rill United States
I—'/jL Environmental Protection
I # % Agency
Office of Water
EPA 823 R 18 001
May 2018
2017 Five-Year Review of the 2012
Recreational Water Quality Criteria
U.S. Environmental Protection Agency
Office of Water Office of Science and Technology
Washington, D.C.
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Disclaimer
This review report discusses the implementation status and scientific basis for the EPA's current
recommended recreational water quality. While this document cites statutes and regulations that
contain legally binding requirements, it does not itself impose legally binding requirements on
the EPA, states, tribes, other regulatory authorities, or the regulated community. The EPA, state,
tribal, and other decision makers retain the discretion to adopt approaches on a case-by-case
basis that differ from those discussed in this document as appropriate and consistent with
statutory and regulatory requirements. This document does not confer legal rights or impose
legal obligations upon any member of the public. This document does not constitute a regulation,
nor does it change or substitute for any Clean Water Act (CWA) provision or EPA regulations.
The EPA could update this document as new information becomes available. The EPA and its
employees do not endorse any products, services, or enterprises. Mention of trade names or
commercial products in this document does not constitute an endorsement or recommendation
for use.
Acknowledgments
The U.S. Environmental Protection Agency would like to thank the following experts for their
willingness to participate in interviews at the 2017 Water Microbiology Conference at the
University of North Carolina (UNC) in Chapel Hill: John Griffith, Southern California Coastal
Water Research Project; Rachel Nobel, University of North Carolina at Chapel Hill; Merek Kirs,
University of Hawaii; and Gary Toranzos, University of Puerto Rico. Other valuable information
was provided by Steve Weisberg of Southern California Coastal Water Research Project;
Shannon Briggs of the Michigan Department of Environmental Quality; Julie Kinzelman, City of
Racine, Wisconsin; Donna Francy, Rebecca Bushon, and Meredith Nevers, United States
Geological Survey; and state Beach Program coordinators in our cooperating BEACH Act states,
territories and tribes.
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Table of Contents
Disclaimer ii
Acknowledgments ii
Acronyms vi
Executive Summary viii
I. Introduction 1
II. Background - 1986 Criteria 3
A. State of the Science in 2000-2010 3
B. Key Points of the 2012 RWQC 4
1. Magnitude, Duration, and Frequency: Geometric Mean and Statistical Threshold
Value 4
2. NEEAR Gastrointestinal Illness Rate 5
3. The 2012 RWQC Includes Two Sets of Recommended Criteria Values 5
4. No Marine/Fresh Water Illness Rate Differential 6
5. A Single Level of Beach Use 6
6. Beach Action Values 6
7. qPCR Rapid Quantitation Methods 6
8. More Tools for Assessing and Managing Recreational Waters 6
III. Scope and Methods of the Review 7
A. Inventory of Scientific Information Published Since 2010 7
B. Recreational Criteria Implementation Tools 7
C. Sources of Information and How Information Was Accessed 7
D. How the Assessment Was Conducted 8
1. EPA Recreational Water Research 8
2. A Systematic Review of Available Peer Reviewed Literature 8
3. Supplemental Review of Relevant Materials by the EPA 8
E. Collection of Information from Practitioners, Academics, and Stakeholders
Involved in Beach Monitoring 8
IV. Findings of the Review 10
A. Inventory and Evaluation of Recreational Water Information 10
1. Introduction 10
2. Water Ingestion and Children 10
3. Health Relationships and Coliphage 13
4. Health Relationships and Additional Alternative Indicators 14
5. Etiologic Agents 15
6. T ropical Waters 15
7. Non-Point Sources 17
8. Wet Weather 18
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9. Health Burden 19
10. Non-Enteric Illness 19
B. Summary of Coliphage Advancements for RWQC 20
1. Introduction 20
2. Literature Reviews 21
3. Methods 21
4. 2016 Coliphage Experts Workshop 22
C. Summary of Scientific Advancements in FIB qPCR 23
1. Introduction 23
2. Enterococcus spp. qPCR EPA Method 1609 24
3. Available Technical Support Information 25
D. Human/Non-Human Fecal Source Identification 27
1. Fecal Source Identification 27
2. EPA MST Research 29
3. Selected External Research Contributions to MST Development 31
E. Emerging Issues: Evidence of Exposure to Antimicrobial Resistant Bacteria in
Recreational Waters 34
1. Introduction 34
2. Antimicrobial Resistance Mechanisms 34
3. Point Sources and Non-Point Sources 35
4. Evidence of Recreational Exposure 36
5. The EPA's Work on AMR for Recreational Waters 37
F. Assessment of Recreational Criteria Implementation and Tools 38
1. Sanitary Surveys 38
2. Statistical Modeling for Predictive Estimates of Water Quality 39
3. Deterministic Process Modeling for Recreational Beach Site Assessment and
Enhancement/Remediation 41
4. Integrated Environmental Modeling and QMRA 41
5. Adoption Status and Perceived Barriers 43
G. Recreational Criteria for the Cyanotoxins: Microcystins and
Cylindrospermopsin 45
Summary and Priorities for Further Work 478
A. Health Studies (see section IV. A for more information) 48
B. Developments for Coliphage, Including Analytical Methods (see section IV.B
for more information) 49
C. Analytical Methods (see section IV. C. for more information) 49
D. Microbial Source Tracking (see section IV. D for more information) 50
E. Antimicrobial Resistance (see section IV. E for more information) 51
F. Implementation Tools and RWQC Adoption (see section IV.F for more
information) 52
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1. Implementation Tools 52
2. Review of RWQC Adoption Status and Perceived Barriers 53
G. Recreational Criteria for Cyanotoxins (see section IV.G for more information) 54
VI. Assessment of the Need to Revise the 2012 RWQC 55
VII. References 57
Appendix A. Advancements in Mitigating Interference in Quantitative Polymerase Chain
Reaction (qPCR) Methods for Microbial Water Quality Monitoring 82
A. Introduction 82
B. Methods 84
1. Systematic Literature Search 84
2. Systematic Literature Screening 85
C. Results 86
1. Literature Screening and Review 86
2. Advancements in Enterococcus spp. qPCR Methods 86
3. Advancements in E. coli qPCR Methods 93
4. Digital PCR 95
D. References 97
Appendix B. Communication with Regional Coordinators on the Implementation of the
RWQC 102
Appendix C. Review of the EPA's 2012 Recreational Water Quality Criteria Health
Study Information Expert Consultation 104
A. BACKGROUND 106
B. CHARGE QUESTIONS 106
C. THE APPROACH TAKEN 107
D. RESPONSES 107
Charge 1—Summary of peer-reviewed studies 107
Charge 2—Children's health 109
Charge 3—New information on health and indicators? 110
Charge 4—Relationships between health and alternative indicators 110
Charge 5—Fecal sources and contamination dynamics, wet/dry weather 112
Charge 6—Outbreak information during the last 10 years 112
E. ACKNOWLEGEMENT 142
F. REFERENCES 142
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Acronyms
AMR antimicrobial resistence
AMRB antimicrobial resistant bacteria
ARG antimicrobial resistant genes
AWQC ambient water quality criteria
BAV Beach Action Value
BEACH Beaches Environmental Assessment and Coastal Health
CAFO concentrated animal feeding operation
CAWS Chicago Area Waterways System
CCE calibrator cell equivalent
CDC Centers for Disease Control and Prevention
cfu colony forming unit
CHEERS Chicago Health, Environmental Exposure, and Recreation Study
CRE carbapenem-resistant Enterobacteriaceae
Ct cycle threshold
C W A CI ean W ater Act
D-HFUF dead-end hollow fiber ultrafiltration
DNA deoxyribonucleic acid
EMM Environmental Master Mix
EnDDaT Environmental Data Discovery and Transformation
EPA Environmental Protection Agency
ESBL extended-spectrum P-lactamase
FIB fecal indicator bacteria
FSI fecal source identification
GBM generalized boosted modeling
GI gastrointestinal
GM geometric mean
HAB hazardous algal bloom
HCGI highly credible gastrointestinal illness
HGT horizontal gene transfer
hr hour
IEM integrated environmental modeling
IFTAR Interagency Task Force on Antimicrobial Resistance
L liter
mL milliliter
MLR multiple linear regression
MRSA methicillin-resistant Staphylococcus aureus
MST microbial source tracking
NEEAR National Epidemiological and Environmental Assessment of Recreational
Water
NGI NEEAR-GI illness
NIST National Institute of Standards and Technology
NPDES National Pollutant Discharge Elimination System
ORD Office of Research and Development (U.S. EPA)
PCR polymerase chain reaction
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pdu
PCR-detectable units
PLS
partial least squares
QMRA
quantitative microbial risk assessment
qPCR
quantitative polymerase chain reaction
RfD
reference dose
RNA
ribonucleic acid
RT-PCR
reverse transcriptase-polymerase chain reaction
RT-qPCR
reverse transcriptase-quantitative polymerase chain reaction
RWQC
Recreational Water Quality Criteria
SAL
single-agar layer
SCCWRP
Southern California Coastal Water Research Project
SSM
single sample maximum
STV
statistical threshold value
TMDL
Total Maximum Daily Load
TSA
temporal synchronization analysis
UCB
University of California, Berkeley
UIC
University of Illinois Chicago
UNC
University of North Carolina
U.S.
United States
USGS
United States Geological Survey
VB
Virtual Beach
WQS
water quality standards
WW TP
wastewater treatment plant
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Executive Summary
The United States (U.S.) Environmental Protection Agency (EPA) has conducted a five-year
review of its 2012 Recreational Water Quality Criteria (RWQC), as required by the Beaches
Environmental Assessment and Coastal Health (BEACH) Act amendments to the Clean Water
Act (CWA) section 304(a)(9)(B). In conducting this review, the EPA considered several factors,
including the availability and evaluation of new science, the review of information related to the
underlying science used to develop the 2012 RWQC, additional implementation support needs,
and perceived barriers to state adoption.
An important goal of this review and report is to document the assessment of whether revisions
to the 2012 RWQC are necessary. The EPA's review included compiling the relevant scientific
information published since 2010, gathering updated information on recreational criteria
implementation tools and summarizing the information received from implementers of
recreational water quality monitoring and improvement programs across the country. The EPA
also conducted outreach to the recreational water quality community in the course of this review.
The report contains extensive information in each of the topic areas, and the conclusions derived
from the report are summarized below.
Science Review
Health Studies. Findings on health studies are generally consistent with the findings of studies
that formed the basis for the 2012 RWQC, and enhance the depth and strength of the evidence
underlying the RWQC. A growing body of evidence suggests that children can be
disproportionately susceptible to health effects resulting from exposure to pathogens in
recreational waters. There are opportunities for further resolution of epidemiological
relationships, especially in the area of children's health protection and wider application of
Enterococcus spp. qPCR.
Priorities for Further Work: Re-analysis of epidemiological data to assess potential differences in
risk to children. Re-analysis of Enterococcus spp. qPCR data for consideration in criteria
development, especially to address effluent sources. Also, evaluate how QMRA can be used to
address risk to children from swimming exposure, and other regulatory purposes.
Coliphage as an indicator. Because evidence strongly suggests most illnesses in recreational
waters are due to enteric viruses, development and implementation of viral indicators, such as
coliphage, may yield advances in public health protection.
Priorities for Further Work: Completion and publication of coliphage methods and development
of coliphage-based RWQC for inclusion into the "tool box."
Indicators and Performance of qPCR Methods. The advances in qPCR methodology since
2010 have brought greater reliability and utility to beach monitoring programs where they have
been implemented, yet opportunities remain for further refinement of qPCR methodologies.
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Enterococcus spp. measured by qPCR is a better predictor of swimming-associated GI illness
and more timely than current culturable bacterial indicators. These factors coupled with a greater
distribution of qPCR-capable laboratories in the future could lead to enhanced public health
protection if implemented under the current criteria.
Priorities for Further Work: Completion of method validation and publication for the E. coli
qPCR method (Draft Method C), development of alternative site-specific criteria for Draft
Method C, additional training and capacity-building in qPCR laboratories in states, tribes, and
localities.
Microbial Source Tracking. Accurate and reliable MST technologies could markedly improve
future water quality management in the U.S., possibly allowing for the development of
alternative site-specific criteria based on pollution sources present, strategic remediation
planning based on fecal pollution levels from human sources. Use of alternative water quality
metrics, such as human-associated MST technologies, may also be helpful to inform public
health risk levels under wet weather conditions.
Priorities for Further Work: Completion and publication of standardized methods for EPA
human-associated MST methods (HF183/BacR287 and HumM2) and completion of a DNA
reference material development with NIST. Development and validation of virus-based human
fecal source identification procedures. Further investigation of MST application in recreational
water quality management settings such as prioritizing polluted sites for remediation based on
human waste levels, identification of non-point pollution sources, and the development of
alternative water quality metrics based on wet and dry weather scenarios.
Antimicrobial Resistance. The complex issue of antimicrobial resistance is becoming of
increasing interest, creating a demand for more data to both inform our understanding of the
forces driving this resistance and the actions needed to preserve bacterial susceptibility to our
first-line medications. There is an increasing body of literature available on the environmental
occurrence of AMRB/ARG and potential exposure in recreational waters. To develop a more
complete picture regarding the threat and risks associated with antibiotic resistance, research is
needed to better understand the role the environment plays in transferring AMRB/ARG to
primary contact recreators. For example, additional research is needed on the incidence,
associated risks, and transfer mechanisms in recreational waters, as well on the removal of
AMRB/ARG by wastewater treatment processes. The EPA is in the early stages of developing a
broader surveillance strategy and looking for meaningful opportunities to improve human health
relating to exposures to AMRB/ARGs.
Priorities for Further Work: Development and standardization of AMRB/ARG detection
methodologies. Collect information on the occurrence of AMRB/ARG in environmental waters,
wastewater influent/effluents, and other potential reservoirs. Develop wastewater treatment and
disinfection processes for AMRB/ARG targets. Characterize potential associated public health
risks and mitigation strategies.
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Implementation Review
Although not essential in terms of their association with current or potentially revised RWQC,
implementation activities are crucial to applying the advancing science to protect public health in
recreational waters.
Sanitary Surveys. Sanitary Surveys continue to serve as an important tool for informing site
remediation, characterizing waters for QMRA and site-specific criteria development, and can be
linked with integrated environmental modeling.
Priorities for Further Work: Conversion of current marine sanitary survey tablet-based
application to a web-based application, additional outreach on available sanitary survey
applications, collaboration with Great Lakes beach programs on fresh water sanitary survey
application and opportunities for integration with environmental modeling.
Predictive/Statistical Modeling. Predictive models offer states, territories, and tribes an
alternative for same-day notification and resulting public health protection with lower capital
investment and unit costs than other rapid methods.
Priorities for Further Work: Additional support to develop predictive models in marine
environments as well as models paired with newer indicators such as qPCR-based indicators.
Deterministic Process Modeling for Recreational Beach Site Assessment and
Enhancement/Remediation. These models provide a means of understanding physical forces
influencing the movement of contaminants for problem definition and remediation and can
include QMRA health-based models to develop site-specific criteria or evaluate remediation.
Priorities for Further Work: Development of additional training and tools to make process
models and integrated environmental modeling more accessible to states, tribes and other
interested stakeholders.
Quantitative Microbial Risk Assessment (QMRA). QMRA can enhance the interpretation and
application of new or existing epidemiological data by characterizing various exposure scenarios,
interpreting potential etiological drivers for the observed epidemiological results, and accounting
for differences in risks posed by various sources of fecal contamination. Progress since 2010
includes new QMRA software infrastructure developed to provide risk estimates within a
standard microbial watershed assessment.
Priorities for Further Work: Development of additional training and tools to make QMRA
models more accessible to states, tribes and other interested stakeholders. Completion and
publication of remaining QMRA guidance.
Criteria Implementation: Adoption Status and Perceived Barriers. The 2012 RWQC include
many new elements that strengthen overall health protection in recreational waters and promote
more consistent implementation. Many states that have some, but not all, of the elements of the
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2012 RWQC in their water quality standards have been reluctant to adopt the new criteria due to
the initial administrative burden associated with rulemaking and other resource concerns.
Priorities for Further Work: Continued funding of BEACH Act grants. Consider additional
implementation guidance and explore reconsideration of addressing differences based on
frequency of use.
Cvanotoxins in Recreational Water
Recreators exposed to cyanotoxins in ambient recreational waters are at risk. The EPA is
working to develop human health recreational ambient water quality criteria or swimming
advisories for microcystins and cylindrospermopsin. The EPA expects to revise and publish a
final criteria document in 2018.
Priorities for Further Work: Completion and publication of recreational criteria for the
cyanotoxins, microcystins, and cylindrospermopsin.
Assessment of the Need to Revise the 2012 RWQC
Based on the review of the existing criteria and developments in the available science described
in this report, and consistent with CWA section 304(a)(9)(B), the EPA has decided not to revise
the 2012 Recreational Water Criteria during this review cycle. The Agency believes, however,
that further research and analysis as identified in this Report will contribute to the EPA's future
review of the 2012 RWQC. The EPA will work with the environmental public health community
as the Agency moves forward with its research efforts. The use of qPCR and ongoing research in
methods and indicators continue to strengthen and augment the tools available to support the
current criteria.
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I. Introduction
The United States (U.S.) Environmental Protection Agency (EPA) has conducted a 5-year review
of its 2012 Recreational Water Quality Criteria (RWQC), as required by the Beaches
Environmental Assessment and Coastal Health (BEACH) Act amendments to the Clean Water
Act (CWA) section 304(a)(9)(B). In conducting this review, the EPA considered several factors,
including the availability of new science and evaluation of the underlying science used to
develop the 2012 RWQC, additional implementation support needs, and perceived barriers to
state adoption. The Agency used the information in this "state-of-the science" report to assess
whether new or revised RWQC are necessary at this time.
The development of the 2012 RWQC and this review are both requirements of the BEACH Act
of 2000, which has provided grants to states, territories, and tribes to implement water quality
monitoring and notification programs for coastal recreation waters1 (including the Great Lakes)
since 2002. The 2012 RWQC included development of a beach advisory threshold for use in
posting swimming advisories and the ambient water quality criteria (AWQC) for use in a variety
of other CWA programs (e.g., deriving National Pollutant Discharge Elimination System
[NPDES] permits). Advisory decisions based on water quality monitoring are intended to reduce
the risk to recreators and other users of these waters from illness associated with exposure to
human fecal contamination and provide the public with information to make decisions about
their actions. AWQC that are developed under CWA section 304(a) are recommendations on the
latest science, which states and authorized tribes can adopt as part of their water quality
standards (WQS). In the case of the 2012 RWQC, the EPA's recommendations were designed to
protect primary contact recreational waters, not just coastal recreation waters. It is important to
note that Congress required states and authorized tribes with coastal recreation waters to adopt
new or revised WQS addressing pathogens in such waters within 36 months of the EPA's
publication of the 2012 RWQC (CWA section 303(i)(l)(B)). The criteria, once adopted by states
and authorized tribes and approved by the EPA under CWA section 303(c), become part of the
regulatory structure of the state/authorized tribe and are intended to protect primary contact uses
for the applicable waters. The recreational criteria values that are part of a state's or authorized
tribe's approved WQS have a direct bearing on the issuance of NPDES discharge permits,
waterbody assessments, the decisions regarding attainment of WQS under CWA sections 303(d)
and 305(b), and the development of targets for Total Maximum Daily Loads (TMDLs) for
restoring impaired waters.
1 The BEACH Act of 2000 defines coastal recreation waters as follows:
The term 'coastal recreation waters' means—
(i) the Great Lakes; and
(ii) marine coastal waters (including coastal estuaries) that are designated under section 303(c) by a State for use
for swimming, bathing, surfing, or similar water contact activities.
The term 'coastal recreation waters' does not include—
(i) inland waters; or
(ii) waters upstream of the mouth of a river or stream having an unimpaired natural connection with the open sea.
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The criteria values specified in the RWQC are for densities of culturable fecal indicator bacteria
(FIB) in water. The FIB, enterococci and Escherichia coli (E. coli), are not pathogenic under
usual circumstances, but their presence in water above specified levels can indicate the presence
of viral, bacterial, or protozoan pathogens associated with an elevated risk of illness. Therefore,
ensuring that the RWQC are consistent with the current state of the science and are protective of
human health is key to protecting the health of users of all waters designated for primary contact
recreation.
The EPA identified the following objectives for this review of the 2012 RWQC:
• Inventory and evaluate health study information published since 2010 on public health
impacts associated with exposure to fecal contamination in recreational waters.
• Review the 2012 RWQC based on internal EPA input on the science, taking into
consideration feedback from the greater beach water quality community and
stakeholders.
• Identify additional indicators and methods, including those that have become more
refined or feasible since the issuance of the 2012 criteria, and assess their applicability for
predicting potential adverse human health effects from recreational exposure.
• Provide information on the state of the science with respect to source tracking methods,
sanitary survey design, predictive modeling for both fresh and marine waters, and other
implementation tools.
• Include the latest science and information pertaining to the development of other criteria,
such as coliphage and cyanotoxin criteria, that have the potential to protect recreational
uses.
• Assess factors affecting state/authorized tribe adoption of the RWQC including perceived
barriers to adoption and how states have implemented the criteria to meet their specific
circumstances.
An important goal of this review and report is to document the basis for the assessment of
whether revisions to the 2012 RWQC are necessary. That assessment was based on the overall
review findings described later in this report, including internal EPA evaluation of the latest
science, input from the greater beach water quality community, state agency representatives, and
feedback from other stakeholders.
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II. Background - 1986 Criteria
FIB densities have long served as the surrogate measure of fecal contamination and, by
inference, the presence of pathogens commonly associated with fecal material. The 1986 Criteria
relied on a series of epidemiological studies that the EPA conducted in the late 1970s and early
1980s to evaluate culturable indicators of fecal contamination and illness in swimmers. These
studies included E. coli, enterococci, and fecal coliforms, which had been the basis of
recreational criteria recommendations before 1986. These epidemiological studies showed that
enterococci are good predictors of gastrointestinal (GI) illnesses in fresh and marine recreational
waters, E. coli is a good predictor of GI illnesses in fresh waters, and fecal coliforms were poor
predictors of GI illness (Cabelli et al., 1982; Cabelli, 1983; Dufour, 1984).
Table 1. The 1986 Criteria Provided Geometric Mean (GM) and Single Sample Maximum
(SSM) (75th %ile) Values
GM
(cfub/100 mL)
SSMa
(cfu/100 mL)
Illness Rate
n/1,000
In Fresh Waters
Enterococci
33
61
8
E. coli
126
235
8
In Marine Waters
Enterococci
35
104
19
a The 1986 Criteria also provided SSM values for three other lower intensity levels of beach use.
b cfu = colony forming unit.
One of the stated goals of the BEACH Act of 2000 was to move beyond the perceived
limitations of the EPA's previously recommended 1986 Ambient Water Quality Recreational
Criteria for Bacteria (the 1986 Criteria) in place at the time. The lag between sample collection
and the receipt of analytical results was considered a potential impediment to public health
protection, and the BEACH Act of 2000 envisioned "improving detection in a timely manner in
coastal recreation waters of the presence of pathogens that are harmful to human health" (106th
Congress of the United States).
A. State of the Science in 2000-2010
The EPA planned and subsequently conducted epidemiological investigations at U.S. beaches in
2003, 2004, 2005, 2007, and 2009, known collectively as the National Epidemiological and
Environmental Assessment of Recreational Water (NEEAR) study. TheNEEAR study enrolled
54,250 participants, encompassed nine locations, and collected and analyzed numerous samples
from a combination of freshwater, marine, tropical, and temperate beaches (U.S. EPA, 2010c;
Wade et al., 2008, 2010). Health studies were also conducted by other entities during the period,
such as the Southern California Coastal Water Research Project (SCCWRP), but not all were
published prior to the development of the RWQC (Colford et al., 2007; Till et al., 2008; Marion
et al., 2010; Sinigalliano et al., 2010).
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The EPA also held a 5-day scientific workshop in 2007 to obtain a broad range of external
scientific input to support the development of the 2012 RWQC. The report from this workshop,
Report of the Experts Scientific Workshop on Critical Research Needs for the Development of
New or Revised Recreational Water Quality Criteria (U.S. EPA, 2007a), served as the scientific
roadmap for new 2012 RWQC and implementation guidance. The EPA used the report from the
Experts Scientific Workshop to develop the Critical Path Science Plan for the Development of
New or Revised Recreational Water Quality Criteria (U.S. EPA, 2007b), which was externally
peer reviewed. The EPA completed 32 projects to inform the development of the 2012 RWQC.
The 2012 RWQC document (U.S. EPA, 2012a) lists these projects and provides a description of
the science used to develop the elements of the 2012 RWQC including:
• Epidemiological studies and quantitative microbial risk assessments (QMRAs)
• Site characterization studies
• Indicators/Methods development and validation studies
• Refining and validating both EPA and other models for fresh and marine beaches
• Developing recommended levels of public health protection.
B. Key Points of the 2012 RWQC
The 2012 RWQC use enterococci and E. coli as predictors of GI illnesses in recreational waters,
and include eight major elements, described below.
1. Magnitude, Duration, and Frequency: Geometric Mean and Statistical
Threshold Value
The 2012 RWQC consist of three primary components: magnitude, duration, and frequency.
Magnitude: The magnitudes of the bacterial indicators are the measured densities of the FIB
from the water quality density distribution used for the criteria, expressed both as a geometric
mean (GM-50th percentile value) and as a statistical threshold value (STV-90th percentile
value).
Duration: The duration is the period over which excursions of the magnitude values are recorded
and calculated. The EPA recommended a duration of 30 days in the criteria for both the GM and
the STV.
Frequency: The frequency is how often the GM or the STV are exceeded. The EPA
recommended no exceedances for the GM over the period of the duration.
Because the STV reflects the 90th percentile of the distribution of values used to determine the
RWQC, the RWQC allowed for a 10-percent exceedance of the STV (1 in 10 samples). The EPA
selected the estimated 90th percentile of the water quality distribution to account for the expected
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variability in water quality measurements, while limiting the percentage of samples allowed to
exceed the STV as a threshold of water quality impairment.
The EPA was clear that "both the GM and the STV would be part of the WQS, and, therefore
both targets would be used to determine whether a waterbody attains the WQS for primary
contact recreation" (U.S. EPA, 2012a).
Table 2. 2012 RWQC Recommended GM and STV Values for 36 and 32 Illnesses/1,000
Recreators (NEEAR-GI Illness [NGI]) for Marine and Fresh Waters
Estimated Illness Rate (NGI): Estimated Illness Rate (NGI):
Criteria Elements 36 per 1,000 Primary Contact 32 per 1,000 Primary Contact
Magnitude Magnitude
Indicator
GM
(cfu/100 mL)a
STV
(cfu/100 mL)a
GM
(cfu/100 mL)a
STV
(cfu/100 mL)a
Enterococci - marine
and fresh water
35
130
30
110
OR
E.coli - fresh water
126
410
100
320
Duration and Frequency: The waterbody GM should not be greater than the selected GM magnitude in any 30-day
interval. These should not be greater than a 10-percent excursion frequency of the selected STV magnitude in the
same 30-day interval.
aThe EPA recommends using EPA Method 1600 (U.S. EPA, 2002a) to measure culturable enterococci, or another
equivalent method that measures culturable enterococci, and using EPA Method 1603 (U.S. EPA, 2002b) to measure
culturable E.coli or any other equivalent method that measures culturable E.coli.
2. NEEAR Gastrointestinal Illness Rate
The EPA's use of the NGI definition for illness rate in the 2012 RWQC reflected a change in the
GI definition of illness to capture a broader range of milder symptoms compared to the definition
the EPA used as the basis for the 1986 Criteria (highly credible gastrointestinal illness or HCGI).
Whereas HCGI required fever along with gastrointestinal symptoms to be considered a case,
fever was not required for NGI. The equivalent rate of occurrence of NGI is approximately 4.5 x
HCGI, so that the comparable base illness rate in the 2012 RWQC is 36 illnesses/1,000
swimmers vs. 8 illnesses/1,000 swimmers in the 1986 Criteria (Wymer et al., 2013). The 36
illnesses/1,000 NGI does not represent an increase in risk of illness over the 8 illnesses/1,000
HCGI, but has led to an incorrect perception of an increase in some instances (NRDC, 2014).
3. The 2012 RWQC Includes Two Sets of Recommended Criteria Values
Criteria values were provided for culture- and quantitative polymerase chain reaction (qPCR)-
enumerated FIB at two illness rates, 32 and 36 illnesses per 1,000 swimmers (NGI illness rate).
Based on the EPA's analysis of the available information, either set of thresholds protects the
designated use of primary contact recreation and, therefore, protects the public from the risk of
exposure to harmful levels of pathogens from fecal contamination. The two sets of numeric
concentration thresholds included in the 2012 RWQC provide states and authorized tribes
flexibility to make risk-management decisions based on local conditions.
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4. No Marine/Fresh Water Illness Rate Differential
The recommendations for criteria illness rate are consistent for both marine and fresh waters,
which was not the case for the 1986 Criteria.
5. A Single Level of Beach Use
The 1986 Criteria included four SSM values appropriate for different levels of beach use
intensity corresponding to the 75th, 82nd, 90th, and 95th percentiles of the distribution of values
from the water quality sampling distributions observed in the EPA's epidemiological studies. In
the 2012 RWQC, the Agency removed those use intensity recommendations. Accordingly, the
2012 RWQC includes criteria values for two different illness rates, but a single level of beach
use intensity. For further discussion of the elimination of the use intensity values in the 1986
Criteria for the 2012 RWQC, please refer to Section 3.6.1 in the 2012 RWQC document (U.S.
EPA, 2012a).
6. Beach Action Values
In addition to recommending criteria values, the EPA also provided states and authorized tribes
with Beach Action Values (BAVs) for use in notification programs. The BAV was defined as the
75th percentile of the water quality distribution of values of E. coli and Enterococcus spp. in the
epidemiological studies. The EPA's intent was to provide the BAV for states and authorized
tribes as a precautionary tool for beach management decisions. The EPA recommended the
BAVs as beach notification values for adoption by the states in their public health programs, but
not as part of the 2012 RWQC recommendations under CWA section 304(a).
7. qPCR Rapid Quantitation Methods
The EPA developed and validated a molecular testing method using qPCR as a rapid analytical
technique for the detection and quantitation of enterococci in recreational water (EPA Method
1611). The EPA included qPCR-based values for the GM, STV, and BAV for both illness rates
in the 2012 RWQC document. Due to potential matrix interference issues in water types other
than those studied at the NEEAR effluent-affected beach sites, the EPA encouraged states and
authorized tribes to conduct a site-specific assessment of the local appropriateness of qPCR
before using this method for purposes of beach monitoring.
8. More Tools for Assessing and Managing Recreational Waters
The EPA provided additional information on tools for evaluating and managing recreational
waters, such as predictive modeling and sanitary surveys, and stressed the need for a tiered
approach to developing beach monitoring plans in the 2014 National Beach Guidance and
Required Performance Criteria for Grants. The Agency also provided Technical Support
Materials for developing site-specific criteria and for adopting the use of alternative indicators or
methods at recreational beaches.
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III. Scope and Methods of the Review
This section describes the measures the EPA has taken to assess advances in the state of the
science supporting the 2012 RWQC since 2010 and the process of its implementation. The
measures include an inventory of the relevant scientific information published since 2010, a
description of recreational criteria implementation tools applied at recreational settings,
information on sources of information and how information was accessed, and a summary of
information received from implementers of recreational water quality monitoring and
improvement programs across the country.
A. Inventory of Scientific Information Published Since 2010
A thorough inventory of scientific information published since 2010 for topics central to
recreational waters monitoring and assessment is the core of this review. Three general
categories of relevant information were identified:
i. Performance and Implementation of qPCR Methods for FIB 2010 to present
ii. Health Studies, including epidemiological studies, refinement of analyses of data
from previous studies, and the application of QMRA to water quality data and
complex settings at recreational beaches
iii. Microbial source tracking (MST), including human and non-human fecal source
markers and tracking.
B. Recreational Criteria Implementation Tools
A further category of activities and tools related to water quality monitoring and contextual
assessment of beach settings was identified as highly relevant to the implementation of the
BEACH Act and activities related to the 2012 RWQC. This category of implementation tools
includes:
i. Sanitary surveys and watershed assessments
ii. Statistical approaches for predictive estimates of water quality
iii. Deterministic modeling for recreational beach site assessment, enhancement, and
remediation of adverse infrastructure impacts to sites.
C. Sources of Information and How Information Was Accessed
The collection and analysis of information in each of these categories included accessing post-
2010 information from three broad sources:
• EPA recreational water research and publications relating to that research
• External (non-EPA) academic research conducted by researchers at academic institutions
and government organizations that have focused on recreational water activities and
science related to the BEACH Act
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• Implementers of recreational water quality monitoring and improvement programs across
the country, including the EPA Regional Beach Program coordinators and state,
municipal, and county officials in health and environmental agencies who often are direct
or indirect recipients of BEACH Act grant funds or whose activities those funds leverage.
D. How the Assessment Was Conducted
1. EPA Recreational Water Research
For this review, offices within the EPA inventoried recreational water research. This information
is presented along with other information in Section IV below.
2. A Systematic Review of Available Peer Reviewed Literature
The EPA performed systematic searches of the peer-reviewed literature for articles pertaining to
qPCR Methods for FIB; health studies, including epidemiological studies of recreational water
contact activities; the application of QMRA to water quality data and complex settings at
recreational beaches; and human and non-human fecal source markers and tracking (MST).
Multiple sets of search terms applicable to the topic were applied to references in Web of
Science and Pub Med (http://www.ncbi.nlm. ni h. gov/pubmed). Abstracts were screened for
relevance to the scope of the search. The literature search was limited to English-language, peer-
reviewed citations, published between 2010 and March 2017. Following the abstract screening,
the full text of articles passing scope was reviewed for specific information related to each topic.
Search terms and databases searched are provided in Appendix A.
For qPCR methods, the literature search returned 337 unique results, of which 54 were relevant
based on the abstract screening. An additional 13 studies were identified through other sources
(e.g., cited in another paper). For the qPCR methods review, 32 studies were summarized. For
the health studies, the literature search returned 2,018 unique results, of which 98 were relevant
based on the abstract screening (15 of these were then excluded based on the full text review).
An additional 23 studies were identified through other sources (e.g., cited in another paper). For
the health study review, 106 studies were summarized. Results of the systematic reviews are
included in Appendix A and Appendix C.
3. Supplemental Review of Relevant Materials by the EPA
The EPA reviewed literature resulting from the systematic searches and from materials available
from other sources such as technical documents from states and the United States Geological
Survey (USGS). Summaries of this review are included in Section IV.
E. Collection of Information from Practitioners, Academics, and Stakeholders
Involved in Beach Monitoring
The EPA conducted informal interviews with recreational water public health practitioners;
members of the academic community, particularly those with expertise in methods and
epidemiology; and federal, state, and local government officials. Topics were discussed
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according to the role those individuals played in applying the RWQC, for example, state
implementer or local beach practitioner of beach program management. In addition, recent
issues, events, and trends in recreational water science were discussed with academic scientists.
The EPA also held discussions with other stakeholders.
The EPA interviewed practitioners from SCCWRP, the State of Michigan Department of Health,
the City of Racine Wisconsin Department of Health, and USGS science centers in the Great
Lakes region. EPA staff also spoke with researchers from the University of South Florida,
University of North Carolina, University of California - Davis, University of Miami, University
of Puerto Rico, and University of Hawaii.
The role of the EPA Beach Program Coordinators in the eight EPA regional offices with
BEACH Act Programs (Regions 1-6, 9, and 10) is central to the ongoing operation, funding, and
technical support of state, territorial, and tribal beach monitoring programs: The EPA Beach
Program coordinators provide technical advice and oversee the BEACH Act grants for the
qualifying entities within their region. Responsible state, tribal, and territorial agency contacts
and managers in those regions not only coordinate and operate monitoring and advisory
programs, but also move regulatory actions pertaining to the adoption of the criteria at the state
or tribal level through the their respective regulatory and, in many cases, legislative processes.
The EPA invited the Beach Program Coordinators and the respective states, tribes, and territories
to discuss the 2012 RWQC, their implementation, and the quality of experiences they had
implementing the RWQC.
The EPA conducted outreach to address the interests of various sectors of the recreational water
stakeholder community. In addition to informal outreach to trade associations and non-
governmental organizations that were key stakeholders in the development of the 2012 RWQC,
The EPA held a public webinar in July 2017 on the review for any interested stakeholders.
Participants included stakeholders from across the spectrum of environmental, industry, local
government, and public health stakeholder groups. The webinar provided an overview of the
review the EPA has undertaken and enabled stakeholders to provide input on the topics included
in the review. The EPA communicated some of the initial findings of the review of the science
and the timeline for completing the review.
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IV. Findings of the Review
A. Inventory and Evaluation of Recreational Water Information
1. Introduction
Between 2002 and 2009, the EPA conducted a series of epidemiological studies at beach sites
across the United States and Puerto Rico, collectively known as the National Epidemiologic and
Environmental Assessment of Recreational (NEEAR) Water Studies. These studies were a
collaboration between the EPA and the Centers for Disease Control and Prevention (CDC). The
studies were designed to address amendments to the CWA known as the BEACH Act. The
BEACH Act includes requirements for the EPA to study new, more rapid measures of fecal
contamination in recreational waters and their associations with health effects among beach-
goers, including non-gastrointestinal effects such as respiratory illness, skin rash, eye irritation,
and ear infection.
The 2012 Recreational Water Quality Criteria were based on literature published before 2010.
Research that EPA investigators have contributed to since 2010 has focused primarily on
publications based on the NEEAR data and publications that used combined datasets from the
NEEAR study and similar studies. These studies were conducted by the University of California,
Berkeley (UCB); SCCWRP; and the University of Illinois Chicago's (UIC) Chicago Health,
Environmental Exposure, and Recreation Study (CHEERS). Although the EPA did not lead most
of these studies, the Agency made significant contributions, including providing data and
assistance in interpretation, analysis, and publication of the studies. Additionally, as part of the
EPA's 5-year review of the 2012 RWQC, the EPA completed the Expert Consultation Report,
summarizing health studies published from 2010 to 2017, which included EPA and non-EPA
epidemiological studies, exposure assessments, and quantitative microbial risk assessments
(QMRAs) (Appendix C). This Chapter summarizes results from health studies by topic area:
water ingestion and children, coliphage, additional alternative indicators, etiologic agents,
tropical waters, non-point sources, wet weather, health burden, and non-enteric illnesses.
2. Water Ingestion and Children
A growing body of scientific evidence suggests that children can be disproportionately
susceptible to health effects from pathogen exposures in ambient waters compared to adults. The
risk differential could be due to one or more of the following: 1) children's immunological,
digestive, and other bodily systems that are still developing; 2) children's greater exposure
because they ingest more water and breathe more air in proportion to their body weight than
adults; and 3) children's behavior, such as increased time spent in water and more vigorous
activity, that might result in increased exposure in comparison to adults.
Historically, risk assessors have had limited data for evaluating children's potential exposures
and health outcomes relative to adults as a result of exposure to fecal pathogens found in
contaminated recreational waters. Few epidemiological studies and microbial risk assessments
have explored child-specific risks from microbial contaminants found in water, although this is
changing in recent years. The EPA identified four publications based on three studies published
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since 2010 that evaluated and reported incidental ingestion while recreating that included
children. These studies are summarized in subsequent paragraphs.
