for the purpose of detecting and quantifying them in wastewater and water. There
is some but as yet only limited evidence that the numbers of coliphages in ambient water are
associated with risks of enteric illness in those exposed to them from recreational activities (that
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is, a dose-response relationship). However, the predictability of coliphages as viral indicators of
risks of viral infection and illness from recreational water exposures requires further study to
better understand and quantify these relationships.
Charge Question 3: Because coliphages are viruses that resemble human enteric viruses in a
variety of relevant properties and because they meet all of the criteria of a good fecal indicator
system for viral pathogens of interest and concern from recreational water exposures, they have
several important advantages over and are more credible than traditional FIB. FIB do not meet as
many of the desired criteria to be viral indicators of fecal contamination of recreational waters.
Among other reasons, this is because coliphages are much more like human enteric virus than are
FIB, they survive and persist more like human enteric viruses in wastewater, ambient waters and in
response to environmental stressors and wastewater treatment process than do FIB and because
unlike many of the FIB, they do not multiply, regrow or otherwise increase in numbers in sewage
and environmental waters. Furthermore, some studies of wastewater treatment systems show
that coliphages are reduced to lesser extents than traditional FIB by conventional primary-
secondary treatment and disinfection. Therefore they are more like the human viral pathogens in
their reductions by wastewater treatment systems. In some studies, coliphages and Clostridium
perfringens spores are at higher concentrations in treated sewage effluents than are any of the
traditional FIB. See Figure 1 below.
Raw
Treated
Figure 1. Microbial indicators in raw and treated wastewater (primary, secondary-treated, and
chlorination; GM values of 24 bi-weekly samples)
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Topic 2: Coliphage as a Predictor of Gastrointestinal Illness
Topic Leads:
• Jack Colford
• Tim Wade
Charge Questions:
In EPA's Coliphage Review, eight epidemiological studies were reviewed. Four of the eight studies
found a statistically significant relationship between F-specific coliphages and gastrointestinal (Gl)
illness. One additional study found a statistically significant association between Gl illness and
somatic coliphage and suggested a no observed adverse effects level (NOAEL) of 10 plaque
forming units (PFU) per 100 milliliters (mL).
1. Comment on the overall strength of the association between coliphage and human health
illness in epidemiological studies conducted in ambient recreational waters.
2. Are there specific characteristics that influence the association between coliphage and human
health illness (i.e., source of contamination, salinity)?
3. Are there specific conditions under which traditional FIB are not adequate to protect public
health (i.e., Lamparelli et al., 2015; Marion et al., 2010) and if so, comment on the potential for
coliphages to be useful in those situations?
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Response: Jack Colford
In my opinion, the EPA has provided a thoughtfully constructed review document summarizing the
published literature to date on the relationship between coliphage and human illness. I am not
aware of additional published literature with evidence to address this specific question that has
been omitted. The EPA review of the eight studies concludes with the statement: "Overall, the
epidemiologic evidence is suggestive of a potential relationship between coliphages and human
health."
Because of the limited peer-review literature addressing this question, this conclusion is based on
few published results and I agree that the wording a "potential relationship" is correct given the
evidence available to this point. This relationship, like all claims about associations between
potential markers of illness and the illnesses themselves needs to be scrutinized critically. The
charge question specifically asks about the overall strength of the association between coliphage
and human health illness. When people ask epidemiologists about the "strength" of an
association, the questioner is most often interested in the magnitude of the measure of
association (such as the odds ratio or rate ratio or relative risk). However, equally important in the
evaluation of the "strength" of an association is an examination of the potential biases that may
have caused a systematic over or underestimation of the relationship. I think this potential for bias
raises the most concern in the small published literature to date on the question of the
coliphage/health relationship. These concerns about potential biases are important enough in my
interpretation of the results to warrant a brief description of them here. The EPA has
(appropriately) not attempted to synthesize the disparate results of the studies and present one
summary ("meta-analytic") numeric estimate of the overall relationship.
It should be mentioned that the conduct of human health studies such as those presented is
operationally difficult. The enrollment and follow-up of participants is not an easy task, the
accurate measurement of coliphage and other indicators is challenging and expensive, and the
analyses of the data can be complicated if not carried out in experienced hands. The potential for
bias exists at each of these steps with resulting impact on the estimate of the strength (or
existence) of the reported relationship. My critique of these studies begins with an
acknowledgement of how difficult they are to complete and with recognition of and
congratulations to the investigators who carried them out. This acknowledgement
notwithstanding, I believe there are a large number of potential biases present in these studies.
Several studies found no relationship between measured coliphage and illness. The reader is left to
wonder if this is simply due to the small study sample sizes (and resulting wide confidence
intervals overlapping the null hypothesis). At times the authors, understandably, are reaching to
describe their results as "positive" associations despite the lack of statistical significance (this
reach is not unique to this field).
Throughout this body of work there is a multiplicity of both exposure and outcome definitions. In
a situation such as this there is the possibility of misclassification bias (of exposure and/or
outcome). Other sessions of this conference will address the question of the proper coliphage
measures that should be employed, but with respect to outcome measurement I would argue that
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it is likely that the slightly different definitions of gastrointestinal illness and other health
outcomes are reasonably aligned to allow for interpretation across studies. I think it unlikely that
our interpretation of the results is impaired by this potential misclassification bias of the outcome
(illness); I cannot comment yet on the potential misclassification of the exposure and will wait until
I hear the presentations addressing this issue.
Very few of the studies employed the use of negative control outcome measures. This refers to
the inclusion of symptoms and/or outcomes that could not plausibly be affected by the exposure
of interest (here the exposure to coliphage). Use of negative control outcomes is a broad
technique for identifying several types of bias. For example, if the swimmers and non-swimming
comparison groups experienced a rate of traffic accidents after leaving the beach that showed the
same strength of association with coliphage level as the association of illness to coliphage
exposure, one would wonder if some sort of reporting bias was present in the data (since traffic
accidents are not plausibly linked to coliphage exposure). I did not find that the authors of the
reviewed studies employed negative control outcome measures.
One feature of these studies that makes it difficult to arrive at a firm conclusion about the strength
of the association is the differing choices of a counterfactual (comparison) group. In some studies,
the counterfactual for the exposed bathers is a group of non-swimmers with no water contact. In
other studies, the counterfactual is a group of swimmers with lower levels of water exposure.
Since non-swimmers and swimmers with low water exposure are plausibly different from each
other with respect to key other variables (current health status, age, gender, etc.) the choice of
which comparison group to use can impact the estimate of the strength of the association and
thus bias the results.
Some of the studies employed the "gold standard" in clinical research, the randomized controlled
trial (RCT) design. Generally, RCTs are believed to be crucial in linking exposures and outcomes
because of their ability to balance characteristics in exposed and unexposed groups and to remove
from the participants any choice about whether or not to undergo exposure. Although RCTs are
powerful tools, I do not believe they are of critical importance for the study of this question (the
association of coliphage and illness) because it is essentially impossible that swimmers and non-
swimmers chose their exposure (or non-exposure) to coliphage in any way based on the coliphage
levels in the water. The two RCTs presented, however, were conducted under experimental
conditions that may not reflect normal beach exposure mechanisms or time...making the
generalizability of the results difficult for me to interpret Additionally, the RCTs were, due to the
costs and logistics required, quite small with respect to the numbers of subjects enrolled. As
described above, these small sample sizes contributed to large confidence intervals which made
the detection of statistically significant results more challenging even if truly present.
It is difficult to assess from the materials presented to us whether or not publication bias might
also impact our understanding of the strength of the coliphage/illness association. It is widely
known that authors who conduct studies with negative results are less likely to submit that work
for publication and that journals are less likely to publish negative studies that are submitted. In
the present context of these coliphage studies, publication bias could be present if authors who
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published recreational water studies omitted coliphage results while publishing their more
traditional indicator results.
Methods in the field of epidemiology are evolving and improving - at least we hope that to be the
case. A major effort is underway in the field to adhere to much more rigorous guidelines with
respect to scientific replication. These guidelines include, for example, the prior publication of
analysis plans to help ensure that authors don't "cherry-pick" the results they choose to present in
their published work. Current replication standards also suggest that investigators make de-
identified datasets available for public download and independent replication. To the best of my
knowledge, none of these authors (myself included) followed these steps in their work because it
was not common practice in the era in which these studies were conducted. Hopefully, this will
occur with subsequent research on this topic. Such replication helps to ensure that the strength of
an association stands up more firmly to outside review and to confirm that the findings are not
due to error.