In 2006, the EPA conducted a pilot study to quantify the rate of water ingested among pool
swimmers (including children six years and older). Water ingestion was quantified by measuring
cyanuric acid in the pool water before swimming and in the urine after swimming (Dufour et al.,
2006). This study was expanded to include a larger sample with a wider range of ages beyond
that of the pilot study, and those results were published in 2017 (Dufour et al., 2017). The main
findings built on the previous study and demonstrated that children between six and 10 years of
age swallowed more water than adults, and male adults swallowed more water than female
adults. In both Dufour studies, the actual amount of time spent swimming in water was a strong
predictor of the volume of water ingested (Dufour et al., 2006, 2017).
To develop additional estimates of the volume of water ingested per swimming event
considering both rate of ingestion and time spent in the water, the EPA applied data distributions
of age, gender and time spent in the water from the over 60,000 observations at 12 freshwater
and marine beaches in the combined NEEAR/UCB data set (excluding tropical beaches). Age-
and gender-specific rates of ingestion from both Dufour studies were combined with these data
in a simulation study to develop detailed, age-specific estimates of the volume of water ingested
per swimming event (DeFlorio-Barker et al., 2017a). The authors reported that children (aged 6-
12 years) swallow a median of 36 mL (90th percentile =150 mL) of water, while adults aged 35
years and older swallow 9 mL (90th percentile = 64 mL) per swimming event, with male
children swallowing more water compared to female children of the same age.
A study by Schets et al. (2011) provides incidental ingestion volumes for children aged 0 to 14
years in different types of waters based on surveys of parents' estimates of the amount their
children incidentally ingested. Of the 8,000 adults who completed the questionnaire, 1,924
additionally provided estimates for their eldest child (<15 years of age). On average, depending
on the water type, children and adult men ingested at a greater rate than women. For example, in
swimming pools, children (38 mL/hour [hr]) ingested at a greater rate than adults (males 30
mL/hr; females 21 mL/hr). The exposure rates were not adjusted for body weight.
Like Dufour et al. (2017), Suppes et al. (2014) used cyanuric acid as an indicator of pool-water
ingestion to evaluate the rate of water ingested by 16 children aged five to 17 years. They found
children, on average, ingested pool water at a higher rate than adult participants. Total time in
water, quantified by viewing videos, was used to adjust pool-water ingestion volumes to obtain
rates. After adjustments for false-positive measurements were applied, the mean rate at which
adults ingested water was 3.5 mL/hr (range 0-51 mL/hr). The mean rate at which children
ingested water was 26 mL/hr (range 0.9-106 mL/hr).
In addition to greater exposure, the EPA NEEAR study provided some evidence that children
were at a greater risk of swimming-associated illness following exposures to fecally-
contaminated recreational water (Wade et al., 2008). Using the combined NEEAR/UCB data set
representing over 80,000 observations from 13 beach sites, UCB researchers led an additional
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analysis to provide summary estimates of gastroenteritis risks and illness burden associated with
recreational water exposure and determine whether children have higher risks. Participants were
classified as non-swimmers, swimmers below culturable enterococci criteria and swimmers
above culturable enterococci criteria (U.S. EPA, 2012a). Authors concluded that children aged
0-4 and 5-10 years had the most water exposure, exhibited stronger associations between levels
of water quality and illness, and accounted for the largest attributable illness burden (Arnold et
al., 2016).
Several other studies also evaluated the risk to children in the beach environment. Cordero et al.
(2012) characterized the variation in the risk of GI illness at an urbanized tropical beach during
the dry and rainy seasons. Enterococci were below the water quality standard during the study,
but were higher in the autumn rainy season. GI illness was reported more often during the rainy
season compared to the dry season and a much higher risk of GI illness occurred among children
<5 years of age compared to other age groups (Cordero et al., 2012).
Lamparelli et al. (2015) conducted a prospective-cohort epidemiological study at five beaches in
Sao Paulo, Brazil affected by human sewage. At all five beaches, children <10 years of age had
increased incidence of GI illness compared to recreators >10 years of age. Rates of GI illness
among children <10 years of age ranged from approximately 10 to 20% at the five beaches. The
pattern of elevated enterococci and elevated illness incidence across the five beaches, however,
was inconsistent.
Sanborn and Takoro (2013) identified children younger than five years as being a high risk group
for illness from recreational water exposure, especially if they have not been vaccinated for
Rotavirus, de Man (2014) noted markedly higher risks of infection with agents of GI illness per
flood event in urban floodwaters for children, relative to adults exposed to the same waters that
were contaminated variously with Giardia spp. (35%, 0.1-142 cysts/liter [L]), Cryptosporidium
(30%), 0.1-9.8 oocysts/L), Noroviruses (29%>, 102—104 PCR-detectable units [pdu]/L) and
Enteroviruses (35%>, 103—104 pdu]/L). Although not comparable to recreational water exposure in
the density ranges of thresholds of the RWQC, these findings underscore the contrast between
adult and children's illness rates in a given setting.
In summary, increased water ingestion among children documented by Dufour et al. (2017) and
DeFlorio-Barker et al. (2017a) support the epidemiological evidence from Arnold et al. (2016)
and Wade et al. (2008) that children are more highly susceptible to swimming-associated GI
illness, likely in part due to increased water ingestion rate per swimming event. Increased
understanding of exposure of children gained since 2010 will be used in conjunction with
epidemiological data and other health studies to further refine estimates of risk to children.
These additional analyses are required to sufficiently quantify risks and to potentially revise the
criteria.
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3.
Health Relationships and Coliphage
Since the 2012 Recreational Criteria were issued, the EPA has been evaluating development of
recreational criteria for coliphage, a viral indicator (See Chapter IV). As part of these efforts,
UCB led a comprehensive reanalysis of the NEEAR/UCB/SCCWRP data using data from beach
sites where coliphage was measured. Questions addressed by this reanalysis were 1) Is coliphage
associated with GI illnesses among swimmers? 2) How does coliphage compare to standard FIB
(culturable enterococci) as a health indicator? and 3) Does coliphage presence affect the
association between culturable enterococci and GI illness?
The studies included observations at six marine beach sites (two from NEEAR and four from
UCB/SCCWRP in California) and over 40,000 beach goers and 1,818 water samples. Four of
these beaches were classified for at least part of the study duration as human-impacted due to the
known presence of fecal discharges. Two beaches were classified as not human-impacted
because of no known sources of fecal discharge at those sites (Benjamin-Chung et al., 2017). The
water samples were assayed for male-specific or somatic coliphage by EPA Method 1601 or
1602. Assays conducted to detect indicators varied by beach.2 Somatic coliphage was detected
more frequently than male-specific coliphage, and some beach sites had a low frequency of
detection. Overall, no association between the presence of coliphage (or culturable enterococci)
and GI illness was found among swimmers nor did the presence of coliphage affect the
association between culturable enterococci and GI illness. Under "high-risk" conditions, defined
as those for which human fecal contamination was likely impacting the beach, however,
associations between both culturable enterococci and coliphage and GI illness among swimmers
were observed.
This pooled analysis represents the largest evaluation to date of the association between
coliphage in recreational water and GI illness. The findings provide evidence that the presence of
coliphage is associated with GI illness among swimmers under conditions when human fecal
contamination is present. Compared to associations with culturable enterococci, associations
were similar for somatic coliphage and there was some evidence for a stronger association with
male-specific coliphage. This work highlights the potential utility of coliphage as a predictor of
GI illness when human fecal contamination is likely present. Potential limitations include a
relatively high frequency of non-detects at all six marine beach sites, which could have been
attributable in part to the use of 100-mL water samples rather than larger volume samples
(Benjamin-Chung et al., 2017).
With regard to the SCCWRP studies (Griffith et al., 2016), male-specific coliphage (EPA
Method 1603) exhibited a stronger association with GI illness compared to culturable
enterococci (EPA Method 1600) at Aval on and Doheny Beaches (Griffith et al., 2016). At
2Somatic coliphage (EPA Method 1601) was analyzed at Avalon, Doheny, and Mission Bay; somatic coliphage
(EPA Method 1602) was analyzed at Avalon and Doheny; male-specific coliphage (EPA Method 1601) was
analyzed at all six beaches; male-specific coliphage (EPA Method 1602) was analyzed at Avalon and Doheny.
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Malibu, a site where septage seeps at one end of the beach, F+ ribonucleic acid (RNA) coliphage
genotype II was the only indicator significantly associated with GI illness (Colford et al., 2012).
4. Health Relationships and Additional Alternative Indicators
Although observations show that development and use of alternative fecal indicators is a rich and
evolving field, no strong case has been made for changing the indicators currently recommended
in the 2012 RWQC. As stated by numerous studies, however, alternative method-indicator
combinations might be supported in certain situations (fecal sources, source dynamics,
waterbody type) (Savichtcheva and Okabe, 2006; U.S. EPA, 2007a,b; Boehm et al., 2009;
Schoen et al., 2011; Ashbolt, 2015; Griffith et al., 2016) and warrant further study especially in
specific settings, such as tropical waters.
In the NEEAR studies, no indicators beyond culturable enterococci and Enterococcus spp.
measured by qPCR were tested at every site. Enterococcus spp. measured by qPCR was strongly
and consistently associated with GI illness among swimmers across the NEEAR studies in both
marine and fresh waters. These associations led to the development of supplemental criteria in
the 2012 RWQC. In addition to the associations with GI illness among swimmers and male-
specific coliphage at two of the NEEAR marine sites, associations between GI illness and
Bacteroidales measured by qPCR (Wade et al., 2010) and GI illness and Clostridium spp.
measured by qPCR were also observed. Archived NEEAR water samples were recently tested
for the presence of human-specific Bacteroides markers of fecal contamination. Although
detections of one marker (BsteriFl) showed patterns of positive associations with swimming-
associated GI illness, consistent associations between the presence of other human-specific
Bacteroides markers and GI illness were not observed among swimmers due to frequent non-
detects and generally low levels of detection (Napier et al., 2017). The authors state that
quantitative measures for the human markers could be needed to assess the relationship between
risk and human fecal pollution.
Studies conducted by UCB and SCCWRP at Doheny, Avalon, and Malibu beaches in California
tested a broader range of indicators than did the NEEAR studies (Colford et al., 2012; Arnold et
al., 2013; Yau et al., 2014; Griffith et al., 2016). Although all three beaches were affected by
human fecal contamination, the contamination dynamics were complex and differed significantly
from site to site. The indicator results are summarized by Griffith et al. (2016). At all three study
sites, F+ coliphage was more strongly associated with GI illness than culturable enterococci (see
Coliphage section). At Doheny Beach and Avalon Beach, associations between swimming-
associated GI illness and Enterococcus spp. measured by qPCR were similar to those observed
for culturable enterococci. For the other multiple indicators assessed, positive associations were
observed only when these beaches were thought to be impacted by human fecal contamination
(e.g., when the berm was open at Doheny Beach and under conditions of high submarine
groundwater discharge at Avalon). Arnold et al. (2016) also observed the positive associations
with health effects for both culture and qPCR-enumerated enterococci at beaches with known
point sources of human fecal pollution, but not at beaches lacking those sources. Arnold et al.
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(2016) also reported increased illness risks for children for Enterococcus spp. measured by both
qPCR and culturable enterococci.
5. Etiologic Agents
Researchers have long suspected viruses as possible etiological agents in swimming-associated
illness (Cabelli et al., 1982; WHO, 2003; Sinclair et al., 2009). Most types of enteric human
viruses are generally unlikely to occur in animal feces (Feachem et al., 1983; Halaihel et al.,
2010), although pigs and birds periodically carry zoonotic waterborne viruses (Meng, 2011;
Raoult, 2011). Moreover, Wong et al. (2009) reported that water samples from both Silver Beach
and Washington Park Beach (both NEEAR beaches) contained human adenoviruses. Other
results indicate that enteric viruses can be highly infectious even at low doses (Teunis et al.,
2008, Wade, et al., 201) and are relatively resistant to standard sewage treatment processes
(Laverick et al., 2004; Lodder and de Roda Husman, 2005; Pusch et al., 2005; van den Berg et
al., 2005; Haramoto et al., 2006). These studies collectively highlight the potential importance of
human enteric viruses as etiological agents of concern in recreational waters contaminated by
human fecal sources and, in particular, treated and disinfected effluent.
The understanding of the human health effects from pathogens in ambient waters has grown
(e.g., detection methodologies, epidemiological study designs, risk assessment approaches,
evaluation of risk management). For example, as part of the 2009 NEEAR study at Boqueron
Beach, Puerto Rico, the EPA collected saliva samples from a subsample of study participants to
test using a multiplex salivary immunoassay for evidence of infection among swimmers. This
assay, developed by EPA scientists, can detect infection from several potentially waterborne
pathogens including common variants of norovirus (Augustine et al., 2017; Griffin et al., 2011,
2015). Of 1,298 participants who provided three samples, 34 (2.6%) had antibody responses
indicative of a potential infection with norovirus genogroup I or II. The infection rate was over
four times higher among swimmers who immersed their heads in water compared to participants
who did not immerse their heads in water. Very few of the infections were associated with self-
reported symptoms, indicating these infections were likely asymptomatic or produced mild
symptoms that were unnoticed or not reported. The findings provide some of the first direct
evidence that enteric viruses (norovirus) are transmitted during swimming even without the
presence of symptoms (Wade et al., 2016). QMRA analyses support these results, which indicate
enteric viruses are likely the most important etiologic agent in waters affected by human fecal
sources (Soller et al., 2010a). A pathogen monitoring program at this location during the
epidemiological study detected enteric viruses in beach water and a QMRA conducted
incorporating the pathogen data showed that enteric viruses could account for almost all of the
illnesses reported (Soller et al 2016).
6. Tropical Waters
Researchers and regulators have long expressed concern regarding the applicability of FIB in
tropical environments due to their potential to regrow and persist in the water, sand, and soil in
these environments (Boehm et al., 2009). Recent studies in tropical locations found levels of
E. coli and enterococci four to five logs higher compared to coliphages and enterophages,
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suggesting a natural source of the FIB indicators in pristine tropical waters (Santiago-Rodriguez
et al., 2016).
An EPA epidemiological study conducted in 2009 in Boqueron Beach, Puerto Rico did not
provide conclusive evidence of the applicability of FIB in tropical environments due to
interference of the qPCR signal and relatively low levels of fecal contamination (Wade et al.,
2010). Similarly, a study in Luquillo, Puerto Rico found a higher risk of GI illness compared to
non-swimmers and a higher risk of GI illness observed among children <5 years of age (Cordero
et al., 2012), but no consistent associations with levels of fecal contamination. Sanchez-Nazario
et al. (2014) also conducted an epidemiological study at three tropical beaches with point and
non-point sources of fecal pollution. Although they found an increased risk of illness among
swimmers compared to non-swimmers, including when water quality met the current microbial
standard, indicators were not predictive of GI illness. Coliphages were found to be the best
predictors of respiratory illness followed by E. coli (Sanchez-Nazario et al., 2014).
Lamparelli et al. (2015) reported the findings of a prospective-cohort epidemiological study at
five beaches in Sao Paulo, Brazil affected by partially (primary treatment and chlorination),
poorly, or non-treated human sewage. Highly significant exposure-response relationships
between levels of E. coli and enterococci bacteria and self-reported GI illness were found among
swimmers. The geometric mean for enterococci ranged from 16 to 64 cfu/100 mL and for is. coli
from 42 to 234 cfu/100 mL, and three of the five beaches had geometric means below the EPA's
current recommendations. The findings of the study provide some of the first published evidence
that FIB are predictive of swimming-associated GI illness in tropical environments at sites
impacted by sources of human fecal contamination. Other measures of fecal contamination (i.e.,
molecular measures, coliphage), however, were not available.
Additionally, conditions in more tropical regions, especially Hawaii, are such that the 2012
RWQC may be more protective when used in conjunction with QMRA. This is due to the
propensity for enterococci to be associated with contaminated soil (Vijayavel et al., 2010) and to
exhibit higher decay rates in the environment (Kirs et al., 2016), and the enhanced possibility of
enterococci regrowth in a tropical setting. One QMRA study reported that GI illness risks from
viral exposures were generally orders of magnitude greater than bacterial exposures in Hawaiian
waters impacted by stream discharges (Viau et al., 2011). Researchers found a positive,
significant association between GI illness rates predicted by QMRA and Clostridium perjringens
densities; no other microbial indicators correlated to risk (Viau et al., 2011). Another QMRA
study found a correlation between densities of indicator bacteria and rainfall in an urbanized
tropical stream, but not between rainfall and a human fecal marker (Kirs et al., 2017). The stream
studied is chronically affected by human sewage inputs, such as illegal cross-connections and
leaking sewer systems, during dry and wet weather periods. Kirs, et al. note that water
management decisions in Hawaii should not rely solely on enumeration of enterococci oris, coh
(Kirs et al., 2017). In Hawaii, where Enterrococci are found at high densities in soils, multiple
lines of evidence, including Clostridium perjringens, indicator bacteria, and F+-specific
coliphage, were required to identify sewage as the cause of water quality impairment in an
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urbanized tropical watershed. Again, this represents an ongoing challenge in tropical waters for
traditional indicators.
7. Non-Point Sources
Beach sites with known human sources of fecal contamination are considered to have the highest
risk of swimming-associated illness due to the large numbers of microorganisms and the high
potential for pathogenic microorganisms in human sewage and human fecal contamination. In
addition, associations with FIB are most reliable and consistent at beach sites dominated by
human point source fecal contamination. Studies with non-point, non-human, diffuse, and
sporadic sources often have failed to identify significant associations between FIB density and
illness. An EPA study conducted in 2009 at a marine beach site (Surfside Beach, South
Carolina), with no identified point sources of human fecal contamination, found no strong or
consistent associations between levels of FIB and swimming-associated illness (Wade et al.,
2010). Similarly, at a beach with no known point sources, a dose-response relationship was
observed between skin infections and culturable enterococci, but was not observed between GI
illness and any FIB (Sinigalliano et al., 2010). Additionally, a series of three large
epidemiological studies by UCB and SCCWRP with EPA contribution confirmed that
associations between FIB and illness are most robust and consistent when human fecal
contamination impacts the beach.
At Malibu Beach, California, Arnold et al. (2013) found no association between any of the fecal
indicator organisms measured and illness among swimmers. The beach is impacted by non-point
source urban runoff, and during the study water quality was good, meeting or exceeding the
EPA's and the State of California's criteria. At Avalon Beach, California, Yau et al. (2014)
reported significant associations between culturable enterococci and GI illness among swimmers
only when submarine groundwater (influenced by human fecal contamination from leaking
septic and sewer systems) was likely impacting the beach. Enterococcus spp. measured by qPCR
was also positively associated with GI illness among swimmers under conditions when
submarine groundwater discharge was high. At Doheny Beach, California, associations between
culturable enterococci and Enterococcus spp. measured by qPCR and GI illness were observed
only when a "sand berm" was open, allowing potentially untreated human contamination from
the San Juan Creek to impact the beach (Colford et al., 2012).
Collectively, these studies provide evidence that when human sources impact marine beach sites,
enterococci (enumerated by both culture and qPCR) are associated with GI illness among
swimmers. When impacts are not associated with known human sources, FIB densities are not as
strongly associated with GI illness.
Regarding non-human sources, QMRA analyses found that exposure to animal fecal sources
such as gull, chicken and pigs might pose a lesser risk compared to human fecal material (Soller
et al., 2010a). Risk from bovine feces directly deposited into a recreational waterbody can result
in risk similar to that posed by secondary treated and disinfected effluent. The EPA conducted a
series of field experiments using land-applied cattle manure, pig slurry, and chicken litter to
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evaluate runoff containing animal fecal material. Simulated rainfall mobilized FIB and pathogens
from the study area. The EPA included the results from the mobilization experiments to modify
the fecal loading of FIB and pathogen parameters in a QMRA analysis. Risk from all non-human
sources including bovine feces can be less when the fecal material is land applied and reaches
surface water via rainfall-induced runoff (Soller et al., 2010a; EPA 2010c; Soller et al., 2015).
Risks from mixed sources are driven predominantly by the proportion of the contamination
source with the greatest ability to cause human infection (potency), which is not necessarily the
most abundant source(s) of FIB (Schoen and Ashbolt, 2010; Soller et al., 2014).
Risks from nonhuman fecal sources can be influenced, however, by the magnitude of
contamination. One study in New Zealand comparing human-impacted waters with waters
impacted by other animal wastes found both types of waters had similar potential for illness risks
and both were higher than non-impacted "control" waters (McBride et al., 1998). This study
included sites that were heavily impacted by animal waste (i.e., non-human) from rural
watersheds. Considered together, this information suggests that both the nature and the
magnitude of the fecal source impacting a waterbody influence the potential for human health
risks.
8. Wet Weather
In recent years, several studies have highlighted the importance of significant rainfall in
determining the degree of water contamination. For example, a recent epidemiological-coupled
QMRA study in California surfers found that FIB measured in seawater (i.e., Enterococcus spp.,
fecal coliforms, and total coliforms) were strongly associated with illnesses, but only during wet
weather. Urban coastal seawater exposure increased the incidence rates of many acute illnesses
among surfers, with higher incidence rates after rainstorms (SCCWRP, 2016; Arnold et al.,
2017). The QMRA component of the aforementioned study found that human enteric viruses are
the pathogens of primary concern, based on site-specific pathogen monitoring data of storm
water, site-specific dilution estimates, and literature-based data for ingestion pathogen dose-
response and morbidity. Norovirus (genogroups I and II), enterovirus, and adenovirus were
detected regularly in the stormwater discharges. No known permitted point-source discharges
affect nearshore coastal waters in southern California; rather, wet weather facilitates discharges
of raw human sewage to leak or overflow from malfunctioning infrastructure. To help improve
water quality in both dry and wet weather conditions in southern California, alternative water
quality metrics, like the human-associated fecal source marker HF183, are being used to inform
decisions (SCCWRP, 2016).
Another study (Abia et al., 2016) noted that ingestion of 1 mL of river water from the Apies
River in Gauteng, South Africa could lead to 0-4% and 1-74% probability of illness during the
dry season and wet season, respectively. Authors noted that activities that disturb sediments lead
to elevated risk of infection to users of the river. In the Chicago Area Waterways System
(CAWS), wet-weather conditions also contributed to elevated pathogen loads (Rijal et al., 2011).
A QMRA in Philadelphia, Pennsylvania waterways found dry-weather risk estimates to be
significantly lower than those predicted for wet-weather conditions (Sunger et al., 2015).
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9. Health Burden
Several studies provided evidence of the costs, burden, and severity resulting from swimming-
associated illness. These studies relied on data collected as part of the NEEAR epidemiological
study, the UCB/SCCWRP epidemiological studies, the UIC CHEERS or a combination of these
data sets.
Collier et al. (2015) used the NEEAR data set to document the overall occurrence of illness and
healthcare utilization among beachgoers and their swimming exposures and social and
demographic characteristics. DeFlorio-Barker et al. (2017a) considered how alternative
definitions of GI illness, including severity of the episode, affected associations among
swimming exposures. In a second paper, DeFlorio-Barker et al. (2017b) developed a range of the
health burden costs resulting from swimming-associated GI illness. In this analysis, the authors
found that each case of swimming-associated GI illness resulted in costs (due to medications,
time lost from work, etc.) ranging from $46 to $263 (U.S. dollars).
In addition to reanalyses of the NEEAR data, CDC and the EPA summarized information on
recent outbreaks in recreational waters for 2010-2011. The National Outbreak Reporting System
(NORS) is a passive reporting system through which state and local health officials voluntarily
report outbreaks to the CDC. During this time, 21 outbreaks associated with untreated
recreational water occurred, resulting in 479 cases and 22 hospitalizations. Seven outbreaks were
caused by E. coli 0157-H7 or 0111; two outbreaks by norovirus; and one outbreak by
adenovirus. Twenty outbreaks were in fresh water (e.g., lakes), and one outbreak was in marine
water (Hlavsa et al., 2015). Due to the voluntary nature of this surveillance system, which relies
on individual states and localities to report outbreaks, the outbreaks reported to CDC are likely
an underestimate of the actual number of outbreaks. In addition, the number of cases reported
due to outbreaks represent only a small fraction of the total cases that occur in the population
because most cases, especially for relatively mild and self-limiting illnesses, are not reported.
10. Non-Enteric Illness
In the 2000 BEACH Act amendments to the CWA, the EPA was required to study illnesses other
than GI illness, the illness most commonly associated with recreational water exposure. Other
endpoints include respiratory symptoms, skin rashes, and ear and eye infections. The NEEAR
study found no associations between these other non-GI symptoms and levels of fecal
contamination at beach sites (Wade et al., 2008, 2010). An analysis of earaches and ear
infections reported from the NEEAR studies confirmed that, although swimmers had higher rates
of earache and ear infections, these were not associated with fecal contamination (Wade et al.,
2013). A meta-analysis by Yau et al. (2009) combined the NEEAR and UCB/SCCWRP data to
study skin-related symptoms and found that although swimmers reported higher rates of skin-
related symptoms, there was no association with levels of fecal contamination.
These studies provide additional evidence that GI illness is the most frequent and most consistent
illness associated with fecal contamination at beach sites. Although other illnesses such as eye,
skin, respiratory, and ear infections can be caused by exposure to fecally contaminated
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recreational waters, they occur less frequently and have inconsistent associations with indicators
of fecal contamination.
B. Summary of Coliphage Advancements for RWQC
1. Introduction
Over the past few years, the EPA has been working to develop RWQC for coliphage, a viral
indicator, to ensure public health protection from water sources that have been influenced by
viral fecal contamination (U.S. EPA, 2015f, 2017a). Increasing evidence through microbial risk
assessments (Schoen and Ashbolt, 2010; Soller et al., 2010a,b, 2015) and epidemiological
studies (Lee et al., 1997; Colford et al., 2005, 2007; Wiedenmann et al., 2006; Wade et al., 2010;
Griffith et al., 2016; Cabelli et al., 1982) illustrate that viruses cause most illnesses associated
with primary contact recreation in surface waters impacted by human sources. Further, U.S.
outbreak surveillance data collected by CDC points to viruses as the leading pathogen group
responsible for untreated ambient recreational water outbreaks (Jiang et al., 2007; Sinclair et al.,
2009; Hlavsa et al., 2015).
Human enteric viruses enter recreational surface waters from both treated and untreated human
sources. A driving issue is that current wastewater treatment and disinfection processes
specifically target the removal and inactivation of bacteria, not viruses (U.S. EPA, 2015f,
2017a). Numerous studies have identified the presence of viruses in wastewater treatment
effluent, often when traditional fecal indicator bacteria are nondetectable (U.S. EPA, 2015f).
Although the EPA recommends coliphage as an option for evaluating fecal contamination in
groundwater, the Agency currently has no coliphage recommendations applied to surface waters
for protecting primary contact recreation. Coliphages are a subset of bacteriophage viruses that
infect E. coli. In particular, male-specific (or F+ specific) and somatic coliphages have been
proposed as more reliable indicators of human viral pathogens associated with fecal
contamination than traditional fecal indicator bacteria (Gerba, 1987; Palmateer et al., 1991;
Havelaar et al., 1993; Cabelli et al., 1982). Coliphages exhibit numerous desirable indicator
characteristics. For example, they are abundant in domestic wastewater, raw sewage sludge, and
polluted waters; are physically similar to viruses causing illnesses associated with primary
contact recreation; originate almost exclusively from the feces of humans and other warm-
blooded animals and undergo only very limited multiplication in sewage under some conditions
(i.e., high densities of coliphages and susceptible host E. coli at permissive temperatures); are
nonpathogenic; amenable to overnight culture methods and can be counted cheaply, easily, and
quickly; in some studies show correlations to GI illness among swimmers; and are similarly
resistant to sewage treatment and environmental degradation as enteric viruses of concern
(Funderburg and Sorber, 1985; Havelaar et al., 1990, 1993; Sobsey et al., 1995; Gantzer et al.,
1998; Grabow, 2001; Mandilara et al., 2006; Nappier et al., 2006; Pouillot et al., 2015; U.S.
EPA, 2001 a,b, 2015f).
As part of the coliphage criteria development process, the EPA has 1) conducted a series of
literature reviews; 2) refined somatic and male-specific (or F+) coliphage enumeration methods
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(culture and molecular), including completing single-laboratory and multilaboratory validation
studies on culture-based methods for wastewater treatment effluent and ambient waters; 3)
conducted the 2016 Coliphage Experts Workshop; and 4) participated in an analysis of
NEEAR/UCB epidemiological data, specifically evaluating coliphage-health associations. This
section summarizes the results from the EPA's literature reviews, information about the
EPA'scoliphage enumeration methods, and conclusions on the 2016 Coliphage Experts
Workshop. The epidemiological studies where coliphage was measured as an indicator are
discussed above.
2. Literature Reviews
In 2015, the EPA published Review of Coliphages as Possible Indicators of Fecal Contamination
for Ambient Water Quality (U.S. EPA, 2015f), a peer-reviewed literature review of the scientific
information that the EPA will evaluate to develop coliphage-based AWQC for the protection of
swimmers. The review generally illustrates the currently available data support the conclusion
that coliphages are good alternative indicators of fecal contamination to the EPA's currently
recommended criteria for E. coli and enterococci. In addition, coliphages are better indicators of
viruses in treated wastewater than bacteria.
Additionally, the EPA has conducted a series of systematic literature reviews of viruses in raw
sewage and in ambient waters (Eftim et al., 2017a; U.S. EPA, 2017a). The work indicates that
pathogenic viruses (norovirus) are found in raw sewage at logio mean densities of 4.7 (logio
standard deviation of 1.5) genome copies/L (Eftim et al., 2017a). The systematic literature
review of male-specific and somatic coliphage densities in raw sewage and ambient waters are in
progress, but the work has been presented at the 2016 and 2017 University of North Carolina
(UNC) Water Microbiology Conferences and 2015 Coliphage Experts Workshop. Collectively,
the data will be used to assist in the criteria derivation for the coliphage-based RWQC (Eftim
2016; 2017b).
Finally, fate and transport of bacteriophage (and other indicators) were reviewed (U.S. EPA,
2015f; McMinn et al., 2017). As part of this effort, the EPA evaluated inactivation through the
wastewater treatment processes based on the published literature (McMinn et al., 2017) and
investigated decay in marine environments (Wanjugi et al., 2016). The results indicate that logio
reduction of coliphage was more similar to that of viral pathogens than FIB to human viruses,
suggesting they might be better surrogates for removal of viral pathogens than FIB (U.S. EPA,
2015f; McMinn et al., 2017). Additionally, bacteriophage exhibit differential decay patterns in
marine waters that appear influenced by several biotic and abiotic factors and by bacteriophage
type (Wanjugi et al., 2016).
3. Methods
The EPA's culture-based assay uses dead-end hollow fiber ultrafiltration (D-HFUF) paired with
the single-agar layer (SAL) procedure as described in EPA Method 1602 to concentrate and
enumerate culturable somatic and F+ specific coliphage from large volumes (>1 L) of surface
waters (McMinn et al., 2017a). Application of the method to a variety of surface waters resulted
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in average percent recoveries of greater than 50%. Analyses to date indicate that D-HFUF-SAL
is a robust and sensitive method that can be used for routine measurements of culturable somatic
and F+ coliphage from surface waters (McMinn et al., 2017a). The D-HUF-SAL method (2 liters
[L]) for the enumeration of F-specific and somatic coliphage has undergone multi-laboratory
validation for use in ambient waters and in advanced treated wastewater effluent. Similarly, EPA
Method 1602 (100 mL) has undergone multi-lab oratory validation for use in secondary
wastewater (no disinfection) effluent.
The EPA has additionally developed molecular assays to target four genogroups of F-specific
(F-RNA [ribonucleic acid]) coliphages via reverse transcriptase-polymerase chain reaction
(RT-PCR) and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), which
can provide information regarding the source of fecal pollution (human vs. other animals)
(Friedman et al., 2009, 2011, 2017). Specificity of the assays for their respective fecal sources
was evaluated on F-RNA coliphages (n = 49) originating from various warm-blooded animals,
sewage and combined sewage overflow, demonstrating its usefulness in discriminating coliphage
from different sources (Friedman et al., 2009). In addition, successful evaluation on a panel of
environmental F-RNA strains demonstrated their utility in the assessment of sanitary quality of
recreational waters (Friedman et al., 2011). Further evaluation of RT-qPCR methods signified
that the F-RNA genotyping procedure successfully indicated possible human fecal
contamination, but the methodology has challenges and would require substantial refinements
and improvements before considering its use for routine measurements of coliphage densities in
surface waters (Paar et al., 2015).
4. 2016 Coliphage Experts Workshop
The EPA held the Coliphage Experts Workshop in March 2016 as part of the Agency's ongoing
efforts to build the scientific basis for developing coliphage-based water quality criteria. The
EPA brought together a group of 12 internationally recognized experts on the state of the science
of coliphages and their usefulness as a viral indicator for the protection of public health in
recreational waters. Experts represented a spectrum of perspectives from academia, federal
agencies (EPA, CDC, Food and Drug Administration), and the wastewater industry. The EPA
recently published a peer-reviewed meeting proceedings report on the workshop (U.S. EPA,
2017a). The goal of the workshop was not to reach consensus; instead, it was designed to be a
critical thinking and information-gathering exercise. Agenda discussion topics included the need
for a viral indicator, coliphage as a predictor of GI illnesses, how coliphage could be useful as an
indicator of wastewater treatment performance, male-specific versus somatic coliphage, a
systematic literature review of viral densities, and future research.
During these discussions, individual experts had common views that viruses are a source of
illness in recreational water exposures and that those viruses enter surface waters via wastewater
treatment plant (WWTP) effluent, especially during wet weather events and when WWTPs
exceed design flows. Additionally, experts noted that coliphages are more similar to human
pathogenic viruses than traditional FIB and they more closely mimic the persistence of human
pathogenic viruses. Experts also suggested that future epidemiological studies specifically
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include coliphages as measured indicators. As an indicator of WWTP performance, coliphages
are consistently present in municipal sewage and provide a baseline for examining different
WWTP processes under varied conditions. Experts indicated that the literature suggests
coliphage and human viruses have more similar log-reductions during wastewater treatment,
compared to traditional FIB. Opinions ranged, however, on whether somatic, male-specific
coliphage, or both would be better for various applications. Evidence shows a relationship to GI
illness in epidemiological studies for both coliphage types in some studies. A fact sheet and the
proceedings for the 2016 Coliphage Experts Workshop are available online (U.S. EPA, 2017a).
C. Summary of Scientific Advancements in FIB qPCR
1. Introduction
U.S. EPA's 2012 RWQC (2012 RWQC; U.S. EPA, 2012a) included qPCR EPA Method 1611
(U.S. EPA, 2012b) as a supplemental indicator method to detect and quantify Enterococcus spp.
in ambient water on a site-specific basis. The qPCR methodology offers the advantage of
providing rapid detection results (2-6 hours), allowing beach managers to make same-day
decisions to protect beachgoers. In contrast, water quality results for traditional culturable
indicator methods are not available until 24-48 hours after sampling. In addition to providing
rapid results, The EPA's Enterococcus spp. qPCR (Method A, Draft of EPA Method 1611) was
more strongly associated with GI illness enterococci measured by culture in the NEEAR study
(Wade et al., 2008; U.S. EPA, 2010c). At the time of the 2012 RWQC publication, however, the
EPA still had limited experience with the method's performance across a broad range of
environmental conditions. The 2012 RWQC contain this cautionary language: "EPA has limited
experience with its performance across a broad range of environmental conditions. States should
be aware of the potential for qPCR interference (see Section 3.1.1) in various waterbodies, which
may vary on a site-specific basis. Thus, the EPA encourages a site-specific analysis of the
method's performance prior to use in a beach notification program or adoption of WQS based on
the method" (U.S. EPA, 2013d).
Interference is any process that results in lower quantitative estimates than actual values. For
qPCR-based enumeration methods, interference can occur when substances bind to the target
deoxyribonucleic acid (DNA), which can prevent the primers from binding, inhibit polymerase
function, or cause the DNA to precipitate prior to amplification. Examples of substances causing
interference include humic acids, coral sands, calcium, and certain types of clay particles;
however, many other unidentified substances likely also contribute to qPCR interference.
Since 2010, however, the EPA has made significant advancements in the performance of the
EPA's FIB qPCR methods. These advancements are articulated through peer-reviewed
manuscripts, EPA Method documents, technical support materials, and a systematic literature
review of qPCR methods (see Appendix A). Two key developments include the publication of an
improved qPCR-based method for enumeration of Enterococcus spp. (EPA Method 1609) and
the development of a draft EPA qPCR-based method for enumeration of E. coli (Draft Method
"C"), both of which were included in the EPA's 2015 Great Lakes Beaches study. Finally,
calculation tools to facilitate data analysis by the user are also available. This section briefly
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describes improvements in EPA qPCR methods (EPA Method 1609 and Draft Method C) and
technical support information available to stakeholders. The EPA notes that no change is needed
in the criteria to apply these revised methods.
2. Enterococcus spp. qPCR EPA Method 1609
To address the potential for high interference levels, the EPA developed EPA Method 1609 (U.S.
EPA, 2013b), which uses a custom designed reagent for environmental sample testing called
Environmental Master Mix (EMM) (TaqMan; Applied Biosystems, Foster City, CA), that results
in lower levels of interference in undiluted samples (Haugland et al., 2012, 2016; Cao et al.,
2012; Sivaganesan et al., 2014). Like EPA Method 1611, EPA Method 1609 requires the sample
processing control (SPC) interference control assay using Sketa 22 and recommends the internal
amplification control (IAC) assay.
Appendix A (Table A-4) summarizes the EPA's systematic literature review results of the
application of EPA Enterococcus spp. qPCR methods in ambient waters. EPA Method 1609 has
a qPCR interference range of 0-14% in undiluted samples in both temperate marine and fresh
waters, based on the SPC and IAC controls. In contrast, EPA Method 1611 has a much higher
interference rate in undiluted samples ranging from 0 to 53% in both temperate marine and fresh
waters, using both SPC and IAC for controls. For both methods, a five-fold dilution of the water
samples reduces the interference rate in fresh and marine waters, and routinely performing this
dilution is recommended in Method 1611.
Overall, EPA Method 1609 is recommended over EPA Method 1611. EPA Method 1609 has an
overall more robust performance, with no sample dilution required in most instances, and a lower
overall interference rate, as compared to other EPA methods (Draft Method A, EPA Method
1611). Sample dilution and use of the EMM addressed inhibition at the nine marine and 23 of the
25 potentially problematic freshwater sites in 10 states comprehensively investigated by the EPA
since 2010 (Haugland et al., 2014, 2016).
Based on these results, use of EPA Method 1609 is appropriate when the required and suggested
controls are employed. Use of the EMM, the Sketa 22 SPC assay, and optional use of the IAC
assay both reduces interference and identifies whether interference was observed in the qPCR
sample. These controls are not available for culture methods.
Revisions to Method 1609 (and 1611) have been published by the EPA as Methods 1609.1 and
1611.1, respectively, that are available online (U.S. EPA, 2015e). These updates were introduced
to further standardize absolute Enterococcus spp. CCE density estimates across laboratories and
to relate them to 2012 RWQC values (Haugland et al., 2014). Greater standardization can be
achieved through the suggested use of EPA-provided DNA reference materials and data
calculation support materials (see below Available Technical Support Information).
The EPA has developed a draft qPCR method for E. coli (Draft Method C; Chern et al., 2011),
which incorporates the same interference modifications and controls as EPA Method 1609. A
multi-laboratory validation study is currently underway, and results are expected by the end of
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2018. Appendix A describes the systematic literature review results of 13 studies by the EPA and
others that have evaluated qPCR methods for E. coli. Overall, results illustrate low rates of
inhibition (<10%) at the locations sampled. The number of sites and samples reported, however,
is significantly smaller than for Enterococcus spp. qPCR. The EPA's Draft Method C shows
promise for use on a site-specific basis, but, no peer-reviewed demonstrations of its use in
routine monitoring are presently available.
3. Available Technical Support Information
Since 2010, the EPA has also developed a series of support materials and information for
stakeholders interested in using qPCR in their waterbodies.
qPCR Standards
Evaluation of reference materials used in the qPCR-based methodology highlighted the
importance of using standardized protocols and reference materials (Shanks et al., 2012; Cao et
al., 2013a; Haugland et al., 2014). Efforts are ongoing with the National Institute of Standards
and Technology (NIST) to establish an interagency agreement to develop DNA reference
materials for the EPA's qPCR methods. In the meantime, Agency laboratories have prepared
DNA reference materials for Enterococcus spp. (EPA Methods 1609.1 and 1611.1) and E. coli
(Draft Method C) that can be used for the standardization of these methods and has made these
materials available to the public. The EPA contact information for obtaining these materials is
currently undergoing revision.
Training Sessions
Successful application of the qPCR-based methods requires sufficient laboratory capability and
proficiency. Proficiency is affected by the experience of laboratory personnel, and sufficient
training of personnel is needed to ensure adequate method performance. The EPA has held
multiple "train-the-trainer" sessions to assist states in learning qPCR techniques. The continued
availability of standards will also help facilitate consistency of results within and between
laboratories. Additionally, the EPA has an ongoing collaboration with Michigan Department of
Environmental Quality to assess the implementation of the E. coli qPCR method in state public
health and water testing laboratories. Additional stakeholder troubleshooting and guidance are
expected to result from this effort.
qPCR Acceptability Criteria
The EPA has provided guidance on how to evaluate the acceptability of EPA Enterococcus spp.
qPCR EPA Methods 1611 or 1609 at a specific beach. The guidance assumes that the testing
laboratory has been able to perform one of these methods within the acceptance criteria, and now
wishes to ascertain whether qPCR would be acceptable for use at a particular site. Site
acceptability is based on the demonstration that a sufficiently high percentage of multiple
samples, collected from the site over time, show an absence of sample matrix interference, as
determined by the qPCR methods controls. It is important to note that EPA Method 1609 reduces
the frequency of interference compared to EPA Method 1611 and allows analyses of undiluted
extracts for greater analytical sensitivity at many sites. A recent multi-laboratory study of
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potentially problematic sites across the United States revealed that 20 of the 22 sites met the EPA
site acceptability guidelines when using EPA Method 1609 (Haugland et al., 2016).
Calculation Spreadsheets
The EPA has provided Excel spreadsheet workbooks that can be used for the standardized for the
calculation of Enterococcus spp. calibrator cell equivalent (CCE) densities in test samples in
Methods 1609.1 and 1611.1 (U.S. EPA, 2015e). The workbooks will automatically perform the
calculations employing formulas that are derived from EPA Methods 1611.1 or 1609.1 and
require only inputs of raw cycle threshold (Ct) measurements of the methods standards, control
samples, and test samples. The workbooks also identify test samples that fail the acceptability
criteria for the interference controls in the methods.
Detection and Quantification of EPA Enterococcus spp. qPCR Methods
The EPA has provided results and conclusions from an EPA Office of Research and
Development (ORD) study to determine the limit of detection and lower limit of quantification
of EPA Method 1611. The analyses were performed on 5 x-diluted DNA extracts of samples (as
specified in this method) containing known quantities of enterococci cells. The lower limit of
quantification was reported at different thresholds of acceptable variability: 10%, 20%, and 33%
coefficient of variation. At 10% coefficient of variation, the estimated lower limit of
quantification was 179 cells/sample and at 33%, 125 cells/sample. The 99% frequency limit of
detection was between 75 and 150 cells/sample. The conclusion from this study was that the
overall method should normally be sensitive enough to support the EPA RWQC values except
possibly when less than recommended sample volumes are collected or when total DNA
recoveries from the samples are extremely low. Extrapolation of these results to EPA Method
1609, which recommends analyses of undiluted extracts, suggests that this method should
support the RWQC values in virtually all samples of recommended volume that pass the
acceptability criteria for the controls in the method (U.S. EPA, 2013d). The 33% coefficient of
variation lower limit of quantification value from this study has been incorporated into the EPA
Excel spreadsheet workbook for Methods 1609.1 or 1611.1.
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D. Human/Non-Human Fecal Source Identification
1. Fecal Source Identification
Background
FIB currently recommended for management of fecal contamination in ambient waters are found
in the feces of warm-blooded and some cold-blooded animals. FIB methods are commonly used
for water quality management because procedures are typically straightforward and inexpensive,
especially cultivation-based protocols. Ideally, FIB information provides valuable information on
the total level of fecal pollution in the waterbody of interest. FIB approaches, however, have
several limitations that can reduce their utility for water quality management. For example,
naturalized FIB populations have been reported to exist in some non-fecal sources, such as beach
sands, soils, and sediments, and are associated with aquatic algae and plants (Badgley et al.,
2010, 2011; Gordon et al., 2012; Byappanahalli et al., 2007, 2012a,b; Eichmiller et al., 2013;
Bradshaw et al., 2016). In addition, FIB results provide no information about different pollution
sources present (Hagedorn et al., 2011; McLellan and Eren, 2014). Many impaired waters are
polluted by multiple fecal sources originating from human waste treatment facilities, agricultural
practices, and wildlife. FIB are not always well correlated with pathogens, potentially limiting
protection of public health (Savichtcheva et al., 2006; Wu et al., 2011; Harwood et al., 2014). In
addition, culturable indicator densities might not reflect potentially high-risk scenarios when
disinfected effluents affect a waterbody (Wade et al., 2008; Wong et al., 2009; Soller et al.,
2010b; Schoenetal., 2011).
Fecal source identification (FSI) techniques are used to characterize different fecal sources
potentially present in polluted waters (McLellan and Sauer, 2009; Harwood et al., 2014). FSI
methods rely on the detection of host-identifiers, which are typically chemical or microbial
targets highly associated with a particular pollution source (Hagedorn et al., 2011). Research
attempts to link FIB occurrence trends to host-identifier measurements often show poor
correlations (Hagedorn and Weisberg, 2009). It is important to note, however, that waters can be
polluted by multiple fecal sources. As a result, FIB can represent a cumulative measure of
multiple fecal pollution sources, and some non-fecal sources of indicator, while a host-identifier
is targeting a particular source. Furthermore, fecal contamination from human and animal
sources contribute different pathogens to ambient waters resulting in variable relationships
between FIB, pathogens, and illness outcomes (Soller et al., 2010b; McLellan and Eren, 2014).
For example, human fecal contamination, such as untreated sewage and even disinfected
effluent, is associated with the highest potential risk of GI illness (Soller et al., 2010b, Schoen et
al., 2011).
The notion that some fecal pollution sources represent a higher public health risk is not new. The
World Health Organization's recreational water guidelines highlight the pollution source risk
differential and incorporate a water classification scheme that emphasizes fecal contamination
from humans (WHO, 2003). This realization has led to a large body of research exploring the
application of host-identifiers for water quality management. For example, Bradshaw et al.
(2016) found that incorporating a combination of FIB, host-identifiers, and other water quality
measurements improved water quality assessment in a mixed land use area in a watershed. Other
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research groups have focused on potential health-predictive associations between host-identifiers
and illness outcomes (Boehm et al., 2015; Brown et al., 2017). The ability to make a direct
association between a host-identifier and the risk of an illness outcome could substantially
improve public health protection in recreational water management scenarios.
Fecal Source Identification with Chemical Host-Identifiers
Many organic and inorganic chemical compounds are reported to be highly associated with
human fecal contamination, including chemicals that are closely associated with human feces
(e.g., fecal steroids, caffeine, or synthetic chemicals found in products specific to household and
community waste streams) (NRC, 2004; Hagedorn and Weisberg, 2009). The steroid used most
frequently as a human host-identifier is coprostanol, produced by catabolism of cholesterol in the
intestinal tract (Leeming and Nichols, 1996). A panel of multiple fecal sterols can provide even
more information than coprostanol alone in the presence of human fecal contamination (U.S.
EPA, 2007a). Caffeine is another common chemical host-identifier of human fecal
contamination. The main sources of caffeine in U.S. waters is likely human fecal waste and
coffee waste disposal activities. Optical brighteners, added to laundry detergents, are also
reported to be useful for detecting sources of human fecal pollution in municipal effluent
(Hagedorn et al., 2005; Hartel et al., 2007). Optical brighteners can be measured with a hand-
held fluorometer, which can provide immediate and relatively inexpensive monitoring results in
the field (Hagedorn and Weisberg, 2009). Linear alkylbenzenes, residues of surfactants
commonly used in detergents, are another potential chemical host-identifier of human
contamination in surface waters (Phillips et al., 1997; Gustafsson et al., 2001). Chemicals from
other personal care products and some pharmaceuticals might also prove useful as human fecal
waste host-identifiers and could be a valuable management tool for groups with the appropriate
resources and expertise (U.S. EPA, 2007a).
Fecal Source Identification with Microbial Host-Identifiers
Microbe-based FSI, often referred to as MST, targets enteric microbial species closely associated
with the gut of a particular animal group. To date, a wide range of technologies is reported to
identify these host-associated microorganisms, ranging from canine scent detection to next-
generation sequencing (Yan and Sadowsky, 2007; U.S. EPA, 2011; Hagedorn et al., 2011; Santo
Domingo et al., 2011; Boehm et al., 2013). The most widely used technologies use molecular
methods such as the polymerase chain reaction (PCR) (Stewart et al., 2013). Molecular methods
refer to protocols used in genetics, microbiology, biochemistry, or other related fields to study
biologically important molecules such as DNA, RNA, and proteins. Before the widespread use of
molecular detection and quantification techniques, studies examined the enumeration of specific
groups of bacteria, such as the fecal anaerobes Bifidobacteria spp. and Methanobrevibacter
smithii, as potential host-identifiers of human and other animal fecal contamination sources
(Harwood et al., 2009; Balleste and Blanch, 2011; McLellan and Eren, 2014).
Over the past decade, the field of molecular biology has advanced significantly. By combining
the concept of host-associated bacteria with molecular methodologies, a central MST hypothesis
has emerged suggesting that host-associated genetic markers can act as a metric of fecal
contamination from a particular animal group. As a result, considerable amounts of time and
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resources have been dedicated to the development, testing, and performance validation of
molecular MST technologies (Shanks et al., 2008, 2009, 2010; Haugland et al., 2010; Boehm et
al., 2013, 2015; Ebentier et al., 2013; Stewart et al., 2013). Researchers have also focused on
examining potential relationships among molecular host-identifiers, FIB, pathogens, and public
health outcomes (Harwood et al., 2014, Dubinsky et al., 2016; Kirs et al., 2017).
Accurate and reliable MST technologies could dramatically improve water quality management
in the United States. Some applications include enhanced characterization of fecal contamination
trends in waterbodies impacted by multiple pollution sources, increased understanding of
potential public health risks in recreational water settings, and targeted remediation of fecal
contamination. To date, MST has aided the identification of fecal pollution sources in impaired
waters (Kirs et al., 2017) and urban-impacted recreational beaches (Molina et al., 2014), and
helped identify pollution sources during wet-weather-related overflows of human sewage
impacting U.S. coastal waters (SCCWRP, 2016). MST tools have been applied in the
development of TMDL management plans as part of CWA requirements and in the evaluation of
best management practice effectiveness (U.S. EPA, 2005). MST methods have also been
combined with high-resolution digital mapping strategies to successfully identify non-point
sources of human fecal pollution in a large watershed (Peed et al., 2011). Significant advances
made in this area now allow the potential for application of MST to facilitate decision-making
for water quality managers. Successful implementation should rely on a "tool box" approach,
where MST methods are combined with other established water quality assessment methods to
enhance management activities. In addition, the growing body of scientific evidence suggesting
that public health risks due to exposure from fecal contamination in recreational waters can be
quite different when pollution sources are human compared to non-human sources suggests MST
methodologies could play a key role in future public health risk assessments (Soller et al., 2010a,
2014, 2015; U.S. EPA, 2014b). While research in this area progresses, with continuing advances
and broader application of these technologies, MST clearly has great potential to improve water
quality management and help protect public health.
2. EPA MST Research
Human sources of fecal contamination pose a greater potential risk to human health compared to
many animal sources (Soller et al., 2010a). Therefore, understanding the sources of fecal
contamination is important to protect beachgoers from exposure to poor microbial water quality.
The 2012 RWQC has provisions that recommend FSI technologies for use as a sanitary
characterization tool (EPA 820-F-12 058) and evidence to support alternative criteria eligibility
(EPA 820-R-14-010). Since 2012, The EPA has made significant progress toward the
implementation of human source identification technologies, particularly in regard to
HF183/BacR287 and HumM2 qPCR methods.
The EPA's ORD maintains an active research program to advance science in human FSI
technologies to support implementation of the EPA's RWQC. U.S. recreational waters may be
affected by human fecal waste originating from numerous sources such as leaking sewer lines,
faulty septic systems, combined sewer overflows (CSOs), sanitary sewer overflows (SSOs), or
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illicit activities. Human fecal waste can harbor disease-causing pathogens that contribute to poor
public health outcomes and economic burdens. Since currently recommended FIB methods do
not discriminate between human and other potential pollution sources in recreational
environments due to local wildlife and agricultural activities, human FSI methods can
complement general fecal indicators to improve recreational water quality management. ORD
has invested considerable resources to develop, validate, standardize, and implement human FSI
technologies.
Since 2010, EPA researchers have published 16 peer-reviewed studies and developed a series of
technology transfer tools focusing on human FSI method validation, standardization, and
implementation.
The EPA collaborated with SCCWRP and 25 other expert laboratories to conduct an
international blinded study to identify top performing human FSI technologies (Boehm et al.,
2003). Findings demonstrated that most experts (>90%) favor PCR-based technologies (Stewart
et al., 2013), that qPCR methodologies are highly reproducible only with standardized protocols
(Ebentier et al., 2013), and that HF183 and HumM2 qPCR technologies are top-performing
human FSI methods (Layton et al., 2013). As a result, the EPA and collaborators performed a
technical review of the HF183 qPCR technology resulting in the optimized HF183/BacR287
method for recreational water applications (Green et al., 2014). HF183/BacR287 and HumM2
qPCR methods were then subjected to an EPA-led 16-laboratory national validation for fresh and
marine recreational water use (Shanks et al., 2016). Currently, draft EPA Methods for human FSI
are under internal review.
A series of studies was conducted to support implementation of qPCR human FSI applications.
Notable contributions include experiments demonstrating the uniformity in sewage microbial
communities across the United States (Shanks et al., 2013), how the unit of measure (e.g.,
enterococci cell count, total DNA mass) for qPCR can alter reported concentrations of human
fecal pollution (Ervin et al., 2013), in situ human waste decomposition varies by pollution source
and indicator type (Wanjugi et al., 2016), and QMRA modeling can be used to predict links
between HF183 and public health risk in raw sewage (Boehm et al., 2015).
The demand for human FSI is rapidly increasing. In response, EPA scientists have developed a
series of tools to help facilitate technology transfer of HF183/BacR287 and HumM2 qPCR
human FSI technologies. Tools include standardized data acceptance metrics, draft EPA Method
procedures, a self-administered method proficiency test, and an automated data analysis tool
(Shanks et al., 2016).
Field demonstrations are necessary to provide real-world examples of human FSI qPCR method
application. EPA scientists have conducted multiple field studies focusing on identification of
diffuse human fecal pollution sources from urban runoff (Molina et al., 2014) and septic field
discharge in recreational beach, stream, and river settings (Peed et al., 2011).
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Key Implementation Gaps
The EPA advances in national method validation, standardization, and implementation combined
with key scientific studies and technology transfer tools provide necessary information to support
the use of human FSI qPCR technologies in recreational settings. Two key implementation gaps
remain, however, including: 1) formal publication of EPA Methods for HF183/BacR287 and
HumM2; and 2) the development of a DNA reference material. To develop a national DNA
reference material, the EPA recently entered into an Interagency Agreement with the National
Institute of Standards and Technology (October 2017).
Emerging Science
The EPA is actively conducting scientific experiments to support human FSI qPCR method
implementation. Current efforts are focused on the development of a standardized procedure to
prioritize recreational sites based on human fecal pollution levels (Cao et al., 2013b, Cao et al.,
2018). In addition, two large-scale field studies are being conducted using FSI in conjunction
with other water quality and climate parameters from nine Great Lake and 29 Tillamook
watershed sites to identify occurrence patterns and impact on water quality management in
recreational settings. Finally, the EPA and collaborators have developed new viral-based human
FSI technologies to complement bacterial HF183/BacR287 and HumM2 methods (Stachler et al.,
2017).
3. Selected External Research Contributions to MST Development
"Tool Box" MST Application Demonstrations
The presence of microbial pollutants in surface waters can originate from several sources (e.g.,
wastewater effluent, sewage leaks, sewer overflows, illegal discharges, wildlife, and agricultural
runoff). The presence of these pollutants can be influenced by several factors such as weather
conditions, adjacent land use, local waste management infrastructure, and watershed
characteristics. Currently no single method is used to define local water quality, discriminate
between pollution sources, provide weather condition information, and include local land use
practices. As a result, many researchers and management experts employ a "tool box" approach
to build a comprehensive framework to interpret water quality conditions. For example, Litton et
al. (2010) reported the use of a quantitative sanitary survey approach combined with a range of
other analytical tools to identify potential sources of FIB contributing to local water impairment.
Water quality metrics included FIB measurements of enterococci and Escherichia coli (IDEXX
methods), select genetic markers (HF183 and Enterococcus spp.) determined by qPCR, and
chemical identifiers of sewage and wastewater. Findings provided important insights on the
benefits and limitations of specific methods, the value of a "tool box" approach to interpret water
quality data, and the promising potential of human-associated MST methods. Another study
performed in collaboration with scientists from the University of South Florida and the
SCCWRP authority paired FIB and bacterial human-associated MST methods with virus-based
water quality metrics to investigate non-point sources of fecal pollution at two California marine
beaches (McQuaig et al., 2012). Findings illustrated limitations of FIB alone to characterize
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sewage pollution sources and the presence of viral pathogens, underscoring the value of a "tool
box" approach for water quality management.
Technology Transfer
An essential component of the successful implementation of MST method is technology transfer.
Molecular MST methods can be technically demanding, requiring specialized equipment,
detailed procedures, and specialized training. A useful MST technology must be transferable,
with a high degree of reliability and reproducibility. Many MST methods have been developed
and validated within individual laboratories. Inter-laboratory testing has been minimal, providing
limited information on MST method reproducibility. Information on key factors that influence
MST method reproducibility across laboratories is vital for successful implementation and public
acceptance. To help address this gap, several organized studies have been conducted to evaluate
molecular MST protocols. For example, scientists from five laboratories situated across the Gulf
of Mexico region conducted a study to characterize the inter-laboratory performance of three
human-associated MST end-point PCR methods (Gordon et al., 2012). Another group evaluated
nine host-associated MST qPCR methods across five laboratories using standardized and non-
standardized procedures (Layton et al., 2013). Findings demonstrated the successful technology
transfer of MST molecular methods and the importance of standardized procedures.
Advances in Virus-Based MST
Most currently available human MST technologies target fecal bacteria. The presence of some
viruses, however may also be used to distinguish human from non-human sources of fecal
contamination. As a result, research efforts have been made to develop virus-based MST
methodologies (McQuaig et al., 2009; Rosario et al., 2009) and compare performance to
bacteria-based approaches (Staley et al., 2012). For example, Staley and colleagues performed
side-by-side testing of sewage diluted in five water types (estuarine, marine, tannic, lake, and
river) to evaluate the suitability of each method to estimate risk of GI illness. Findings
demonstrated strengths and limitations of bacteria- and virus-based MST approaches and
included a recommendation for a "tool box" approach incorporating both bacterial and viral
methodologies in future studies.
The Source Identification Protocol Project
The Source Identification Protocol Project was an international effort to identify top performing
MST methods and characterize the current state of the science. This effort was led by the
SCCWRP authority and scientists from Stanford, the University of California - Los Angeles,
University of California - Santa Barbara, and EPA-ORD. The effort was designed to engage the
international scientific community to identify top performing MST technologies for human,
ruminant, cattle, dog, and swine pollution sources by expert consensus; demonstrate the
importance of procedure standardization; and explore emerging technologies based on microbial
community methodologies. The study was centered on the construction of a 64-sample, blinded
sample set challenging expert laboratories to correctly detect and quantify fecal pollution
sources. Participating in the study were 27 expert laboratories from seven countries applying 41
MST methods. Findings were published in a special edition of the International Water
Association journal of Water Research (Reis and Wuertz eds., 2013). The Source Identification
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Protocol Project represents a critical step toward the public acceptance of MST technologies for
water quality management by identifying top methods based on expert consensus.
Emerging MST Technologies
New technologies such as next-generation sequencing and digital PCR represent potentially
important advances for MST. As a result, the research community has begun to explore the
application of these new technologies for MST. For example, next-generation sequencing has
been successfully used to track the movement of CSO events in the Great Lakes (Newton et al.,
2013). Some MST methods have also been adapted to a digital PCR platform to explore potential
advantages of this new technology for molecular testing of environmental samples (Cao et al.,
2015). Emerging technologies will likely harness the power of high-throughput nucleic acid
sequencing and other methodologies for the rapid and simultaneous measurements of multiple
MST host-identifiers. These novel technologies will likely provide future water quality
managers, public health officials, and researchers with powerful tools to improve water quality
management.
Importance of MST Genetic Marker Decay
Understanding the decay of microorganisms associated with specific fecal pollution sources is
necessary for implementation of MST molecular methods for water quality management. Unlike
culture-based FIB methods, MST molecular methods target nucleic acids instead of live cells that
need to be cultivated in a laboratory. This fundamental difference in method analyte between
cultivated FIB measurements and genetic testing with MST technologies can result in different
persistence behaviors in environmental settings. Thus, understanding how different
environmental stressors influence FIB and MST genetic marker decay is imperative to interpret
water quality results properly. As a result, researchers have investigated factors such as sunlight
(Green et al., 2011), water type (Jeanneau et al., 2012), temperature (Kreader et al., 1998; Okabe
and Shimazu, 2007; Dick et al., 2010), and influence of indigenous microbiota (Kreader et al.,
1998; Dick et al., 2010) on FIB and MST genetic marker decay. Findings suggest that cultivated
FIB decay trends are significantly different from MST genetic markers. Most studies agree that
persistence of MST genetic markers is typically longer in colder water and in marine waters
compared to fresh water.
First State Manual on Implementation of MST Methods
The state of California has spent approximately $100 million since 2001 to improve beach water
quality at impaired recreational sites. Despite these efforts, several locations still frequently
exceed local WQS based on FIB measurements alone, mostly due to poor information on
contamination sources leading to inadequate cleanup strategies. Advances in science and the
need for fecal source pollution information led the California Clean Beach Initiative program to
fund research efforts with the aim to develop a state manual for implementing MST methods
(Griffith et al., 2013). This pioneering manual outlines a tiered, "tool box" approach to
implement MST methodologies based on a hypothesis-driven, science-based strategy that
provides a foundation for implementing MST technologies on a national level.
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E. Emerging Issues: Evidence of Exposure to Antimicrobial Resistant Bacteria
in Recreational Waters
1. Introduction
Within the past several years, an increasing body of research indicates the environment has
become not only a recipient of drug-resistant bacteria, but also a reservoir for and a source of
resistance genes (Martinez, 2009; Wright, 2010; U.S. EPA 2013e; Berendonk et al., 2015). Drug-
resistant bacteria and associated genes have become an emerging concern regarding the
protection of human health during recreational activities in surface waters. Contaminated
wastewater effluents, a variety of non-point sources, and even naturally occurring bacteria, are
potential origins of drug-resistant or antimicrobial resistant bacteria (AMRB) and antimicrobial
resistant genes (ARG) in recreational waters. Environmental surveillance is a key tool in
furthering the understanding of AMRB/ARG and in participating in the One Health approach to
this growing global issue of concern that incorporates human health, animals, and the
environment, as recommended in the National Antimicrobial Resistance Monitoring System3
program. Additionally, future research on recreational waters should include: 1) human health
risk assessment strategies for various exposure scenarios; 2) removal of AMRB/ARG from
wastewater treatment processes and disinfection; 3) environmental selection for resistance; and
4) mitigation strategies for preventing the loading of AMRB/ARG into recreational waters.
2. Antimicrobial Resistance Mechanisms
Although drug-resistance genes are naturally occurring, anthropogenic releases of antibiotics and
ARG through clinical and agricultural use represent a much larger concern for human and
ecological health. Contaminants such as heavy metals and pharmaceuticals are also reported to
exert selective pressure that can result in co-selection for antibiotic resistance in the environment
(Wellington et al., 2013; Baker-Austin et al., 2006). The dispersion of resistance throughout the
environment occurs through loading of wastewater effluent and discharges, application of animal
waste to land, horizontal gene transfer (HGT) among bacteria, and gene selection via
environmental conditions. HGT is the primary mechanism of concern in the environmental
dispersion of drug-resistant bacteria. When resistant bacteria are released into the environment,
they can share their resistance genes with native bacteria and pathogens via HGT. HGT occurs in
one of three ways: (1) uptake of genetic material from the environment—transformation;
2) direct transfer of genetic material from one cell to another—conjugation; or 3) movement of
genetic material from one cell to another via a bacteriophage vector (Burmeister, 2015). Very
small concentrations of antibiotics in polluted environments could enable the selection for
resistant and multi-resistant genes using HGT mechanisms, which could result in the
maintenance or increase of concentration of ARGs (Gullberg et al., 2014; Baquero and Coque,
2014).
3The National Antimicrobial Resistance Monitoring System is a Food and Drug Administration program to promote
and protect public health. This ongoing collaboration with CDC and U.S. Department of Agriculture aims to work
within a One Health approach to address AMR challenges.
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3. Point Sources and Non-point Sources
The EPA categorizes pollutant sources as either point sources or non-point sources for regulatory
purposes. Point sources can include discharges from WWTPs, industrial facilities, and
concentrated animal feeding operations (CAFOs) (U.S. EPA, undated). Municipal sewage, which
is treated in WWTPs, can contain waste from households, storm drains, and hospitals. A wide
variety of products that contain antibacterial compounds, such as triclosan, are now sold and
used in households beyond the more common antibacterial hand soaps and cleaning products,
ranging from toys to kitchen tools (Aiello and Larson, 2003). Studies indicate that antibacterial
agents in soap and other household products select for environmental microflora that allow
ARGs to thrive (Levy, 2001).
Hospital wastewater carries diverse communities of pathogens and pharmaceuticals, entering
surface waters, either directly or indirectly through municipal sewage systems or after hospital
pretreatment. Originating in hospitals, pathogenic bacteria with resistance against all or almost
all of our existing antibiotic treatments are of increasing concern. For example, carbapenem-
resistant Enterobacteriaceae (CRE) has been declared the highest priority by CDC, a critical
pathogen of concern. A healthcare-associated infection that infects hospitalized patients, CRE is
caused by Klebsiella and E. coli bacteria (CDC, 2013) and has been found in significantly higher
concentrations in hospital wastewater as compared to municipal wastewater (Lamba et al., 2017).
CRE and other drug-resistant microbes can spread into surface water and from there into
recreational waters, through insufficient treatment of hospital wastes. In some cases, before
hospital wastes are discharged into sewage systems, local authorities may regulate pollutant
levels via the National Pretreatment Program to prevent overloading publicly owned water
infrastructure with heavy loads of contaminants.
Regarding WWTP controls, the extent to which drug-resistant bacteria might survive wastewater
treatment or pretreatment processes is not well established. The potential is multifaceted—
mobile elements, bacteriophage, naked DNA of killed cells, or bacterial cells that have survived
chlorination could be released into waterways (Rizzo et al., 2013; Gantzer et al., 1998). Tertiary-
treated effluent has been found to contain high levels of antibiotic resistance determinants, at
20 times the concentrations of background levels (Lapara et al., 2011). One study in northern
China found that wastewater effluent contained a higher concentration of ARG than influent at
the same plant (Mao et al., 2015). The effect was associated with heavy metals and antibiotic
residues in wastewater, indicating that these conditions might select for a proliferation of
resistance, not removal of it (Mao et al., 2015). Seasonal disinfection practices (e.g., chlorinating
only in summer) at certain WWTPs could influence levels of bacteria (Mitch et al., 2010) and
AMRB entering surface waters via effluent discharge. In addition, sewer overflows are of
concern due to the concentration of untreated pharmaceuticals and AMRB/ARG flowing
unrestricted into surface waterbodies during rain events (Scheurer et al., 2015).
CAFOs may receive high volumes of antibiotics used for animal growth promotion, regular
disease prevention, and treatment (U.S. EPA, 2013e). Here, high usage of antibiotics selects for
resistant bacteria both within the animals and environment (Hribar, 2010). Additionally, of all
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antimicrobials used in food-animal production, an estimated 25-75% of the drugs is excreted
unchanged into waste (Kummerer, 2004). In 2005, the U.S. Department of Agriculture reported
that CAFOs produced roughly 44 times more solid waste than WWTPs (Graham and Nachman,
2010). The EPA estimates "nearly all" of produced CAFO waste is used for land application
(U.S. EPA, 2004), which creates a potential risk that bacteria and antibiotic residues may run off
to surface waters depending on compliance with established permitting requirements. Of the
surface water samples taken in a farm environment study, 81% of E. coli isolates exhibited
resistance to cephalothin (Sayah et al., 2005). Near a concentrated swine operation, Sapkota et al.
(2007) found down-gradient surface water contained 33-fold higher median levels of enterococci
and E. coli compared to up-gradient. These down-gradient samples also had a higher percentage
of erythromycin and tetracycline resistance (Sapkota et al., 2007).
Non-point sources constitute diffuse sources of antibiotics, resistance genes, or resistant bacteria
that generally enter a waterbody via runoff, drainage, seeping, or precipitation (U.S. EPA,
2017b). Animal feeding operations (facilities or lots that do not meet the regulatory definition of
a CAFO are not considered a point source) may further contribute to the contamination and
spread of AMRB/ARG in the environment (U.S. EPA, 2013e). Land application of waste and
manure from animal feeding operations adds antibiotics and resistant genes to soils and water
(Hamscher et al., 2002; De Liguoro et al., 2003). High-volume usage of these antibiotics, such as
tetracycline, results in leakage to groundwater and surface water supplies, disturbing bacterial
communities and promoting resistance (De Liguoro et al., 2003). Although non-point sources
carry a smaller concentration of resistant strains compared to point sources, their role remains an
important consideration (Parveen et al., 1997).
Birds, particularly seagulls, play a role in the movement of and exposure to AMRB/ARG.
Seagulls have been shown to carry extended-spectrum P-lactamase (ESBL) producing E. coli
(Simoes et al., 2010), and multidrug resistance has been found in other wild birds (Sjolund et al.,
2008; Cole et al., 2005). Compared to other bird species, gulls (Larus spp.) are significantly
more likely to carry disease-causing pathogens, due to their tendency to forage on anthropogenic
waste (Fenlon, 1981; Aim et al., 2018; Belant et al., 1998). Bacterial transport by gulls is
associated with sewage outfalls, indicating that effluents containing AMRB are more likely to
cause dissemination of that bacteria via gulls (Fenlon, 1981). A study in France found that 9.4%
of the gulls observed were carrying ESBL-producing bacteria (Bonnedahl et al., 2009).
Additionally, Aim et al. (2018) found evidence that gulls act as transport vectors, picking up
human pathogens from anthropogenic waste sites at landfills and wastewater outputs, and
spreading these pathogens to recreational waters and beaches.
4. Evidence of Recreational Exposure
Exposures to AMRB/ARG have been documented at beaches and in recreational waters globally.
Prospective cohort epidemiological studies on three California beaches correlated the detection
of a variety of indicators, AMRB, and pathogens with incidence of gastrointestinal (GI) illness
(Griffith et al., 2016). Methicillin-resistant Staphylococcus aureus (MRSA) was highly
associated with GI illness, showing a stronger correlation than the EPA's current culture method
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(EPA Method 1600) at the beach where it was measured, which was impacted by human sewage
from faulty infrastructure (Griffith et al., 2016). This work highlights potential risks associated
with AMRB in recreational waters impacted by human sewage and indicates that recreators
could in some cases be exposed to MRS A (Griffith et al., 2016).
More recently, AMRB surveillance was combined with human exposure estimates to quantify
the probability of exposure to E. coli harboring biaCTx-M genes in coastal waters. These genes
represent nearly 80% of all ESBL-producing Enterobacteriaceae, which confer resistance to
multiple antibiotics, such as fluoroquinoloenes, aminoglycosides, and tetracyclines. Authors
conducted a cross-sectional epidemiology study comparing regular surfers and non-surfers to
evaluate the association between water exposure and gut colonization by E. coli harboring gene
biaCTx-M. Results indicated that 6.3% of surfers were colonized by the gene, compared to 1.5% of
non-surfers (risk ratio = 4.09; CI 1.02-16.4) (Leonard et al., 2018).
Schijven et al. (2015) measured concentrations of ESBL-E. coli (ESBL-EC) in recreational
waters and in source waters, including ditches surrounding poultry farms and municipal
wastewater. Using this information, they modeled the potential of EBSL-EC to reach recreational
waters and thus the and the probability of human exposure through swimming. They found
exposure to ESBL-EC by swimming is likely, when recreational waters are located downstream
of wastewater treatment plants or livestock farms and noted that research is warranted for the
evaluation of public health effects, such as colonization, infection, or horizontal gene transfer,
upon exposure.
Studies have also shown E. coli and enterococci persisting in sand are capable of not only
surviving in sandy environments but also replicating (Hartz et al., 2008: Haack et al., 2003:
Whitman and Nevers, 2003: Aim et al., 2006). In recent years, evidence has grown that
AMRB/ARG also survive and replicate in sand. Studies in Puerto Rico and the United Kingdom
found recreational waters and sand could be reservoirs for resistance genes and estimated human
exposures to resistant bacteria while swimming (Santiago-Rodriguez et al., 2013; Leonard et al.,
2015). In 2014, a Michigan-based study captured HGT of resistance genes among E. coli within
sand microcosms of recreational freshwater beaches of Lake Huron (Aim et al., 2014).
5. The EPA's Work on AMR for Recreational Waters
In 2001, the EPA and 10 other federal agencies formed the Interagency Task Force on
Antimicrobial Resistance (IFTAR, 2001; Colson, 2010). The EPA has conducted surveillance
research related to AMRB/ARG in wastewater and ambient waters. Initially, the EPA studied the
occurrence of E. coli resistant to a variety of clinically relevant antibiotics in primary wastewater
effluents (Boczek et al., 2006, 2007). Subsequent research has focused on the occurrence of E.
coli isolates in primary wastewater effluents that meet the CDC definition of CRE and the
presence of different carbapenemase genes associated with CRE globally (estimated completion
date: Summer 2018).
As part of the National Rivers and Streams Assessment (NRSA 2013-2014), the EPA is
enumerating ARG in rivers and streams across the United States using droplet digital PCR. The
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EPA has begun investigating gene targets, including but not limited to, beta-lactam, carbapenem,
tetracycline, and colistin, and other genetic markers of AMRB.
F. Assessment of Recreational Criteria Implementation and Tools
This section discusses the advances in implementation tools, the status of 2012 RWQC adoption,
and perceived barriers to adoption. In Section 6.0 of the 2012 RWQC document, the EPA
discussed two important tools for evaluating and managing recreational waters, sanitary surveys,
and predictive models. With the publication of the criteria document and the National Beach
Guidance and Required Performance Criteria for Grants, 2014 Edition (U.S. EPA, 2014a), the
EPA has encouraged states and beach managers to use both tools to protect public health.
1. Sanitary Surveys
As a widely used tool for investigating the sources of fecal contamination impacting a
waterbody, sanitary surveys are important to understanding watersheds and beaches. Sanitary
surveys involve collecting environmental, meteorological, physical, and water quality data at the
beach and in the surrounding watershed. Sanitary surveys help state and local beach program
managers and public health officials identify and characterize sources of beach water pollution.
By identifying, assessing, and mitigating pollution sources, states can reduce or eliminate beach
advisories and closures.
Beach managers can use sanitary survey results to prioritize state or local resources to help
improve recreational beach water quality. Routine or daily sanitary survey data (e.g., bacteria
levels, source flow, turbidity, rainfall) also can be used to develop models for predicting beach
water quality using readily available data.
Since the publication of the 2012 RWQC, the EPA has published two new sanitary survey tools:
a paper and an electronic version of the Marine Beach Sanitary Survey. In 2013, the Agency
published the Marine Beach Sanitary Survey (U.S. EPA, 2013a) for marine waters. This survey
is based on the Great Lakes Beach Sanitary survey (U.S. EPA, 2008) for fresh waters with minor
modifications to include factors important in marine environments (e.g., tidal phase and flow, rip
currents). Like the Great Lakes Beach Sanitary Survey, the Marine Beach Sanitary Survey
consists of two forms—the Routine On-site Sanitary Survey (U.S. EPA, 2013b) and the Annual
Sanitary Survey (U.S. EPA, 2013c) to help states conduct both short- and long-term beach
assessments. The Routine On-site Sanitary Survey is conducted at the same time water quality
samples are taken and includes a form for documenting the methods used to collect data during
the survey. The Annual Sanitary Survey records information about factors in the surrounding
watershed that might affect water quality at the beach, including, for example, information on
septic tank locations and conditions, land use information, and other observations relating to
long-term of water quality impacts within the watershed. Like the Great Lakes Sanitary Survey,
the Marine Beach Sanitary Survey can be used to address a variety of beach management uses
including:
• Characterizing risk and prioritizing beaches
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• Beach and watershed planning
• Remediation
• Predictive modeling
In September 2016, the EPA released a mobile app of the routine marine beach sanitary survey
form for use on Android and Apple tablets. Sanitary surveys routinely provide valuable and
useful environmental information that can be paired with water quality data and to develop
predictive models. The EPA's goal with the development of this app is to provide a tool that
would make both the collection and transfer of environmental and water quality data easy for
model development purposes.
Sanitary surveys are now a widely used element of state beach programs on both coasts and in
the Great Lakes. With the EPA's Great Lakes Restoration Initiative and BEACH Act grant
funds, Great Lakes states have been able to reduce significantly the percentage of sources
previously identified as "Unknown" that impact their beaches using sanitary surveys. Once
identified and characterized, sources can then be targeted and prioritized for remediation, leading
to fewer exceedances and advisories and to overall improved local water quality.
2. Statistical Modeling for Predictive Estimates of Water Quality
In the 2012 RWQC, the EPA encouraged the use of predictive models to supplement water
quality monitoring using culture methods and to enable timely beach notification decisions.
Predictive modeling uses past water quality data and current observed hydrometeorological data
to estimate water quality at a given time. Predictive models enable assessment of the risk to
human health from exposure to both human and non-human sources impacting beaches and their
associated watersheds.