In epidemiology the term "effect modification" (or "interaction") is often used to refer to factors
that influence the relationship between an exposure of interest and an outcome. For example, the
(strong) effect seen between asbestos and lung cancer is modified by the presence of smoking in a
way such that the presence of both smoking and asbestos is more strongly associated with lung
cancer than would be expected by the "sum" of the two exposures independently. In the studies
reviewed for the relationship between coliphage exposure and illness, there is some evidence to
suggest that the relationship of coliphage to illness may be modified by subject age and by the
presence of a point source discharge. This would have the immediate effect of calling into question
whether coliphage levels alone, without specification of the age of the population and the location
of the exposure, could be an accurate risk monitoring tool. Two papers presented in the review
(Lamparelli and Marion) show associations between other fecal indicators and illness in disparate
settings including an inland lake and a tropical beach. The association of coliphage or other viral
indicators with illness in these settings cannot be determined with currently available data.
The confidence of the field in the use of fecal indicator bacteria for recreational water monitoring
grew out of the replication of a large number of studies under various conditions; a similar
pathway to understanding is likely to be required to properly understand the relationship between
coliphage and illness. The studies to date have inserted coliphage measurement into studies of
fecal indicator bacteria; there will likely need to be large studies focused on coliphage as the
exposure of interest. In conclusion, the evidence presented is suggestive but not conclusive about
the relationship between coliphage exposure in recreational water and human illness. Although I
am not able to draw a firm conclusion about the relationship at this point, the current evidence
does suggest that this is a promising relationship that deserves further evaluation because of its
biologic plausibility and the attractive operational features of coliphage measurement that are
being discussed in other sessions of this conference.
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Response: Tim Wade
Charge Question 1: FIB such as Enterococcus and E. coli have been broadly and successfully used
as indicators of water quality at beach sites, and have for the most part been established as
predictors of swimming associated Gl illness in epidemiological studies. However, in some notable
circumstances, studies have failed to demonstrate an association, or the association has been
weaker than expected. Moreover, as noted in EPA's Coliphage review, researchers have believed
for many years that enteric viruses may be responsible for the excess illnesses commonly observed
among swimmers. As a result, an indicator better suited to viral waterborne pathogens is
theoretically well-justified. However, epidemiology studies of coliphage and human health in
recreational waters are relatively few and generally limited in terms of scope and diversity
compared to studies focusing on fecal indicator bacteria such as Enterococcus and E. coli. Despite
the limited number and scope of the studies, several of those that have evaluated an association
between coliphage and swimming associated illnesses have found fairly convincing evidence that
coliphage presence, or in some cases density, was associated with illnesses among swimmers. In
addition to the studies noted in EPA's review, a report by Pike et al. in marine waters also
appeared to find an association with coliphage and gastrointestinal illness (Pike at al., 1994).
Because this study was a precursor to randomized exposure studies by Fleisher and Kay (2006)
presumably this study was at a site impacted by human sewage.
In evaluating the overall strength of the association, a first critical question concerns the illness
endpoint. This should be based in part on the strength and consistency of observed associations,
but biological plausibility should guide which endpoints are considered in the first place. Because
the general idea behind using coliphages is that they may be a more specific indicator of fecal
contamination, and in particular a better indicator of viral pathogens associated with feces, the
most plausible endpoint for consideration is Gl illness, and to a lesser extent respiratory illness.
Endpoints such as skin rashes, ear and eye infections, could also feasibly be associated with fecal
contamination, they are less strongly linked to viral waterborne pathogens, so should be
considered secondary outcomes.
The epidemiology studies considered in the review are quite heterogeneous in terms of study
population, design, location, water type (marine vs. fresh), water sampling, data analysis and
illness outcome definition and assessment, which limits the ability to combine and assess the
literature as a cohesive group or to make broad ranging general conclusions regarding coliphage
and swimming associated illness. Even when a common endpoint, such as Gl illness is considered,
the specific definition can vary across studies, sometimes significantly, which can affect
interpretation of the results. For example norovirus associated infections often produce a limited
or no fever. Some definitions of Gl illness used in epidemiology studies require fever in addition to
diarrhea or other symptoms as part of an episode- this could result in norovirus associated illness
episodes being underestimated or not counted.
Although the specific definitions varied, the most frequent endpoint associated with coliphage was
Gl illness which is consistent with the biological plausibility discussed above and the current
understanding of coliphage as an indicator. However, the studies as a whole are varied and
somewhat inconsistent in the findings. Critically, there is inconsistency in the types of coliphage
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measured- while some measured multiple types, some studies only measured somatic, some F-
specific, and at least two only measured F-specific RNA coliphage. Only a few studies tested
multiple coliphages, so for most studies associations with, for example somatic and F-specific
coliphage cannot be compared. Often, sufficient detail is not provided, for example Von Schirnding
et al. (1992) indicates "coliphages" were tested in addition F-specific bacteriophages, and does not
report numbers of samples detected and range, only "insignificant densities...were detected".
Similarly, Van Asperen et al. (1998) only tested for F-specific RNA bacteriophage, and although the
densities are reported, quantitative measures of illness associations are not reported, only a
mention that an exposure response relationship was not observed. This limits the ability to assess
trends across studies of potentially positive but non-significant trends and the possibility of
combining data in a formal meta-analysis.
One of the strongest associations observed between coliphage and Gl illness was Lee et al. (1997)
in a study of canoeists and rafters in a man-made river which was highly contaminated by treated
and untreated sewage and runoff. In this study, increased risk of Gl illness were observed for
canoeists and rafters exposed to F specific RNA coliphage between 230-320 PFU/100 ml and 690-
3080 PFU/100 ml compared to 10-30 PFU/100 ml. Note that there is some question on whether
these levels are correct, because one table reports the results as per 100 ml and the other as per
10 ml. Regardless, because of the high levels of contamination and because the study focused on
canoeists and rafters, the generalizability of these findings to other sites and populations is
questionable.
Wade et al. (2010) and Colford et al. (2007) each found positive associations with F-specific
coliphage at marine beaches among a beachgoing population using a similar "design at a human
impacted and a primarily non-human impacted beach. However, especially for Colford et al.
(2007), the findings are limited due to few swimmers exposed on days when coliphage was
present. Griffith et al. (2016) appears to provide some additional evidence under conditions when
a human impact was likely but is impossible to fully evaluate because the study is unpublished and
full details are not publicly available.
The study with perhaps the strongest and most convincing findings of an association and potential
exposure-response was the randomized exposure study by Wiedenmann et al. (2006). This study
found excess Gl illness, or a NOAEL, above 10 somatic coliphage per 100 ml (note that this level is
below the reference range for Lee et al. (1997) for F-specific RNA coliphage). Unfortunately, F-
specific coliphage was not measured. Interestingly, no other studies have reported an association
with somatic coliphage. Abdelzaher et al. (2011) also conducted a randomized exposure study, but
somatic and F-specific coliphage were detected infrequently and no associations with illness were
observed.
In summary, there is some epidemiological evidence that coliphage is associated with Gl illness
among swimmers in both marine and fresh recreational waters. However, overall, the evidence is
rather inconsistent and a coherent synthesis is limited due to a wide range of methods, sites, study
designs, measurement methods, etc. There is not, however, strong evidence to establish exposure
response associations based on these studies as a whole. A main concern is the numbers of studies
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that did not detect coliphage or had few detects- this poses major issue for establishing exposure
response associations or establishing threshold values.
Charge Question 2: The epidemiological studies alone conducted to date do not provide a clear
answer to this. Positive associations have been found in a range of conditions, although
inconsistently. Studies have found positive associations at both marine and freshwater beaches,
and at sites impacted by human sources (Lee et al., 1997; Wiedenmann et al., 2006; Wade et al.,
2010) as well as sites impacted by non-human sources (Colford et al., 2007). Comments by Griffith
et al. (2016) would also indicate that human vs. non-human source is important as an association
was only observed on days of "high risk", or likely human impact. As discussed above, the lack of
detection or low concentrations of coliphage in some studies is concerning. Sites where coliphage
are detected infrequently would influence the ability to detect an association with health, unless
the conditions under which they are not-detected can be clearly understood. In these cases,
coliphage may be less favorable than standard FIB which can usually be commonly detected. If, for
example, the extent to which salinity or other factors impact the ability to detect coliphages, this
could be a concern but I am not aware if these methodological limitations are an issue. In most
cases, however, the low detections were at sites with non-point and likely non-human sources of
fecal contamination.
As with most general indicators, it is expected that coliphages would be best associated with Gl
illness in a consistent manner at sites impacted by human sources of fecal contamination. Because
they can also be found in the feces of other animals, which are unlikely to carry waterborne
enteric viruses, this lack of human-specificity would likely limit their application at predominantly
animal impacted sites, which seems to be supported in the study by Abdelhazer et al. (2011) and
from the comments noted by Griffith et al. (2016). However, the study by Colford et al. (2007)
seems to contradict this where some association was found with only coliphage and no other
indicators at a beach impacted predominantly by birds.
One condition which may influence the association with coliphage and illness is the type of
treatment sewage receives prior to discharge. In conditions where there is a relatively consistent
source of raw or untreated sewage, it may be that coliphage adds little compared to standard FIB,
though it certainly may perform at least as well. However at sites impacted by treated sewage or
partially treated sewage, which disproportionally kills FIB and bacterial pathogens over viruses
such as chlorination, coliphage may be a preferred indicator as it will likely better mimic the
survival of viral pathogens.