Virtual Beach
In developing the 2012 RWQC criteria, the EPA conducted research and published a two-volume
survey report to advance the use of predictive models. Volume I (U.S. EPA, 2010a) describes the
types of predictive tools (e.g., statistical models, rainfall thresholds, and notification protocols)
that can be used to make beach notification decisions and how they are being used as part of
beach management programs across the United States. Volume II (U.S. EPA, 2010b) provides
the results of EPA research on the development of statistical models at research sites. Volume II
also introduces Virtual Beach (VB), a software package and statistically based decision tool that
allows users to build site-specific statistical models to predict FIB levels at recreational beaches.
VB reads input data from a text or Excel file, assists users in preparing data for statistical
analysis, and provides three analytical techniques for model development.
Since the publication of criteria, the EPA has released several versions of VB and has made
several enhancements to this tool. The current version of VB, VB3.0, includes statistical methods
that provide users more flexibility in modeling datasets. In addition to multiple linear regression
(MLR), which was the only original option, users can now use partial least squares (PLS)
regression and generalized boosted modeling (GBM) algorithm to fit their data and make
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predictions. PLS regression reduces overfitting in the presence of correlated predictors, an issue
that can arise with MLR. GBM is a machine-learning technique that uses decision/regression
trees instead of linear equations. It enables accurate predictions for new observations without
overfitting and handles nonlinear relationships between response and independent variables
without having to transform the independent variables. One drawback of GBM is that the model
cannot be inspected graphically or expressed mathematically. Both PLS and GBM modeling use
cross-validation to evaluate real-world prediction accuracy (Cyterski et al., 2013). GBM has
been shown across a wide range of datasets to outperform an array of other statistical methods in
recent years (Corsi et al., 2016).
Other improvements to VB include automated interaction with the USGS Environmental Data
Discovery and Transformation (EnDDaT) system for site-based data acquisition; a genetic
algorithm for intelligent search of parameter space for MLR modeling; probability of health
criteria exceedance calculations for model predictions; and cross validation of MLR models to
assess predictive capabilities (Cyterski et al., 2013).
Recursive Partitioning Based Models
Recursive partitioning is an alternative to parametric regression methods. In recursive
partitioning, a decision tree is used to model the response variable by splitting the observations
into subgroups that share similar values of the response variable and similar values of associated
covariates nodes. Modeled outcomes are obtained by answering an ordered sequence of
questions, with the question asked at each step dependent on answers to previous questions.
Recursive partitioning can be used to predict pathogen occurrence (categorical response) or
concentration (continuous response) in a variety of applications using covariates that are easier to
measure (e.g., FIB, water quality parameters) than direct pathogen analysis (Bradshaw et al.,
2016; Wilkes et al., 2011).
Other Research and Guidance
Other methods to achieve better predictions of FIB densities in recreational waters include
temporal synchronization analysis (TSA). A paper by Cyterski et al. (2012) investigated
improvements in empirical modeling performance using independent variables that had been
temporally synchronized with the FIB response variable. TSA investigates whether the
dependent (response variable) and independent (covariates) data series are temporally phase-
shifted and examines if function (e.g., mean, sum, standard deviation) of the independent
variables over some temporal window can produce a better correlation to the dependent variable.
Using data collected from South Shore Beach in Milwaukee, Wisconsin, results show that TSA
was useful for reducing mean square error of fitted data and improved predictive model
performance (as measured by the mean square error of prediction) (Cyterski et al., 2012).
In 2016, the EPA published new guidance to encourage state and local beach managers to
investigate the utility of predictive models for their beach monitoring and notification programs
and to assist with developing these tools. The guidance, Six Key Steps to Developing and Using
Predictive Tools at Your Beach (U.S. EPA, 2016b), provides a simple, straightforward approach
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on how to develop a predictive model for beach water quality. The guidance walks the user
through each step in the process, from deciding whether a model is needed or appropriate for the
user's beach (Step 1) to developing (Step 4), validating (Step 5), and evaluating the model over
time (Step 6). It also provides useful hints and tips.
The benefits of predictive models for beach monitoring and notification programs have been
further demonstrated by the other studies (Shively et al., 2016; Francy et al., 2013) conducted
since the publication of the 2012 RWQC. Predictive models offer states, territories, and tribes the
potential for same-day notification and public health protection with considerably lower capital
investment and unit costs than other rapid methods.
3. Deterministic Process Modeling for Recreational Beach Site Assessment and
Enhancement/Remediation
Whereas predictive statistical models typically rely on the regression of predictive
(observational) variables against water quality data determined in a related timeframe, benefit to
understanding the occurrence and timing of pollution events also can be derived from the use of
deterministic process models in recreational water settings. The models used to simulate and
predict contaminant transport, attenuation, and concentration are hydrodynamic process models
that apply fully understood and documented process equations populated with appropriate
observed variables. Model outputs are useful in demonstrating variations and movement of
contaminants in response to currents, wind, and other weather variables.
Nevers and Boehm (2010) provide an overview of using deterministic models to predict FIB
densities in surface waters. Nevers and Boehm underscore the value of fate and transport models
for increasing and refining the understanding of mechanisms that lead to observed variations in
water quality but that are not well defined. Other instances where deterministic models have
been applied to water quality at recreational venues are described in U.S. EPA (2010a).
4. Integrated Environmental Modeling and QMRA
Epidemiological and QMRA studies have shown that elevated levels of FIB in surface waters
can be associated with an increased risk of illness. The epidemiological relationships are not
consistent among different water and fecal source types, however, and QMRA analyses have
shown that risks can differ depending on the source of fecal material that predominates in a
waterbody (Fewtrell and Kay, 2015; Soller et al., 2010a,b, 2014, 2015). The pathogens
responsible for the illnesses vary in their type (e.g., viruses, bacteria, protozoa) and occurrence in
the source of fecal material affecting the waterbody (e.g., wastewater effluent, sewage overflows,
feces from agricultural animals, wildlife). Additionally, the fate and transport characteristics of
the various pathogens can differ and be affected by the way feces enters a waterbody and is
transported within the waterbody and by the pathogen-host interactions at the receptor location.
Integrated environmental modeling (IEM) is a framework that allows the characterization of
these complex patterns by linking models, databases, and visualization tools in various ways to
provide comprehensive and flexible solutions to environmental and risk management questions.
IEM provides a science-based structure that develops and organizes multidisciplinary knowledge
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and applies it to explore, explain, and forecast environmental system responses to natural and
human-induced stressors (Whelan et al., 2014).
EPA scientists incorporated the health modeling associated with a QMRA into an IEM
framework that includes automated data access retrieval and processing; integrated model
databases; approaches for performing sensitivity, variability, and uncertainty analyses; and risk
quantification, on a watershed scale (Whelan et al., 2014). A QMRA software infrastructure has
been developed to automate the manual steps associated with a standard microbial watershed
assessment (e.g., TMDL, sanitary surveys), as much as possible, to expedite the process (make
faster), minimize resource requirements (save money), increase ease of use, and bring more
science-based processes into the analysis. It supports watershed-scale microbial source-to-
receptor modeling by focusing on animal- and human-impacted watersheds, and links to a user
interface and workflow that automates data collection, collation of microbial sources, watershed
delineation, and flow and microbial calibration at downstream receptors (Kim et al., 2013; Kim
et al., 2016; Whelan et al., 2018). A process that normally has taken months or even years to
complete now can be completed within days, depending on source data availability. The software
contains source information to support a sanitary survey by linking pollution sources, physical
features, land-use, etc., as they vary with time (Whelan et al., 2017a). By integrating watershed
fate and transport models with the health models describing health risks and exposure, policy-
related issues can be iteratively explored in ways that the traditional empirical approaches do not
allow. Furthermore, the IEM framework with QMRA provides a platform that facilitates
transparency and reproducibility, supporting the evaluation and management of watersheds.
Finally, the software can be used to help develop site-specific water quality criteria that differ
from the EPA's recommended criteria.
Some of the components necessary to integrate the QMRA into the IEM framework include:
Microbial Source Module
The Microbial Source Module determines microbial loading rates associated with 1) land-applied
manure on undeveloped areas from domestic animals; 2) direct shedding (excretion) on
undeveloped lands by domestic animals and wildlife; 3) urban or engineered areas; and 4)
discharge to streams from leaking septic systems and from domestic animals in animal feeding
operations (AFOs) including NPDES permitted facilities (Whelan et al., 2015a; Whelan et al.,
2018).
Microbial Release Model
Mathematical models were developed to describe the physics to predict the release of microbes
from fresh and aged manure—cattle solid pats, poultry dry litter, and liquid waste from swine—
during rainfall events, which helps improve microbial loading estimates in mixed-use watersheds
(Kim et al., 2013; Whelan et al., 2017b).
Microbial Properties Database Editor
A Microbial Properties Database Editor is an interface to a database that bridges the gap between
monitoring and modeling, as it was developed to capture microbial-relevant data used by
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QMRA-related process-based source, fate, transport, and risk models. Users can modify physico-
microbial properties related to indicators and pathogens, such as mass of a microbe, excretion
density of microbes in animal feces, prevalence, etc. Microbial properties are related to changes
in or with the microbe such as inactivation rate, dose-response coefficients,
attachment/detachment rates, etc. (Whelan et al., 2015b).
Microbial Inactivation Model
The solar photo-inactivation model provides FIB, virus, and pathogen inactivation rates required
by predictive models and QMRA in recreational waters, accounts for ultraviolet wavelength
effects, is based on aquatic and atmospheric parameters, and accounts for variability of photo-
inactivation over space and time in Great Lakes and other recreational waters. For example,
depth dependence and time dependence of inactivation rates of indicator enterococci were
estimated following rainfall events in the Manitowoc River and Manitowoc, Wisconsin beaches
using the model and data collected at river and beach sites. This model was also used to predict
photo-inactivation rates at three beaches and tributaries located in southern Lake Michigan and
Lake Erie.
EPA scientists have been applying the IEM framework to characterize fecal sources and water
quality in specific watersheds (e.g., Manitowoc, Wisconsin, Tillamook, Oregon), including
evaluating seasonal dynamics of sources and relative risks from various source in the watershed.
These efforts have supported the development of the framework and calibration of the model
with real, site-specific data.
5. Adoption Status and Perceived Barriers
As mentioned earlier in this report, Congress directed states and tribes with coastal recreation
waters to adopt new or revised WQS within 36 months of the EPA's publication of the 2012
RWQC. (CWA Section 303(i)(l)(B)). WQS are the foundation for a wide range of programs
under the CWA. They serve multiple purposes including establishing the water quality goals for
a specific waterbody, or waterbody segment, and providing the regulatory basis for deriving
water quality-based effluent limits beyond the technology-based levels of treatment required by
CWA sections 301(b) and 306. WQS also serve as a target for CWA restoration activities such as
TMDLs. The WQS regulation at 40 Code of Federal Regulations part 131 describes the
requirements and procedures for states and authorized tribes to develop, adopt, review, revise,
and submit WQS and the requirements and procedures for the EPA to review, approve,
disapprove, or promulgate WQS as authorized by section 303(c) of the CWA.
In addition to recommending the FIB and criteria values for magnitude, duration, and frequency
in the 2012 RWQC document, the EPA also provided states with BAVs for use in notification
programs. The state, tribal, or local government entity responsible for ensuring public health
typically uses BAVs to make decisions about whether swimming or engaging in other primary
contact reaction in their waters is safe.
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Status of Adoption of the 2012 RWQC into WQS
The EPA reviewed the current recreational WQS in effect for CWA purposes in all 50 states,
five U.S. Territories, and the District of Columbia and for the three authorized tribes that receive
BEACH Act grant funds. For this discussion, the term "jurisdiction" refers to these entities.
• Seventeen jurisdictions have adopted and the EPA has approved revised RWQC for all
primary contact waters; three additional jurisdictions have adopted and the EPA has
approved revised RWQC for just their coastal recreation (i.e., BEACH Act) waters).
• Of the 38 BEACH Act jurisdictions, 14 are using the recommended 2012 RWQC BAV
(e.g., 70 enterococci, 235 E. colt) for their coastal recreation waters and the remaining 24
are using an alternative Beach Notification Threshold (BNT), often the SSM value
derived from the 1986 Criteria (e.g., 104 cfu/100 mL enterococci, 235 cfu/100 mL
E. coli).
• Four jurisdictions have only fecal coliform in their WQS as their FIB; nine additional
jurisdictions use fecal coliform as the only FIB for some but not all of their waters
designated for primary contact recreation (e.g., they use enterococci as the FIB in their
marine waters and fecal coliform in their fresh waters).
For the elements of the 2012 RWQC (magnitude, duration, and frequency and FIB), the
replacement of fecal coliform with either coli or enterococci as the FIB is the most commonly
identified need. Most jurisdictions, however, have revised the FIB for their BEACH Act waters
and are considering revising FIB for all waters in the near future. The STV is the second element
most commonly found to need revision, and that is related to multiple use categories discussed in
the next section. Use of a BAV/Alternate Beach Notification Threshold for swimming advisories
has been almost universally implemented in BEACH Act jurisdictions.
Barriers to Adoption of the 2012 RWQC
The EPA interviewed 34 of the 38 BEACH Act jurisdictions and several additional inland states.
The goal of these interviews was to discuss the status of the adoption of 2012 RWQC into their
standards and to identify any barriers to adoption.
• Adoption is a lengthy process
Jurisdictions noted that revisions to WQS are an administrative burden, and jurisdictions have
limited resources. The CWA requires revisions to be scientifically defensible and protective of
public health. Once a jurisdiction has evaluated the revisions based on these factors and any state
regulatory administrative requirements, they then seek public and EPA input on the draft
revisions. In addition, because the RWQC are intended to protect public health, state/tribal
processes often require two agencies to be involved in the revisions: the public health department
and the state/tribal environmental agency.
• Pre-2012 RWQC are protective
Jurisdictions believe that RWQC based on the 1986 Criteria do not significantly differ from the
2012 RWQC. They have expressed concern that they offer little or no benefit to public health as
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compared to the administrative burden needed to adopt because their current criteria are similar
to the 2012 RWQC. The EPA continues to believe that all the elements of the 2012 RWQC
meaningfully strengthen them as compared to previous criteria recommendations.
• Multiple magnitude values based on use intensity
Another barrier to adopting the jurisdictions mentioned is related to eliminating the use intensity
paradigm that was part of the 1986 Criteria and the EPA's 2004 promulgation for coastal
recreation waters. As discussed earlier in this document, in the 2012 RWQC, the EPA removed
the use intensity categories that had differing magnitude values depending on how likely the
waterbody was to be frequently visited. The categories included designated bathing beach,
moderate use, light use, and infrequent use. Some jurisdictions have designated all their waters
as primary contact recreation. States and tribes (and their stakeholders) are concerned that
primary contact recreation might not be attainable in their less frequently used waters and these
waters will have to be listed as impaired under CWA section 303(d). Revising a waterbody's
designated use, however, is also a potentially administratively burdensome process. Therefore,
these jurisdictions find the elimination of the use intensity categories as a barrier to statewide
adoption of the 2012 RWQC.
• Need for additional criteria guidance
A few jurisdictions mentioned that the EPA's implementation documents released with the 2012
RWQC were helpful, but they are struggling with development of site-specific criteria and
alternative Beach Notification Thresholds. They also noted that the release of the EPA's
expected guidance documents on QMRA would help them evaluate the specific pathogenic risks
at a particular primary contact recreation waterbody and support site-specific RWQC
development.
Based on these discussions, the EPA concludes that additional support to jurisdictions through
the continuation of BEACH Act grants that support program implementation and additional EPA
guidance would encourage additional adoption of the 2012 RWQC.
G. Recreational Criteria for the Cyanotoxins: Microcystins and
Cylindrospermopsin
In December 2016, the EPA published draft recommended values for microcystins and
cylindrospermopsin under CWA 304(a) for states to consider as the basis for swimming
advisories for notification purposes in recreational waters to protect the public or for adopting
new or revised WQS. The Human Health Recreational AWQC or Swimming Advisories for
Microcystins and Cylindrospermopsin focuses on the health risks associated with recreational
exposures in waters containing these cyanotoxins produced by cyanobacteria.
Cyanobacteria, also commonly referred to as blue-green algae, are photosynthetic bacteria that
are ubiquitous in nature, including occurrence in surface waters. Microcystins, a class of
cyanotoxins including over 100 congeners, and cylindrospermopsins can be produced by
multiple genera of cyanobacteria commonly found in fresh waters of the United States (U.S.
EPA, 2016a).
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Cyanotoxin-producing cyanobacteria fit the definition of pathogenic {i.e., disease-causing)
organisms (Stewart et al. 2006). Cyanotoxins have the potential to cause direct damage to
multiple targets within the body (e.g., liver toxicity, kidney toxicity, adverse neurologic and
reproductive effects, etc.) and can result in severe adverse outcomes for people who are exposed.
For microcystins, the primary adverse health effect of concern is liver toxicity and for
cylindrospermopsins, kidney toxicity; other potential health endpoints have been noted for both
toxins (U.S. EPA, 2015c,d). Direct contact with cyanobacterial cells, either dermally, ingestion
or by inhalation, can elicit an allergenic response in those exposed resulting in itchy rashes, eye
irritation, gastrointestinal distress and respiratory symptoms (Bernstein et al. 2011; Levesque et
al. 2014; Geh et al. 2015).
Environmental conditions that promote excessive growth of cyanobacteria in surface waters can
lead to situations in which cyanotoxins are produced or cyanobacterial cell density is high, or
both, known as harmful algal blooms (HABs). Environmental factors that play an important role
in the development of cyanobacterial blooms and their production of cyanotoxins include the
levels of nitrogen and phosphorus, the ratio of nitrogen to phosphorus, temperature, organic
matter availability, light attenuation, and pH. Cyanotoxins can be produced before an HAB
reaches visibly high cell densities and, once produced, these cyanotoxins can persist even after a
bloom is no longer visible. Given that cyanobacterial blooms typically are seasonal events and
can be short term, recreational exposures are likely to be episodic.
The EPA evaluated the health effects of microcystins and derived a reference dose (RfD) in its
2015 Health Effects Support Document for the Cyanobacterial Toxin Microcystins (U.S. EPA,
2015c). Exposure to elevated levels of microcystins could lead to liver damage. The critical
study for the derivation of the microcystins RfD was conducted by Heinze et al. (1999) based on
rat exposure to microcystin-LR in drinking water. The critical effect dose from this study was
slight-to-moderate liver necrosis, and levels of liver enzymes associated with tissue damage. The
EPA established the RfD based on microcystin-LR and used it as a surrogate for other
microcystin congeners. The RfD was used to derive the EPA's previously published Drinking
Water Health Advisories (U.S. EPA, 2015a,b) and the recommended values in this document.
The critical dose and effects used to establish the RfD from Heinze (1999) are supported by a
Guzman and Solter (1999) study, also conducted in rats.
The EPA evaluated the health effects of cylindrospermopsin and derived an RfD in its 2015
Health Effects Support Document for the Cyanobacterial Toxin Cylindrospermopsin (U.S. EPA,
2015d). The kidneys and liver appear to be the primary target organs for cylindrospermopsin
toxicity. The critical study for the derivation of the cylindrospermopsin RfD was conducted by
Humpage and Falconer (2002, 2003) based on drinking water exposure to mice. The critical
effect was kidney damage, including increased kidney weight and decreased mouse urinary
protein. Mouse urinary proteins are synthesized in the liver (U.S. EPA, 2015c).
Exposure to cyanobacterial cells has also been linked to multiple inflammatory health effects. At
this time, available data are insufficient to develop nationally recommended recreational values
for cyanobacterial cell density related to inflammatory health endpoints. The reported
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epidemiological relationships in the literature are not consistent for specific health outcomes
(e.g., dermal symptoms, eye/ear irritation, fever, GI illness, and respiratory symptoms) or for
those health outcomes associated with specific cyanobacterial cell densities. The uncertainties
related to the epidemiological study differences, such as study size, species, and strains of
cyanobacteria present, and the cyanobacterial cell densities associated with significant health
effects, do not support the development of a single cell value applicable to all recreational
waters. For more information on HABs, see https://www.epa.gov/nutrient-policv-
data/ cyan ob acteri al -h arm ful-al gal -bloom s-water. See https://www.epa.gov/wqc/microbial-
pathogenrecreational-water-qualitv-criteria for information on the draft Recreational AWQC for
microcystins and cylindrospermopsin and the final AWQC when they become available. The
EPA also recently made available information for recreational water managers to use for
monitoring and responding to cyanobacteria and cyanotoxins in recreational waters, see
https://www.epa.gov/niitrient-policv-data/monitoring-and-responding-cvanobacteria-and-
cyan otoxins-recreati on al - waters.
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V. Summary and Priorities for Further Work
In this review of the 2012 RWQC, the EPA has assessed the extent of scientific progress in the
field of human microbial health risks associated with exposure to fecal pollution from swimming
and other use of recreational waters. There has been considerable progress in many areas. The
EPA and other organizations have invested heavily in the science that was the basis for the 2012
RWQC. The progress since publication in 2012 has led to increases in the utility and level of
function of technologies and approaches that provided data for developing the 2012 RWQC. A
clear example of this progress is the development of qPCR EPA Method 1609 for the
enumeration of Enterococcus spp. This refined method greatly reduces the impact of method
interference encountered with EPA Method 1611. Further, the development of an qPCR method
for E. coli, the indicator more commonly used and preferred by states in the Great Lakes, will
leverage the use of qPCR in those important waters. Developments such as these will lead to
more timely estimates of water quality and reduced risk to swimmers. This section provides
highlights of the scientific and implementation reviews and describes the conclusions and
priorities for further work. The additional work described below will help inform future reviews
of the RWQC.
A. Health Studies (see section IV.A for more information)
Additional health studies pertaining to the basis for the 2012 RWQC provided confirmation of
these findings:
• Both epidemiological and QMRA-based studies provide scientifically defensible
estimates of human health effects from exposure to waters contaminated by feces.
• In waters affected by human fecal contamination, GI illness is the most sensitive health
endpoint reported in epidemiological studies.
• Children can be more highly exposed and have greater susceptibility to swimming-
associated GI illness.
• Waters affected by some non-human sources could pose less risk compared to human
fecal contamination.
• Enterococcus spp. qPCR and coliphage are associated with GI illness at sites impacted by
human sources.
• Norovirus infection and transmission are associated with swimming.
Findings on health studies are generally consistent with the findings of studies that formed the
basis for the 2012 RWQC, and enhance the depth and strength of the evidence underlying the
RWQC. A growing body of evidence suggests that children can be disproportionately susceptible
to health effects resulting from exposure to pathogens in recreational waters. There are
opportunities for further resolution of epidemiological relationships, especially in the area of
children's health protection and wider application of Enterococcus spp. qPCR.
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Priorities for Further Work: Re-analysis of epidemiological data to assess differences in risk to
children. Re-analysis of Enterococcus spp. qPCR data for consideration in criteria development,
especially to address effluent sources. Also, evaluate how QMRA can be used to address risk to
children from swimming exposure, and other regulatory purposes.
B. Developments for Coliphage, Including Analytical Methods (see section IV.B
for more information)
An area in which the EPA has invested considerable resources is the exploration of a viral
indicator-based RWQC. The EPA has conducted a literature review for the prospects of
coliphage RWQC and published Review of Coliphages as Possible Indicators of Fecal
Contamination for Ambient Water Quality (U.S. EPA, 2015). Other important milestones
completed include:
• Systematic literature reviews of viral densities in raw sewage and ambient waters
• Development of quantitation methods for coliphage
• Methods for culturable coliphage enumeration
- Draft Method 1642 - ultrafiltration + single agar layer.
- Draft Method 1643 - single agar layer.
• Presenting findings (e.g., at 2016/2017 UNC Water Microbiology Conferences)
• Application of methods to 2015 Great Lakes Study, and consulting outside experts in the
field
2016 Coliphage Experts Workshop.
- Fact Sheet and Peer-reviewed Meeting Proceedings.
Because evidence suggests most illnesses in recreational waters are due to enteric viruses, the
development and implementation of coliphage as a viral indicator will likely yield improvements
in public health protection.
Priorities for Further Work: Completion and publication of coliphage methods and development
of coliphage-based RWQC for inclusion into the "tool box."
C. Analytical Methods (see section IV. C. for more information)
When introduced with the 2012 RWQC, EPA Method 1611 represented a major advance in
microbial detection and quantitation methodology. Advances that produced EPA Method 1609
eliminated much of the concern about method interference and greatly increased the acceptance
of qPCR methods by the recreational water community. The advances in methods include
specific improvements:
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EPA Method 1609for Enterococcus spp.
• Provides the same results as EPA Method 1611 but with less sample interference in most
situations and is recommended over EPA Method 1611. Recommendation to use
undiluted samples compared to EPA Method 1611 for enhanced sensitivity.
• Updated EPA Method 1609.1 (and EPA Method 1611.1) facilitate standardization of
results by referring to standardized DNA reference materials available from the EPA and
a standardized Excel workbook for performing calculations.
EPA method for E. coli (draft Method C)
• Incorporates the same interference control modifications as EPA Method 1609.
• Recent nationwide field studies have suggested similar low frequencies of interference as
with Method 1609.
The advances in qPCR methodology since 2010 have brought greater reliability and utility to
beach monitoring programs where they have been implemented, yet opportunities remain for
further refinement of qPCR methodologies. Enterococcus spp. measured by qPCR, is more
predictive of swimming-associated GI illness and more timely than current culturable bacterial
indicators. These factors coupled with a greater distribution of qPCR-capable laboratories in the
future are likely to lead to enhanced public health protection.
Priorities for Further Work: Completion of method validation and publication for the E. coli
qPCR method (Draft Method C), development of alternative site-specific criteria for Draft
Method C, additional training and capacity-building in qPCR laboratories in states, tribes, and
localities.
D. Microbial Source Tracking (see section IV. D for more information)
Some of the most significant advances in RWQC research have occurred in the field of MST. A
limitation of the current FIB paradigm for assessing the risk of illness in recreational settings is
that indicator methods do not distinguish between pollution sources, which may indicate
different human health risk. FIB from non-human sources could be present in recreational waters
representing a potentially lower illness risk compared to the same pollution level originating
from a human source alone. In such situations, the 2012 RWQC might be over-protective.
Similarly, disinfected WWTP effluent could pose a higher risk of illness than reflected by the
RWQC due to the survival of disease-causing viral pathogens. Under these circumstances, the
2012 RWQC thresholds may be under-protective. Measuring enterococci with qPCR methods at
the threshold values included in the 2012 RWQC document can currently be used to address this
concern. The EPA continues to develop criteria for coliphage, a viral indicator, which could also
be used to address this concern. Information on fecal pollution sources becomes an essential
element for management and protection of public health at beaches and other recreational waters.
Numerous advances in MST such as the development of host-associated genetic technologies
(e.g., HF183/BACR287, and HumM2) with a high degree of specificity and sensitivity are
improving recreational water management. A growing list of MST studies are demonstrating the
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potential role of rainfall in fecal water pollution. In addition, multiple studies show that genetic
MST methods can be highly reproducible when standardized procedures are used. It is clear that
fecal source identification technologies are a valuable addition to the recreational water quality
assessment "tool box."
Accurate and reliable MST technologies could markedly improve water quality management in
the United States by allowing the development of alternative site-specific criteria based on the
differences in risk across sources and identifying opportunities for source remediation. Use of
alternative water quality metrics, such as human markers, might be helpful to inform risk levels
in wet weather conditions.
Priorities for Further Work: Completion and publication of standardized methods for EPA
human-associated MST methods (HF183/BacR287 and HumM2) and completion of a DNA
reference material development with NIST. Development and validation of virus-based human
fecal source identification procedures. Further investigation of MST application in recreational
water quality management settings such as prioritizing polluted sites for remediation based on
human waste levels, identification of non-point pollution sources, and the development of
alternative water quality metrics based on wet and dry weather scenarios.
E. Antimicrobial Resistance (see section IV. E for more information)
The complex issue of antimicrobial resistance is becoming of increasing interest, creating a
demand for more data to both inform our understanding of the forces driving this resistance and
the actions needed to preserve bacterial susceptibility to our first-line medications. There is an
increasing body of literature available on the environmental occurrence of AMRB/ARG and
potential exposure in recreational waters. To develop a more complete picture regarding the
threat and risks associated with antibiotic resistance, research is needed to better understand the
role the environment plays in transferring AMRB/ARG to primary contact recreators. For
example, additional research is needed on the incidence, associated risks, and transfer
mechanisms in recreational waters, as well on the removal of AMRB/ARG by wastewater
treatment processes. The EPA is in the early stages of developing a broader surveillance strategy
and looking for meaningful opportunities to improve human health relating to exposures to
AMRB/ARGs.
Priorities for Further Work: Development and standardization of AMRB/ARG detection
methodologies. Collect information on the occurrence of AMRB/ARG in environmental waters,
wastewater influent/effluents, and other potential reservoirs. Develop wastewater treatment and
disinfection processes for AMRB/ARG targets. Characterize potential associated public health
risks and mitigation strategies.
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F. Implementation Tools and RWQC Adoption (see section IV.F for more
information)
1. Implementation Tools
Sanitary Surveys
As a widely used tool for investigating the sources of fecal contamination impacting a water
body, sanitary surveys are important to understanding watersheds and beaches.
Following a wave of Sanitary Survey development and promotion in the Great Lakes 2005-2010,
recent developments in sanitary surveys include the publication of the:
• Marine Beach Sanitary Survey.
• Marine Beach Sanitary Survey Mobile Application (App)
Sanitary Surveys continue to serve as an important tool for informing site remediation,
characterizing waters for QMRA and site-specific criteria development, and can be linked with
integrated environmental modeling.
Priorities for Further Work: Conversion of current marine sanitary survey tablet-based
application to a web-based application, additional outreach on available sanitary survey
applications, collaboration with Great Lakes beach programs on fresh water sanitary survey
application and opportunities for integration with environmental modeling.
Predictive/Statistical Modeling
Predictive/Statistical models provide a means for expanding the scope of coverage of water
quality measurement in area and time. There have been substantial recent developments in this
area, including:
• Virtual Beach model building tool has been enhanced with data acquisition (EnDDaT), and
PLS (Partial Least Squares) and GBM (Generalized Boosted Modeling) predictive
calculation capabilities.
• The EPA released new guidance, Six Key Steps to Developing and Using Predictive Tools at
Your Beach (March 2016).
Predictive models offer states, territories, and tribes an alternative for same-day notification and
resulting public health protection with lower capital investment and unit costs than other rapid
methods.
Priorities for Further Work: Additional support to develop predictive models in marine
environments as well as models paired with newer indicators such as qPCR-based indicators.
Deterministic Process Modeling for Recreational Beach Site Assessment and
Enhancement/Remediation
Deterministic Process Models are useful to simulate and characterize contaminant transport and
attenuation. Integrated Environmental Monitoring provides a science-based structure useful in
developing and organizing information to explore and forecast environmental system responses
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to varying conditions. The EPA is developing several new modules related to microbial sources,
release, and inactivation. Progress since 2010 includes development of new QMRA software
infrastructure developed to provide risk estimates within a standard microbial watershed
assessment.
These models provide a means of understanding physical forces influencing the movement of
contaminants for problem definition and remediation and can include QMRA health-based
models to develop site-specific criteria or evaluate remediation.
Priorities for Further Work: Development of additional training and tools to make process
models and integrated environmental modeling more accessible to states, tribes and other
interested stakeholders.
Quantitative Microbial Risk Assessment (QMRA)
QMRA is a tool for assessing and managing risks to humans from exposure to pathogens in
recreational waters. This tool is an alternative to assessing microbial risk in recreational waters
based on epidemiology studies, which are costly and time-consuming. QMRA can also enhance
the interpretation and application of new or existing epidemiological data by characterizing
various exposure scenarios, interpreting potential etiological drivers for the observed
epidemiological results, and accounting for differences in risks posed by various sources of fecal
contamination. Integrated environmental modeling provides a means of understanding physical
forces influencing the movement of microbial contaminants for problem definition and
remediation and can include QMRA health-based models to develop site-specific criteria or
evaluate remediation.
Priorities for Further Work: Development of additional training and tools to make QMRA
models more accessible to states, tribes and other interested stakeholders. Completion and
publication of remaining QMRA guidance.
2. Review of RWQC Adoption Status and Perceived Barriers
States and authorized tribes have taken a range of approaches in adopting the RWQC. States
have used the flexibility of the RWQC to adopt a variety of protective strategies appropriate to
local conditions. Great Lakes states had only minor adjustments to beach implementation. The
adoption of the 2012 RWQC has been relatively slow (17 of 38 BEACH Act jurisdictions),
despite the fact that use of an approved beach action threshold was widely accepted. As a result,
no states or tribes had to forgo receiving BEACH Act grant funds for lack of an approved beach
notification threshold. RWQC adoption in some states is lagging due to state processes such as
involvement of two agencies (e.g., department of health and department of environment) or
requirements for legislative approval. Some states expressed that changes between 1986 and
2012 RWQC are not substantive enough to take on the administrative burden of criteria adoption
in WQS. In addition, many states still desire the paradigm that allows different criteria for
different use intensities (high use beaches vs. infrequently used recreational waters).
Jurisdictions also emphasized that they need BEACH Act grants to operate and maintain
monitoring programs. The message from the managers of state recreational waters programs is
that for some states, adoption is only justifiable with a more pronounced change in criteria
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magnitude values. The EPA notes that although the criteria magnitudes were not drastically
different in the 2012 RWQC as compared to the 1986 criteria, the 2012 RWQC included
additional elements that strengthen overall health protection in recreational waters and promote
more consistent implementation (https://www.epa.gov/sites/production/files/2015-
10/documents/rec-factsheet-2012. pdf).
Priorities for Further Work: Continued funding of BEACH Act grants. Consider additional
implementation guidance and explore reconsideration of addressing differences based on
frequency of use.
G. Recreational Criteria for Cyanotoxins (see section IV.G for more information)
Another critical water quality issue that the environmental community is facing is the growing
number of water bodies in the U.S. and elsewhere affected by cyanotoxins and other products of
harmful algal blooms (HABs). Recreators can be exposed to cyanotoxins in ambient recreational
waters leading to increased health risks. Distinct from the 2012 RWQC for fecal indicators, The
EPA is working to develop Human Health Recreational AWQC or Swimming Advisories for
microcystins and cylindrospermopsin, having published a Draft in December 2016 and taken
public comment (period closed 3/20/17). The EPA expects to revise and publish a final
document in 2018. Other researchers have found that predictive models may be useful for
estimating the probability of exceeding cyanobacterial levels related to HABs.
Additionally, the EPA has made materials available for Recreational Water Managers on public
messaging and notification, monitoring plans, and means of networking with key partners. Water
Quality Criteria Materials available with final criteria will include frequently asked questions for
assessment, listing/TMDLs/NPDES permits, and information on adoption and implementation
flexibilities for the criteria.
Priorities for Further Work: Completion and publication of recreational criteria for the
cyanotoxins, microcystins, and cylindrospermopsin for inclusion into the "tool box".
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VI. Assessment of the Need to Revise the 2012 RWQC
The scientific studies and progress described in this document detail robust continuing advances
in the capability of the environmental public health community to protect primary contact
designated uses at beaches and in other recreational waters. This scientific progress is the
continuation of a major effort by the U.S. EPA and other entities to apply modern tools, such as
molecular quantification (qPCR) methods for FIB, extensive epidemiological studies, and other
integrative approaches that formed the scientific basis for the 2012 RWQC.
A foundational element of the 2012 RWQC is the use of the indicator paradigm. This approach
employs FIB to detect the presence of fecal material and, therefore, the risk of illness from
exposure to fecal pathogens. The studies described in this report underscore the protectiveness of
the 2012 RWQC and its threshold values, especially when coupled with the use of qPCR as a
means of quantification as is fully described in Section IV. A. Detection of FIB using qPCR
methods represents a major advance in supporting this paradigm. Advances continue to unfold
with refinements to existing methods and qPCR methods for additional organisms (e.g., E. coli).
Efforts to develop viral indicators such as coliphage (described in Section IV.B) represent a
further work-in-progress to develop indicators for other pathogens of concern.
Although this report includes descriptions of many areas of evolving scientific knowledge, it is
clear that there needs to be further work to allow use of this new and emerging information in
recreational water quality criteria development. For example, we describe data demonstrating
children ingest greater volumes of water, given their body weight, relative to adults, and may be
potentially at greater risk than the general population. Going forward, the EPA will further
evaluate epidemiology data in combination with other health studies and exposure information
regarding risks to children to determine if changes are needed in the future.
In another example, there is notable progress in the area of microbial source tracking (MST) and
fecal source identification which is employing qPCR for identification of the presence of
species-specific gene segments for pollution source identification (see Section IV.D). The use of
reliable human source FIB markers brings with it the possibility to resolve the ambiguity and
limitations of the indicator paradigm. However, although this technology continues to advance,
further work still needs to be completed (e.g., completion of analytical methods and
standardization of DNA reference material) before the EPA could use it in support of CWA
304(a) recommendations for recreational water quality criteria.
Additionally, new work is in progress, but not yet completed, to develop recreational
criteria/swimming advisory values for cyanotoxins, which are contaminants of emerging concern
in recreational waters. While not envisioned in the BEACH Act of 2000, these contaminants
directly affect users of recreational waters, and their inclusion represents an integrative approach
to health protection.
Specific areas remain where additional progress is needed to support potential future revisions of
the 2012 RWQC, as are described in this review report:
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> Re-analysis of epidemiological data and use of QMRA to assess differences in risk to
children.
> Re-analysis of Enterococcus spp. qPCR data for consideration in criteria development,
especially to address effluent sources.
> Completion and publication of coliphage methods and development of coliphage-based
RWQC for inclusion into the recreational waters "tool box."
r- Completion of method validation and publication for the E. coli qPCR method {Draft
Method C).
> Completion and publication of standardized methods for EPA human-associated MST
methods (HF183/BacR287 and HumM2) and completion of a DNA reference material
development with NIST. Development and validation of virus-based human fecal source
identification procedures.
> Conversion of current marine sanitary survey tablet-based application to a web-based
application.
> Development of predictive models in marine environments as well as models paired with
newer indicators such as qPCR-based indicators.
> Development of additional training and tools to make process models and integrated
environmental modeling more accessible to stakeholders.
> Development of additional training and tools to make QMRA models more accessible.
> Completion and publication of recreational criteria for cyanotoxins (microcystin and
cylindrospermopsin).
Based on the EPA's review of the existing criteria and developments in the available science, the
EPA has decided not to revise the 2012 Recreational Water Criteria during this review cycle. The
Agency believes, however, that further research and analysis as identified in this Report will
contribute to the EPA's future review of the 2012 RWQC. The EPA will work with the
environmental public health community as the Agency moves forward with its recreational water
research efforts. The use of qPCR and ongoing research in methods and indicators continue to
strengthen and augment the tools available to support the current criteria.