Charge Question 3: The epidemiology literature provide little information to address this issue.
Only a limited number of epidemiology studies have been conducted in a tropical environment and
those that have did not test for coliphage. Theoretically, as several authors have speculated,
coliphage may be a better indicator in tropical environments where FIB can persist, or potentially
regrow in the environment, since coliphages do not. This may be especially true at tropical sites
where sewage is at least partially disinfected, such as Hawaii. However, at this time this is
speculation as no studies have addressed this directly. The EPA Coliphage Review cites one paper
which observed no association between coliphages and pathogens in tropical coastal streams,
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which provides some evidence that coliphages may not in fact provide additional benefit in
tropical environments, but the evidence is very limited in this area.
The conclusion of the authors of the Lamparelli et al. (2015) study was that FIB, Enterococcus and
E. coli were in fact were well-associated with Gl illness among swimmers in tropical environments
where inputs were primarily raw or untreated sewage. Absolute illness rates differed and may
have been higher than allowable under EPA regulations but the authors do not directly report
swimming associated illness and differences could be a result of the study population, illness
definition or study design, differences in the study population and the high level of contamination
observed at these beaches. As discussed above, when impacts are a constant source of raw
sewage, coliphage may add little as an indicator compared to general fecal indicators.
Marion et al. (2010) also observed associations with E. coli and in a subsequent paper found that
human adenovirus in combination with E. coli explained more Gl illness than either indicator
alone. So although Marion et al. (2010) provides some evidence of the benefit of a viral pathogen-
indicator, since they did not measure coliphage it is difficult to draw any conclusions directly
regarding coliphages.
As discussed above, when the sources are treated sewage, or diffuse and mixed sources, coliphage
may be a better indicator as it is less affected by disinfection and better mirrors the fate and
survivability of waterborne pathogenic viruses. Although the NEEAR freshwater studies, which
were conducted at sites with treated sewage, (Wade et al., 2008) did not measure coliphage, they
speculated that the reason the Enterococcus qPCR measure was better associated with Gl illness
compared to culture was the persistence of the genetic material compared to the culturable
measures, potentially better mirroring waterborne viruses' survival through the treatment
process.
In summary, provided they can be detected, I would expect coliphage should be at least as
effective as FIB in sites with a high level of human fecal contamination from untreated sources.
However, if they are undetected at sites with moderate levels of contamination, this may be
problematic. Finally they may provide an advantage when sites are impacted by treated sewage
effluent or at sites with sporadic impacts of human fecal contamination (due to their longer
persistence in the environment). However, epidemiology studies alone only provide partial and
inconsistent support for these expectations.
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Topic 3: Coliphage as an Indicator of Wastewater Treatment Performance
Topic Leads:
• Bill Burkhardt and Kevin Calci
• Naoko Munakata
• Joan Rose
Charge Questions:
In EPA's Coliphage Review, EPA summarized indicator attributes and treatment removal
efficiencies of FIB, coliphages, and enteric viruses. EPA concluded that coliphages are likely a
better indicator of viruses across wastewater treatment, compared to the currently recommended
FIB (i.e., enterococci and E. coli).
1. Comment on EPA's conclusion that human pathogenic viruses are entering surface waters via
wastewater treatment effluent.
2. Summarize the most important reasons that coliphages might be useful models or indicators
of the behavior of enteric viruses in wastewater treatment and disinfection processes.
3. Comment on EPA's conclusion that monitoring for coliphages would be more useful than
enterococci and E. coli in predicting human viral pathogens in wastewater treatment effluent.
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Response: Bill Burkhardt and Kevin Calci
Charge Question 1: Human pathogenic viruses were documented in treated wastewater effluent
three decades ago by Cabelli, Dufour, Havelaar, Gerba, Goyal, Bitton, and Sobsey to name a few.
Contemporary data from da Silva et al. (2007); Flannery et al. (2012); Katayama et al. (2008); and
Tian et al. (2012), are several that have not only detected but quantified Norovirus from treated
wastewater effluent with adequate extraction and RT-qPCR inhibition controls. Data from those
types of peer reviewed journals articles and USFDA data were the basis of the AEM manuscript
entitled, Meta-Analysis of Norovirus and Male-specific coliphage (MSC) Concentration in WWTPs
by Pouillot et al. (2015). The manuscript supports the conclusions of the EPA, by modeling the
influent, pre-disinfected effluent and final effluent virus loads and demonstrating that, on average,
wastewater treatment plants contribute human enteric viruses into the marine environment.
In addition, decades of outbreaks of viral gastroenteritis associated with molluscan shellfish, in
many cases, supports the conclusion that treated wastewater can impact shellfish growing areas.
Recently, the FDA has documented the impact of a variety of wastewater treatment designs, flows,
and disinfection types on shellfish sentinels placed at various dilutions of effluent in the estuarine
environment. The results were consistent with the EPA conclusion that secondary treatment with
chlorination does not sufficiently reduce or inactivate enteric viruses in the final effluent discharge.
Furthermore, the human enteric viral impact from the treated wastewater effluent can be
quantified in the sentinel shellfish tissue.
Charge Question 2: Coliphages and more specifically MSC meet many of the "ideal indicator"
requirements discussed by Berg, Cabelli and Goyal. Based on six facts, Kott (1981) proposed
coliphage as indicators of human enteric viruses; 1) Coliphages are found in abundance in
wastewater and polluted water, 2) The population of coliphages exceed those of enteric viruses, 3)
Coliphages are incapable of reproduction outside the host organism, 4) Coliphages can be isolated
and counted by simple methods, 5) The time interval between sampling and final result is shorter
than that for enteric viruses, and 6) Many coliphages are more resistant to inactivation by adverse
environments and disinfection than enteroviruses. Although dated they still hold true today.
One of the limitations of using coliphage as a surface water pollution indicator and trying to
correlate them with the occurrence of human enteric virus is they do indeed have different
ecologies. That issue is circumvented if coliphage are used directly as a treatment process
indicator. MSC are well suited as an indicator of treatment because they are roughly the same
shape, size and nucleic acid composition as human norovirus. Although coliphage and enteric virus
tend to have a high affinity to sewage solids, most mechanical secondary treatment plants are not
designed to slow the water enough to achieve greater than a two log reduction during the
treatment process. FDA data demonstrate a substantial reduction in MSC from tertiary WWTPs.
MSC have been shown to be very resistant to chlorination as applied by most WWTPs. The amount
of free chlorine in a high demand environment limits the efficacy. Combine that with relatively
short contact times and the impact is further reduced to less than a log reduction. In contrast, a
greater than two log reduction of MSC will be observed with UV disinfection on a tertiary system
working at or below design flow. One of the great benefits of using MSC is that not only is the
Coliphage Experts Workshop
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method facile, cheap and relatively quick, but it informs you of the viability of the organism. Unlike
the molecular test which show the genetic material been conserved but cannot distinguish if the
protein coat or receptors are intact. In general, it seems enteric virus inactivation by WWTP
disinfection is more similar to MSC than anything else we have to compare as a process indicator.
Charge Question 3: Treatment standards and technologies have steadily improved from their
inception. A large leap occurred in the 1950s by disinfecting effluent with chlorine to stay off
Typhoid fever outbreaks linked to swimming in and harvesting shellfish from sewage impacted
waters. The bacterial pathogen was mitigated from the effluent but the viral risk remained. In the
following decades treatment plants standards continued to improve but FIB were still found
commonly in effluent especially under higher than designed flows. Catchments had many
combined sewage overflows that kept WWTP impacted waters easily identified by FIB analysis at
water quality stations.
Currently, standards and design technologies can produce bacterial-free effluent underflow
capacity well over their design. FDA data demonstrate it is specifically these instances that the
MSC outperform the FIB in assessing the increased contribution of enteric viral load from the
WWTP. As sanitary indicators E. coli and enterococci serve us well, but with respect to
performance indicators of modern day WWTP viral reduction efficiencies, they do not come close.
Coliphage Experts Workshop
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Response: Naoko Munakata
This response focuses on Charge Questions #2 and 3, specifically on the behavior of various
organisms across disinfection processes. Most data presented below is given numerically in the
literature, but some were estimated based on graphs.
Because treatment depends on the process and the organism of interest, the following sections
are organized by process. A 2008 Water Environment Research Foundation (WERF) report (Leong
et al., 2008) estimated that in the United States, there are 4,450 major POTWs, defined by the EPA
as facilities owned by a state or municipal entity and have an average dry weather design flow of
>0.95 MGD. Of these plants, 70-75% were estimated to use chlorine or chloramines for
disinfection; most of these plants likely use chloramines, because wastewater effluents typically
contain ammonia, which reacts with chlorine to form chloramines. Approximately 20-25% of the
facilities were estimated to use UV, approximately 3-4% did not disinfect, and 7 plants used ozone.
Therefore, this report focuses on these disinfection technologies.