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Appendix A. Advancements in Mitigating Interference in Quantitative
Polymerase Chain Reaction (qPCR) Methods for Microbial Water Quality
Monitoring
A. Introduction
The U.S. EPA's 2012 Recreational Water Quality Criteria (2012 RWQC; U.S. EPA, 2012a)
included qPCR Method 1611 (U.S. EPA, 2012b) as a supplemental indicator to detect and
quantify Enterococcus spp. in ambient water on a site-specific basis. The qPCR methodology
offers the advantage of providing rapid detection results (2-6 hours), allowing beach managers to
make same-day decisions to protect families and their children. In contrast, water quality results
for traditional culturable indicator methods are not available until 24-48 hours after sampling. In
addition to providing rapid results, the EPA's Enterococcus spp. qPCR (Method A) was
significantly associated with gastrointestinal (GI) illness in the human-impacted EPA NEEAR
studies (Wade et al., 2006, 2008, 2010). At the time of the 2012 RWQC publication, however,
the EPA still had limited experience with the method's performance across a broad range of
environmental conditions. States were cautioned to be aware of the potential for qPCR
interference in various waterbodies, which may vary on a site-specific basis. The EPA
encouraged a site-specific analysis of the method's performance prior to use in a beach
notification program or in the adoption of qPCR-based WQS (U.S. EPA, 2013a).
As defined in this report, interference is any process that results in lower quantitative estimates
than expected or actual values (Haugland et al., 2012). For qPCR-based enumeration methods,
interference occurs when substances in the test sample inhibit polymerase function, or cause the
DNA to be lost or unavailable for amplification. Examples of substances causing interference
include humic acids, coral sands, calcium, and certain types of clay particles; however, there are
likely many other unidentified substances that can also contribute to qPCR interference. From a
public health standpoint, this interference can result in false negative results of the sample.
However, several method controls have been created and refined over the past few years to
estimate and control sample interferences (Table A-l).
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Table A-l. Common qPCR Interference Controls
Interference Controls
Abbreviation
Application
Common Types
Sample Processing Control
(EPA Method A, 1611, and
1609)
SPC
A non-target DNA sequence used to
estimate recovery efficiency. The control
involves spiking a known quantity of non-
target DNA into the sample prior to
processing.
Sketa 2
Sketa 22
Internal Amplification
Control
(EPA Method 1611 and
1609)
IAC
A non-target DNA sequence added to the
reaction mix prior to the qPCR reaction. If
the non-target DNA does not amplify as
expected, a problem with the qPCR
reaction is indicated (e.g., DNA
polymerase inhibition).
IAC5
Dilution
(Caoetal., 2012)
dilution
Dilution of the sample can result in
dilution of other compounds that interfere
with DNA amplification. Different
dilutions can be compared (i.e., serial
dilutions).
5x
25x
Ratio spiked test
matrix/spiked control
matrix
(Haugland et al., 2016)
STM/SCM
The recovery of target DNA sequences
(gene copies) from target organisms
spiked into the water samples (STM) can
be compared to the recovery of DNA from
spiked target organisms in control samples
(SCM). The STM/SCM ratio can provide
an additional measure of interference
caused by inhibitors in the water matrix.
Not applicable
Addition of higher salmon
DNA concentrations to
samples during extraction
(Haugland et al., 2012)
Not applicable
Demonstrated at one tropical site (PR) to
reduce interference due to DNA loss
during sample extraction (Haugland et al.,
2012)
25x increase in
salmon DNA
concentration
Calculation using delta-
delta cycle threshold
(EPA Method A, 1611, and
1609)
AACt
A method to estimate Enterococcus spp.
in a water sample, accounting for recovery
and partial inhibition. The AACt is
calculated from the ACt (Enterococcus
assay Ct - Sketa SPC assay Ct value) for
the water sample and for the
calibrator/positive control sample and then
subtracting the calibrator/positive control
ACt from the water sample ACt.
Not applicable
In the 2012 RWQC document, the EPA also noted other rapid qPCR methods, such as the draft
EPA Bacteroidales qPCR Method B (U.S. EPA, 2010), which demonstrated a significant
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association with illness at the NEEAR marine beaches (Wade et al., 2010), and emerging qPCR
methods for E. coli. An example of the latter has been recently evaluated against culturable
methods and demonstrated utility on a site-specific basis (Lavender and Kinzelman, 2009). As
part of the five-year review of the 2012 RWQC, the EPA is interested in understanding
information about the status of Enterococcus spp., E. coli, and Bacteroidales qPCR methods.
The specific objectives of this work are to identify: 1) where qPCR methods have been applied
since 2010; 2) the rate of interference when using molecular methods in those waterbodies; 3)
method improvements that have reduced interference; and 4) method or water matrix attributes
(e.g., turbidity) and dynamics of fecal contamination that may continue to contribute to poor
performance or increased interference/inhibition. Additionally, we want to provide information
on an upcoming enumeration tool, digital droplet PCR (ddPCR).
B. Methods
1. Systematic Literature Search
We performed a systematic literature search of the peer-reviewed literature for articles reporting
qPCR monitoring data in recreational water in PubMed (http://www.ncbi.nlm.nih.gov/pubmed)
and Web of Science. The search included the keywords shown in Table A-2. The literature
search was limited to English language peer-reviewed citations published between 2010 and
March 2017.
Table A-2. Literature Search Terms
PubMed Set
Search Terms
Set 1
(ambient-water[tiab] OR Beach[tiab] OR Beaches[tiab] OR estuaries[tiab] OR estuaries[mh] OR
Estuarine[tiab] OR Estuary[tiab] OR freshwater[tiab] OR fresh-water[mh] OR fresh-water[tiab]
OR Lake[tiab] OR lakes[tiab] OR lakes[mh] OR Marine[tiab] OR recreational-water[tiab] OR
Reservoir[tiab] OR reservoirs [tiab] OR River[tiab] OR Rivers [tiab] OR rivers [mh] OR
stormwater[tiab] OR storm-water[tiab] OR Stream[tiab] OR streams[tiab] OR surface-water[tiab])
Set 2
AND rapid-method[tiab]
OR molecular-method[tiab] OR qPCR[tiab] OR quantitative-PCR[tiab] OR quantitative-
polymerase-chain-reaction[tiab] OR Real-Time-Polymerase-Chain Reaction[mh] OR RT-
PCR[tiab] OR digital-droplet-PCR[tiab] ORMethod-1609[tiab] OR Method-1611 [tiab] OR
Method-B[tiab]
Set 3
AND fecal-indicator[tiab]
OR l'jiierococcus\tiab | OR Escherichia-coli[tiab] OR enterococci[tiab] OR it. co//'[tiab] OR
Bacteroidales[tiab]
Set 4
AND detection[tiab]
Set 5
AND PCR-inhibitory-compounds[tiab]
OR inhibition[tiab] OR inhibitor[tiab] OR inhibitory-effects [tiab]
84
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NOT terms
NOT Pool[tiab]
ORPools[tiab] OR hot-tub[tiab] OR hot-tubs[tiab] OR spa[tiab] OR spas[tiab] OR sauna[tiab] OR
saunas[tiab] OR seeded[tiab] OR spiked[tiab] OR bench-top[tiab]
Limit:
Language
AND (English[lang])
Limit: Date
AND ("2010/01/01"[PDAT] : "3000/12/31"[PDAT])
Web of
Science Set
Search Terms (searched in Title, Abstract, and Keywords)
Set 1
(ambient-water OR Beach OR Beaches OR estuaries OR Estuarine OR Estuary OR freshwater
OR fresh-water OR Lake OR lakes OR Marine OR recreational-water OR Reservoir OR
reservoirs OR River OR Rivers OR stormwater OR storm-water OR Stream OR streams OR
surface-water)
Set 2
AND rapid-method
OR molecular-method OR qPCR OR quantitative-PCR OR quantitative-polymerase-chain-
reaction OR Real-Time-Polymerase-Chain Reaction OR RT-PCR OR digital-droplet-PCR OR
Method-1609 ORMethod-1611 ORMethod-B
Set 3
AND fecal-indicator
OR Enterococcus OR Escherichia-co//' OR enterococci OR E. coli OR Bacteroidales
Set 4
AND detection
Set 5
AND PCR-inhibitory-compounds
OR inhibition OR inhibitor OR inhibitory-effects
NOT terms
NOT Pool
OR Pools OR hot-tub OR hot-tubs OR spa OR spas OR sauna OR saunas OR seeded OR spiked
OR bench-top
2. Systematic Literature Screening
Abstracts were screened for relevance to the scope, including papers using the following
methods: Enterococcus spp. qPCR (Method 1609); Enterococcus spp. qPCR (Method 1611);
Bacteroidales qPCR (Method B); E. coli qPCR, and digital droplet PCR. Following the abstract
screening, the full text of articles passing scope was reviewed for specific information related to:
study location, sampling time, waterbody type, analytical method(s) applied, how inhibition was
controlled, contamination source(s) and dynamics (e.g., wet-weather driven), water quality
results, percent of samples inhibited, limit of quantitation, and percent recovery. Studies had to
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provide information on the occurrence and/or evaluation of inhibition to be included in the
review.
C. Results
1.
Literature Screening and Review
The literature search returned 337 unique results, of which 54 were relevant based on the abstract
screening (Figure 1). An additional 13 studies were identified through other sources (e.g., cited
in another paper). A total of 32 studies included Enterococcus qPCR, 22 included E. coli qPCR,
and 18 included Bacteroides qPCR. Tables 4 and 5 summarize the final subset of the
Enterococcus and E. coli qPCR papers. No studies were found that evaluated the EPA
Bacteroidales qPCR Method between 2010 and 2017 in fresh or marine waters. However,
multiple studies were found that investigated Bacteroides for microbial source tracking (MST)
purposes. Advancements in MST are discussed elsewhere in the 2017 Review.
Figure A-l. Summary of Number of Articles Screened and Reviewed
Literature Search:
337 Unique Results
Primary Screening
(Title and Abstract):
54 Relevant Results
Plus 13 from other
qPCR Literature
(number of papers)
Full-text Review
Sort by Target Organism
Enterococcus: 32
E. coli: 22
aSome studies report
multiple organisms
Full-text Review
17 Included in Table 3b
EPA Method 1609: 6
EPA Method 1611: 3
EPA Method A:!
Scorpion: 5
Other: 3
13 Included in Table 4
EPA Method C: 3
Scorpion: 4
Other: 6
bSome studies report
multiple methods
2.
Advancements in Enterococcus spp. qPCR Methods
EPA Methods: The EPA's first published qPCR method tor Enterococcus spp. (Method A) was
successfully applied to the EPA's NEEAR study (Haugland et al., 2005). Based on the literature
review only, the freshwater sites in the Great Lakes and four temperate marine beaches
demonstrated minimal to no interference, but the tropical marine beach site samples from Puerto
Rico exhibited significant interference (U.S. EPA, 2010c; Haugland et al., 2012). Prior to the
publication of the 2012 RWQC, The EPA updated Method A as the published Method 1611
(Table A-3). Updates included: 1) a requirement of the SPC assay to use Sketa 22; and 2) a
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recommendation for using the IAC assay. As in EPA Method A, the method used a reagent
called Universal Master Mix (UMM) (TaqMan; Applied Biosystems, Foster City, CA) (U.S.
EPA, 2012b). However, EPA Method 1611 was found to result in high levels of interference in
some waters, unless samples were diluted five-fold or more (Haugland et al., 2012, 2016;
Sivaganesan et al., 2014). Dilution is a standard methodological approach to lessen interference
or other negative amplification effects that can occur when utilizing undiluted extracts.
To address the potential for high interference levels, particularly due to inhibition, the EPA
developed EPA Method 1609 (U.S. EPA, 2013b), which uses the Environmental Master Mix
(EMM) reagent (TaqMan; Applied Biosystems, Foster City, CA), resulting in lower levels of
interference in undiluted samples (Cao et al., 2012; Haugland et al., 2012, 2016; Sivaganesan et
al., 2014). Like EPA Method 1611, EPA Method 1609 requires the SPC interference control
using the Sketa 22 assay and recommends the IAC assay. Table A-3 summarizes analytical
details related to reducing interference and the strategies for controlling for interference in the
various qPCR methods.
Non-EPA Methods: Most other qPCR methods for measuring Enterococcus spp. in ambient
water have been applied by a single research laboratory. The exception is the Scorpion-based
qPCR assay from Noble et al. (2010). The Scorpion qPCR technology uses a different master
mix (OmniMix, Cepheid, Inc., Sunnyvale, CA) and processing controls (Smartbeads, Cepheid
Inc., Sunnyvale, CA) and was designed to be faster than other qPCR chemistries. The Scorpion-
based method was included in Tables A-3 and A-4 because multiple papers evaluated the method
in ambient waters.
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Table A-3. Enterococcus spp. qPCR Methods
Method
(reference)
Analytical
Permutations
Master Mix
Recommended
Sample Extract
Dilution
Performance/Interference
Evaluation Analyses
SPC: Acceptance range
IAC: Acceptance
range
TSC or CE
spike
recovery:
Acceptance
range
EPA Method
1609
(Haugland et
al., 2016)
EMM
Undiluted
(5x diluted
optional)
Sketa 22: Test sample Ct
within 3 units of calibrator
samples (mandatory in
method)
IAC 5: Test sample
Ct within 1.5 units
of negative control
samples
(recommended in
method)
TSC: 50-
200%b
EPA Method
1611
(Haugland et
al., 2016)
UMM
5x diluted
Sketa 22: Test sample Ct
within 3 units of calibrator
samples (mandatory in
method)
IAC5: Test sample
Ct within 1.5 units
of negative control
samples
(recommended in
method)
TSC: 50-
200%b
EPA Method
A
(EPA-821-R-
10-004, 2012)
UMM
5x or 25x
diluted
Sketa 2a: Test sample Ct
within 3 units of
uninhibited reference
samples
Not evaluated
CE:
Acceptance
ranges
defined by
study
results0
Fresh water:
detect -
333%
Marine
water: detect
-1123%
Scorpion
method
(Noble et al.,
2010)
OmniMix
lOx diluted, if
needed
Lactococcus (SmartBeads):
1.5 Ct shift
Enterococcus IC,
Lactococcus IC
(SmartBeads): 1.5
Ct shift
Not
evaluated
a Sketa 22 was also evaluated.
b Recovery ratio of spiked test matrix (filters and retentates from collected water samples spiked with Enterococcus cells) to
spiked control matrix (clean filters spiked with Enterococcus cells).
c Recovery ratio of estimated qPCR cell equivalents in spiked test matrix to estimated CFU in the spikes. Spiking done with 550
CFU Bioballs™.
Acronyms and Abbreviations: CE = cell equivalent; EMM = Environmental MasterMix; IAC = Internal Amplification Control;
IC = propriety PCR positive internal control template; UMM = Universal MasterMix; SPC = Sample processing control; TSC =
target sequence copy
Table A-4 summarizes results from 16 papers that included information on the selected
Enterococcus qPCR methods. In a recent national study focusing primarily on potentially
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problematic sites, EPA Method 1609 showed an average qPCR interference rate of 10% (range
0-22%) and 11% (range 0-24%) in undiluted samples from 9 and 12 individual temperate marine
and freshwater sites, respectively, based on the SPC and IAC controls (Haugland et al., 2016).
Average interference rates from other studies were lower (Table A-4). A five-fold dilution of the
water sample extracts from the national study reduced the average interference rates to 4% and
3% for temperate marine and freshwaters and reduced the interference rates at most sites (9/9
marine and 10/12 freshwater) to acceptable frequencies of <10% (U.S. EPA, 2013a; Haugland et
al., 2016).
Table A-4. Summary of Interference Rates for Enterococcus spp. qPCR Methods
Citation
Water
Type
Location
(# of sites)
Fecal
Source
# of Samples
Undiluted
(% interference)
# of Samples
Diluted 5X
(% interference)
Strategies to
Test
Interference
EPA Method 1609 (EMM)
Dorevitch et al.,
2017
FW
MI (9)
WW, NPS
1256 (1.1)
540 (0.37)
SPC (Sketa 22) (Ct
3)
Haugland et al., 2016
M
FL, CA, NC
(9)
Not
reported
241 (10)
356 (4)
SPC (Sketa 22) (Ct
3)a
IAC (IAC 5) (Ct
1.5)
Haugland et al., 2016
FW
WI, OH, FL
(13)
Not
reported
491 (11)
419(3)
SPC (Sketa 22) (Ct
3)a
IAC (IAC 5) (Ct
1.5)
Sivaganesan et al.,
2014
FW
OH, KY, IN,
PA, IA (7)
NPS, SS,
WW, AW,
HW
221 (5)
221 (3)
SPC (Sketa 22) (Ct
3)
IAC (IAC 5) (Ct
1.5)
Haugland et al., 2012
FW
OH, KY (5)
NPS, SS,
WW, AW,
HW
268b (0)
268b (0.7)
SPC (Sketa 22) (Ct
3)a
IAC (IAC 5) (Ct
1.5)
Cao et al., 2012
M, FW
CA, IL (52)
NPS, HW
133(0)
Not reported
SPC (Sketa 2)c (Ct
3)a
Cao et al., 2012
M, FW
CA, IL (52)
NPS, HW
133(1)
133(0)
IAC (IAC 5) (Ct
1.7)a
89
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Citation
Water
Type
Location
(# of sites)
Fecal
Source
# of Samples
Undiluted
(% interference)
# of Samples
Diluted 5X
(% interference)
Strategies to
Test
Interference
EPA Method 1611
(VMM)
Haugland et al., 2016
M
FL, CA, NC
(9)
Not
reported
240b (>40)d
359 (7)
SPC (Sketa 22) (Ct
3)a
IAC (IAC5) (Ct
1.5)
Haugland et al., 2016
FW
WI, OH, FL
(13)
Not
reported
490b (>40)d
419(6)
SPC (Sketa 22) (Ct
3)a
IAC (IAC 5) (Ct
1.5)
Cao et al., 2013
M
CA (9b)
HW, SS,
WW
12(0)
Not reported
SPC (Sketa 22) (Ct
threshold not
indicated)
Converse et al.,
2012a
FW
WI(1)
AW,
80 (0)
Not reported
SPC (Sketa 22) (Ct
3)
Converse et al.,
2012b
M
CA (3)
NPS
1,200 (7)
Not reported
SPC (Sketa 22) (Ct
3)
Haugland et al., 2012
FW
OH, KY (5)
NPS, SS,
WW, AW,
HW
268b (18)
268b (1.5)
SPC (Sketa 22) (Ct
3)a
IAC (IAC 5) (Ct
1.5)
Haugland et al., 2012
M
PR (6b)
Not
reported
Not reported
684 (32f
SPC (Sketa 22) (Ct
3)
IAC (IAC 5) (Ct
1.5)
EPA Method A (or Haugland et al, 2005)
Raith et al., 2014
M
CA (9)
Not
reported
Not reported
306 (5)i
SPC (Sketa 2) (Ct
3)e
Zimmer-Faust et al.,
2014
M, FW
CA, Mexico
(18)
AW, NPS,
WW, SS
82 (0)
Not reported
SPC
Cao et al., 2012
M, FW
CA, IL (52)
NPS, HW
133(7)
133(1)
SPC (Sketa 2) (Ct
3)a
90
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Citation
Water
Type
Location
(# of sites)
Fecal
Source
# of Samples
Undiluted
(% interference)
# of Samples
Diluted 5X
(% interference)
Strategies to
Test
Interference
Cao et al., 2012
M, FW
CA, IL (52)
NPS, HW
133 (42)
133(7)
IAC (IAC5) (Ct
1.7)a
Haugland et al., 2012
FW
Ohio River
OH, KY (5)
NPS, SS,
WW, AW,
HW
268b (30)
268b (7)
SPC (Sketa 2) (Ct
3)a
IAC (IAC 5) (Ct
1.5)
Haugland et al., 2012
M
PR (6b)
Not
reported
Not reported
895 (36)a
SPC (Sketa 2) (Ct
3)
IAC (Ct 1.5)
Haugland et al., 2012
FW
AZ, CA,
GA,HI,IA,
IN, LA,
MD,MN,
NC,NJ,NY,
WA,WI (27)
Not
reported
Not reported
108 (7)
SPC (Sketa 2) (Ct
3)
Saueretal.,2011
FW
WI
NPS, WW
214 (< 1)
Not reported
IAC
Abdelzaher et al.,
2010
M
FL (1)
NPS, HW
12(0)
Not reported
SPC
Scorpion* (Noble et al. 2010)
Raith et al., 2014
M
CA (9)
Not
reported
Not reported
306 (5)1
SPC (Ct> 1.7)
Cao et al., 2013
M
CA (9b)
HW, SS,
WW
Not reported
12(0)
SPC (Sketa 22
Converse et al.,
2012b
M
CA (3)
NPS
1,200(16)
Not reported
SPC (Ct 1.6)
Cao et al., 2012
M, FW
CA, IL (52)
NR, NPS,
HW
133 (42)
133(4)
SPC (Sketa 2) (Ct
3)a
Cao et al., 2012
M, FW
CA, IL (52)
NR, NPS,
HW
133 (56)
133(18)
IAC (Ct 1.7)a
Noble etal., 2010
M, FW
CA
WW, UR,
SS
238(<5)
Not reported
SPC (Lactococcus)
(Ct 1.5)
IAC (Ct 1.5)
91
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Citation
Water
Type
Location
(# of sites)
Fecal
Source
# of Samples
Undiluted
(% interference)
# of Samples
Diluted 5X
(% interference)
Strategies to
Test
Interference
Other
Cao et al., 2012
M, FW
CA, IL (52)
NPS, HW
TF: 133(9)
TF: 133(1)
SPC (Sketa 2) (Ct
3)a
Cao et al., 2012
M, FW
CA, IL (52)
NPS, HW
TF: 133 (53)
TF: 133(8)
IAC (IAC 5) (Ct
1.7)a
Cao et al., 2012
M, FW
CA, IL (52)
NPS, HW
TFF: 133 (23)
TFF: 133 (2)
SPC (Sketa 2) (Ct
3)a
Cao et al., 2012
M, FW
CA, IL (52)
NPS, HW
TFF: 133 (90)
TFF: 42 (37)
IAC (IAC 5) (Ct
\lf
Bergeron et al., 2011
M
Spain,
France
HW, SS,
WW
85 (0)
Not reported
qPCR control
Santiago-Rodriguez
et al., 2012g
FW
PR
NPS, AW,
WW
130 (0)
130 (0)11
Not reported
Wang et al., 2016g
FW, M
CA (8)
Not
reported
24 (0)
Not reported
Compare to digital
PCR results
a Other interference controls evaluated (dilution and/or STM/SCM).
b Interference rates shown are based on SPC assay only, IAC assay results were generally in agreement when available.
c Deviated from Method 1609 by using Sketa2 rather than Sketa22 SPC assay.
d Average interference rate was not reported separately for M and FW in undiluted samples. However, separate rates are reported
for 5x diluted samples.
e The SPC control was evaluated using Ct shift acceptance thresholds of 3.0 and 1.7. When using the 1.7 Ct acceptance threshold,
22% interference was found.
f Scorpion is a proprietary qPCR primer and probe chemistry.
g qPCR conducted with lx TaqMan Universal PCR Master Mix and the Enterol 23S rRNA gene assay.
h 10-fold dilution
1 Composite of undiluted and 5X dilution results. 5X dilutions analyzed only for undiluted samples that failed Sketa2 assay
acceptance criterion.
Abbreviations and Acronyms: Master Mixes: EMM = environmental master mix; UMM = universal master mix; Waterbodv
Types: M = marine (Pacific Ocean, Atlantic Ocean, brackish stream); FW = Freshwater (river, stream, inland lake, Great Lakes);
Fecal Sources: NPS = non-point source/urban runoff; HW = human waste; AW = animal waste; SS = spiked samples; WW =
waste water; Interference Controls: SPC = sample processing control; IAC = internal amplification control; Ct shift = Difference
in Cycle Threshold values for between control and environmental samples; STM/SCM = recovery ratio of spiked test matrix
(STM) (filters and retentates from collected water samples spiked with Enterococcus cells) to spiked control matrix (SCM) (clean
filters and buffers spiked with Enterococcus cells); Sketa 22 = primers for salmon sperm DNA; Sketa 2 = primers for salmon
sperm DNA; TF = TaqFast method; TFF = Taq Fastfast method
In contrast, EPA Method 1611 exhibited a much higher average interference rate in undiluted
samples, ranging from 18-53%, in studies of corresponding temperate marine and freshwater
sites. A five-fold dilution of the water sample extracts again significantly reduced the
interference rate in both freshwaters and marine waters to acceptable levels of <10% at most
sites studied. For EPA's qPCR Method A, the interference rate was significantly higher when
92
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using Sketa 2, as compared to using Sketa 22 in Method 1611 for analyses of Ohio River water
samples (Table A-4).
Only one of the studies shown in Table A-4 addressed the potential reason for interference in the
water samples tested (Haugland et al., 2012). Haugland et al. (2012) suggest that the
predominance of polymerase inhibitory compounds (such as calcium, iron, iron containing
compounds, and tannic acid) affecting amplification that would affect both IAC and SPC assay
results in the Ohio River, and DNA binding compounds (such as humic acid and melanin) that
would primarily affect the SPC assay results in Boqueron Bay could explain the discrepancy in
failure rates observed for these two control assays among samples from the two locations.
Kinzelman et al. (2011) speculated that runoff from land during precipitation events could have
been a factor for interference in that particular study. Additionally, Wang et al. (2016) spiked
qPCR reactions with organic (humic acid, 5 ng/[j,L) and inorganic (calcium, 2.0 mM) matter to
test their inhibitory effects on PCR reactions. The study found that small concentrations of both
caused significant inhibition. Additionally, too few studies provided adequate information on
fecal source dynamics to draw any meaningful conclusions on how sources might impact the
likelihood of interference (Table A-4).
Overall, EPA Enterococcus qPCR (Method 1609) resulted in fewer interfering samples, as
compared to other methods (EPA Method A, Method 1611, and the Scorpion-based method).
Use of the EMM and, when necessary, sample dilution addressed interference at the 9 marine
and 23 of the 25 freshwater sites in 10 states that were comprehensively investigated in EPA
studies (Haugland et al., 2012, 2016; Sivaganesan et al., 2014). Based on these results, use of
EPA Method 1609, including the required and suggested controls, is appropriate on a site-
specific basis.
3. Advancements in E. coli qPCR Methods
The EPA has developed a draft qPCR method for E. coli (Method C, using EC23S857 primers)
(Chern et al., 2011). Three studies were found that referred to using the aforementioned E. coli
primers (Table A-5). In addition to using Sketa 22 for an SPC, two of the studies (Peed et al.,
2011; Molina et al., 2014) used the CowM2 plasmid as an IAC, which was originally developed
by EPA researchers for bovine-specific microbial source tracking (Shanks et al., 2008). The
method also employs the EMM reagent, which minimizes interference.
Over the past few years, other researchers have developed qPCR methods for E. coli and tested
those methods in ambient waters, using a variety of available primers and probes specific to E.
coli (Table A-5). These methods have not been directly compared to EPA's E. coli qPCR method
in ambient waters, and thus advantages are unclear.
The 13 studies included in Table A-5 all illustrate low rates of interference (<10%). However,
the number sites and samples reported is significantly smaller than for Enterococcus qPCR. EPA
Method C shows promise to have similar performance characteristics as Method 1609 based on
its current use of the same reagents (EMM), controls (Sketa22, SPC and IAC5, IAC assays) and
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target genes (23 S rRNA) for use on a site-specific basis. However, there are no peer-reviewed
demonstrations of its use for routine monitoring..
Table A-5. Summary of Interference Rates for E. coli qPCR Methods
Citation
Water
Type
Location
(# of sites)
Fecal
Source
# of Samples
Undiluted
(% interference)
# of Samples 5x
Diluted
(% interference)
Strategies to
Evaluate
Inhibition
EPA draft Method C [EC23SS57/a
Chernetal.,2011
M, FW
MA, PR (12)
NPS
25 (0)
Not reported
SPC (Sketa 2)
(Ct> 3)
Peed etal., 2011
FW
OH (9)
NPS, WW
215(2.1)
Not reported
IAC (CowM2)b
(Ct 35.1 ±1.8)
Molina etal., 2014
M
SC, FL (5)
NPS
471c (7)
Not reported
IAC (CowM2)b
(Ct 33.8 ±1.6)
Scorpion (Noble etaL, 2010)
Krometis et al., 2013
FW
NC (4)
NPS
94 (31)
29 (20)d
SPC (Sketa 2)
(Ct> 1.5)
Painter et al., 2013
FW
TX (1)
HW,AW
Not reported
102 (19)e
IAC
Converse et al., 2012b
FW
WI(1)
AW
80 (0)
Not reported
SPC (Sketa 22)
(Ct 3)
Noble etal., 2010
M, FW
CA (6)
WW, UR,
SS
226 (<5)
Not reported
Ct shift (> 1.5)
Other
Cloutier and McLellan,
2017
FW
WI (6)
Not
reported
124
Not reported
Byappanahalli et al., 2015
FW
IN (1)
Not
reported
5C (0)
Not reported
SPC
Walker etal., 2013
M, FW
PR, Trinidad
(44)
NPS, WW
210(0)
Not reported
IAC (Ct <2)
Zhang et al., 2012a
FW
MO (1)
SS,NPS
10c (8)
Not reported
IAC
Bergeron et al., 2011
M
Spain, France
(3)
HW, SS,
WW
80
0
Ct shift (24.5 ±
0.5 cycles)
Sauer etal., 2011
FW
WI (4)
NPS, WW
220 (<1)
Not reported
IAC
a Current draft method calls for the use of salmon DNA SPC with Sketa22 assay, IAC5 plasmid and assay for inhibition control,
56 degrees Celsius annealing temperature for thermal cycling, and EMM reagent. Some of these provisions were not followed in
the reported studies.
b IAC using CowM2 plasmid DNA
c Estimated
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d lOx dilution
e DNA not diluted but cleaned using post-extraction technique (Chelex® 100 and solubilization/binding buffer QX1).
Abbreviations and Acronyms: Waterbodv Type: M = marine (Pacific Ocean, Atlantic Ocean, brackish stream); FW = Freshwater
(river, stream, inland lake, Great Lakes); Fecal Source: NPS = non-point source/urban runoff; HW = human waste; AW = animal
waste; FC = fecal contamination; SS = spiked samples; WW = waste water; Interference Controls: SPC = sample processing
control; Sketa 22 = primers for salmon sperm DNA; Sketa 2 = primers for salmon sperm DNA; IAC = internal amplification
control
4. Digital PCR
Digital PCR (dPCR) is an emerging technology for determining the quantity of target DNA
sequences in a sample. While traditional qPCR involves measuring DNA products in a single
tube after each qPCR cycle, dPCR partitions the sample into thousands to millions of smaller
reactions that are examined individually for binary endpoint results (presence/absence). The
DNA density is then estimated from the fraction of positives using Poisson statistics. The dPCR
method can be conducted in chambers or droplets, the latter is known as ddPCR. The discussion
below does not differentiate between these types of dPCR. The dPCR methods may offer several
possible advantages over qPCR discussed below. However, it should be noted that there are few
publications to-date that have evaluated the method in ambient waters (Cao et al., 2015, 2016a,
b; Wang et al., 2016). Thus, the method is not broadly recommended for routine monitoring, at
this time.
First, dPCR does not require a standard curve, thus eliminating some of the labor and materials
associated with regularly running batch standards and the biases associated with calibration
model variability (Wang et al., 2016). However, it is important to note that a positive standard
control is still recommended by dPCR experts (Bustin et al., 2009). As a result, practitioners will
still need to create and maintain a standard reference material as a positive control for routine
testing.
Second, dPCR may have improved repeatability and reproducibility compared to qPCR (Cao et
al., 2016a) for some applications. Repeatability refers to the precision of an assay among
replicates of the same sample over a short period of time (short-term precision). Reproducibility
refers to the consistency in results among operators, runs, or laboratories (long-term precision).
The higher precision associated with dPCR allows for the detection of a 1.25-fold difference in
the DNA template, whereas qPCR can typically only detect a two-fold difference in clinical gene
expression applications (Cao et al., 2016a). However, it remains unclear whether this small but
important difference in precision will prove useful in environmental sample applications where
additional variability in concentration estimates is possible and likely. Although dPCR may
provide some advantages to qPCR with respect to repeatability and reproducibility, issues with
accuracy may arise in samples requiring DNA extraction from a complex mix of biological
materials (Huggett et al., 2013), such as environmental samples. Some ambient water samples
may be characterized as complex and, in these instances, it is important that experimental
replication and the number of replicates are appropriate for measurement of the desired target
(Huggett et al., 2013). An increase in the associated error of a DNA target concentration estimate
may occur in a given dPCR assay if replication includes variability from extractions. To date, it
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remains unclear whether these issues potentially nullify any precision advantage of dPCR over
traditional qPCR approaches for ambient water sample applications.
Third, because of sample partitioning, it has been reported that dPCR may be less prone to
environmental amplification inhibition compared to qPCR applications although other types of
interference may still occur (Cao et al., 2016a). In general, inhibitors within a sample matrix will
either completely prevent or partially reduce amplification, the latter scenario resulting in an
underestimation of the true target DNA concentration. Wang et al. (2016) found that humic acid
caused a similar level of amplification inhibition in both dPCR and qPCR experiments, however
inhibition of dPCR was partially relieved when the number of thermal cycles was increased. In
addition, others have found that dPCR is able to tolerate PCR inhibitor concentrations that are
one to two orders of magnitude higher than those in paired qPCR tests with conventional
reagents (Cao et al., 2016a). However, the incidence of amplification inhibition in ambient
surface water qPCR samples is reported to be extremely rare when using customized DNA
polymerases such as Environmental Mastermix (Cao et al., 2012; Haugland et al., 2012, 2016).
It is also important to note that both dPCR and qPCR are susceptible to partial amplification
inhibition, where the presence of inhibitors could either reduce the percentage of positive
partitions (dPCR) or lower the quantification cycle (qPCR) leading to an underestimation of the
true DNA target concentration. As a result, it is useful to employ a quantitative control with each
test sample to monitor for both complete or partial amplification inhibition to validate findings
(Bustin et al., 2009; Huggett et al., 2013).
Finally, dPCR may be superior to qPCR for multiplex reaction applications, amplification of two
or more different DNA templates in one reaction (Cao et al., 2016b). Multiplexing in traditional
qPCR can lead to an underestimation of the less abundant target if not properly optimized,
whereas dPCR may provide more robust quantification of multiple DNA targets. Using dPCR,
Cao et al. (2016b) duplexed Enterococcus spp. and HF183 (EntHF183 dPCR assay), which
provided accurate and repeatable information on both general and human-associated fecal
contamination in environmental waters, without the need to run two separate qPCR tests.
There are also several potential limitations of dPCR as compared to qPCR. First, given this is a
new technology, there would likely be additional costs associated with implementing it in a
laboratory and obtaining the necessary instrumentation and supplies (Huggett et al., 2013).
Secondly, the quantifiable range is smaller for dPCR, and currently the upper limit of
quantitation of dPCR is four orders of magnitude lower than that of qPCR. Thus, sample dilution
is required when measuring samples with a high concentration of a DNA target, like those
potentially found in sewage spills (Cao et al., 2016b). Additionally, Poisson statistics require
uniformity in the partitions for accurate results. Viscous DNA, due to high concentrations or long
templates, may result in uneven distributions, biasing the partitions and leading to potentially
inaccurate results. Finally, if double-stranded DNA is denatured into single strands, the template
is effectively increased because single-strands can occupy different partitions, which could lead
to up to a two-fold overestimation by dPCR (Cao et al., 2016b).
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L.E. 2010. Presence of pathogens and indicator microbes at a non-point source subtropical
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Cao, Y., Raith, M.R., Griffith, J.F. 2015. Droplet digital PCR for simultaneous quantification of
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337-349.
Cao, Y., Griffith, J.F., Weisberg, S.B. 2016a. The next-generation PCR-based quantification
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Chern, E.C., Siefring, S., Paar, J., Doolittle, M., Haugland, R.A. 2011. Comparison of
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Letters in Applied Microbiology, 52(3): 298-306.
Cloutier, D.D., McLellan, S.L. 2017. Distribution and Differential Survival of Traditional and
Alternative Indicators of Fecal Pollution at Freshwater Beaches. Applied and Environmental
Microbiology 83(4).
Converse, R.R., Kinzelman, J.L., Sams, E.A., Hudgens, E., Dufour, A.P., Ryu, H., Santo-
Domingo, J.W., Kelty, C.A., Shanks, O.C., Siefring, S.D., Haugland, R.A., Wade, T.J. 2012a.
Dramatic improvements in beach water quality following gull removal. Environmental Science
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Converse RR, Griffith JF, Noble RT, Haugland RA, Schiff KC, Weisberg SB. 2012b.
Correlation between quantitative PCR and culture-based methods for measuring Enterococcus
spp. over various temporal scales at three California marine beaches. Applied and Environmental
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Dorevitch, S., Shrestha, A., DeFlorio-Barker, S., Breitenbach, C., Heimler, I. 2017. Monitoring
urban beaches with qPCR vs. culture measures of fecal indicator bacteria: Implications for public
notification. Environmental Health 16:45.
Haugland, R.A., Siefring, S.C., Wymer, L.J., Brenner, K.P., Dufour, A.P. 2005. Comparison of
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Haugland, R.A., Siefring, S., Lavender, J., Varma, M. 2012. Influences of sample interference
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by the qPCR method. Water Research, 46(18): 5989-6001.
Haugland, R.A., Siefring, S., Varma, M., Oshima, K. H., Sivaganesan, M., Cao, Y., Raith, M.,
Griffith, J., Weisberg, S.B., Noble, R.T., Blackwood, A. D., Kinzelman, J., Ananeva, T., Bushon,
R.N., Harwood, V. J., Gordon, K.V., Sinigalliano, C. 2016. Multi-laboratory survey of qPCR
enterococci analysis method performance in U.S. coastal and inland surface waters. Journal of
Microbiological Methods, 123: 114-125.
Huggett, J.F., Foy, C.A., Benes, V., Emslie, K., Garson, J.A., Haynes, R., Hellmans, J., Kubista,
M., Mueller, R.D., Nolan, T., Pfaffl, M.W., Shipley, G.L., Vandesompele, J., Wittwer, C.T.,
Bustin, S.A. 2013. The Digital MIQE guidelines: Minimum information for publication of
quantitative digital PCR experiments. Clinical Chemistry, 59(6): 892-902.
Krometis, L.A., Noble, R.T., Characklis, G.W., Blackwood, A.D., Sobsey, M.D., 2013.
Assessment of E. coli partitioning behavior via both culture-based and qPCR methods. Water
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Lavender, J. S., Kinzelman, J.L. 2009. A cross comparison of qPCR to agar-based or defined
substrate test methods for the determination of Escherichia coli and enterococci in municipal
water quality monitoring programs. Water Research 43: 4967-4979.
Molina, M., Hunter, S., Cyterski, M., Peed, L.A., Kelty, C.A., Sivaganesan, M., Mooney, T.,
Prieto, L., Shanks, O.C. 2014. Factors affecting the presence of human-associated and fecal
indicator real-time quantitative PCR genetic markers in urban-impacted recreational beaches.
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Noble, R.T., Blackwood, A.D., Griffith, J.F., McGee, C.D., Weisberg, S.B. 2010. Comparison of
rapid quantitative PCR-based and conventional culture-based methods for enumeration of
Enterococcus spp. and Escherichia coli in recreational waters. Applied Environmental
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Painter, S.M., Pfau, R.S., Brady, J.A., McFarland, A.M.S. 2013. Quantitative assessment of
Naegleria fowleri and Escherichia coli concentrations within a Texas reservoir. Journal of Water
and Health 11(2): 346-357.
Peed, L.A., Nietch, C.T., Kelty, C.A., Meckes, M., Mooney, T., Sivaganesan, M., Shanks, O.C.
2011. Combining land use information and small stream sampling with PCR-based methods for
better characterization of diffuse sources of human fecal pollution. Environmental Science and
Technology, 45: 5652-5659.
Raith, M., Ebentier, D., Cao, Y., Griffith, J., Weisberg, S. 2014. Factors affecting the
relationship between quantitative polymerase chain reaction (qPCR) and culture-based
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116:737-746.
Santiago-Rodriguez, T.M., Tremblay, R.L., Toledo-Hernandez, C., Gonzalez-Nieves, J.E., Ryu,
H., Santo Domingo, J.W., Toranzos, G.A. 2012. Microbial quality of tropical inland waters and
effects of rainfall events. Applied and Environmental Microbiology 78(15): 5160-5169.
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specific Bacteroides genetic marker provides evidence of widespread sewage contamination of
stormwater in the urban environment. Water Research 45(14): 4081-4091.
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Sivaganesan, M., Haugland, R.A. 2008. Quantitative PCR for detection and enumeration of
genetic markers of bovine fecal pollution. Applied and Environmental Microbiology, 74(3): 745-
752.
Sivaganesan, M., Siefring, S., Varma, M., Haugland, R.A. 2014. Comparison of Enterococcus
quantitative polymerase chain reaction analysis results from midwestern U.S. river samples using
EPA Method 1611 and Method 1609 PCR reagents. Journal of Microbiological Methods, 101: 9-
17. Corrigendum 115, 166
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Reaction (qPCR) Assay. Office of Water. EPA-822-R-10-003.
U.S. EPA. 2012a. Recreational Water Quality Criteria. EPA 820-F-12-058. Office of Water,
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U.S. EPA. 2012b. Method 1611: Enterococci in Water by TaqMan® Quantitative Polymerase
Chain Reaction (qPCR) Assay. EPA-821-R-12-008. Office of Water, Washington, DC.
U.S. EPA 2013a. Acceptability of the EPA qPCR Test at Your Beach. Office of Water. EPA-
820-R-13-012.
U.S. EPA 2013b. US Environmental Protection Agency: Method 1609: Enterococci in Water by
TaqMan® Quantitative Polymerase Chain Reaction (qPCR) with Internal Amplification Control
(IAC) Assay.
Wade, T.J., Calderon, R.L., Sams, E., Beach, M., Brenner, K.P., Williams, A.H., Dufour, A.P.,
2006. Rapidly Measured Indicators of Recreational Water Quality Are Predictive of Swimming-
Associated Gastrointestinal Illness. Environmental Health Perspectives 114(1): 24-28.
Wade, T.J., Calderon, R.L., Brenner, K.P., Sams, E., Beach, M., Haugland, R., Wymer, L.,
Dufour, A.P. 2008. High sensitivity of children to swimming-associated gastrointestinal illness:
results using a rapid assay of recreational water quality. Epidemiology, 19(3): 375-383.
Wade, T.J., Sams, E.A., Haugland, R., Brenner, K.P., Li, Q., Wymer, L., Molina, M., Oshima,
K., Dufour, A.P. 2010. Report on 2009 National Epidemiologic and Environmental Assessment
of Recreational Water Epidemiology Studies. EPA 600-R-10-168. Office of Research and
Development, Washington, DC.
Walker, T.J., Bachoon, D.S., Otero, E., Ramsubhag, A. 2013. Detection of verotoxin producing
Escherichia coli in marine environments of the Caribbean. Marine Pollution Bulletin 76(1-2):
406-410.
Wang, D., Yamahara,K.M., Cao, Y., Boehm, A.B. 2016. Absolute Quantification of
Enterococcal 23S rRNA Gene Using Digital PCR. Environmental Science & Technology 50:
3399-3408.
Zhang, Y.Y., Riley, L.K., Lin, M.S., Hu, Z. 2012. Determination of low-density Escherichia coli
and Helicobacter pylori suspensions in water. Water Research 46(7): 2140-2148.
Zimmer-Faust, A.G., Thulsiraj, V., Ferguson, D., Jay, J.A. 2014. Performance and specificity of
the covalently linked immunomagnetic separation-ATP method for rapid detection and
enumeration of enterococci in coastal environments. Applied and Environmental Microbiology
80(9): 2705-2714.
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Appendix B. Communication with Regional Coordinators on the
Implementation of the RWQC
4/X/17
To: Regional Beach Coordinators
From: JohnWathen& Samantha Fontenelle
Re: Barriers and experiences of states' implementation of 2012 RWQC and adoption of
BAVs
Regional Beach Program folks:
This is not another call for an update on the status of states adoption of the criteria, although
current information on that topic is always welcome. As we have discussed on Beach Program
calls, OST is conducting a 5-year review of the 2012 RWQC as required by the BEACH Act. In
addition to assessing the continued scientific currency of the RWQC, we are examining a range
of issues pertinent to the RWQC.
One of those issues relates to barriers or other issues encountered by states that have adopted or
are adopting the RWQC and BAVs and now have some experience with their application. For
example, one exception that we heard from some states when the RWQC were issued was that
the lower BAV would lead to more advisories, which would require more re-sampling to lift, and
would lead to fewer beaches being monitored. We are interested in hearing from the states in
your regions as to what their experiences have been to generally answer these questions:
1. What barriers to implementation of the 2012 RWQC, if any, has your state beach
program encountered?
2. Have any adverse consequences to adoption been experienced?
3. Have there been positive experiences or outcomes as a result of adoption?
4. Without undoing any of the significant elements of the RWQC and implementation
guidance, is there anything that could be addressed in the guidance that should be
included in the review report and be subsequently changed in the guidance to improve the
operation of the beach monitoring and advisory programs in the states.
We would like to schedule individual calls with each regional beach coordinator(s), HQ Beach
Program staff, and state beach program leads in the region together on the phone during the
month of April. Just to be clear, this is region by region and not with everyone on the line
together, which would likely be unwieldy. This would be an informal opportunity for the states
to be heard. We will be in touch with you the week of April 10 to schedule calls in the near
future.
Thanks for your cooperation on this.
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Appendix C. Review of the EPA's 2012 Recreational Water Quality Criteria
Health Study Information Expert Consultation
Review of EPA's 2012 Recreational
Water Quality Criteria Health Study
Information Expert Consultation
Graham McBride (Graham.McBride@niwa.co.nz)
Principal Scientist, Water Quality-Aquatic Pollution
NIWA (National Institute of Water and Atmospheric Research,
Hamilton, New Zealand
June 30, 2017
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List of Abbreviations
AGI
Acute gastrointestinal illness
BAV
Beach Action Value
BEACH Act
Beaches Environmental Assessent and Coastal Health Act
CAT
Catellicoccus marimammalium
CAWS
Chicago Waterways System
EPA
U.S. Environmental Protection Agency
Epi
Epidemiological
ETEC
enterotoxigenic E. coli
FIB
Fecal indicator bacteria
FIO
Fecal indicator organism
GI
gastrointestinal
HAdV
human adenovirus
MST
Microbial Source Tracking
NEEAR
National Epidemiological and Environmental Assessment of Recreational Water
QMRA
Quantitative Microbial Risk Assessment
qPCR
quantitative Polymerase Chain Reaction
RWQC
Recreational Water Quality Criteria
STEC
Shigatoxigenie coli
WRP
Water Reclamation Plant
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A. BACKGROUND
The 2012 U.S. Environmental Protection Agency (EPA) Recreational Water Quality Criteria
(RWQC) are designed to protect the public from exposure to pathogens in waters designated for
primary contact recreational uses. Criteria development included an analysis of research up to
2012, and an evaluation of the association between illness and extent of fecal contamination in
these waters. The 2012 RWQC provide two sets of numeric concentration thresholds based on
the use of two bacterial indicators, E. coli and enterococci. Illness rates upon which these
recommendations are based include the outcomes from the National Epidemiological and
Environmental Assessment of Recreational Water (NEEAR) study and earlier epidemiological
studies used to support the 1986 Ambient Water Quality Criteria.
The Beaches Environmental Assessment and Coastal Health (BEACH) Act of 2000 requires that
the EPA review and, as necessary, revise recreational water quality criteria within five years of
publication. The EPA is currently doing that. The overall goal of this review is to develop an
EPA report that describes available information and includes the Agency's assessment of
whether revisions to the 2012 criteria are necessary to ensure the protection of recreational
waters. The EPA is requesting expert consultation to facilitate two of the EPA's main objectives
for this project:
• Inventory and evaluate health study information available since 2010 on public health
impacts from exposure to fecal contamination in recreational waters.
• Assess new information regarding existing recommended and alternative
indicator/methods combinations, and their relationship to health assessment for the
general population and children
To assist this expert consultation. The EPA, via their consultants (ICF, www.icf.com) has
provided the following charge questions.
B. CHARGE QUESTIONS
1. Please provide a summary review of peer-reviewed health studies published since 2010
describing human health risks from exposure to recreational waters affected by fecal
contamination. This can be presented in tabular form including the following headers:
Reference, location, study type and design, contamination source(s), water quality metrics,
health effects evaluated, health linkages reported, and conclusions. Epidemiological
(including NEEAR), exposure, and microbial risk assessment studies should be considered.
2. Based on the above review of health studies, please separately summarize any new
information as it pertains to children's health (e.g., exposure, ingestion rate, health risk
studies). Regarding the quantitative polymerase chain reaction (qPCR)-health relationships
(e.g., Enterococcus qPCR or Bacteroides qPCR), does the relationship between the molecular
indicator and the health outcome (i.e. gastroenteritis) for adults/general population differ for
children?
3. Based on the above review of health studies, please separately summarize any new
information on relationships between health and the following currently-recommended
indicators of water quality: culturable coli, culturable enterococci, and Enterococcus spp.
qPCR. In what scenarios of fecal contamination are culture-based indicators and/or qPCR-
based indicators predictive?
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4. Based on the above review of health studies, please separately summarize information on
relationships between health and alternative indicators (e.g., Clostridiumperfringens, human
or animal source markers, pepper mild mottle virus), with the exception of coliphage since the
EPA has a separate ongoing effort with that specific indicator.
5. Based on the above review of health studies, describe the specific fecal sources and
contamination dynamics (e.g., differential effects between wet and dry weather) impacting
waters in studies with a statistical relationship between health and water quality.
6. Please summarize any outbreak information during the last 10 years including, etiological
agent, symptoms reported, and water quality information that may be relevant to this overall
health study review.
C. THE APPROACH TAKEN
A list of 98 potentially appropriate references was forwarded to the author by ICF for
consideration. Selection of these references considered recent investigations using both
epidemiological (Epi) and Quantitative Microbial Risk Assessment (QMRA) methods. A few of
the papers selected were more in the nature of a microbiological experiment or survey - these
were included to highlight issues arising from laboratory methodology.
Of these 98 documents, 15 were considered but not included in detailed analysis (judged to be
not particularly relevant), and a further 23 were included, including nine papers published (or in
press) in 2017. That is, 104 references in total were considered. A few of the included references
predate 2010 (e.g., Harrington etal. (1993), Ferley etal. (1989)), because they are particularly
relevant, but are not widely known.
Each reference was perused according to the items required by the charges. This entailed detailed
review of texts to record responses to the charges, and checking of preliminary material provided
by ICF. Full copies of all references are held by the author in electronic form.
To facilitate clarity in the following text, longer lists of references and detailed technical
information in support of an inference are given in footnotes.
D. RESPONSES
Charge 1—Summary of peer-reviewed studies
The response, after addressing all the topics stated for this Charge, are summarized in Table
lError! Reference source not found.. Use of a small font allows all the charges' components to
be presented on the same page. Accordingly it appears after responding to Charge 6, with
headers repeated on each page.
The Conclusions column (the last in the table) includes the health metrics item (the basis for the
response to the third Charge). The content of cells in that column comprise materials derived
from the document's abstract and conclusions. Information on water quality metrics and
contaminant sources were obtained from the methods sections of the reviewed documents.
Synthesis of those water quality metrics are included in the Conclusions column, in which the
most important points taken from these information cells are underlined.
The main observations and inferences drawn, other than those in response to Charges 2-6, are
guided by the Conclusions column in Table 1 and are as follows.
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a) Epidemiological studies and QMRA are quite different approaches to the task of setting
health-related water quality criteria, yet they are complementary. In particular, their relative
strengths vary from one location to another. For example, Epi studies capture the actual water
ingestion or inhalation volumes during exposure events at its site(s), whereas QMRA has to
estimate those volumes. On the other hand, Epi studies are restricted to the location(s) and
times at which they were carried out, whereas QMRA can be applied to many other situations
for which Epi study results are scarce (such as locations not impacted by human wastes).
b) Epidemiological studies can gain an extra advantage by including in their monitoring a
selection of fecal indicators and pathogens, as do some of the reviewed documents. This
includes different ways of measuring a given indicator, e.g., enterococci by culture and by
PCR. In contrast, including several pathogens in a QMRA is relatively straightforward and
less expensive than in epidemiological studies.
c) It should always be recognised that environmental waters can contain a mix of pathogens,
only some of which may be analysed. So the calculated risk for those for the selected
pathogens may not capture the water's overall pathogenicity.
d) Many epidemiological studies report an increase in health risk for the exposed (e.g.,
swimmers) versus the non-exposed, but fail to find a relationship between some water quality
variable and health risk, e.g., "Epidemiological studies show a generally elevated risk of
gastrointestinal illness in bathers compared to non-bathers but often no clear association with
water quality as measured by fecal indicator bacteria; this is especially true where study sites
are impacted by non-point source pollution" (Fewtrell & Kay 2015). In contrast, QMRA
models are built on pathogen dose-response curves which, in general, exhibit a monotonic
increase in risk of infection or illness with increasing dose—so association of health risk and
water quality is always evident.
e) The form of language used by Fewtrell & Kay (2015)-"no clear association"-is appropriate
and is in common use in the studies reviewed. But it is common for science interpreters to
make a stronger claim: That the absence of a statistically significant result admits a finding of
"no association". However, failure to attain statistical significance is not the same as
establishing the veracity of the tested hypothesis.1 In the case of the phraseology used by
Fewtrell & Kay (2015), that "failure" merely means that the relative strength of association is
higher in one case ("exposed versus not-exposed") than in the other ("association of health
risk with water quality"). It would be helpful to make this point in the revised criteria.
f) In general, risks posed by animal sources such as gull, chicken and pigs may pose a lesser risk
compared to human fecal material, but not so for bovine cattle2 and possibly for ovine
ruminants. This inference has mostly been established using QMRA models.
1 These hypotheses are all two-sided, positing that there is exactly zero change in some statistic of the population
being sampled. So the lack of statistical significance merely means that the sample size was insufficient to obtain a
finding of statistical significance because, in general, P-values for such tests decrease with increasing sample size,
e.g., see McBride et al. (2014)—and many other statistical writings on this matter, such as those referenced therein.
So failure to attain significance may merely reflect that the study size was simply not "big enough". In that regard it
is notable that the earlier epidemiological studies that underpinned the "Ambient Water Quality for Bacteria-1986"
were based on much larger sample sizes (on the order of 30,000) than many more recent studies have used: The
"Dufour freshwater study" had 34,598 participants and the "Cabelli marine study" had 26,686.
2Schoen & Ashbolt 2010, Ehsan et al. 2015, Soller et al. 2015, Brown et al. 2017).
108
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g) Dose-response models for Norovirus, as used in a number of QMRA models,3 are remarkably
dependent on the degree of aggregation of virions present in the low concentrations typical in
environmental waters. For example, without loss of generality, take a simplified case where
10 people each ingest 100 mL of water from a container in which there are 10 Norovirus
particles. If these pathogens are aggregated into one clump of ten, then only one of the ten
people can be affected; the other nine are exposed to "no dose". At the other extreme, up to
ten people could be affected if there is no aggregation—each particle is independent of the
other. Many, if not all, of the ten subjects then receives a "low dose", thus increasing the
average risk faced by these 10 people. Studies of the dynamics and effects of aggregation
phenomena are therefore warranted (Soller et al. 2017 is an excellent start).
h) A meta-analysis of studies with two different gastrointestinal endpoints (NGI vs. HCGI)
would be helpful, to enable comparisons between Epi studies to be "on common ground"
(Wymere^a/. 2013).
i) Future studies should consider including more emerging pathogens, especially anti-microbial
pathogens (Leonard et al. 2015, Young 2016).
j) Eventually, MST (Microbial Source Tracking) markers may support source apportionment as
well as risk assessment, given additional epidemiological data and/or empirical descriptions
of pathogen-Bacteroidales relationships (Bambic et al. 2015).
k) Risks from mixed sources are driven predominantly by the proportion of the contamination
source with the greatest ability to cause human infection (potency), which is not necessarily
the greatest source(s) of fecal indicator bacteria (FIB) (Schoen & Ashbolt 2010, Soller et al.
2014).
1) Conditions in more tropical regions, especially Hawaii, may require more use of QMRA
given the propensity for enterococci to be associated with contaminated soil (Vijayaval et al.
2010) and to exhibit higher decay rates in the environment (Kirs et al. 2016)-and the
enhanced possibility of enterococci regrowth.
Charge 2—Children's health
In response to Charges 2-6, epidemiological studies (Sanborn & Takoro 2013, de Man et al.
2014, Arnold et al. 2016) show that children appear to be at higher risk (cf. adults) when
swimming/playing in water. There are two possible causes:
a) Children may have a higher rate of ingestion or inhalation of ambient water.
b) Children may be more susceptible to pathogen infection.
Regarding a), the innovative swimming pool studies reported by Dufour et al. (2017) show that
children may ingest water at rates four times greater than adult rates. Increasingly, such data are
being included in QMRA models. On the other hand, in some settings children may ingest at a
lower rate (but still more than adults), depending on their swimming behaviour (Suppes et al.,
2014). The choice of exposure data, particularly in terms of duration, has a substantial effect on
risk predicted by QMRA.
3Soller et al. (2010a&b), Schoen et al. (2011), Viau et al. (2011), Francy et al. (2013), McBride et al. (2013), de
Man et al. (2014), Sales-Ortells & Medema (2014), Schijven et al. (2015), Zlot etal. (2015), Eregno el at. (2016),
Soller et al. (2016), Soller et al. (2017).
109
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Regarding b), it is commonly held in health risk modelling that children are born with inherent
susceptibility that reduces over time4 as some immunity is developed and maintained. Studies
reported herein do not take explicit account of this aspect, yet it seems highly desirable to do so,
especially as children appear to be the most at-risk group.
Charge 3—New information on health and indicators
a) In general, Norovirus is likely to be the most important pathogen for humans in waters
affected by discharges of treated sewage (Soller et al., 2010a).
b) Prior-day E. coli culture testing was no better than chance in predicting the exceedance of
the qPCR Beach Action Value (BAV). E. coli culture testing of beaches (on the same
day) led to three times the number of BAV exceedance as did enterococci qPCR testing
of beach water (Dorevich et al. 2017).
c) In French rivers (Ardeche Basin), fecal streptococci were best correlated to
gastrointestinal morbidity, fecal coliforms less so. Swimmers suffer skin ailments much
more frequently than non-swimmers.
d) Coupling QMRA with an epidemiological study at a single study site provides a unique
ability to understand human health risk and illnesses, especially under conditions where
water quality, as measured by traditional fecal indicator organisms (FIOs) is good and/or
average illness rates are lower than can be quantified via epidemiological methods (Soller
et al. 2016).
e) The fecal indicator bacteria Enterococcus spp., estimated by qPCR, is well-associated
with gastrointestinal illness among swimmers (Wade et al. 2010, Wyer et al. 2013).
f) Statistically significant trends of increasing proportions of human adenovirus (HAdV)-
positive results in categories of increasing FIO concentration were found in freshwater
but not seawater samples (Wyer (2013).
These observations can be interpreted to imply that there is no strong case for changing the
indicators currently recommended in the RWQC.
Charge 4—Relationships between health and alternative indicators
a) A benchmark illness rate of 30 gatrointestinal (GI) illnesses per 1000 swimmers occurred
at median concentrations of 4,200 copies of HF183 and 2,800 copies of HumM2 per 100
mL of recreational water (Boehm et al. 2015).
b) When the level of CAT (Catellicoccus marimammalium, a gull feces marker) exceeds 4 x
106 copies/100 mL of water, the median predicted illness exceeds 3 illnesses/100
swimmers (Brown et al. 2017).
c) Associations between GI and traditional and rapid methods for Enterococcus have been
observed at marine beaches (Colford et al. 2012).
d) QMRA results reported by Corsi et al. (2016) highlight the importance of investigating
multiple pathogens within multiple categories to avoid underestimating the prevalence
and risk of waterborne pathogens.
e) Predictive models were not effective at estimating human health risks associated with
recreation at all inland lake sites; however, their use at two lakes with high swimmer
4Pond, K. (2005). Water Recreation and Disease. World Health Organization, US Environmental Protection
Agency, International Water Association, London, pp. 7, 29.
110
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densities provided better estimates of public health risk than current methods, and will be
a valuable resource for beach managers and the public (Francy et al. 2013).
f) Griffith el al. (2016) report that no indicator combinations consistently had a higher odds
ratio5 than EPA Method 1600, but one composite indicator, based on the number of
pathogens detected at a beach, was significantly associated with gastrointestinal illness at
both Avalon and Doheny when freshwater flow was high. These results suggest that site-
specific conditions at each beach determine which indicator or indicators best predict GI
illness.
g) Potential EPEC strains were readily isolated from contaminated marine recreational water
and may represent a public health risk to swimmers and beach users. The frequency of
detection of potential EPEC strains varied considerably by sample. Neither
Shigatoxigenic E. coli (STEC) nor enterotoxigenic coli (ETEC) strains were detected
(Hamilton et al. 2010).
h) Kirs et al. (2016) argue for the inclusion of HF183Taqman human fecal marker in future
epidemiological studies.
i) Nnane et al. (2011) report a "very small" correlation coefficient between presumptive E.
coli and phages of Bacteroides (GB-124).
j) Performance of a calibrated qPCR total enterococci indicator was compared to a culture-
based assay to index infectious human enteric viruses released in treated human
wastewater. Results illustrate that the pathogen source contributing the majority of risk in
a mixture may be overlooked (when only assessing fecal indicators using a culture-based
method (Schoen etal. 2011).
k) At a beach with no known point sources (e.g., discharge of treated sewage), a dose-
response relationship was observed between skin infections and enterococci enumerated
using membrane filtration methods. No other significant dose-response relationships
between reports of human illness and any of the other FIB or environmental measures
were observed (Sinigalliano etal. 2010).
1) Viau et al. (2011) report that GI illness risks from viral exposures were generally orders
of magnitude greater than bacterial exposures in Hawaiian waters impacted by stream
discharges. The median risk associated with each stream was positively, significantly
correlated to the concentration of Clostridium perfringens in the stream water.
m) Bacteroides phages were considered potential markers of sewage because they also
survived for three days in fresh stream water and two days in marine water (Viiayavel et
al. 2010).
n) Yau et al. (2014) noted that associations between GI illness incidence and FIB levels
{Enterococcus EPA Method 1600) among swimmers who swallowed water were not
significant when not accounting for submarine groundwater discharge, but were strongly
associated when submarine groundwater discharge was high compared to when it was
low.
These observations show that development and use of alternative fecal indicators is a rich and
evolving field. Given that, it seems premature to promulgate any form of directives on their
selection and use.
5Odds ratios are a measure of relative risk and take the same values as the coefficients of a logistic regression
statistical model (relating health outcomes to selected covariates), as used by Griffith et al. (2016).
Ill
-------
Charge 5—Fecal sources and contamination dynamics, wet/dry weather
a) Fecal indicator bacteria measured in seawater (Enterococcus spp., fecal coliforms, total
coliforms) were strongly associated with incident illness only during wet weather. Urban
coastal seawater exposure increases the incidence rates of many acute illnesses among
surfers, with higher incidence rates after rainstorms (Arnold etal. 2017).
b) Ingestion of 1 mL of river water could lead to 0%-4% and l%-74% probability of illness
with E. coli during the dry and wet season, respectively. Activities that cause disturbance
of sediments lead to elevated risk of infection to users of the river (Abia et al. 2016).
c) Swimming in natural swim environments and in pools following a recent fecal
contamination event pose significant public health risks (Pintar 2010).
o Wet weather conditions contribute to elevated pathogen loads in the Chicago
Waterways System (CAWS) to such an extent that disinfecting the effluents of three
major Water Reclamation Plants (WRPs) that discharge to the CAWS would reduce
the aggregate recreation season risk to incidental contact recreators negligibly (Rijal
et al. 2011).
d) Dry-weather risk estimates were found to be significantly lower than those predicted for
wet-weather conditions (Sunger et al. 2016).
These observations highlight the importance of significant rainfall in determining the degree of
water contamination. Note that contact recreation does occur during, and shortly after, rainfall
events.
Charge 6—Outbreak information during the last 10 years
The illnesses that may arise after contact with fecally-contaminated water are generally "mild".
As such they are usually substantially under-reported (unless the outbreak is "large"), even if the
illness in question is "notifiable". Also, contact with water is usually only one of several
potential causes. Accordingly, it seems best to rely mostly on reports where careful
investigations have identified the illness and its source.
a) An outbreak among white-water rafters provides evidence of the changing epidemiology
of leptospirosis and suggests consideration of a wider range of risk exposures, including
those related to recreational activities of more affluent urban populations, in addition to
the well-recognized occupational hazards of rural farming (Agampodi et al. 2014).
b) During a seven-year period, illness outbreaks reported to the Australian OzFoodNet, were
predominantly classified as being transmitted person-to-person or from an unknown
source. Fifty-four (0.83%) outbreaks were classified as either 'waterborne' or 'suspected
waterborne', of which 78% (42/54) were attributed to recreational water and 19% (10/54)
to drinking water (Dale & Kirk, 2010).
c) The infection risks resulting from swimming in Belgian waters were above 50% for
several days in waters near an accidental spillage of wild poliovirus type 3 (Duizer et al.
2016).
d) Approximately 5,700 outbreak-related cases were identified across the state of Utah in
2007. Of 1,506 interviewed patients with laboratory-confirmed cryptosporidiosis, 1,209
(80%>) reported swimming in at least one of approximately 450 recreational water venues
during their potential 14-day incubation period (Edwards etal. 2012).
112
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e) Outbreaks, especially the largest ones, were most frequently associated with treated
recreational water and characterized by acute gastrointestinal illness (AGI).
Cryptosporidium remains the leading etiologic agent (Hlavsa 2011).
These observations show that while the endemic pattern of infectious disease generally accounts
for the majority of illness cases, outbreaks cause public concern, especially for cases such as
reported above for Belgian beaches. Outbreaks can serve as a warning against complacency.
113
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Table 1. Detailed response to the first charge
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Conclusions (includes health linkages)
Abdelzaher et
al. (2010)
Florida, Virginia
Key
Epi:
Randomized
trial
Urban, dogs,
storm water
GI illness,
skin illness,
acute febrile
respiratory
illness
Enterococci
(culture), E. coli,
Fecal coliform, C.
perfringens,
Enterococci
(qPCR), F-specific
coliphage, somatic
coliphage,
Cryptosporidium,
Giardia,
Enterovirus, V.
vulnificus, S.
aureus, DogBac,
BacHum-UCD, B.
thetaiotaomicron,
polyomavirus
4
No statistically significant correlations between health outcomes and any of the
indicator organisms, including colinhages. were identified in this investigation. Average
daily excess illness percentage rates (calculated by subtracting the daily illness rate for
non-swimmers from that for swimmers) for gastrointestinal, skin, and acute febrile
respiratory illness were 2.0% (standard deviation [SD] = 3.3), 5.6% (SD = 4.7), and
1.2% (SD = 2.9), respectively.
Abia, et al.
(2016)
Apies River, South
Africa
QMRA
Informal
settlements,
wastewater
treatment
plants, animal
farms
Caused by
measured
pathogens
E. coli, V.
cholerae,
Salmonella spp.,
Shigella spp.
Ingestion of 1 mL of river water could lead to 0%-4% and l%-74% nrobabilitv of
illness uring the dry and wet season, resnectivelv. Activities that cause disturbance of
sediments would lead to elevated risk of infection to users of the river.
Agampodi et
al. (2014)
Sri Lanka
Follow-up,
exposures for
white-water
rafting
Rural runoff
Leptospirosis
None. Clinical
6
Exnosure from white-water rafting. This outbreak nrovides evidence of the changing
enidemiology of leDtosnirosis and suggests a wider range of risk exnosures including
those related to recreational activities of more affluent urban nonulations in addition to
the well-recognized occupational hazards of rural farming.
Almeida et al.
(2012)
Argentina
Epi
City
wastewater
treatment plant
General
Enterococci, E.
coli, total
coliforms, fecal
coliforms
Following the RWQI values classification, most of the Potrero de los Funes water
samnles fell in the good duality range during the study neriod. Advocates conjoint use
of microbial and Dhvsical/chemical components in a recreational water duality index.
Arnold et al.
(2013)
USA, Malibu
Beach, CA
Epi:
prospective
cohort
Dry weather
runoff and non-
point sources
Diarrhea and
GI illness
Culturable
Enterococcus
3,
4
n = 5,674. Diarrhea was more common among swimmers than non-swimmers (adjusted
odds ratio = 1.88 [95% confidence interval = 1.09-3.24]) within 3 days of the beach
visit. Water quality was generally good (fecal indicator bacteria levels exceeded water
quality guidelines for only 7% of study samples). Fecal indicator bacteria levels were
not consistently associated with swimmer illness. Sensitivity analyses demonstrated that
114
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Reference
•-c
d
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J
35
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^ i
£ I
Conclusions (includes health linkages)
overall inference was not substantially affected bv the choice of exposure and outcome
definitions.
Arnold et al. USA-wide Epi; combined Many Diarrhea, Culturable 2 Combined data from 13 prospective cohort studies (n = 84,411). Water exposure
(2016) 13 prospective gastrointestin Enterococcus accounted for 21% of diarrhea episodes and 9% of missed daily activities but was
studies al illness unassociated with gastroenteritis leading to medical consultation. Children aged 0 to 4
and 5 to 10 years had the most water exposure, exhibited stronger associations between
levels of water quality and illness, and accounted for the largest attributable illness
burden. Conclusions. The higher gastroenteritis risk and associated burden in young
children presents important new information to inform future recreational water quality
guidelines designed to protect public health.
Culturable - Study of surfers (n = 654). Fecal indicator bacteria measured in seawater (Enterococcus
Enterococcus, fecal species, fecal coliforms, total coliforms) were strongly associated with incident illness
coliforms, total only during wet weather. Urban coastal seawater exposure increases the incidence rates
coliforms of many acute illnesses among surfers, with higher incidence rates after rainstorms.
Arnold et al. USA, San Diego, Epi: Urban runoff Gastrointestin
(2017) two beaches longitudinal after storms aland
(Tourmaline study respiratory
Surfing ark, Ocean illness
Beach.
Ashbolt et al.
USA
QMRA
Many
General
FIOs (generally) -
Exploration of various scenarios with the aid of quantitative microbial risk assessment
(2010)
models has been shown to assist in identifying issues, research gaps and management
goals. Major gaps that need to be filled before further real progress can be made with
OMRA and predictive models include: defining the relationships between reference
pathogens and a range of potential indicators, be thev culture or PCR endpoint assays.
Bambic et al.
USA, Callegus
Data
Municipal
-
E. coli. Real-time 4
Results demonstrate that MST based on Bacteroidales assays can inform watershed
(2015)
Creek, CA
summaries,
wastewater
QPCR for
managers seeking to develop strategies to comply with criteria, but it is critical to
including
(dry
surrogate PP7,
handle non-detects with appropriate statistical methods and to acknowledge the
Kaplan-Maier
conditions),
Adenovirus and
underlying assumptions of qPCR-based MST. While MST shows promise for providing
treatment for
agricultural and
Enterovirus, and
quantitative source apportionment, there are still data gaps including relative decay rates
non-detect
municipal
four fecal
of FIB, Bacteroidales and pathogens in effluent-impacted surface waters and lack of
data. Monte
storm water
Bacteroidales
qPCR assays for viruses that reflect viable/infective concentrations (e.g., using PMA).
Carlo model.
(wet
conditions)
assays (universal)
BacUni, (human-
associated)
BacHum,
(ruminant-
associated)
BacCow, and (dog-
associated)
BacCan.
Eventually, MST markers may support not only source apportionment but also risk
assessment, given additional epidemiological data and/or empirical descriptions of
\>at\iogsa-Bacteroidales relationships.
Betancourt et
Venezuela
QMRA
Human
Gastrointestin
Cryptosporidium, 2,4
Potential risks of Cryptosporidium and Giardia infection from recreational water
al. (2014)
sewage,
al
Giardia,
exposure were estimated from the levels of viable (oo) cysts (DIC+, DAPI+, PI ) found
115
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variously-
enterococci, C.
in near-shore swimming areas using an exponential dose response model. The study
treated
Perfringens,
revealed the potential risk of parasite infections via primary contact with tropical marine
Bacteroidales
waters contaminated with sewage; higher risk estimates for Giardia than for
marker (HF183)
Cryptosporidium were found. Mean risks estimated by Monte Carlo methods were
and Clostridium
below the U.S. EPA upper bound on recreational risk of 0.036 for cryptosporidiosis and
coccides.
giardiasis for both children and adults. However, 95th percentile estimates for giardiasis
for children exceeded the 0.036 level.
Boehm et al.
USA
QMRA
Human
GI
Bacteroidales'.
4, 5
Simulated GI risk increased with concentration of the human quantitative PCR markers
(2015)
Human markers
in recreational waters. A benchmark illness rate of 30 GI illnesses per 1000 swimmers
HumM2 and
occurred at median concentrations of 4200 conies of HF183 and 2800 copies of
HF183Taqman
HumM2 per 100 mL of recreational water.
Brown et al.
Six California
QMRA
Gulls
GI
"CAT":
3,4
Considered densities of CAT and infectious zoonotic pathogens Salmonella and
(2017)
beaches,
Catellicoccus
Campylobacter in gull feces, volume of water ingested during bathing, and
marimammalium
dose-response relationships. CAT densities measured in 37 fresh gull fecal droppings.
Losm densities ranged from 4.6 to 9.8 loglO copies CAT/g of wet feces. When the level
of CAT exceeds 4x10- copies/100 mL of water, the median predicted illness exceeds 3
illnesses/100 swimmers.
Colford et al.
United States
Cohort,
Small craft
20 health
30 different
3, 4
n = 9.525. Sienificant increases in risk of diarrhoea (from swallowine water) and other
(2012)
(Doheny Beach,
prospective
harbor,
outcomes,
microbial
outcomes in swimmers compared to non-swimmers. Exposure fbodv immersion, head
2007-08)
WWTP, San
including GI
indicators,
immersion, swallowed water) was associated with increasing risk of GI illness. Daily GI
Juan Creek
and skin rash
including rapid
illness incidence patterns differed: swimmers (2-day peak) and non-swimmers (no
(when berm
methods and new
peak"). With berm-open. associations between GI and traditional and rapid methods for
open),
microbial
Enterococcus were observed: fewer associations occurred when berm status was not
indicators
considered.
Collier et al.
USA: five marine
Epi:
Human
GI
-
2
Combined studv: n = 54.250. Overall, swimmers reported a hieher unadjusted incidence
(2015):
and four freshwater
prospective
wastewaters
of GI illness and earaches than non-swimmers. Current surveillance systems might not
NEEAR study
cohort
detect individual cases and outbreaks of illness associated with swimming in natural
water. Water quality analysis not included.
Corsi et al.
Three Lake
QMRA (2ntl-
Wastewater
GI
22 pathogens
4
Detections of human and bovine viruses and pathogenic bacteria at all beaches,
(2016)
Michigan beaches
order, i.e.,
effluent,
(Human viruses,
indicating influence of multiple contamination sources: occurrence 40 to 87% for
performing a
impervious
bovine viruses,
human viruses, 65- 87% for pathogenic bacteria, and 13-35% for bovine viruses.
set of iterations
runoff, agric.
protozoa,
Enterovirus, Adenovirus A, Salmonella spp., Campylobacter jejuni, bovine
for each
runoff, rural
pathogenic bacteria
polvomavirus. and bovine Rotavirus A were present most frequentlv. Risk assessment
random sample
septic systems
done for C. ieiuni. Salmonella spp.. and Enteroviruses to estimate risk of infection and
from the dose-
illness. Median infection risks for one-time swimming events were approximately 3 x
response curve)
10~5, 7 x 10~9, and 3 x 10~7 for C. jejuni, Salmonella spp., and Enteroviruses,
respectively. Results hiehlieht the importance of investieatine multiple pathoeens
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Reference
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Conclusions (includes health linkages)
Dale et al. Australia, Survey - GI
(2009) Melbourne
within multiple categories to avoid underestimating the prevalence and risk of
waterborne pathogens
2, 6 n = 2,811. The relationship between sporadic gastroenteritis and recreational swimming
considered temporality between reported swimming (in public or private pools/spas and
in marine or freshwater settings) and a highly credible gastroenteritis (HCG) event.
Overall. HCG events were more likely in participants who had swum in a public
pool/spa during the previous week or had swum in a public pool/spa during the previous
2 weeks. Sub-analysis by age showed that HCG episodes were also more likely in adults
who had swum in a private pool/spa during the previous week or swum at an
ocean/beach during the previous 2 weeks, demonstrating significant associations
between all swimming locations and gastrointestinal symptoms. This study showed that
although the incremental risk of recreational swimming is significant, it is relatively
small.
Dale & Kirk Australia Survey Pool water, GI Cryptosporidium, 6 n = 6,515. During seven years, outbreaks were reported to OzFoodNet, most of which
(2010) public Salmonellae were classified as being transmitted person-to-person or from an unknown source. Fiftv-
swimming pool four ("0.83%~) outbreaks were classified as either 'waterborne' or 'suspected
waterborne'. of which 78% (42/54) were attributed to recreational water and 19%
(10/54) to drinking water. Conclusions: There have been few waterborne outbreaks
detected in Australia, and most of those reported have been associated with recreational
exposure. However, there are difficulties in identifying and categorising gastroenteritis
outbreaks, as well as in obtaining microbiological and epidemiological evidence, which
is likely to result in misclassification or underestimation of water-associated events.
Ae.Ma.net al. Netherlands QMRA Urban E. coli, intestinal 5 23 flood events (2011 & 2012). The water contained Campylobacter jejuni (prevalence
(2014) floodwaters enterococci, 61%, range 14 to >103 MPN/L), Giardia spp. (35%, 0.1 -142 cysts/L),
Campylobacter, Cryptosporidium (30%, 0.1 - 9.8 oocysts/L), Noroviruses (29%, 102 - 104 pdu/L) and
Cryptosporidium, Enteroviruses (35%, 103 - 104 pdu/L). The mean risk of infection per event for children
Giardia, enteric was 33%. 23% and 3.5%. respectively, and for adults it was 3.9%. 0.58% and 0.039%.
viruses, An exposure frequency of once every 10 years to flooding originating from combined
Noroviruses (GI sewers resulted in an annual risk of infection of 8%.
and Gil),
Enterovirus
DeFlorio- USA, NEEAR and Epi Mostly urban GI
Barker et al. CHEERS data
(2017)
Dorevitch et USA Epi (review) Inland waters GI
al. (2010) (IW) and
The Cost-of-Illness (COI) provides more information than the frequency of illness, as it
takes into account disease incidence, health care utilization, and lost productivity. Use
of monetized disease severity information should be included in future studies of water
quality and health.