Chloramines
Chloramine disinfection is generally given as a function of the contact time (CT) value, which is the
product of the total chlorine residual concentration and contact time. The total chlorine residual
decays over time in wastewater effluents; CT values can use the added dose (which over-estimates
exposure to the disinfectant), the final residual (which under-estimates exposure), or an
integration (rarely used). The residual chosen can make a large difference in CT value; for example,
if the added dose is 5 milligrams (mg)/L and the residual after 90 minutes is 2 mg/L, the "dose CT"
is 450 mg-min/L, but the "residual CT" is 180 mg-min/L. For compliance with regulations such as
the California Title 22 requirements for recycled water, the residual CT is used. For some of the
data collected, both the dose and residual were given, but in others, only one value was provided.
Figure 1 presents disinfection data for various organisms for both the dose and residual CT values.
¦ Poliovirus
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Figure 1. Inactivation of various organisms by chloramines as a function of
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In Figure 1, some data were not shown because they were outside the graphed time scale:
• 1(a) Somatic coliphage log reductions of 2.0-2.2 at CT values of 2700-4900 mg-min/L.
(Worley-Morse, 2015).
• 1(b) MS2 log reductions of 0.3-0.8 at CT values of 1700-7500 mg-min/L (Shang et al., 2007).
Based on CT data, poliovirus disinfection appears to be highly variable. E. coli disinfection was
similar to or slightly greater (less conservative) than poliovirus disinfection, and Enterococcus and
F+ coliphage disinfection was within the range of poliovirus disinfection. MS2, somatic coliphage,
and "enteroviruses" appear to be more resistant to chloramines than poliovirus. The enterovirus
data are unexpected, because poliovirus is a member of the enterovirus genus. However, the data
are from a single study that investigated seeded poliovirus and indigenous enterovirus in primary
effluent, while most of the other data were collected with secondary or tertiary effluent; the
authors also hypothesized that the high resistance of the indigenous virus was due to association
with particles, which shielded them from the chloramines.
Overall, Enterococcus or F+ coliphage may be the best indicator; their disinfection was similar to
poliovirus. E. coli appear to be less resistant than poliovirus based on the (limited) residual CT data
and may therefore be non-conservative. MS2 coliphage are clearly more resistant (and therefore
conservative), and it is tempting to identify them as the best indicator. However, several authors
suggest that chloramines have no effect on MS2, and attribute any observed disinfection to free
chlorine that exists immediately after dosing, before it reacts with ammonia to form chloramines.
Consequently, MS2 may not show any disinfection by chloramines, regardless of the true level of
inactivation of pathogenic viruses. The data on somatic coliphage are limited but suggest that they
are similarly resistant to chloramines.
Free Chlorine
Free chlorine disinfection is also generally given as a function of the CT value. Figure 2 presents
disinfection data for various organisms for the residual CT values; there were insufficient data for
comparison with the dose CT values. Based on these data, MS2 appears to be a reasonable
indicator of poliovirus and adenovirus disinfection; F+ coliphage appear to be more resistant.
Coliphage Experts Workshop
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0
0.1 1 10 100 1000
Free Chlorine Residual CT (mg-min/L)
Figure 2. Inactivation of various organisms by free chlorine as a function of residual CT
UV
Over the past 10-20 years, the use of UV for disinfection has been increasing at wastewater
treatment plants across the country. Figure 3 shows inactivation of various organisms by UV
disinfection, primarily with low pressure UV lamps. E. coli was the least resistant of the organisms
shown. Poliovirus, other viruses (coxsackievirus, reovirus, rotavirus, hepatitis A, Echovirus), and
somatic coliphage showed similar resistance to UV; Enterococcus and MS2 were slightly more
resistant than these organisms. Adenovirus is notoriously resistant to UV disinfection and showed
a lower level of inactivation than any of the other organisms. Based on this study, E. coli is not an
ideal indicator organism because it is not conservative relative to the pathogenic viruses.
Enterococcus, somatic coliphage, and MS2 appear to be slightly more conservative than the
pathogenic viruses, and would be better indicator organisms of most enteric viruses (except
Adenovirus).
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8
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Figure 3. Inactivation of various organisms by UV disinfection.
"Other viruses" include coxsackievirus, reovirus, rotavirus, hepatitis A, Echovirus
Ozone
Ozone disinfection is often reported as a function of the transferred ozone dose, or less
commonly, the applied ozone dose. In the presented study, MS2 is less resistant (conservative)
than poliovirus, and therefore is not a good indicator. Enteroviruses were disinfected to below
detection (>~2.8-log) at a transferred ozone dose of 5 mg/L. Thus, based on the limited data
presented here, MS2 is not a good indicator organism because it is less conservative than
poliovirus; Enterococcus may be a viable indicator.
Summary
Based on the data presented here, it is difficult to conclude that coliphage are better indicators of
the behavior of enteric viruses in wastewater treatment.
• F+ coliphage (mostly tested with MS2) were slightly conservative indicators of most enteric
viruses (except adenovirus) for UV disinfection, and behaved similarly to poliovirus for free
chlorine. MS2 was conservative for chloramines, but may be so resistant that it shows no
disinfection, regardless of the level of enteric virus disinfection; this is particularly
problematic given the high percentage of POTWs using chloramines for disinfection. Finally,
for ozone, MS2 was less conservative/resistant than poliovirus.
• Very limited data on somatic coliphage suggest that they may behave similarly to MS2, with
little to no disinfection by chloramines, even at doses where poliovirus is disinfected. For
UV, it behaved similarly to most of the enteric viruses (except adenovirus).
• E. coli appears to be slightly less resistant than poliovirus to chloramines, and is much less
resistant than enteric viruses to UV disinfection. Therefore, it is not an appropriate
indicator organism.
¦ Poliovirus
• Other Viruses
•Adenovirus
X Enterococcus
XE. coli
~ MS2
A Somatic Coliphage
->0<-
K- i ¦ J*—~~
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• Enterococcus may be the most promising indicator. Its behavior was similar to poliovirus for
chloramines and to enteric viruses for UV (except adenovirus), and it was more
conservative than enteroviruses to ozone.
• Overall, significantly more data are needed to better evaluate the ability of various
organisms to serve as appropriate indicators under the different disinfection systems in use
at POTWs around the country.
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Response: Joan Rose
Charge Question 1: There is ample evidence that viruses are found in secondary effluent, yet the
percentage of samples positive and concentrations are highly related to the concentrations found
in raw sewage, the treatment processes and whether there is disinfection used. One of the issues
is that design and operations of sewage treatment plants around the United States is highly varied.
A full scale study of six wastewater facilities in the United States concentrations of coliphagel5597
ranged from 104 to 106 PFU/100 mL. The concentrations of coliphage700891 (f-amp) were more
variable within each plant and ranged from 103 to 107 PFU/100 mL. Figure 1
A comparison of the concentrations of enteric viruses isolated from the untreated wastewater
(MPN) ranged from about 102 to 105 MPN/100 L. Figure 2. (Enteric viruses. Samples were filtered
through Virusorb 1MDS Filters (Cuno Inc.) as per EPA (1994) methodology. Filters were eluted with
1 L of 1.5% beef extract (BBL V) in 0.05 M glycine (pH 9.5, ~25°C) (EPA/Information Collection Rule
[ICR]). The eluted sample was concentrated by organic flocculation and assayed for enteric viruses
by the observation of cytopathic effects (CPE) on recently passed (<4 days) cell lines. MPN
determinations were performed using EPA released software).
7
6
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Figure 1. Coliphage in full scale raw sewage in six facilities in the United States
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8
7
J 6
5
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Figure 2. Cultivatable enteric viruses in raw sewage from six full scale systems in the United States
The concentrations of the cj)phagei5597 host (somatic and MS) ranged from about 10 to 105
PFU/100 mL and were more variable than the concentrations of the 4)phage7oo89 f-amp (male-
specific [MS]). For the four activated sludge facilities (A,B,C,D), coliphage levels were lowest in the
secondary effluents from plant A, which has a longer mean cell resident time (MCRT, the average
time that the mixed liquor suspended particles remain in the activated sludge process).
In secondary effluent the concentrations of viruses ranged from below 10 to about 103 MPN/100 L
with the lowest median concentrations of enteric viruses associated with plants A, E, and F. Enteric
virus detection limits for the secondary effluent samples ranged from 2.9 to 11 MPN/100 L,
depending on the sample volume processed. The concentrations of enteric viruses in about 33% of
the secondary effluent samples were below detection limits, with nondetects associated with all
facilities, except plant C (shortest MCRT) (Figure 3).
These facilities were reclamation systems and after secondary treatment the effluent underwent
sand filtration and was then disinfected so turbidities were down that might have interfered with
the inactivation. After disinfection concentrations of enteric viruses were below detection limits
(0.3 to 1.5 MPN/100L) in 69% of the samples. They were never detected in the disinfected effluent
from facility E (UV) and rarely detected in facility B (prechlorination of filter) or D. In most cases,
the detected concentrations of enteric viruses were below 1 MPN/100L (Figure 4).