4, 5 The distinction of IW versus CW is of less importance than more fundamental variables
such as the scale of the body of water, the source of the pollutant, and the effects of
sediment, which translate into differences in the densities, transport, and fate of
117
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coastal waters
(CW)
indicators and pathogens. This may translate into weaker indicator-pathogen and
indicator-health risk relationships for IW compared with CW. It remains an open
question whether sediment in IW changes the relationship between enterococci aPCR
measures and health risk, which has been described at coastal beaches impacted by
human fecal pollution. In IW with limited dilution capacity and close proximity to
sources, outbreaks of severe disease may be difficult to prevent bv the application of
coastal-derived criteria.
Dorevitch et Chicago
al. (2011)
Experiment Urban
(Amount of water
ingested)
The mean volume of water ingested during limited-contact recreation activities, about
3.5^1 mL is about 35-40% of that observed during swimming (about 10 mL). The
frequency of swallowing at least one teaspoon amount of water during limited-contact
recreation (about 1% of study participants') is about 1/50— the frequency observed
during swimming in a pool (51% of participants').
Dorevitch et Chicago
al. (2012)
Epi
Urban
GI
n = 11,297. Limited-contact recreation, both on effluent-dominated waters and on
waters designated for general use, was associated with an elevated risk of
gastrointestinal illness.
Dorevitch et Chicago
al. (2015)
Epi
Urban
GI
E. coli,
enterococci,
somatic coliphages,
F+ coliphages,
Giardia spp. and
Cryptosporidium
spp. (oo)cysts,
turbidity
3, 4 n = 4,694. Gastrointestinal illness following incidental contact with water during
recreation was not readily predicted bv measures of water quality in the settings studied.
Protozoan pathogens, while frequently detected, were not useful as predictors of illness.
Dorevitch et Chicago
al. (2017)
Epi
Urban GI E. coli, enterococci Monitoring multiple beaches using qPCR methods can generate precise and accurate
by qPCR data for timely public notifications regarding beach water quality. Results of prior-dav
E. coli culture testing were no better than chance in predicting the exceedance of the
qPCR BAV. E. coli culture testing of beaches (on the same day-) led to three times the
number of BAV exceedance as did enterococci qPCR testing of beach water. It is not
known whether similar results would have been obtained at marine beaches or those
significantly impacted by wastewater.
Dufour et al. USA, Hong Kong, Epi
(2012) New Zealand
Birds, animals GI
Reviewed epidemiological studies do not provide evidence for associations between
swimming-associated gastrointestinal illness and exposures to bathing waters
contaminated with feces from animals or birds. Other studies, such as outbreak
investigations and case-control studies, have provided logical linkages to human
infections with zoonotic pathogens and recreational or occupational exposures to water,
but thev have not established a definitive link between water contamination and specific
animal sources. These conclusions do not completely answer the question whether
exposure to animal-contaminated waters poses a health risk to swimmers. The exposure
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to zoonotic pathogens is unlikely to have occurred at beaches meeting local beach water
quality standards.
Dufour et al.
(2017)
Columbus, Ohio
Exposure
experiment
Swimming
pools
chloroisocyanurate 2
(cyanuric acid)
n = 549. Swimming pools disinfected by chloroisocyanurate used to determine the
amount of water swallowed by swimmers. It is in equilibrium with chlorine and
cyanuric acid in the pool water thus provides a biomarker: cyanuric acid that once
swallowed passes through the body into the urine unchanged. The 549 participants,
about evenly divided by gender, and young and adult swimmers, indicated that
swimmers ingest about 32 mL per hour (arithmetic mean) and that children swallowed
about four times as much water as adults during swimming activities. Males had a
tendency to swallow more water than females. Children spent about twice as much time
in the water than adults.
D'Ugo et al. Six countries Experiment
(2016)
Open space,
Grazing land,
Urban
qPCR for
Adenovirus 41,
Mammalian
Orthoreoviruses,
Noroviruses
A 2-year survey showed that Norovirus, Mammalian Orthoreovirus and Adenoviruses
were the most frequently identified enteric viruses in the sampled surface waters.
Although it was not possible to establish viability and infectivity of the viruses
considered, the detectable presence of pathogenic viruses may represent a potential risk
for human health. The methodology developed may aid in rapid detection of these
pathogens for monitoring quality of surface waters.
Duizer et al. Belgium
(2016)
QMRA
Accidental
Infection risk Wild poliovirus 2, 6
release of wild from
poliovirus type swimming
3 and raw
shellfish
consumption
type 3
Accidental release of 1,013 infectious wild poliovirus type 3 particles by a vaccine
production plant in Belgium into the sewage system and associated wastewater
treatment plant (WWTP), and subsequently into rivers that flowed to the Western
Scheldt and the North Sea. QMRA showed that the infection risks resulting from
swimming in Belgium waters were above 50% for several days and that the infection
risk by consuming shellfish harvested in the eastern part of the Western Scheldt
warranted a shellfish cooking advice. Showed that relevant data on water flows were
not readily available and that prior assumptions on dilution factors were overestimated.
Edwards et al. Utah
(2012)
Epi
Recreational
water venues
GI
Cryptosporidium
During the summer of 2007, Utah experienced a state-wide outbreak of gastrointestinal
illness caused by Cryptosporidium, Approximately 5,700 outbreak-related cases were
identified across the state. Of 1,506 interviewed patients with laboratory-confirmed
cryptosporidiosis, 1.209 (80%) reported swimming in at least one of approximately 450
recreational water venues during their potential 14-dav incubation period.
Ehsan et al. Belgium
(2015)
QMRA
Swimming
pools, lakes,
splash parks,
fountains
GI
Giardia and
Cryptosporidium
Cryptosporidium oocysts and/or Giardia cysts were detected in swimming pools,
recreational lakes, splash parks and water fountains in Belgium. Although in
recreational lakes (oo)cysts were frequently present, most positive samples belonged to
species/genotypes that were either animal-specific or predominantly found in animals.
suggesting that the risk of infection during recreation is relatively low. Lower
contamination rates were found in swimming pools, splash parks and water fountains,
but assuming that humans are the most probable source of contamination for these
waterbodies, these findings suggest a potential risk for human infection.
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Eregno et al.
(2016)
Norway, Sandvika QMRA
Sandvika
recreational
beaches
GI
E. coli
Investigated the public health risk from exposure to infectious microorganisms at
recreational beaches, Norway, and dose-response relationships by combining
hydrodynamic modelling with QMRA. Meteorological and hydrological data were
collected to produce a calibrated hydrodynamic model using Escherichia coli. Based on
average concentrations of reference pathogens (Norovirus, Campylobacter, Salmonella,
Giardia and Cryptosporidium) relative to E. coli in Norwegian sewage from previous
studies, the hydrodynamic model was used for simulating the concentrations of
pathogens at the local beaches during and after a heavy rainfall event. The simulated
concentrations were used as input for QMRA and the public health risk was estimated
as probability of infection from a single exposure of bathers during the three
consecutive days after the rainfall event. The level of risk on the first day after the
rainfall event was acceptable for the bacterial and parasitic reference pathogens, but
high-to-severe for the viral reference pathogen at all beaches.
Ferley et al.
(1989)
France Ardeche
basin
Retrospective
Epi
Rural summer GI
camps
Total coliforms,
fecal coliforms,
fecal streptococci,
Aeromonas,
Pseudomonas
n = 5.737. While dated, this often-overlooked paper is included because it focuses on
rivers, not freshwater lakes. 5,737 tourists in eight holiday camps were questioned as to
the occurrence of illness and their bathing habits during the week preceding the
interviews. The rate-ratio contrasting swimmers and non-swimmers for total morbidity
is 2.1. Fecal streptococci were best correlated to gastrointestinal morbidity, fecal
coliforms less so. Swimmers suffer skin ailments much more frequently than non-
swimmers (RR-= 3.7V This type of morbidity is well correlated with the concentration
of fecal coliforms, Aeromonas and Pseudomonas. Produced relationship between
person-days and fecal coliforms/streptococci.
Fewtrell &
Kay (2015)
worldwide
Epi &QMRA
Epidemiological studies show a generally elevated risk of gastrointestinal illness in
bathers compared to non-bathers but often no clear association with water quality as
measured bv fecal indicator bacteria (Wade et al. 2010 is an exception). This is
especially true where study sites are impacted bv non-point source pollution. Evidence
from OMRAs support the lack of a consistent water quality association with health risk
for non-point source-impacted beaches. Future Epi studies should include source
attribution through quantified microbial source apportionment.
Francy et al. 22 Eight Ohio Experiment
(2013) inland recreational
lakes
Birds and other
wildlife; septic
tanks (1) and
treated
wastewater
(l)d
Culture: E. coli &
enterococci;
end point PCR:
Shigella,
Salmonella, STEC,
C. Jejuni and coli
(PCR),
Cryptosporidium,
Giardia
Investigate using predictive models for Escherichia coli and to understand the links
between E. coli concentrations, predictive variables, and pathogens. Based upon results
from 21 beach sites, models were developed for 13 sites, and the most predictive
variables were rainfall, wind direction and speed, turbidity, and water temperature.
Models were not developed at sites where the E. coli standard was seldom exceeded.
Models were validated at nine sites during an independent year. Cryptosporidium,
Adenovirus, eaeA (E. coli), ipaH {Shigella), and spvC {Salmonella) were found in at
least 20% of samples collected for pathogens at five sites. The presence or absence of
the three bacterial genes was related to some of the model variables but was not
consistently related to E. coli concentrations. Predictive models were not effective at all
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qPCR and qRT-
PCR:
Adenovirus,
Enterovirus,
Norovirus
inland lake sites: however, their use at two lakes with high swimmer densities will
provide better estimates of public health risk than current methods and will be a
valuable resource for beach managers and the public.
Galfi et al.
(2016)
Sweden
Four urban
sewers
Total coliforms, E. 4, 5 Covers baseflow and snowmelt periods in storm sewers of four urban catchments in a
coli, enterococci, northern Swedish city. Catchment- and season- (i.e., rainy or snowmelt periods) specific
C. perfringens variations were investigated. Compared to dry weather baseflow, concentrations of
these three indicators in storm water were 10 (snowmelt runoff) to 102 (rain runoff)
times higher. C. verfringens mean concentrations were practically constant regardless of
season and catchment. The list of variables associated with bacteria included the flow
rate, solids with associated inorganics (Fe and Al), and phosphorus, indicating similar
sources of constituents regardless of the season. Although the study findings do not
indicate any distinct surrogates to indicator bacteria, the inclusion of flow rate, solids
and total phosphorus for all seasons, water temperature for rainfall runoff, and total
nitrogen and pFl for snowmelt only) in sanitary surveys of northern climate urban
catchments would provide additional insight into indicator bacteria sources and their
modeling.
Goodwin et al. Three California
(2012) beaches
Experiment Urban
Staphylococcus
aureus, MRSA,
enterococci
The frequent detection (>50%) ofS. aureus in seawater and beach sand samples and the
correlation with water temperature supports the concern that bacterial pathogens exist
and may persist in the environment, including at beaches. Although the correlation
between S. aureus and the number of swimmers was weak and apparent only for S.
aureus in seawater and not sand, the correlation held for data analysed by individual
beach and combined across beaches. These data support the possibility that beach-goers
are one source of this organism, but suggests that other sources not identified in this
study are important as well. Although the prevalence of MRSA was much lower f<3%
of samples') than for S. aureus, these data indicate the potential for virulent and
antibiotic resistant strains to be encountered in this environment. S. aureus was
correlated to enterococci. even though S. aureus is not considered a typical fecal
organism. Perhaps the finding that S. aureus can sometimes be found in wastewater and
in companion animal feces explains this observation.
Gorham& Lee
(2016)
General
Literature
Various
5, 6 Pathogens of potential concern include Campylobacter jejuni, Salmonella Typhimurium,
Listeria monocytogenes, Helicobacter canadensis, Arcobacter spp., Enterohemorragic
Escherichia coli pathogenic strains, Chlamydia psitacci, Cryptosporidium parvum and
Giardia lamblia. Scenarios presenting potential exposure to pathogens eluted from feces
include bathers swimming in lakes, children playing with wet and dry sand impacted by
geese droppings, and other common recreational activities associated with public
beaches. Recent recreational water-associated disease outbreaks in the US support the
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plausibility for some of these pathogens, including Cryptosporidium spp. and C. jejuni.
to cause human illness.
Griffith et al. Three beaches Epi Watershed
(2016) (CA): Doheny, (prospective runoff, point
Malibu, Avalon cohort) sources
GI 41 target indicators 3,4
using 6 different
methodologies
n = 10,785. F1 coliphage (measured using EPA Method 1602) exhibited a stronger
association with GI illness than did EPA Method 1600 at the two beaches where it was
measured, while a molecular method. F1 RNA Coliphage Genotype II. was the only
indicator significantly associated with GI illness at Malibu. MRSA. a known pathogen-
had the strongest association with GI illness of any microbe measured at Avalon. There
were two methods targeting human-associated fecal anaerobic bacteria that were more
strongly associated with GI illness than EPA Method 1600, but only at Avalon. No
indicator combinations consistently had a higher odds ratio than EPA Method 1600. but
one composite indicator, based on the number of pathogens detected at a beach, was
significantly associated with gastrointestinal illness at both Avalon and Dohenv when
freshwater flow was high.
While EPA Methodl600 performed adequately at two beaches based on its consistency
of association with gastrointestinal illness and the precision of its estimated
associations, F+ coliphage measured by EPA Method 1602 had a stronger association
with GI illness under high risk conditions at the two beaches where it was measured.
One indicator. F1 Coliphage Genotype II was the only indicator significantly associated
with GI illness at Malibu. Several indicators, particularly those targeting human-
associated bacteria, exhibited relationships with GI illness that were equal to or greater
than that of EPA Method 1600 at Avalon, which has a focused human fecal source.
Results suggest that site-specific conditions at each beach determine which indicator or
indicators best predict GI illness.
Hall et al.
(2017)
London, River
Thames
Epi
(retrospective
cohort)
Urban
GI
GI surveyed for 1,100 swimmers in a Thames River event in London, UK, to describe
the outbreak and identify risk factors. Associations were tested using robust Poisson
regression. Survey response was 61%, and attack rate 53% (338 cases). Median
incubation period was 34 h and median symptom duration 4 days. Microbiological
diagnoses were confirmed in five cases (four Giardia, one Cryptosporidium).
Hamilton et al. Avalon Bay, CA
(2010)
Survey
Urban
Genomic 4 Potential EPEC strains were readily isolated from contaminated marine recreational
composition and water and may represent a public health risk to swimmers and beach users. Neither
frequency of STEC nor ETEC strains were detected. The frequency of detection of potential EPEC
virulence genes strains varied considerably bv sample, suggesting a strong temporal component. Results
present in E. coli indicate that potential EPEC strains in Avalon Bay were genetically diverse. Since
isolated from beach genotypically identical EPEC strains were detected repeatedly, on successive dates and
water years, these data suggest that E. coli in Avalon Bay were likely due to continual
deposition from an unknown reservoir or through persistence of E. coli in the
environment.
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Harder-
Lauridsen et
al. (2013)
Copenhagen,
Denmark
Epi
(retrospective
cohort)
Urban
GI
E. coli
n = 1,769. A triathlon was held shortly after rainfall. An established model of bacterial
concentration in the water was used to examine the level of pollution in a
spatiotemporal manner. Investigation was repeated after a triathlon competition held in
non-polluted seawater in 2011. Results showed that the 3.8 kilometre open water
swimming competition coincided with the peak of post-flooding bacterial contamination
in 2010, with average concentrations of 1.5x104 E. coli per 100 mL water. The attack
rate of disease among 838 swimmers in 2010 was 42% compared to 8% among 931
swimmers in the 2011 competition (relative risk RR = 5.0). Confirmed aetiologies of
infection included Campylobacter, Giardia lamblia and diarrhoeagenic E. coli. Results
suggest a significant risk of disease in people ingesting small amounts of flood water
following extreme rainfall in urban areas.
Harrington et
al. (1993)
Sydney, Australia,
six popular marine
beaches.
Epi,
longitudinal
Urban
GI,
respiratory
Fecal coliforms,
fecal streptococci,
C. perfingens
Beaches located north and south of Sydney Harbor: 2003 recruits were enrolled,
recording 43,175 swimming events. Of these, 5,879 (14%) had possibly attributable
illness. A rise in relative risks was noted for total illness and respiratory illness but not
for gastrointestinal illness. Females showed an increase in reported illness when beach
swimming was combined with non-ocean swimming. This study lends no support to the
concept of correlating health risk in swimmers with threshold levels of currently used
bacterial indicator organisms. The value of farther exploring the role of Clostridium
perfringens as an indicator organism is supported.
Helmi et al.
(2011)
Reservoir in
Luxembourg, used
for recreation and
drinking-water
supply.
Survey, QMRA Not stated
PCR Giardia
lambla,
Cryposporidium
parveum
Giardia lamblia and Cryptosporidium parvum was monitored for 2 years in the largest
drinking water reservoir in Luxembourg using microscopy and qPCR techniques.
Parasite analyses were performed on water samples collected from three sites. Results
show that both parasites are present in the reservoir throughout the year with a higher
occurrence of G. lamblia cvsts compared to C. varvum oocysts. Only 25% of the
samples positive by microscopy were confirmed by qPCR. (Oo)cyst concentrations
were 10 to 100 times higher between sites and they were positively correlated to the
water turbidity and negatively correlated to the temperature. Highest (oo)cyst
concentrations were observed in winter. No relationship between the concentrations of
(oo)cysts in the reservoir and rain events could be established. In summer 2007. the
maximal risk of parasite infection per exposure event for swimmers in the reservoir was
estimated to be 0.0015% for C. varvum and 0.56% for G. lamblia. Finally, no (ootcvsts
could be detected in large volumes of finished drinking water.
Hlavsa et al.
(2011)
USA-wide
Survey
Various
Diseases
Numerous
Outbreaks, especially the largest ones, were most frequently associated with treated
recreational water and characterized bv AGI. Cryptosporidium remains the leading
etiologic agent. Pool chemical-associated health events occur frequently but are
preventable. Data on other select recreational water-associated health events farther
elucidate the epidemiology of U.S. waterborne disease bv highlighting less frequently
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implicated types of recreational water (e.g.. oceans') and detected types of recreational
water-associated illness ("i.e.. not AGP.
Hokajarvi et
al. (2013)
Finland, freshwater,
17 locations
Survey
Land runoff
Campylobacter, 4, 5 50 Finnish bathing water samples and 34 sewage effluent samples originating from 17
Adenoviruses locations studied in of 2006 and 2007 summers. Campylobacter present in 58% and
(qPCR), E. coli, Adenoviruses in 12% of all bathing water samples; 53% of all sewage effluent samples
intestinal were positive for Campylobacter spp. and 59% for Adenoviruses. C. jejuni was the
enterococci most common Campylobacter species found and human Adenovirus serotype 41 was
the most common identified Adenovirus type. Bathing water temperature displayed a
significant negative relationship with the occurrence of Campylobacter. The counts of
fecal indicator bacteria were not able to predict the presence of Campylobacter spp. or
Adenoviruses in the bathing waters.
Kent & Bayne Chattooga River, Epi
(2010) Southeastern USA
WWTPS, Perceptions
construction of skin
sites infection
Although bacterial skin infections are a chronic problem among Whitewater rafters on
the Chattooga River in the southeastern United States, little is known about the source
of such infections. The Chattooga River is a federally designated "Wild and Scenic"
river. Riverine water quality can be negatively impacted by tributaries that are not
protected by federal guidelines. Water quality in Stekoa Creek, a major tributary of the
Chattooga River, is degraded by sediment derived from construction sites near the
creek, as well as fecal coliform contamination from wastewater treatment facilities. A
survey of Whitewater raft guides was conducted to collect data on incidence of skin
infection, and to assess perceived health risk from recreation activities. Whitewater
rafting guides working on the Chattooga River reported concerns about their personal
health related to degraded water quality and microbial contamination from Stekoa
Creek. Incidence of bacterial skin infection and perceived health risk was strongly
correlated among the Whitewater rafting guides.
Kirs et al.
(2016)
Flawaiian waters
Experiment,
epi-related
Treated
wastewater,
streams, marine
Bacteroides spp. 4 Evaluated human-associated Bacteroides spp. (FlF183TaqMan) and human
(FlF183TaqMan) polyomavirus (FlPyV) markers for host sensitivity and specificity. Both markers were
and human strongly associated with sewage, although the cross-reactivity of the FlF183TaqMan
polyomavirus (also present in 82% of canine [n = 11], 30% of mongoose [n = 10], and 10% of feline
(FlPyV) markers, [n = 10] samples) needs to be considered. Concentrations of FlF183TaqMan in human
enterococci, E. coli fecal samples exceeded those in cross-reactive animals at least 1,000-fold. In the
absence of sunlight, the decay rates of both markers were comparable to the die-off
rates of enterococci in experimental freshwater and marine water microcosms.
However, in sunlight, the decay rates of both markers were significantly lower than the
decay rate of enterococci. Limitations can be mitigated by using both markers
simultaneously; ergo, this study supports the concurrent use of HF183TaaMan and
HPvV markers for the detection of sewage contamination in coastal and inland waters in
Hawaii. Both markers are more conservative and more specific markers of sewage than
fecal indicator bacteria (enterococci and Escherichia coli).
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Analysis of HF183TaqMan (or newer derivatives') is recommended for inclusion in
future epidemiological studies.
Kundu et al.
(2013)
Calleguas Creek, QMRA
Southern CAUSA
Urban (upper
areas), else
agriculture.
Three tertiary-
treated
effluents
GI
PCR: Human 3, 4 Used site-specific QMRA to assess the probability of Adenovirus illness for three
Adenovirus and groups of swimmers: adults with primary contact, children with primary contact, and
Enterovirus: total secondary contact regardless of age. Adenovirus type 40/41 was detected in 11% of 73
and fecal samples, ranging from 147 to 4117 genomes per liter. Enterovirus was detected only
coliforms, once (32 genomes per liter). Seven of eight virus detections occurred when E. coli
enterococci (tidal concentrations were below the single sample maximum water quality criterion for
area only) contact recreation, and five of eight virus detections occurred when fecal coliforms were
below the corresponding criterion. Dose-harmonization was employed to convert viral
genome measurements to TCID50 values needed for dose-response curves. The mean
illness risk in children based on Adenovirus measurements obtained over 11 months
was estimated to be 3.5%. which is below the 3.6% risk considered tolerable by the
current United States EPA recreational criteria for GI. The mean risks of GI illness for
adults and secondary contact were 1.9% and 1.0%, respectively. Risk was lowered
considerably when a small proportion of Adenovirus 40/41 (3%) was assumed as
infectious as Adenovirus type 4, compared to the assumption that all genomes were
Adenovirus 4.
Lamparelli et
al. (2015)
Brazil
Epi
Prospective,
cohort
Wastewater
effluent-
impacted
waters
GI, diarrhoea,
nausea, fever,
vomiting
E. coli and
enterococci
Swimming and sand contact associated with increased risk of GI illness in highly
exposed swimmers. Increases in E. coli and enterococci associated with increased GI
risk—more pronounced children age 0-10.
Lee et al.
(2014)
Ohio
Experiment
3 FWlake
beaches. Non-
point, crop
culture, pasture
qPCR: Human
Adenovirus,
Enterovirus and
Norovirus. E. coli
and Bacteroides.
Human Adenovirus, Enterovirus and Norovirus were monitored using qPCR assays at
freshwater beaches during the swimming season. Human Adenovirus (40%) and
Enterovirus (17%) were detected, but Norovirus was not detected. Enteric virus
densities exhibited no relationships with densities of fecal indicators or culture-
independent genetic markers. Densities of human Enterovirus were correlated with
water inflow rates into reservoirs of freshwater beaches.
Leonard et al.
(2015)
England and Wales
coastal waters
Survey- Coastal
QMRA-related discharges
via estimates of
doses.
E. coli, 3GCs
(prevalence of
3GC-resistance)
determined using
culture-based
methods.
The role the natural environment plays in the spread of antibiotic resistant bacteria
(ARB) and antibiotic resistance genes is not well understood. ARB have been detected
in natural aquatic environments, and ingestion of seawater during water sports is one
route whereby many people could be exposed directly. The aim was to estimate the
prevalence of resistance to one clinically important class of antibiotics (third generation
cephalosporins (3GCs)) amongst E. coli in coastal surface waters. Prevalence data were
used to quantify ingestion of 3GC-resistantZ?. coli (3GCREC) by people participating in
water sports. A further aim was to use this value to derive a population level estimate of
exposure to these bacteria during recreational use of coastal waters in 2012. 0.12% of E.
coli isolated from surface waters were resistant to 3GCs. This value was used to
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estimate that in England and Wales over 6.3 million water sport sessions occurred in
2012 that resulted in the ingestion of at least one 3GCREC. Despite the low prevalence
of resistance to 3GCs amongst E. coli in surface waters, there is an identifiable human
exposure risk for water users, which varies with the type of water sport undertaken. The
relative importance of this exposure is likely to be greater in areas where a large
proportion of the population enjoys water sports. This is the first study to use volumes
of water ingested during different water sports to estimate human exposure to ARB.
Lim et al. Baby Beach, QMRA Undeveloped GI infection
(2017) southern CA open space and
urban. No
obvious point
sources of
human waste.
Enterococci
The utility of FIB as indicators of recreational water illness (RWI) risk has been
questioned, particularly in coastal settings with no obvious sources of human sewage.
The authors employed a source-apportionment QMRA (SA-QMRA) to assess RWI risk
at a popular semi-enclosed recreational beach. SA-QMRA results suggest that, during
dry weather, the median RWI risk at this beach is below the U.S. EPA recreational
water quality criteria (RWQC) of 36 illness cases per 1000 bathers. During wet weather,
the median RWI risk predicted by SA-QMRA depends on the assumed level of human
waste associated with stormwater; the RWI risk is below the EPA RWQC illness risk
benchmark 100% of the time provided that <2% of the FIB in stormwater are of human
origin. However, these QMRA outcomes contrast strongly with the RWQC for 30-day
geometric mean of enterococci bacteria. These results suggest that SA-QMRA is a
useful framework for estimating robust RWI risk that takes into account local
information about possible human and non-human sources of FIB.
Loge et al.
(2009)
Worldwide
QMRA
Any
GI infection
Studies the relative significance of: (1) active shedding of microorganisms from bathers
themselves, and (2) the type and concentration of etiological agent on the observed
heterogeneity of the incidence of illness in epidemiological studies that have been used
to develop ambient water quality criteria. The etiological agent and corresponding dose
ingested during recreational contact was found to significantly impact the observed
incidence of illness in an epidemiological study conducted in recreational water. In
addition, the observed incidence of illness was found not to necessarily reflect
background concentrations of indicator organisms, but rather microorganisms shed
during recreational contact. Future revisions to ambient water quality criteria should
address the etiological agent, dose, and the significance of microbial shedding relative
to background concentrations of pathogens and indicator organisms in addition to the
incidence of illness and concentration of indicator organisms.
Magill-Collins
etal. (2015)
Colorado River,
Grand Canyon
rafting
Epi
Non-point
Norovirus
illness
Norovirus by RT-
qPCR
Confidential illness reports were completed by all individuals with symptoms of AGI,
and samples of fecal matter and vomitus, surface swabs of rafting equipment, and
environmental swabs at stops along the hiking corridor were collected and tested for the
presence of Norovirus using reverse transcription-quantitative polymerase chain
reaction (RT-qPCR). During the active outbreak 97 rafters (1.4%) from 10 trips (2.9%
of all trips) declared AGI symptoms. AGI incidence within the 10 infected trips varied
from 6% to 88%. Outbreaks occurred in 3 distinct temporal clusters that involved 2
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different genogroups of Noroviras. All available toilet fecal samples (5 samples) were
positive for Noroviras RNA: 1 with genogroup I (GI) and 4 with GIL The vomitus
sample tested positive for GI. None of the fomite samples from rafting equipment or
from the hiking corridors were confirmed for Noroviras.
The results suggest that Noroviras may have been introduced by ill or asymptomatic
individuals actively shedding the virus in their vomitus or feces, and spread within, or
between, river trios bv different modes of transmission.
Mallin et al. Wrightsville beach, Survey
(2010) North Carolina.
Urban
Fecal coliforms, 4, 5, 6 From 2007-2009 a study was undertaken to determine the sources of fecal bacteria
enterococci, 16S contamination to the marine waters adjoining Wrightsville Beach. Sampling for optical
rDNA genes of brighteners was included, along with dye studies, and use of molecular bacterial source
Bacteroides- tracking techniques including polymerase chain reaction (PCR) and terminal restriction
Prevotella markers fragment polymorphism (T-RFLP) fingerprinting of the Bacteroides-Prevotella group.
Of the 96 samples collected from nine locations during the study, the water contact
standard for Enterococcus was exceeded on 13 occasions. The T-RFLP fingerprint
analyses demonstrated that the most widespread source of fecal contamination was
human, occurring in 38% of the samples, with secondary ruminant and avian sources
also detected. Optical brightener concentrations were low, reflecting negligible sewer
leakage or spills. A lack of sewer leaks and lack of septic systems in the town pointed
toward discharge from boat heads into the marine waters as the major cause of fecal
contamination: this was supported bv dve studies.
Mannocci et
al. (2016)
Worldwide, meta-
analysis
Epi
Respiratory
A meta-analysis conducted to assess the association between swimming in recreational
water and the occurrence of respiratory illness. Fourteen independent studies that
included 50,117 patients with significant heterogeneity were reviewed. The meta-
analysis reports that people exposed to recreational water (swimmers/bathers) present a
higher risk of respiratory illness compared to non-swimmers/non-bathers. This
percentage increases if adjusted RR by age and gender are considered. A clear
association between swimming in recreational water and the occurrence of respiratory
illness was found. The surveillance of water quality monitoring systems is crucial not
only for gastrointestinal illness, but also for respiratory ones.
Marion et al.
(2010)
USA, East Fork
Lake, Ohio
Epi,
prospective
cohort
Likely
influenced by
non-point
source human
fecal
contamination;
GI Illness
E. coli
Examined relationships between water quality indicators and reported adverse health
outcomes among users of a beach at an inland U.S. lake. Human health data were
collected over 26 swimming days during the 2009 swimming season in conjunction
with water quality measurements. Adverse health outcomes were reported 8-9 days
post-exposure via a phone survey. Wading, playing or swimming in the water was
observed to be a significant risk factor for GI illness (adjusted odds ratio (APR) of 3.2).
Among water users. E. coli density was significantly associated with elevated GI illness
risk where the highest £ coli auartile was associated with an APR of 7.0. GI illness
associations are consistent with previous freshwater epidemiology studies. A unique
finding was observations of positive associations with GI illness risk based upon a
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single daily E. coli measurement. Lastly, this study focused on an understudied issue,
illness risk at inland reservoirs. These results support the usefulness of E. coli as a
health-relevant indicator of water quality for this inland U.S. beach. Includes an E. coli
- HCGI regression.
Marion et al. USA, East Fork Epi: likely GI illness, HAdV, Data pertaining to genetic marker exposure and 8 or 9-day health outcomes were
(2014) Lake, Ohio prospective influenced by diarrhoea, Enterovirus,Norovi available for a total of 600 healthy susceptible swimmers, and with this population we
cohort non-point vomiting rus, E. coli, observed a significant positive association between human Adenovirus (HAdV)
source human enterococci, exposure and diarrhea (odds ratio = 1.6) as well as gastrointestinal illness (OR = 1.5)
fecal Bacteroides upon adjusting for culturable E. coli densities in multivariable models. No significant
contamination; associations between bacterial genetic markers and swimming-associated illness were
observed. Positive association between increasing densities of HAdV and E. coli:
increased odds of GI and HCGI among swimmers.
McBrids et al. USA-wide QMRA Stormwater GI, 'Reference 2,5,6 Data were collected from 12 sites representative of seven discharge types (including
(2013) Respiratory pathogens': residential, commercial/industrial runoff, agricultural runoff, combined sewer
Giardia overflows, and forested land), mainly during wet weather conditions during which times
Cryptosporidium, human health risks can be substantially elevated. Using an example waterbody and
Adenovirus, mixed source, pathogen concentrations were used in QMRA models to generate risk
Enterovirus and profiles for primary and secondary water contact (or inhalation) by adults and children.
Salmonella A number of critical assumptions and considerations around the QMRA analysis are
highlighted, particularly the harmonization of the pathogen concentrations measured in
discharges during this project with those measured (using different methods) during the
published dose-response clinical trials. Norovirus was the most dominant predicted
health risk, though further research on its dose-response for illness (cf. infection) is
needed. Even if the example mixed-source concentrations of pathogens had been
reduced 30 times (bv inactivation and mixing), the predicted swimming-associated
illness rates (largely driven bv Norovirus infections) can still be appreciable. Rotavirus
generally induced the second-highest incidence of risk among the tested pathogens
while risks for the other reference pathogens were considerably lower. Secondary
contact or inhalation resulted in considerable reductions in risk compared to primary
contact.
Mika et al.
(2014)
Southern
California; Santa
Monica Channel
(SMC); Ventura
Harbor, Keys and
Experiment
Natural state
(SMC); mixed
use (residential,
commercial)
Total coliforms, E.
coli, enterococci,
Bacteroides 16s
gene marker
(HF183), byqPCR.
The variability of levels of FIB and a human-associated genetic marker (HF183) during
wet and dry weather conditions was investigated. Seventy-eight to 86% of the samples
collected from SMC sites exceeded standard water quality standards for FIB (n=59 to
76). At SMC, HF183 was present in 58%of the samples (n = 78) and was detected at
least once at every sample site. No individual site at SMC appeared as a hotspot for the
measured indicators, pointing to a likely chronic issue stemming from urban runoff in
wet and dry weather. In Ventura, the Arundell Barranca, which drains into Ventura
Harbor and Marina, was a source of FIB, and HF183 was most frequently detected off a
dock in the Marina. Rainfall significantly increased FIB levels at both SMC and
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Ventura. Sample locations with elevated FIB were geographically distinct from the sites
with elevated HF183 in Ventura, which supports the importance of measuring host-
associated parameters along with FIB in chronically impaired watersheds to guide water
quality managers in pollution remediation efforts.
Ming et al.
(2014)
China (Bohai Bay) QMRA
Domestic
sewage,
aquaculture
industry,
domestic
sewage, non-
point source
runoff, besides,
ship waste
holding tanks
GI
ICC-qPCR
Rotavirus
Dose-response data indicate that Rotavirus (RV) may be one of the more infective
agents among enteric viruses. The major limitation at present in the assessment of
infection from Rotavirus is lack of quantitative data on viral infectivity. In this work, an
integrated cell culture and real-time quantitative polymerase chain reaction (ICC-qPCR)
method and a Beta-Poisson model for risk assessment were employed. A set of 28
surface seawater samples was collected from December 2010 to September 2011 in
Bohai Bay, China, to enable a seasonal risk assessment of infective RV at recreational
beaches. Thirty-two percent of the samples were positive for Rotavirus, and the
estimated concentration range of infectious human Rotavirus was 1 to 279 PFU/L.
Contamination of seawater with Rotavirus was higher in autumn and winter, in
reasonable agreement with the trend observed in a prior epidemiological study. A
preliminary risk assessment indicated the daily risk of illness at almost all the
contaminated sites exceeded an acceptable threshold of marine recreational water
quality (19 illnesses per 1000 swimmers').
Nevers &
Whitman
(2011)
USA, Lake
Michigan, 50
beaches
Survey
Various
Examined whether re-evaluation of the U.S. EPA ambient water quality criteria
(AWQC) and the epidemiological studies on which they are based could increase public
beach access without affecting presumed health risk. Single-sample maxima were
calculated using historic monitoring data for 50 beaches along coastal Lake Michigan
on various temporal and spatial groupings to assess flexibility in the application of the
AWQC. No calculation on either scale was as low as the default maximum (235
CFU/100 mL) that managers typically use, indicating that current applications may be
more conservative than the outlined AWQC. It was notable that beaches subject to point
source FIB contamination had lower variation, highlighting the bias in the standards for
these beaches. Until new water quality standards are promulgated, more site-specific
application of the AWQC may benefit beach managers by allowing swimmers greater
access to beaches.
Nnane et al.
(2011)
UK, River Ouse
Survey
Predominantly
rural
(agriculture),
7% urban.,
E. coli,
intestinal
enterococci;p
hages of
Bacteroides
GB-124,
Clostridium
perfringens,
Heterotrophic
Investigated the integration and application of a novel and simple MST approach to
monitor microbial water quality over one calendar year, thereby encompassing a range
of meteorological conditions. A key objective of the work was to develop simple low-
cost protocols that could be easily replicated. Bacteriophages (viruses) capable of
infecting a human specific strain of Bacteroides GB-124, and their correlation with
presumptive Escherichia coli, were used to distinguish sources of fecal pollution. The
results reported here suggest that in this river catchment, non-human sources of fecal
pollution predominate. During storm events, presumptive E. coli and presumptive
intestinal enterococci levels were 1.1-1.2 logs higher than during dry weather
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plate count,
somatic
coliphage
conditions, and levels of the fecal indicator organisms (FlOs) were closely associated
with increased turbidity levels (presumptive E. coli and turbidity, r = 0.43). The
correlation coefficient between presumptive E. coli and phages of Bacteroides GB-124
was very small (r= 0.05). whilst that between turbidity and suspended solids was high (r
= 0.62). Variations in climate, animal and anthropogenic interferences were all, either
directly or indirectly, related to fecal contamination.
Papastergiou et
al. (2011)
Greece, three
marine beaches
Epi
Septic tanks,
river,
Gl,
respiratory
E. coli, fecal
coliforms, total
coliforms,
enterococci, S.
aureus
n = 4293 incl. 3,805 bathers; 149 samples from the beaches. Despite all beaches being
characterized as of high quality, the levels of bacterial indicators differed among them.
Flealth effects among bathers were not associated with bacterial indicators. A
statistically significant increased risk for symptoms related to respiratory illness,
gastroenteritis, medical consultation and use of medication was observed among bathers
at beaches with higher bather density.
Pintar et al.
(2010)
Canada, Ontario
community level,
public swimming
pool, river, lake.
QMRA
Various
Cryptosporidi
osis
Cryptosporidium
2
Simulated the role of recreational water contact in the transmission of cryptosporidiosis.
Stochastic simulations were based on plausible modes of contamination of a pool
(literature derived), river (site-specific), and recreational lakes (literature derived). The
highest estimated risks of infection were derived from the (highly contaminated)
recreational lake scenario, considered the upper end for risk of infection for both
children C10 infections per 1.000 swims and adults ("four infections per 1.000 swims.
Simulating the likely Cryptosporidium oocyst concentration in a lane pool that a child
would be exposed to following a diarrheal fecal release event resulted in the third
highest mean risk of infection ("four infections per 10.000 swims 15%o: three infections
per 100.000: 95%o: 10 infections per 10.000 swims!). Findings illustrate the need for
systematic and standardized research to quantify Cryptosporidium oocyst levels in
Canadian public pools and recreational beaches. There is also a need to capture the
swimming practices of the Canadian public, including most common forms and
frequency measures. The study findings suggest that swimming in natural swim
environments and in pools following a recent fecal contamination event pose significant
public health risks. When considering these risks relative to other modes of
cryptosporidiosis transmission, they are significant.