Coliphage Experts Workshop
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8
7
6
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~i r
Secondary
Enteric viruses
I
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B C D E F
Figure 3. Enteric viruses in secondary effluent (from Rose, 2004)
4
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ed effl
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Figure 4. Enteric viruses in disinfected effluent (post filtration) (from Rose, 2004)
Many other studies have used molecular techniques and it is clear that the viral particles are
present at high concentrations.
In studies in the Chicago river (Asian et al., 2011) which received non-disinfected effluent,
culturable viruses were detected in 100% of the small number of samples assayed by cell culture
(13) on both BGM and A549 cell lines. The infectious virus numbers ranged from 0.12 to 33 MPN/L
using BGM cells and 0.11 to 26 MPN/L using A549 cells. Average virus concentrations in limited
contact recreational waters as determined by cell culture using BGM cells and A549 cells were 5.5
MPN/L and 7.6 MPN/L, respectively. For bathing beaches, average virus concentrations as
Coliphage Experts Workshop
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determined by cell culture using BGM cells and A549 cells were 8.8 MPN/L and 2.5 MPN/L,
respectively.
Charge Question 2: The viral morphology and size are associated with persistence and resistance
to treatment. One of the issues is the wide range of enteric viruses and phage found in
wastewater. Determining the relationships will require much more data.
We have been working with the City of Toledo on a study to examine waterborne pathogen levels
in urban watersheds during wet weather events. The City of Toledo approximately doubled the
wet-weather flow treatment capacity of the Bay View Water Reclamation Plant (WRP) by adding
auxiliary treatment facilities that include DensaDeg® High Rate Clarification (HRC) with effluent
chlorination. The objective of this study is to evaluate the disinfection efficacy of the HRC auxiliary
wet weather treatment process, by analyzing pathogen removal by the HRC train in comparison to
the activated sludge (AS) train.
We have found that coliphage removal was very similar to Enteric viruses in this study and it was
different then the bacteria and protozoa.
Toledo rain event 5-31-2015 logarithmio reduction values of bacteria, protozoa, and viruses by
conventional and high rate clarification treatment processes.
ORGANISM
CONVENTIONAL
HRC
Influent to
Activated
Sludge
Effluent (pre-
chlorination)
Activated Sludge
Effluent (pre-
chlorination) to
Disinfected Effluent
(post-dechlorination)
Influent to
Disinfected
Effluent (post-
dechlorination)
Influent to HRC
Effluent (pre-
chlorination)
HRC Effluent
(pre-
chlorination) to
Disinfected
Effluent (post-
chlorination)
Influent to
Effluent (post-
chlorination)
Somatic phage3
0.38
1.48
1.85
0.39
0.53
0.93
Male-specific
phage3
1.21
0.64
1.85
0.39
0.09
0.48
Enterococci3
1.73
0.72
2.46
0.48
1.59
2.07
Campylobacter3b
0.00
-0.02
-0.02
0.02
0.00
0.02
Salmonellac
1.27
0.22
1.48
0.57
1.16
1.73
Cryptosporidiumc
0.49
0.12
0.62
1.35
-0.30
1.05
Giardiac
1.20
-0.18
1.02
1.69
-0.38
1.32
Total Cultivable
Virusc
2.84
-1.23
1.61
0.27
0.60
0.87
Adenovirusc
2.84
-0.84
2.00
-0.21
0.60
0.39
a Reduction values calculated from geometric mean values of microbial concentrations (Table 1). Non-detects
calculated as detection limit.
b As a result of low initial concentration, reduction values will appear low. For samples returning a positive result,
detection of Campylobacter was at or just above the limit of detection of the assay.
c Reduction values calculated from composite values of microbial concentrations (Table 4 and Table 6). Non-detects
calculated as detection limit.
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Charge Question 3: The phage in many studies have been able to address viral reductions, yet the
ability to predict presence/absence or concentrations will require more data and analysis.
The presence of human enteric cultivatable viruses were correlated to phage in a coastal system in
a binary regression analysis. Where the human viruses were found only after the phage reached
100 PFU/mL. This relationship may be watershed specific.
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Topic 4: F-Specific Coliphages vs Somatic Coliphages
Topic Leads:
• Nicholas Ashbolt
• JuanJofre
• Rachel Noble
Charge Questions:
EPA's Coliphage Review provides background information relevant to the use of both F-specific
and somatic coliphages, as indicators of viral fecal contamination. EPA is considering these two
possible viral indicators for use in future recreational water quality criteria (RWQC).
1. What are the most important advantages and disadvantages of using these two types of
coliphages as (a) predictors of human health illness in recreational waters; (b) indicators of
wastewater treatment performance?
2. Are there specific attributes of these coliphages or conditions (i.e., source) that influence the
usefulness of the indicator, or would favor the use of one type of coliphage over the other?
3. EPA is aware that other countries have considered coliphages for various purposes. Please
provide summaries and commentaries for any of these efforts you deem helpful.
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Response: Nicholas Ashbolt
Charge Question 1; Point 1: What are the most important advantages and disadvantages of using
these two types of coliphages as:
a) predictors of human health illness in recreational waters;
b) indicators of wastewater treatment performance?
Given that the overall review is to "...summarize the scientific literature on coliphage properties to
assess their suitability as indicators of fecal contamination in ambient water" there is an apparent
misunderstanding as to the value of any general fecal indicator, which coliphages are, versus
"...usefulness of the indicator..." with respect to indicator uses identified in points 4.1 (a) and (b)?
Point 1(a) focusing on F-specific and somatic coliphages, the review fails to clarify that predictors
of human health illness can only be identified by epidemiology studies, whereas predictors of
recreator risk can be undertaken by epidemiology and/or QMRA. The second apparent
misunderstanding arises from epidemiology studies in the United States largely focusing on water
known to be impacted by sewage. For sewage-impacted waters is seems reasonable that enteric
"viral pathogens are the leading causative agents of recreational waterborne illnesses" [pi, para
3]. However, in non-sewage(human)-impacted recreational waters, where epi data typically shows
no ill effects (likely due to the lack of sensitivity in epi studies used, that can be overcome by
QMRA risk estimates), clearly human enteric viruses are not likely to be the main class of pathogen
causing illness, [pi, para 3 goes on to state, leaving it ambiguous as to what fecal sources by...]
"This review considers coliphages as possible indicators of fecal contamination in ambient water."
Hence the review needs to clarify it is limited in scope to sewage-impacted recreational
waters or state otherwise if that is the case. This question is fundamental to answering
Topic 4.1(a)
The fact that Abdelzaher et al. (2011) non-point study [P23] is included suggests more than
just sewage-impacted is to be considered?
As predictors of human enteric viruses, both F-specific and somatic coliphages are consistently
present in sewage, hence what the review needed to do was to clarify fate and transport scenarios
to compare data for these (culturable) coliphages against specific human enteric virus infectivity's
(rather than a meta-analysis of publish data). Then the question (a) of predicting human health
illness could be answered.
The review appears to just provide a general context background [bottom of P2 to first para of P3];
and the misleading statement [P3, para 1] "They originate almost exclusively from the feces of
humans and other warm-blooded animals and can undergo limited multiplication in sewage under
some conditions (i.e., high densities of coliphages and susceptible host E. coli at permissive
temperatures) (Sobsey et al., 1995; Grabow, 2001)." This is potentially misleading as while F-
specific coliphages required the F-pilus in E. coli (or other bacteria) to enter their host and this is
thought to only be expressed at warm-blooded animal temperatures, somatic coliphages enter via
the cell wall so in theory, any cold-blood animal or environmentally adapted E. coli or related
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bacterium could amplify and release somatic coliphages (which represent a very broad range of
bacteriophage families).
We simply do not know what range of somatic coliphages may grow outside of warm-
blooded animal's gastrointestinal tracts.
This concern is backed up by the 540 indicator-pathogen pair study of Wu et al. (2011)
[P27-29] where coliphages and pathogens were not significantly correlated, except for F-
Specific coliphages-adenovirus pairs, which was confounded by salinity level; and by
Lodder et al. (2010) [P31] both coliphage groups sig correlated with enteroviruses but not
with other enteric viruses (norovirus, rotavirus and reovirus).
Comments like [P7, para 2] "Lack of replication in the environment is partially because coliphages
do not replicate below a bacterial host density of 104 colony-forming units per mL (Wiggins and
Alexander, 1985; Woody and Cliver, 1997)" portray a lack of ecological understanding, where
environmental E. coli grow with Cladophora glomerata mats or in sediments at higher densities
(Davies et al., 1995; Verhougstraete et al., 2010).
Yet the comment [P7, end second last para] "However, additional research to test whether the
coliphages detected on environmental E. coli strains can also infect the E. coli strains used in
laboratory assays is needed." is a good point.
Also, F-specific deoxyribonucleic acid (DNA) coliphages are much less studied and hence
poorly understood as to their environmental sources and persistence, making the broad
charge question 'F-Specific coliphages' too broad - suggest the authors are generally
describing F-Specific RNA coliphages.
The term 'microflora' [e.g. used P3,1.3 first bullet] was replaced in the 1980's with the
term 'microbiota' by microbial ecologists and others, given Bacteria and Archaea are not
little plants, but two separate domains of life, more distant than plants are from animals!
Please correct throughout.
[P4, bullet two] about coliphages "they are present in greater numbers than pathogens (Havelaar
et al., 1990; Debartolomeis and Cabelli, 1991; Leclerc et al., 2000)" is not so clearly evident for
environmental waters. For example, somatic coliphages and F-Specific RNA coliphages across U.S.
sewage range 2.1-3.9 and 2.1- 3.7 logio PFU/mL respectively against qPCR human adenoviruses of
1.9-3.4 genome copies/mL; but F-RNA coliphages more rapidly lost infectivity against the others,
so over time in the environment higher ratios are less likely (McMinn et al., 2014). These recent
U.S.-reported coliphages concentrations appear significantly lower that reported for Europe [P 6,
para 2] "Coliphages are present in large numbers in sewage (approximately 107 plaque forming
units [PFU] per milliliter [mL]) (Ewert and Paynter, 1980; Lucena et al., 2004; Lodder and de Roda
Husman, 2005)." Also, [P56, para 3] lower ranges and more variable concentrations reported for
the United States and Canada by Rose et al. (2004) and Payment and Locas (2010).
Furthermore, seems the reviewers and authors of Abdelzaher et al. (2011) [P23, Section
3.7] were unaware that F-Specific coliphages vs somatic coliphages being more rapidly
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inactivated in warm water, as in that Florida epi study, which could have accounted for
non-detects of F-Specific coliphages, whereas in the colder Avalon/Doheny waters F-
Specific coliphages were detected and associated with sewage pollution and health
outcome [P24 Section 3.8], but not by Boehm et al. (2009) [P32, para 2] between qPCR
enteric viruses and coliphages- possible method sensitivity issues in that study? And Viau
et al. (2011) [P31, para 4] found no sig correlation between F-Specific coliphages and
various human enteric viruses in tropical coastal streams. Hence, as a National ambient
water quality criterion, F-Specific coliphages may not be a good target group, which is a
view generally backed up in the review [Environmental Factors and Fate P37-45;
particularly P42, para 1 Love et al. (2010) and P43-44 Romero et al. (2011) temperature-
sunlight synergistic effects].
Point 1 (b) focusing on F-specific and somatic coliphages as indicators of wastewater treatment
performance
At a first pass, the review provides good evidence consistent with this reviewer's view that the
highest concentration, standard method somatic coliphage group provides the largest array of
viron types and highest concentrations to estimate general human enteric virus removal by
wastewater treatment processes [P57-58 specifically provide this evidence]. Given all the
uncertainties in pathogen assays, it seems unlikely that additional work in this area will yield a
different conclusion.
As reported for Australian, coliphages are used as indicators of wastewater treatment efficacy
[P59-60, by Keegan et al. (2012)]. However, what is missing in the review is the recent data from
tropical Singapore on the value and development of a recreational freshwater somatic coliphages
standard (Vergara, 2015; Vergara et al., 2016).
What is missing in the review is some description of the target log reduction required for safe
recreational in receiving waters, so as to clarify what level of sensitivity is required to demonstrate
satisfactory wastewater treatment performance, and therefore what type(s) of methods maybe
applicable.
Charge Question 2: As described above, somatic coliphages provide the most dynamic range in
concentration reduction by wastewater treatment and in viron types to represent a broad range of
human enteric viruses. They also persist longer in the environment, so may provide a more useful
conservative surrogate role compared to F-Specific coliphages.
Charge Question 3: In addition to Australian wastewater treatment identified in the review,
Singapore's developing use was missed (e.g., Vergara et al., 2015)).
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Response: Juan Jofre
Methodological considerations
Standardized methods, EPA and International Organization for Standardization (ISO), are available
for the detection of both somatic and F-specific coliphages. ISO method for F-specific adds the
protocol for differentiating F-specific RNA phages. Strains used for the detection of F-specific are E.
coli HS and S. typhimurium WG49 respectively. The qualifications of the methods:
• Both methods give similar numbers of counts (Schaper and Jofre, 2000; Guzman et al.,
2008)
• Strain HS seems more stable than WG49, but the stability (and re-selection) of strain WG49
is easier to check since it has explicit genetic markers whereas strain HS has not.
• Plaque reading of somatic coliphages is easier than that of F-specific.
• Somatic coliphages need a shorter time for results than F-specific phages.
• The possibility of counting both at once exist. For this purpose strain E. coli C3000 (Rose et
al., 2004) and E. coli CB390 (Guzman et al., 2008) have been used as host strains. Strain
C3000 detects lesser numbers of somatic coliphages as compared to strains CN13 (EPA)
and WG5 (ISO). Strain CB 390 according to results reported first in Spain (Guzman et al.,
2008, and already validated in the US (Sobsey et al., 2014), counts numbers similar to the
sum of those detected by WG5 or CN13 and those detected by strains WG49 or F(amp) HS.
• Methods based in the detection of host lysis by determination of a molecule released by
cell lysis are also available and offer the possibility of fast methods adapted to friendly use
kits.
Relation of coliphages to health risk
Seven epidemiological studies conducted to evaluate the relationships between the presence of
indicator bacteriophages in surface waters and swimming illnesses have been performed with
disparate outcomes (U.S. EPA, 2015). Three of them only studied F-specific phages, and whereas
two of them found some correlation, the third one did not. In one of the studies only considered
somatic coliphages and found some relationship. In the studies where both phages were tested
results are incongruent, since one of them failed to show any relationship with either somatic or F-
specific phages, a second found relationship with F-specific phages and a third found the
correlation with somatic coliphages.
No epidemiological studies to correlate phages and disease in drinking water have been
performed. However, an overlapping between a jaundice outbreak and a high incidence of somatic
coliphages in potable water occurred in a municipality of West Bengala, India in 2014 (Mookerjee
et al., 2014).
Overall, the epidemiological evidence, though without sound statistical correlations, suggests a
likely relationship between coliphages and human health. Also available data do not show a clear
better performance of any of the two groups of bacteriophages.
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Coliphages' abundance
The inputs of somatic coliphages in the environment by feces is greater than that of F-specific
coliphages.
Other than feces, the main contributors of water contamination, this is raw wastewaters,
secondary effluents, sludges, slurries and manure. With little exceptions, when counting in these
matrixes both groups of phages by standardized methods (EPA or ISO) in the same samples,
somatic coliphages overnumber F-specific phages by a factor of 5 to 10.
Thus, values of somatic coliphages in raw municipal wastewater range between 5xl05-107 per 100
mL and those of F-specific between 5xl04-5xl06 per 100 mL (Grabow et al., 1993, Contreras-Coll et
al., 2002, Lucena et al., 2003, Yahya et al., 2015, Zang and Farahbakhsh, 2007). In samples from
septic tanks, somatic and F-specific coliphages were detected with values ranging from 106 to 107
PFU per 100 mL and 105 to 106 PFU per 100 ml respectively (Lucena et al., 2003).
Regarding animal contamination sources, the most reported values of somatic coliphages range
from 104- 105 PFU per 100 mL in slurries and manures to 4xl08 in abattoir wastewaters, whereas
the most reported values for F-specific bacteriophages range from 104 PFU per 100 mL in animal
slurries and manures to 2xl08 in abattoir wastewaters (Grabow et al., 1993, Hill and Sobsey, 1998,
Blanch et al., 2006).
The most frequent values of somatic coliphages in secondary effluents range mostly from 103 to
105 PFU per 100 mL and those of F-specific and F-specific RNA phages from 102 to 5xl04 PFU per
100 mL (Aw and Gin, 2010, Grabow, 1993, Costan-Longares et al., 2008, Zang and Farahbakhsh,
2007, Yahya et al., 2015; Gomila et al., 2008).
The concentrations of somatic coliphages and F-specific phages detected in effluents released by
lagooning are variable depending on the extent of the treatment and the season. In any case, the
relative proportion of somatic coliphages to F-specific RNA phages remains as in incoming water
(Hill and Sobsey, 1998, Alcalde et al., 2003, Lucena et al., 2004, Gomila et al., 2008).
The same trend, this is somatic coliphages overnumbering F-specific phages, is true for primary
and raw sludge. Concentrations above 107 PFU of somatic coliphages and well over 106 PFU per g
of dry weight had been reported for primary and raw sludge (Guzman et al., 2007, Mandilara et al.,
2006b).