Pintar et al. Canada, Ontario QMRA Various Campylobact Campylobacter 2 A comparative exposure assessment was developed to estimate the relative exposure to
(2017) community level, eriosis Campylobacter, the leading bacterial gastrointestinal disease in Canada, for 13 different
public swimming transmission routes within Ontario, Canada, during the summer. Exposure was
pool, river, lake. quantified with stochastic models at the population level, which incorporated measures
of frequency, quantity ingested, prevalence, and concentration, using data from
FoodNet Canada surveillance, the peer-reviewed and gray literature, other Ontario data,
and data specifically collected for this study. The mean number of cells of
Camvvlobacter ingested per Ontarian per day during the summer, ranked from highest
to lowest is as follows: household pets, chicken, living on a farm, raw milk, visiting a
farm, recreational water, beef, drinking water, pork, vegetables, seafood, petting zoos.
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and fruits. The study results identify knowledge gaps for some transmission routes, and
indicate that some transmission routes for Campylobacter are underestimated in the
current literature, such as household pets and raw milk. Many data gaps were identified
for future data collection consideration, especially for the concentration of
Campylobacter in all transmission routes.
Reddy et al.
(2011)
UK (Cornwall)
Epi
"Surfer's ear"
(n = 92. 78 males and 14 females, mean age 27 years, standard deviation 7.9 years).
Participants were grouped according to their awareness of the preventability of surfer's
ear (55 aware, 37 unaware). These groups were comparable in age, surfing history and
gender mix. Surfers aware of the preventability of exostoses (66 per cent) were more
likely to use water precautions than those who were not (38 per cent) (p < 0.01). Two
surfers used water precautions regularly and 48 used them occasionally. Sixtv-one of
the 76 surfers who did not use water precautions (ear plugs') suggested thev would
consider doing so in the future.
Rijal et al.
(2011)
Chicago Area
Waterway
QMRA
Urban, treated
wastewater,
land runoff
G1
pathogenic E. coli
[estimated],
Giardia,
Cryptosporidium,
Adenovirus,
Norovirus, enteric
A microbial risk assessment was conducted to estimate the human health risks from
incidental contact recreational activities such as canoeing, boating and fishing in the
Chicago Area Waterway System (CAWS) receiving secondary treated, but non-
disinfected, effluent from three municipal water reclamation plants. Results under the
current treatment scheme with no disinfection indicated that the total expected
gastrointestinal illness (Gl) rate per 1000 incidental contact recreational exposure events
during combined weather (dry and wet) conditions ranged from 0.10 to 2.78 in the
CAWS. Wet weather conditions contribute to elevated pathogen load in the CAWS; this
study determined that disinfecting the effluents of three major WRPs that discharge to
the CAWS would result in an extremely small reduction in the aggregate recreation
season risk to incidental contact recreators.
Sales-Ortells
& Medema.
(2014)
playing in a water playground (3.7%) and in the pluvial flood from storm water sewers
(4.7%). At these locations, the Gl probability was above the EU Bathing Water
Directive threshold for excellent water quality (3%). All the annual risk medians were
belowthe national incidence of legionellosis of 0.002%. The illness probability was
most sensitive to the pathogens concentration (particularly Campylobacter, Norovirus,
and Legionella) and exposure frequency.
Watergraafsmeer, QMRA Urban Gl, Cryptosporidium, 2 Event and annual Gl probability and Legionnaires' disease were analysed in QMRA
Amsterdam, 20 Legionnaires' Campylobacter, models using selected literature data. Highest mean event probabilities of Gl were found
waterbodies/ disease Norovirus andL. for playing in pluvial flood from a combined sewer overflow (34%), swimming (18%),
features pneumophila and rowing (13%) in the river, swimming (8.7%) and rowing (4.5%) in the lake, and
Sanborn & Canada Epi PubMed Acute Gl
Takoro (2013)
2, 5 There is a 3% to 8% risk of acute gastrointestinal illness (AGI) after swimming. The
high-risk groups for AGI are children younger than 5 years, especially if they have not
been vaccinated for Rotavirus, and elderly and immunocompromised patients. Children
are at higher risk because they swallow more water when swimming, stay in the water
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longer, and play in the shallow water and sand, which are more contaminated.
Participants in sports with a lot of water contact such as triathlon and kite surfing are
also at high risk, and even activities involving partial water contact such as boating and
fishing carry a 40% to 50% increase in risk of AGI compared with non-water
recreational activities.
Sanchez-
Puerto Rico, Three
Epi,
Creeks,
GI,
Coliphage, E. coli
n = 641. Increased risk of illness in swimmers as compared to non-swimmers, even
Nazario et al.
beaches
prospective
wastewater
Respiratory,
(two methods),
when waters met current microbial standards for recreational water quality. Illnesses
(2014)
cohort
treatment
Skin,
Staphylococcus
included GI. skin and respiratory symptoms, earache and fever. Odds ratios (ORs)
plants, septic
Earache,
spp., enterococci
ranged from 0.32 to 42.35 (GI illness), 0.69 to 3.12 (skin infections), 0.71 to 3.21
tanks, animal
Headache,
(respiratory symptoms), 0.52 to 15.32 (earache) and 0.80 to 1.68 (fever). The indicators
feces
Fever
that better predicted the risks of symptoms (respiratory) in tropical recreational waters
were total ("somatic and male-specific") coliphages COR = 1.56. p<0.10. R2 = 3.79%) and
E. coli (OR = 1.38. p<0.10. R2 = 1.97%). Study indicates that coliphages are potentially
good predictors of risks of respiratory illness in tropical recreational waters.
The Netherlands revealed 742 outbreaks during 1991-2007 mainly comprising of skin
conditions (48%) and gastroenteritis (31%), involving at least 5,623 patients. The
number of outbreaks per bathing season correlated with the number of days with
temperatures over 25 °C (r=0.8-0.9), but was not reduced through compliance with
European bathing-water legislation (r=0.1), suggesting that monitoring of fecal
indicator parameters and striving for compliance with water-quality standards may not
sufficiently protect bathers. Bathing sites were prone to incidental fecal contamination
that favoured the growth of naturally occurring pathogens.
n = 8,000 of whom 1924 additionally answered the questions for their eldest child (<15
years). Differences between men and women were small, but children behaved
differently: they swam more often, stayed in the water longer, submerged their heads
more often and swallowed more water. Swimming pools were visited most frequently
(on average 13-24 times/year) with longest duration of swimming (on average 67-81
min). On average, fresh and seawater sites were visited 6-8 times/year and visits lasted
41-79 min. Dependent on the water type, men swallowed on average 27-34 mL per
swimming event, women 18-23 mL, and children 31-51 mL.
QMRAcatch, was developed to simulate pathogen concentrations in water. The model
domain encompasses a main river with wastewater discharges and a floodplain with a
floodplain river. Difiiise agricultural sources not vet included. The floodplain river is
fed by the main river and may flood the plain. Fecal deposits from wildlife, birds, and
visitors in the floodplain are resuspended in flood water, runoff to the floodplain river,
or infiltrate groundwater. Fecal indicator and MST marker data facilitate calibration.
Infection risks from exposure to the pathogens by swimming or drinking water
consumption are calculated, and the required pathogen removal by treatment to meet a
Schets et al. Netherlands Epi - - - 6
(2011a)
Schets et al. Netherlands EPI (exposure
(2011b) study)
Schyven et al. Netherlands
(2015)
QMRA
GI infection
E. coli, a human-
associated
Bacteroidetes)
marker,
Enterovirus,
Norovirus,
Campylobacter,
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and
Cryptosporidium
health-based quality target can be determined. Applicability is demonstrated by
calibrating the tool for a study site at the River Danube near Vienna, Austria, using field
data, including a sensitivity analysis and evaluation of the model outcomes.
Schoen & Worldwide
Ashbolt (2010)
QMRA
Seagulls,
treated sewage
GI
Seagulls:
Campylobacter
jejuni and
Salmonella
enterica
Sewage:
Norovirus, Giardia
intestinalis,
Cryptosporidium
spp., Salmonella
enterica
QMRA estimated probability of illness for accidental ingestion of recreational water
with a specific concentration of fecal indicator bacteria-the geometric mean enterococci
limit of 35 cfu/100 mL, from either a mixture of sources or an individual source. Using
seagulls as a non-sewage fecal source example, the predicted median probability of
illness was less than the illness benchmark of 0.01. When the fecal source was changed
to poorly treated sewage, a relativity small difference between the median probability of
illness and the illness benchmark was predicted. For waters impacted by a mixture of
seagull and sewage waste, the dominant source of fecal indicator was not always the
predicted dominant source of risk.
Schoen et al. World-wide QMRA Human sewage GI
(2011) variously-
treated
Norovirus,
enterococci
(culture and PCR)
Evaluated the relative contribution of fecal indicators and pathogens when a mixture of
human sources impacts a recreational waterbody. Used Norovirus as the reference
pathogen and enterococci as the reference fecal indicator. Contribution made by each
source to the total waterbody volume, indicator density, pathogen density, and illness
risk was estimated for a number of scenarios that accounted for pathogen and indicator
inactivation based on the age of the effluent (source to-receptor), possible sedimentation
of microorganisms, and the addition of a non-pathogenic source of fecal indicators
(such as old sediments or an animal population with low occurrence of human-
infectious pathogens). Enterococci was held constant at 35 cfii/100 mL to compare
results across scenarios. For the combinations evaluated, either the untreated sewage or
the non-pathogenic source of fecal indicators dominated the recreational waterbody
enterococci density assuming a culture method. In contrast. The results support the use
of a calibrated qPCR total enterococci indicator, compared to a culture-based assay, to
index infectious human enteric viruses released in treated human wastewater, and
illustrate that the source contributing the majority of risk in a mixture may be
overlooked when only assessing fecal indicators bv a culture-based method.
Seto et al. Oakland, CA QMRA Urban treated GI Fecal coliform, E. A static QMRA was used to estimate the incremental risk to public health from
(2016) wastewater, coli, Enterococcus, recreational exposure to Adenovirus and the protozoan Giardia spp. in San Francisco
wet-weather male specific Bay for wet season (generally between October and March) blending and non-blending
flows coliphage, events. The mean risks of infection per recreational exposure event during the wet
Adenovirus, season for all of the modeled scenarios were more than an order-of-magnitude below
Enterovirus, the USEPA's illness level (36 illnesses per 1000 contact events) associated with
Giardia spp., recreational water quality. While the QMRA results showed discernible differences in
per event estimated risks between blending and non-blending scenarios, the estimated
incremental increase in the annual number of infections due to blending (based on
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Cryptosporidium
spp.
median estimates') resulted in an estimated combined increase of less than one infection
annually. These estimates are subject to various uncertainties, including the potential for
secondary transmission, assumptions on the extent of exposures, and the number of
blending days required in the future due to climate change.
Sidhu et al.
(2012)
Brisbane, Australia,
urban stormwater
runoff
Survey
Broad range of
urban
Culture: E. coli,
Enterococcus,
PCR:
Adenovirus,
polyomavirus, S.
enterica,
Campylobacter
spp. and
Bacteroides HF183
gene detected with
published primer
and probe sets.
4, 5 Water samples (20 L) were collected after storm events and during the dry weather from
six sites in Brisbane, Australia. Samples were analyzed for FIB, and then concentrated
using hollow fiber ultrafiltration followed by molecular detection of selected enteric
pathogens. The levels of FIB were found to frequently exceed the upper limit of
Australian guidelines for managing risks in recreational water, during the dry periods
and by further several orders of magnitude in the stormwater runoff. Enterococcus spp.
numbers as high as 3x104 /100 mL were detected in the stormwater runoff. Human
Adenovirus and polyomavirus were frequently detected from all six sites during wet and
dry weather conditions suggesting their wide spread presence in the urban aquatic
environments. Campylobacter jejuni, Campylobacter coli and S. enterica were also
detected during both dry and wet weather conditions. Presence of human-specific
HF183 Bacteroides marker in most of the samples tested suggests ubiquitous sewage
contamination. Since stormwater runoff routinely contains high numbers of FIB and
other enteric pathogens, some degree of treatment of captured stormwater would be
required if it were to be used for non-potable purposes.
Sinigalliano et
al. (2010)
United States
EPI:
Prospective
randomized
exposure
Recreational
marine waters
with no known
point source of
sewage
Included GI,
skin illness,
acute febrile
respiratory
illness
Enterococcus
(culture methods
and qPCR),
Bacteroidales
(qPCR, human and
dog markers), gull
Catellicoccus
marker, S. aureus
n = 1.341. 15 study davs. No known point source (e.g.. discharge of treated sewage).
The study reported symptoms between one set of human subjects randomly assigned to
marine water exposure with intensive environmental monitoring compared with other
subjects who did not have exposure. Among the bathers, a positive dose-response
relationship was observed for skin illness and enterococci enumeration by membrane
filtration. Skin illness was positively related to 24 hour antecedent rainfall, while acute
febrile respiratory illness was inversely related to water temperature. There were no
significant dose-response relationships between report of human illness and any of the
other FIB or environmental measures.
Soldanova et
al. (2013)
Europe
Review
Snails and
birds
Schistosomiat
is
6
Summarizes current knowledge about the occurrence and distribution of swimmer's
itch, with a focus on Europe. Relevant studies from the past decade are analyzed to
reveal an almost complete set of ecological factors as a prerequisite for establishing the
life cycle of bird schistosomes. Based on records of the occurrence of the parasite
infective agents, and epidemiological studies that investigate outbreaks of swimmer's
itch, this review concentrates on the risk factors for humans engaged in recreational
water activities.
Soller et al.
(2010a)
World-wide
QMRA
Various
GI
Norovirus, 2,6)
Rotavirus,
Adenovirus,
Epidemiology studies of recreational waters have demonstrated that swimmers exposed
to fecally-contaminated recreational waters are at risk of excess gastrointestinal illness.
Epidemiology studies provide valuable information on the nature and extent of health
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c x 2 Conclusions (includes health linkages)
M *c
II s
Cryptosporidium
effects, the magnitude of risks, and how these risks are modified or associated with
spp., Giardia
levels of fecal contamination and other measures of pollution. However, such studies
lamblia,
have not provided information about the specific microbial agents that are responsible
Campylobacter
for the observed illnesses in swimmers. Considers epidemiological results from studies
jejuni, Salmonella
conducted on the Great Lakes in the US during 2003 and 2004 bv identifying pathogens
enterica, and E.
that could have caused the illnesses observed. Two QMRA-based approaches were used
coli 0157:H7.
to estimate the pathogen combinations that would be consistent with observed illness
rates: in the first, swimming-associated gastrointestinal (GI) illnesses were assumed to
occur in the same proportion as known illnesses in the US due to all non-foodborne
sources, and in the second, pathogens were assumed to occur in the recreational waters
in the same proportion as in disinfected secondary effluent. The results indicate that
human enteric viruses and in particular, Norovirus. could have caused the vast maioritv
of the observed swimming associated GI illnesses during the 2003/2004 water
epidemiology studies. Evaluation of the time-to-onset of illness strongly supports the
principal finding and sensitivity analyses support the overall trends of the analyses even
given their substantial uncertainties.
This work was conducted to determine whether estimated risks following exposure to
recreational waters impacted by gull, chicken, pig, or cattle fecal contamination are
substantially different than those associated with waters impacted by human sources
such as treated wastewater. Published QMRA methods were employed and extended to
meet these objectives w.r.t. GI. Illness risks from these pathogens were calculated for
exposure to fecally contaminated recreational water at the U.S. regulatory limits of 35
cfu/100 mL enterococci and 126 cfli/100 mLE. coli. Three scenarios were simulated,
representing a range of feasible interpretations of the available data. The primary
findings are that: 1) GI illness risks associated with exposure to recreational waters
impacted bv fresh cattle feces may not be substantially different from waters impacted
bv human sources: and 2) the risks associated with exposure to recreational waters
impacted bv fresh gull, chicken, or pig feces appear substantially lower than waters
impacted bv human sources. These results suggest that careful consideration may be
needed in the future for the management of recreational waters not impacted bv human
sources.
Simulated the influence of multiple sources of enterococci (ENT) by considering waters
impacted by human and animal sources, human and non-pathogenic sources, and animal
and non-pathogenic sources. Thev illustrate that risks vary with the proportion of
culturable ENT in waterbodies derived from these sources and estimate corresponding
ENT densities that yield the same level of health protection that the recreational water
quality criteria in the United States seeks (benchmark risk! The benchmark risk is
based on epidemiological studies conducted in waterbodies predominantly impacted by
human fecal sources. The key result is that the risks from mixed sources are driven
Reference
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Soller et al. World-wide QMRA gull, pig, GI Norovirus,
(2010b) chicken, cattle Rotavirus,
Cryptosporidium
spp., Giardia
lamblia,
Campylobacter
jejuni, Salmonella
enterica, and E.
coli 0157:H7
Soller et al. World-wide QMRA Human and GI enterococci
(2014) animal
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predominantly bv the proportion of the contamination source with the greatest ability to
cause human infection (potency), not necessarily the greatest source(s) of FIB.
Predicted risks from exposures to mixtures comprising approximately 30% ENT from
human sources were up to 50% lower than the risks expected from purely human
sources when contamination is recent and ENT levels are at the current water quality
criteria levels (35 cfii/100 mL). For human/non-pathogenic, human/gull, human/pig, and
human/ chicken fecal mixtures with relatively low human contribution, the predicted
culturable enterococci densities that correspond to the benchmark risk are substantially
greater than the current water quality criteria values. These findings are important
because they highlight the potential applicability of site-specific water quality criteria
for waters that are predominantly un-impacted by human sources
Soller et al.
(2015)
World-wide
QMRA
animals (cattle, GI
pigs, chicken)
Literature values 5
of:
E. coli 0157,
Campylobacter,
Salmonella,
Cryptosporidium,
Giardia spp.
Epidemiological studies conducted at locations impacted bv non-human fecal sources
have provided ambiguous and inconsistent estimates of risk. QMRA is another tool. The
potential risk differential between human and selected non-human fecal sources was
characterized previously for direct deposition of animal feces to water. In this
evaluation, the human illness potential from recreational exposure to freshwater
impacted bv rainfall-induced runoff containing agricultural animal fecal material was
examined. Risks associated with these sources would be at least an order of magnitude
lower than the benchmark level of public health protection associated with current US
recreational water quality criteria, which are based on contamination from human
sewage sources.
Soller et al. Boqueron beach in QMRA/EPI GI
(2016) Puerto Rico (prospective
cohort study,
Puerto Rico)
Various
Indicators
Bacteroidales, C.
perfingens,
Coliphage (male-
specific), E. coli,
Enterococcus spp.
(CFU, CCE)
Pathogens
Norovirus,
Adenovirus,
Cryptosporidium,
Giardia,
Salmonella
Estimated the GI illness levels associated with recreational water exposures. The
previously reported epidemiological study had sufficient statistical power to detect an
average illness rate of approximately 17 swimming associated GI illnesses per 1000
recreation events or greater, and found no consistent relationships between water quality
measured bv fecal indicator organisms (FIO~) and swimming-associated illnesses. The
QMRA incorporated monitoring data for pathogens and fecal indicators collected
during the epidemiological study period and calculated average swimming-associated
illness levels that were approximately two GI illnesses per 1000 recreation events. To
the authors' knowledge, this is the first time that a comprehensive water quality
monitoring program and QMRA analysis has been conducted in parallel with a
recreational water epidemiological study. The QMRA results were consistent with the
low rate of reported illnesses during the 2009 epidemiological study (i.e. < 17 GI
illnesses per 1000 recreation events), and provide additional context for understanding
the epidemiological results. The results illustrate that coupling QMRA with an
epidemiological study at a single study site provides a unique ability to understand
human health illnesses especially under conditions where water quality, as measured bv
traditional FIO is good and/or average illness rates are lower than can be quantified via
epidemiological methods alone.
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11
bx
San Diego
QMRA
Various
GI
Indicators
4, 5
Conclusions (includes health linkages)
Soller et al.
(2017, in
press)
(Norovirus
illness)
total coliform, E.
coli and
enterococci
Pathogens
Norovirus Gl,
Norovirus G2,
Enterovirus,
Adenovirus,
Campylobacter,
Salmonella invA,
Salmonella ttr
Modeled GI risk associated with recreational exposures to marine water following storm
events. Estimated GI illness risks via QMRA techniques by consolidating site specific
pathogen monitoring data of stormwater, site specific dilution estimates, literature-based
water ingestion data, and literature based pathogen dose-response and morbidity
information. The authors' water quality results indicated that human sources of
contamination contribute viral and bacterial pathogens to streams draining an urban
watershed during wet weather that then enter the ocean and affect nearshore water
quality. A series of approaches to account for uncertainty in the Norovirus dose-
response model selection and compared our model results to those from a concurrently
conducted epidemiological study that provided empirical estimates for illness risk
following ocean exposure were evaluated. The preferred Norovirus dose-response
approach yielded median risk estimates for water recreation associated illness (15 GI
illnesses per 1000 recreation events) that closely matched the reported epidemiological
results (12 excess GI illnesses per 1000 wet weather recreation events'). The results are
consistent with Norovirus, or other pathogens associated with Norovirus, as an
important cause of GI among surfers in this setting. This study demonstrates the
applicability of QMRA for recreational water risk estimation, even under wet weather
conditions and describes a process that might be useful in developing site-specific water
quality criteria in this and other locations.
Sunger & Haas Philadelphia
(2015)
QMRA
Urban
GI
QMRA under dry and wet weather conditions. Stochastic exposure models were
generated for each exposure scenario and Monte Carlo techniques were applied to
characterize uncertainty in final risk estimates. The drv-weather risk estimates were
significantly lower than those predicted for wet-weather conditions. Moreover, the
predicted risk, calculated in proportion of the frequency of use, was elevated at 6 out of
10 sites (ranging from 9 to 52 illnesses/1000 users/day). Activities contributing most to
the risk of GI illness at creeks were identified as wading and playing (81%), while
fishing was the potential risk contributor (65%) at rivers.
Suppes et al. Four pool sites in Experiment Drinking water
(2014) Tuscon Alabama supply
n = 126. Less frequent head submersion appears to be associated with greater pool
water ingestion rates, which may conflict with one's intuition: These swimmers were
younger and likely less skilled than adults. Video observations suggest children are
'bobbing' with mouths open at water surface to stay above water, and intentionally
spitting and spouting water. Outbreak tools should assess leisure activity engagement
and number of splashes received to the face among cases and non-cases, as both
activities were associated with increased pool water ingestion. Assessing skill as an
ingestion predictor is recommended for future swimming exposure assessments to
clarify why children ingest more pool water than adults. Quantifying and comparing
pool water ingestion with frequency of spitting.
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Tong et al.
(2011)
Treated and
untreated
wastewater; 16 sites
around O'ahu
Experiment
Sewage, land
runoff
The current recreational water quality criteria using growth-based measurements of FIB
concentration have their limitations for swimmer protection. Human Norovirus
(huNoV) was tested as a model in this study. To establish a highly sensitive protocol for
effective huNoV detection in waters, 16 published and newly designed reverse
transcription polymerase chain reaction (RT-PCR) primer pairs specific for huNoV
genogroup I (GI) and genogroup II (Gil) were comparatively evaluated side-by-side
using single sources of huNoV RNA stock extracted from local clinical isolates. Under
optimized conditions, these RT-PCR protocols shared a very different pattern of
detection sensitivity for huNoV. The primer sets COG2F/COG2R and QNIF4/NV1LCR
were determined to be the most sensitive ones for huNoV Gil and GI, respectively, with
up to 105 and 106-fold more sensitive as compared to other sets tested. These two
sensitive protocols were validated by positive detection of huNoV in untreated and
treated urban wastewater samples. In addition, these RT-PCR protocols enabled
detection of the prevalence of huNoV in 5 (GI) and 10 (Gil) of 16 recreational water
samples, confirmed by cDNA sequencing and sequence analysis. Findings from this
study support the possible use of enteric viral pathogens for environmental monitoring
and argue the importance and essentiality for such monitoring activity to ensure a safe
use of recreational waters.
Tseng & Jiang Southern California QMRA
(2012)
Mostly urban GI
beaches
FIO data obtained
from monitoring
results by a number
of agencies
QMRAs were applied to eight popular Southern California beaches using readily
available Enterococcus and fecal coliform data and dose-response models to compare
health risks associated with surfing during dry weather and storm conditions. The
results showed that the level of gastrointestinal illness risks from surfing post-storm
events was elevated, with the probability of exceeding the US EPA health risk guideline
up to 28% of the time. The surfing risk was also elevated in comparison with swimming
at the same beach due to ingestion of greater volume of water. The study suggests that
refinement of dose-response model, improving monitoring practice and better surfer
behavior surveillance will improve the risk estimation.
Viau et al.
(2011)
O'ahu, Hawaii
QMRA
Land runoff,
septic tanks
GI
Viruses
Adenovirus,
Enterovirus,
Norovirus GI, and
Norovirus Gil;
Markers
human, ruminant,
and pig
Bacteroidales
This study used molecular methods to measure concentrations of four enteric viruses
(Adenovirus, Enterovirus, Norovirus GI, and Norovirus Gil) and fecal source tracking
markers (human, ruminant, and pig Bacteroidales) in land-based runoff from 22 tropical
streams on O'ahu, Hawai'i. Each stream was sampled twice in the morning and
afternoon during dry weather. Viruses and human Bacteroidales were widespread in the
streams. Watershed septic tank densities were positively associated with higher
occurrence of human Bacteroidales and Norovirus. There were no associations between
occurrence of viruses and fecal indicator concentrations. Virus concentrations and
previously reported culturable Salmonella and Campylobacter were used as inputs to a
QMRA model to estimate the risk of acquiring GI illness from swimming in tropical
marine waters adjacent to discharging streams. Monte Carlo methods were used to
incorporate uncertainties in the dilution of stream discharge with seawater, swimmer
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ingestion volumes, pathogen concentrations, and dose-response parameters into the
model. Median GI illness risk to swimmers from exposure to coastal waters adjacent to
the 22 streams ranged from 0 to 21/1000. GI illness risks from viral exposures were
generally orders of magnitude greater than bacterial exposures. The median risk
adjacent to each stream was positively, significantly correlated to the concentration of
Clostridium verfrineens in the stream.
Vijayavel et al.
(2010)
O'ahu, 3 STPs to
represent sewage
(variously-treated),
three coastal sites
and a harbor
Survey
Land runoff,
treated sewage
Bacteroides, HB- 3, 4 Previous studies have shown E. coli and enterococci to be unreliable indicators of fecal
73 phage contamination in Hawaii because of their ability to multiply in soils. In this study, the
method of detecting Bacteroides phages as specific markers of sewage contamination in
Hawaii's recreational waters was evaluated because these sewage-specific phages
cannot multiply under environmental conditions. Bacteroides hosts (GB-124, GA-17),
were recovered from sewage samples in Europe and were reported to be effective in
detecting phages from sewage samples obtained in certain geographical areas. However,
GB-124 and GA-17 hosts were ineffective in detecting phages from sewage samples
obtained in Hawaii. Bacteroides host HB-73 was isolated from a sewage sample in
Hawaii, confirmed as a Bacteroides sp. and shown to recover phages from multiple
sources of sewage produced in Hawaii at high concentrations (5.2-7.3 x 105 PFU/100
mL). These Bacteroides phages were considered to be potential markers of sewage
because thev also survived for three days in fresh stream water and two days in marine
water. Water samples from Hawaii's coastal swimming beaches and harbors, which
were known to be contaminated with discharges from streams, were shown to contain
moderate (20-187 cfu/100 mL) to elevated (173-816 cfii/100 mL) concentrations of
enterococci. These same samples contained undetectable levels (<10 PFU/100 mL) of
F+ coliphage and Bacteroides phages and provided evidence to suggest that these
enterococci may not necessarily be associated with the presence of raw sewage. These
results support previous conclusions that discharges from streams are the major sources
of enterococci in coastal waters of Hawaii and the most likely source of these
enterococci is from environmental soil rather than from sewage.
Wade et al.
(2006)
Two Great Lakes
beaches
Epi:
Prospective
cohort
Wastewater
treatment
plants
GI
Enterococcus
(qPCR),
Bacteroides
n = 5,717. Methods to measure recreational water quality in < 2 hr have been
developed. We conducted a prospective study of beachgoers at two Great Lakes beaches
to examine the association between recreational water quality, obtained using rapid
methods, and GI illness after swimming. We tested water samples for Enterococcus and
Bacteroides species using the quantitative polymerase chain reaction (PCR) method.
We observed significant trends between increased GI illness and Enterococcus at the
Lake Michigan beach and a positive trend for Enterococcus at the Lake Erie beach. The
association remained significant for Enterococcus when the two beaches were
combined. We observed a positive trend for Bacteroides at the Lake Erie beach, but no
trend was observed at the Lake Michigan beach. Enterococcus samples collected at
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0800 hr were predictive of GI illness that day. The association between Enterococcus
and illness strengthened as time spent swimming in the water increased.
Wadeetal. Four Great Lakes Epi:, Wastewater GI, Enterococci 2,3 n = 5,717. Swimmers at two beaches had a higher incidence of GI illness when
(2008) beaches prospective effluent- respiratory, (qPCR) and compared to non-swimmers. A statistically significant relationship was observed
impacted rash, eye Bacteroides between an increased rate of GI illness and enterococci at the Lake Michigan beach, and
waters ailments, measured via a positive trend for enterococci at the Lake Erie beach was noted. Association between
earache qPCR enterococci (aPCRt and increased risk of GI illness was significant when results for the
two beaches were combined. Positive trend was observed at the Lake Erie beach for
Bacteroides, but no trend was observed at the Lake Michigan beach.
Wade et al.
(2010)
United States
(Mississippi. Rhode
Island, Alabama
Epi: Cohort,
prospective
Wastewater
effluent-
impacted
waters
GI
enterococci (qPCR) 4
and Bacteroides
measured via
qPCR
n = 6,350. Swimmers at two beaches had a higher incidence of GI illness when
compared to non-swimmers. A statistically significant relationship was observed
between an increased rate of GI illness and enterococci at the Lake Michigan beach, and
a positive trend for enterococci at the Lake Erie beach was noted. The association
between enterococci and increased risk of GI illness was significant when results for the
two beaches were combined. A positive trend was observed at the Lake Erie beach for
Bacteroides, but no trend was observed at the Lake Michigan beach.
Wade et al.
(2013a)
South Carolina
EPI:
Prospective
cohort
Storm water
runoff
diarrhea
n = 11,159. The association between diarrhea among swimmers and rain events at a
beach in South Carolina impacted by stormwater runoff was investigated. During the
summer of 2009, 11,159 beachgoers were enrolled and interviewed. Information about
swimming exposures was obtained, followed by telephone contact 10-12 days later to
ascertain the incidence of diarrhea (3 or more loose stools in a 24 hour period), and
other symptoms. Rainfall was classified as none; low-moderate (<0.39 inches); or high
(>0.4 inches, 90th percentile). Unadjusted incidence of diarrhea was 3.0%, 4.0%, 4.4%,
and 6.5% among non-swimmers; swimmers (body-immersion) following no rainfall in
the previous 24 hours; swimmers following low-moderate rainfall and swimmers
following high rainfall, respectively. Adjusted Odds Ratios and 95% Confidence
Intervals compared to non-swimmers were: 1.33 (0.95-1.86); 1.55 (1.07-2.25); and 2.14
(1.32-3.48) for swimmers with no rainfall, low, and high rainfall in the prior 24 hours,
respectively. There was also a significant trend across categories among swimmers (p =
0.003). Rainfall the day of swimming and during the 24-48 hour lag were not as
consistently associated with diarrhea. In conclusion, diarrhea among swimmers was
associated with rainfall in the 24 hours prior to swimming at a beach impacted bv urban
runoff.
Wadsetal. 9 USA beaches Epi:
(2013b) (four freshwater, 5 Prospective
marine, incl. Puerto cohort
Rico)
Various
earache
FIO (not identified) 5
n = 50,000. Excess risk and health burden of earaches due to swimming in natural fresh
and marine waters was estimated using for nine beaches across the United States.
Economic and physical burdens were also obtained. Model results were used to
calculate excess risk for earaches attributable to swimming. The overall incidence of
self-reported earache was 1.6% in the 10-12 days after the beach visit. Earaches were
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more frequent in head immersion swimmers compared to non-swimmers for all beach
sites and age groups. Earaches were un-associated with water sample measures of fecal
contamination and turbidity. After adjustment for covariates, we calculated 7.12 excess
earaches among head immersion swimmers per 1.000 swimming events.
Wyer et al.
(2012)
Europe-wide
Experiment Various
Escherichia coli,
intestinal
enterococci and
somatic coliphage
During the EU FP6 Project VIROBATHE a database of over 290 HAdV analyses with
corresponding fecal indicator organism (FIO) determinations was gathered and used to
explore statistical associations between HAdV and FIO results. The FIOs measured
werei?. coli, intestinal enterococci and somatic coliphage. Statistically significant trends
of increasing proportions of HAdV-positive results in categories of increasing FIO
concentration were found in freshwater but not seawater samples. The analysis of these
trends in freshwater samples was refined, the trends remaining statistically significant
when using categories of 0.5 loglO intervals of FIO concentration. Logistic regression
models were then developed to predict the probability of a HAdV-positive outcome
from FIO concentration.
Wymer et al. NEEAR sites, USA EPI (combining Various NGI (vs. total coliforms,
(2013) previous HCGI) fecal coliforms, E.
studies) coli, Enterococcus
The US EPA and its predecessors have conducted three distinct series of
epidemiological studies beginning in 1948 on the relationship between bathing water
quality and swimmers' illnesses. Keeping pace with advances in microbial technologies,
these studies differed in their respective microbial indicators of water quality. Another
difference, however, has been their specific health endpoints. The latest round of
studies, the National Epidemiological Assessment of Recreational (NEEAR) Water
studies initiated in 2002, used a case definition, termed "NEEAR GI illness" (NGI), for
gastrointestinal illness corresponding closely to classifications employed by
contemporary researchers, and to that proposed by the World Health Organization. NGI
differed from the previous definition of "highly credible gastrointestinal illness"
(HCGI) upon which the USEPA's 1986 bathing water criteria had been based, primarily
bv excluding fever as a prerequisite.
Incidence of NGI from the NEEAR studies was compared to that of HCGI from earlier
studies. The ratio of NGI risk to that of HCGI is estimated to be 4.5 with a credible
interval 3.2 to 7.7. Conclusions: A risk level of 8 HCGI illnesses per 1000 swimmers, as
in the 1986 freshwater criteria, would correspond to 36 NGI illnesses per 1000
swimmers. Given a microbial DNA-based (qPCR) water quality vs. risk relationship
developed from the NEEAR studies. 36 NGI per 1000 corresponds to a geometric mean
of 475 qPCR cell-equivalents per 100 ml. Figure 1 shows marine and freshwater
relationships combined.
Xiao et al.
(2013)
Three Gorges
Reservoir, China
(TGR)
QMRA
City wastes,
storm water,
land runoff
GI
Culture
E. coli, total
coliform, fecal
During two successive 1-year study periods (July 2009 to July 2011), the water quality
in Wanzhou watershed of the TGR was tested with regard to the presence of fecal
indicators and pathogens. Salmonella, Enterohemorrhagic E. coli (EHEC), Giardia and
Cryptosporidium were detected in the watershed. Prevalence and concentrations of the
pathogens in the mainstream were lower than those in backwater areas. The estimated
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coliform, fecal
streptococci
MPN-PCR
Salmonella,,
EHEC, enterococci
risk of infection with Salmonella. EHEC. Crvvtosvoridium. and Giardia per exposure
event ranged from 2.9 x 10—to 1.68 x 10—. 7.04 x 10—to 2.36 x lQ^2. 5.39 x 10—to
1.25 x 1Q— and 0 to 1.2 x 10^. respectively, for occupational divers and recreational
swimmers. The estimated risk of infection at exposure to the 95% upper confidence
limit concentrations of Salmonella, Cryptosporidium and Giardia may be up to 2.62 x
10~5, 2.55 x 10~4 and 2.86 x 10~3, respectively.
Yau et al. United States Epi, Wastewater GI, skin Enterococcus, total 4 n = 7,317. Swimmers who swallowed water were more likely to experience GI illness
(2014) (Avalon) prospective effluent- infection, eye coliforms, fecal within 3 days of a beach visit than non-swimmers. Risk elevated when either submarine
cohort impacted infection, ear coliforms, E. coll, groundwater discharge was high or solar radiation was low. The risk of GI illness was
waters, infection incl three rapid not significantly elevated for swimmers who swallowed water when groundwater
groundwater methods for discharge was low or solar radiation was high. Associations between GI illness
Enterococcus incidence and FIB levels (Enterococcus EPA Method 1600) among swimmers who
swallowed water were not significant when not accounting for groundwater discharge-
but were strongly associated when groundwater discharge was high compared to when
it was low.
Young (2016) Marine, Worldwide Survey
Point vs. non-
point
GI,
respiratory,
skin
enterococci
Numerous studies have demonstrated increased GI risk with marine swimming -
typically defined as head immersion: Potential emerging marine threats include
Shewanella and Vibrio bacteria, and the presence of human pathogens in the marine
environment that are resistant to antimicrobials.
Zeigler et al.
(2014)
Las Vega, Nellis
Air Force Base
Epi: case-
control
Cattle ranch
fever,
vomiting,
hemorrhagic
diarrhea
On October 12, 2012, the Nellis Air Force Base Public Health Flight (Nellis Public
Health), near Las Vegas, Nevada, was notified by the Mike O'Callaghan Federal
Medical Center (MOFMC) emergency department (ED) of three active duty military
patients who went to the ED during October 10-12 with fever, vomiting, and
hemorrhagic diarrhea. Initial interviews by clinical staff members indicated that all
three patients had participated October 6-7 in a long distance obstacle adventure race on
a cattle ranch in Beatty, Nevada, in which competitors frequently fell face first into mud
or had their heads submerged in surface water. There were 22 cases (18 probable and
four confirmed) of Campylobacter coli infection among active duty service members
and civilians. A case control study using data provided by patients and healthy persons
who also had participated in the race showed a statistically significant association
between inadvertent swallowing of muddy surface water during the race and
Campylobacter infection ("odds ratio = 19.4: pcQ.OOl').
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Zlot et al. Oregon, Lake Epi, Lake, cause Norovirus
(2015) Regional Park retrospective unknown illness
cohort
Conclusions (includes health linkages)
6 In July 2014, Multnomah County public health officials investigated a Norovirus
outbreak among persons visiting Blue Lake Regional Park in Oregon. During the
weekend of the reported illnesses (Friday, July 11-Sunday, July 13) approximately
15,400 persons visited the park. The investigation identified 65 probable and five
laboratory-confirmed cases of Norovirus infection (70 total cases'). No hospitalizations
or deaths were reported. Analyses from a retrospective cohort study revealed that
swimming at Blue Lake during July 12-13 was significantly associated with illness
during July 13-14 (adjusted relative risk = 2.3: 95% confidence interval TCI1 = 1.1-
64.9). Persons who swam were more than twice as likely to become ill compared with
those who did not swim in the lake.
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E. ACKNOWLEGEMENT
Dr. Audrey Ichida (ICF Consultants) provided the initial set of potential references and was
unfailing in her support of this project. Manuscript reviews were conducted by Dr. Neale
Hudson, NIWA, Hamilton, New Zealand, and by Professor Alexandria Boehm (Department of
Civil and Environmental Engineering, Stanford University, California, USA).
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