Regarding surface waters, reports from diverse parts of the world regarding concentrations of
both groups of phages measured by either EPA or ISO methods, after analyzing equal volumes of
sample and using exactly the same procedure for phage detection in surface waters indicate
numbers of somatic coliphages being greater than numbers of F-specific phages in either fresh or
marine waters. With little exceptions, the gap between the numbers of somatic and those of F-
specific is equal or greater than the gap in sources (Lucena et al., 2003; Rezaeinejad et al., 2014;
Ibarluzea et al., 2007; Contreras- Coll et al., 2002; Choi et al., 2005; Skraber et al., 2002, Moce-
Llivina et al., 2005; Lodder et al., 2010, Love et al., 2014).
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Persistence in water environments
There are numerous data reported on persistence and resistance experiments with laboratory
grown model somatic coliphages and F-specific coliphages. Nevertheless, I will limit the discussion
to naturally occurring coliphages.
Experiments performing in situ inactivation with sewage and/or waste stabilization pond effluent
mixed with river or seawater in a proportion of 10 % placed in a 560 millimeter (mm) depth (300
liters) open-top chambers were done. Experiments were performed in winter (approx. 12-C) and
summer (approx. 169C), in the dark, and with sunlight. The calculated T90 for somatic coliphages
ranged from 7 hours in seawater, summer and sunlight to 2303 hours in river water, winter and
the dark; and the calculated T90 for F-specific RNA bacteriophages ranged from 4.8 hours in
seawater, summer and sunlight to > 2303 hours in river water, winter and the dark (Sinton et al.
1999, 2002).
In other experiments, authors mixed raw municipal wastewater at a proportion of 2% with either
river or sea water, placed the mixture in dialysis tube, with a cut-off of 14.000 Daltons, that once
sealed were placed at a 20-25 centimeter (cm) depth in the same site where the river and sea
water were collected (Duran et al., 2002; Moce-Llivina et al., 2005). These sites were in the shadow
during part of the day. For somatic coliphages T90 ranged from 53 hours in seawater and summer
(>25-C) to 385 hours in winter (6-109C) and river water, whereas F-specific RNA T90 ranged from 14
hours in seawater and summer (>25-C) and 323 in river water and winter (6-109C).
Following land application of liquid pig manure Gessel et al. (2004) found that the numbers of
somatic coliphages after application are always higher than the values of F-specific, and that the
proportion somatic coliphages to F-specific phages rather increases in the following days.
In my opinion, it is worth to mention that the great majority of data regarding persistence and
presence of F-specific and F-specific RNA bacteriophages in receiving waters have been
obtained in cold and temperate climates. However, some evidences indicating that in areas or
periods with surface water temperatures higher than 259C the picture might be different. Thus,
in the experiments of in situ inactivation in the summer time, the T90 of F-specific RNA phages
becomes clearly shorter than those of somatic coliphages (Duran et al., 2002). In addition, in
oxidation ponds that can be viewed as an in situ inactivation experiment, Alcalde et al. (2003)
reported that in a stabilization pond series in the dry and warm desert of Judea (Israel), the F-
specific bacteriophages were removed more efficiently than somatic coliphages, mostly during
the summer. In the other hand, F-specific RNA bacteriophages occurring in secondary effluents
are significantly more sensitive to temperatures ranging from 259C to 409C than E. coli and
somatic coliphages (Agullo-Barcelo, 2013). A pair of studies performed in the United States
determined the numerical ratios between F-DNA phages and F-RNA phages in surface waters
and observed that in summer the F-specific RNA phages were minority, whereas in the other
seasons were more than 90% (Cole et al., 2003; Rahman et al., 2009). The abatement of F-
specific RNA phages in sludge mesophilic (30-359C) anaerobic digestion is faster (approximately
two logio units versus one log 10 unit) than that of somatic coliphages (Mandilara et al., 2006b;
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Guzman et al., 2007). Thus, in my opinion, the persistence of F-specific and F-specific RNA
bacteriophages need some extra verifications before rating their persistence in warm areas.
Resistance to treatments
Primary sedimentation, flocculation-aided sedimentation, activated sludge digestion, activated
sludge digestion plus precipitation and trickling filters remove both somatic coliphages and F-
specific bacteriophages in numbers ranging from 50% to 99.9 %, without significant differences in
the extent of reduction of the numbers of both groups of bacteriophage (Aw and Gin, 2010,
Grabow, 1993, Costan-Longares et al., 2008, Zang and Farahbakhsh, 2007, Yahya et al., 2015;
Gomila et al., 2008).
Depth filtration, precipitation/flocculation plus filtration and even membrane filtration does not
remove differently somatic and specific coliphages found in secondary effluents (Nieustadt, 1988,
Rose et al., 2004, Hill and Sobsey, 1998, Zang and Farahbakhsh, 2007, de Luca et al., 2013 , Marti
et al., 2011).
Chlorination of secondary effluents in water reclamation seems to cause a major effect on somatic
coliphages, but this differential effect is not great enough to change the relative proportions and
somatic coliphages follow overnumbeing F-specific phages (Rose et al., 2004, Costan-Longares et
al., 2008, Montemayor et al., 2008, Mandilara et al., 2006a).
Regarding radiations, UV irradiation has a significant major effect on somatic coliphages than in F-
specific, and after strong UV treatments F-specific numbers surpass those of somatic coliphages
(Costan-Longares et al., 2008, Montemayor et al., 2008).
Foto-oxidation (H2O2, HO2) uses to be a very effective treatment for bacteriophages, as it has been
described for specific bacteriophages; but when foto-oxidizing a secondary effluent a greater
elimination of F-specific bacteriophages is noticed (Agullo-Barcelo et al., 2013).
Most sludge treatments required prior to its release in soil are founded in heat treatment. A major
inactivation of F-specific phages can already be observed in mesophilic digestion, and all the
treatments with higher temperatures increase the difference in the ratio somatic coliphages:F-
specific phages (Mignotte-Cadierges et al., 2002. Guzman et al., 2007, Mandilara et al., 2006b).
Regarding the effect of soil filtration in the abatement of both groups of phages, again studies
comparing both are lacking. Sinton et al. (1997) reported that in an alluvial gravel aquifer F-specific
RNA phages showed greater attenuation than somatic coliphages.
General conclusion
Detection of somatic coliphages is easier than that of F-specific phages
No clear difference between somatic and F-specific phages regarding relation to risk
No clear differences regarding overall persistence in the environment (except at water
temperatures over 259C)
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No clear differences regarding overall resistance to treatments (except UV and heath)
Regarding abundance, the general trend is that somatic coliphages overnumber F-specific
coliphages in the great majority of the settings, including surface, and hence recreational, waters.
However, there are some matrixes such as reclaimed water in which UV plays the main role in
inactivation, clayey sediments and groundwater from certain aquifers where F-specific phages
have been described to become predominant.
Having these considerations in mind, the convenience of detecting both groups of bacteriophages
at once has to be considered.
PROJECT IN THE EUROPEAN UNION
In 1996 we were commissioned by the European Union to lead a study aimed to determine
whether methods based in bacteriophages were feasible for being applied in routine for the
determination of water quality in bathing waters and to make a preliminary study of concentration
of bacteriophages in a number of bathing areas through Europe (Jofre et al., 2000). More than 20
labs across Europe participated in the study. In this study, other than assessing the feasibility of
methods permitted us to complete the ISO methods for the detection of bacteriophages in waters
and to have.
First conclusion was that the application of all methods in routine was feasible, but that the
method for detecting somatic coliphages is easier, faster and more cost effective than the
methods for detecting F-specific RNA phages and phages infecting Bacteroides (Mooijman et al.,
2005). As well some preliminary data on numbers of the different groups of bacteriophages in
fresh and marine bathing waters through Europe were provided (Contreras-Coll et al., 2002).
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Response: Rachel Noble
The discussion regarding selection of either MSC or somatic coliphage (SC) for potential criteria
development for recreational waters is a complex one. MSC are typically single stranded RNA
viruses (found as linear ssRNA) as Leviviridae, and when found as Inoviridae (ss DNA) or
Tectiviridae (ds DNA) are DNA viruses. As such they are typically suggested to be biochemically
similar to other human enteric pathogens such as noroviruses and enteroviruses. Under an
electron microscope, enteroviruses such as Coxsackie virus, and MSC cannot be distinguished from
one another. SC are DNA viruses, from the families Podoviridae, Microviridae, Myoviridae and
Siphoviridae. Their genome can be arranged as either linear ds DNA, or circular ss DNA, they are
thought to approximate the biochemical and persistence attributes of DNA-based human viral
pathogens such as adenoviruses. Somatic phages have been considered in the literature to
potentially replicate more readily in aquatic environments as replication does not require the F
pilus interaction. In addition, somatic coliphages generally outnumber MSC in wastewater and raw
water sources by about a factor of 5. To some this has been seen as a benefit (less likely to suffer
from non-detect results when quantifying SC), and to some these features have been seen as
strong negatives. This document is not intended as a comprehensive review of all of the past
literature conducted on the two types of coliphage, rather it frames any consideration of either
type or both types for criteria development in the context of the top issues featuring the most
recent published (and some as yet unpublished) literature. It also highlights areas where little
research has been conducted, identifying data gaps that are vital for study prior to any final
decision.
1. Advantages and Disadvantages: A. Prediction of human health
In summary of studies featured in the EPA Coliphage Review. Several epidemiological studies
conducted in the past decades noted a relationship with MSC. In many of those studies, somatic
coliphages were not measured. There were also several studies, such as Wiedenmann et al. (2006)
a relationship was observed between somatic coliphages and human health. In southern
California, it was reported (Colford et al., 2007) that F+ coliphage was weakly predictive of human
health outcomes. However, this result was based on a very small sample size, and there was
concern by the PI group that these results should not be over-interpreted. In later epidemiological
studies in Avalon and Doheny State Beach, F+ coliphage (measured using EPA Method 1602)
exhibited a stronger relationship with Gl symptoms than Enterococcus quantified by EPA 1600 at
the two beaches where it was measured, but the odds ratio when considering the entire study (all
risk conditions) were not statistically different from 1 for either marker. During high risk
conditions, the relationships for the F+ coliphage were statistically stronger than for EPA 1600 but
it must be noted that this was a small subset of the time over which the study was conducted. A
molecular method, which was specific for F+ RNA Coliphage Genotype II. Interestingly, when only
the high risk conditions were considered the relationship disappeared. The relationship was weak,
but it was the only indicator that was statistically significantly associated with Gl illness at Malibu.
It should be mentioned that no indicator combinations across the studies consistently stronger
predictive relationship to human health than EPA 1600. In southern California, none of the
beaches are impacted by known, permitted sewage discharges, but there are confirmed human
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sources of fecal contamination at both Doheny and Avalon Beaches. Finally, only EPA1602 resulted
in the significant relationships for F+ coliphage, not EPA1601, which calls into question the
enrichment approach.
1.B. As indicators of wastewater treatment performance or as proxies of human viral pathogens
(need to raise the question of quantitative comparisons of viral pathogen infectivity (cell culture)
versus viral indicator infectivity (plaque formation).
There are a host of issues that prevent the ability to use coliphage enumeration as true proxies of
infection by human viral pathogens. While the biochemical traits of the viruses may be similar, and
therefore it is speculated that patterns of degradation and persistence would be similar, there are
significant methodological hurdles to comparing human infection by viruses to coliphage infectivity
as enumerated by plaque formation. The details on the methodological limitations are beyond the
scope of this document, however this document will address and report relationships that have
been observed in recent studies.
A completely different study design was utilized by Pouillot et al. (2015) to assess relationships of
MSC loss in sewage influent versus norovirus genogroups I and II. They found that in certain types
of wastewater treatment plants (e.g. lagoon systems with chlorination), norovirus loss rates were
very close to those measured by MSC loss rates. Within plant systems, they observed correlations
between the two groups of viruses and their loss rates. While this study was not relevant to the
question at hand, it did demonstrate in a large meta-analyses context a strong correlative
relationships between the mean reductions of norovirus genogroup II and MSC was observed
(r=0.8). Given the scale of this data analyses, and the fact that they "judged" the quality of the
reduction data by the presence of censored values, this supports the use of MSC as an index of
treatment success. Whether this usefulness extends into receiving waters is still in question.
Unfortunately, to date, there have been no similar meta-analyses of somatic coliphage disinfection
loss rates to this reviewer's knowledge. Therefore, it is difficult to evaluate. However, in this
framework, given that it is postulated that the main concern to public health at beaches is human
enteric viruses such as norovirus, it may be that MSC are the more useful proxy between the two.
2. Differences such as source, degradation characteristics.
As far as replication in the environment, somatic coliphages have been suggested to be the most
likely to replicated naturally in aquatic environments. Their replication does not require the F pilus
interaction that is necessary for MSC replication. It has been previously thought that little
replication occurs in the natural environment, especially at temperatures below 30C, but this has
been challenged in the recent literature by Ravva and Sarreal (2016). These researchers have
conducted research in waters used for produce and have lines of evidence indicating that the host
utilized to assess replication in the natural system matters (environmental hosts versus laboratory
provided hosts), and they have also demonstrated that fluctuations in temperatures (winter versus
summer) and the presence of host during decay experiments had dramatic effects on persistence
and degradation. In this published research, the study design included assessment of
growth/replication of the phages in the presence of host and at different temperatures. Contrary
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to previously held dogma, the three of the four MSC evaluated were documented to replicate
during incubation at 10 C.
There have been two recent studies that were conducted on the rates of loss of viral pathogens in
relation to MSC and/or SC. In the first, as yet unpublished manuscript (Wu et al., 2016), both a
first-order decay rate approach and a new Bayesian approach were used to compare rates of loss.
The study showed two main results, first that SC degraded more rapidly than MSC in summer
(25°C) months), and that sunlight degradation for both was more rapid than shaded. The most
astounding differences in this particular study were seen between winter and summer months. In
the winter condition, SC still degraded more rapidly than MSC, but the overall rates of degradation
were less than one-fourth that which had been measured in the summer conditions.
Other considerations in the recently reported literature include a publication by Ogorzaly et al.
(2010) that demonstrated that adenoviruses remained infective for much longer than their studied
MSC counterparts, even when studied at 20°C. This study indicated that the selection of MSC alone
as an indicator, at least of the highly prominent adenovirus group, was not suitable.
From Virobathe, Wyer et al. (2012), demonstrated relationships between human adenoviruses and
somatic coliphage. MSC were not part of this assessment. So no further comparison is possible.
Other recently featured articles highlight issues that play into the process of examining correlative
relationships between viral indicators and viral pathogens. Jones et al. (2014) compared 5
currently published methods for recovery of coliphage via either plaque assay or RT PCR and found
that the recovery of the spiked samples varied from <1 to 52%.
A final note is the seasonality of SC and MSC in human sewage, as compared to the load of viral
pathogens such as adenovirus, norovirus, and enterovirus in human sewage. It is unfortunate, but
to date many studies have been conducted in the "off season" months for a particular pathogen
and have not yielded any predictive or quantitative relationships. It is possible that measures of
"pan viral pathogen" DNA or RNA could potentially be quantitatively related to MSC and SC
concentrations, but to date, this type of assessment has not been conducted.
Skraber et al. (2004) considered the relationships between somatic coliphages and human viral
pathogens in river waters of France and noted that the number of river water samples that were
positive for viral pathogens increased with somatic coliphage concentration. However, little formal
quantitative relationship was observed and was hampered due to the use of highly variable
methods.
Issues for discussion:
It has been noted in the literature that there are strong limitations to some of the data analyses
conducted, and those bear mentioning here.
1. Censored data, and data for norovirus and like enteric viruses at low concentrations is a major
problem for data analyses. Many studies include an approach to turn non-detect values into a
reported low value. Some of the assumptions utilized are faulty and that fully quantitative data for
enteric viruses is a vital need prior to setting any future criteria for either MSC or SC.
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2. Extreme events: researchers have almost no way of determined whether extreme events have
played into the values that are observed during a study and incorporated into risk assessment
frameworks, in beach water quality these can present themselves as wind erosion events,
resuspension events, tidal events, to name a few. In the sewage treatment and disinfection
literature, this can present itself in the volumes of untreated stormwater that are contributing to a
system (thereby causing dilution), or volumes of water that are bypassing the system. Either way,
the variability observed for these comparative studies is rarely all methodological and these
factors must be considered for any future deployment.
3. Too little is known about "environmental strains" of MSC and SC. That is, many of the laboratory
based studies that have been conducted and reported in the literature have repeatedly utilized the
same strains. The diversity of SC and MSC in human and other warm blooded animal feces and the
technology available warrants further examination of the true viral dynamics. Many of the strains
used for degradation studies have been shared over the years from research laboratory to
research laboratory and it bears in mind that the diversity of MSC and SC is huge. We need to
include a range of different strains in a laboratory and field analyses decay setting. This was
demonstrated recently by Ravva and Sarreal (2016).
4. A final note of consideration for the MSC versus SC discussion is that EPA has considered the
possibility to utilize both as viral indicators. There is a dearth of literature comparing the two
equally to constituents of interest. As a matter of fact, in all of the literature examined for this
short review, less than 15% of the recent literature included even comparisons of both. This should
serve as an indication of the fact that the effort required, even by the academic or agency
researcher, to conduct full quantification of both, is prohibitive. Furthermore, it is much more
difficult for a non adept technician in the laboratory to conduct MSC quantification. Somatic
coliphage quantification is far easier because of ambient concentrations and necessary laboratory
approaches. For molecular methods, the difficulty associated with quantification of MSC versus SC
is even more magnified, because of the vital importance of any laboratory technician to be familiar
with an array of RNA isolation and extraction techniques, the lability of RNA in a laboratory
environment, and the issues arising from poor attention to detail for the all-important reverse
transcription step that initiates any RT-PCR reaction. If a viral indicator is selected for criteria
development, careful consideration and then the selection between MSC and SC should be one of
the first actions in that process.
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