Thursday, April 14
10:30 a.m.-12:00 p.m.
Session 6:
Advances in Alternative Indicators
and Measurement
101
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U.S. EPA's 2016 Recreational Waters Conference
Evaluation of Three Culture-Based Methods
for Enumeration of Coliphage from Ambient
Waters
Asja Korajkic, PhD
U.S. Environmental Protection Agency
Abstract
Despite a long history of use, many uncer-
tainties exist about the ability of fecal indica-
tor bacteria to predict the presence of enteric
pathogens in ambient waters. Coliphage densi-
ties could provide a useful alternative approach.
Here, we evaluate the ability of three methods
to enumerate F+ and somatic coliphage from
1-liter samples from Lake Michigan and Trail
Creek waters (n=37 each) collected in 2015.
Methods tested include direct membrane filtra-
tion (MF) on 0.45-um pore size nitrocellulose
filters, deadend hollow-fiber ultrafiltration
combined with single agar overlay (D-HFUF-
SAL) and U.S. Environmental Protection Agency
method 1602 (1602). Overall, somatic coliphage
levels ranged from nondetectable (ND) to 4.38
loglO plaque forming units (PFU) per liter and
were consistently higher compared to F+ (ND to
3.35 loglO PFU per liter), irrespective of method
or water type. Concentrations of both coliphage
types were significantly higher (P < 0.0001)
in creek samples than in lake waters. The MF
method recovered significantly less somatic
(lake and creek) and F+ coliphage (creek)
(P < 0.0001) than either 1602 or D-HFUF-SAL.
Coliphage levels in creek water detected by
D-HFUF-SAL and 1602 were not significantly
different (p > 0.05). In lake waters, D-HFUF-
SAL recovered significantly more F+ coliphage
than 1602 (p > 0.0001), but there was no signifi-
cant difference in levels of somatic coliphage
detected by either method (p > 0.05). Results
suggest that D-HFUF-SAL and 1602 perform
in a similar manner, consistently recovering
greater levels of somatic and F+ coliphage than
the MF method, regardless of water type.
Biosketch
Dr. Asja Korajkic is a microbiologist in
the U.S. Environmental Protection Agency's
Office of Research and Development, National
Exposure Research Laboratory in Cincinnati,
Ohio. She received her bachelor of science
degree in microbiology and doctorate in envi-
ronmental and ecological microbiology from
the University of South Florida. Dr. Korajkic's
research interests include characterizing fate
and transport of bacterial and viral indicator
organisms and microbial source tracking mark-
ers, as well as pathogens in aquatic habitats.
More recently, she has been involved in the
development and optimization of viral concen-
tration/detection methods.
102
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f*"!
Day Two: Session 6
J
iL2a
Coliphage Levels in the Effluents of Wastewater
Treatment Plants Discharging Nearby Great
Lakes Beaches
Marirosa Molina, PhD
U.S. Environmental Protection Agency
Abstract
Coliphages are being considered as poten-
tial surrogates for human viruses and thus an
alternative indicator of fecal contamination
in recreational waters. During the summer
of 2015, the U.S. Environmental Protection
Agency conducted a field study to determine
the seasonal and temporal variability of coli-
phages in selected Great Lakes (GL) beaches.
Complementary to this work, a study was
conducted to determine coliphage levels in
the effluents of wastewater treatment plants
(WWTPs) in close proximity to the selected
beaches, with the objective of obtaining point
source information for future modeling exer-
cises of coliphage densities. The study was con-
ducted from June 9 through September 9, 2015.
Effluents from four WWTPs serving the Great
Lakes region were analyzed for somatic and
male-specific (F+) coliphages on a weekly basis
using both the double agar overlay (DAL) and
the deadend hollow-fiber ultrafiltration with
single agar overlay (HFUF-SAL) methods. Based
on the variable concentrations across treatment
plants, both analytical methods were neces-
sary to properly detect the presence of phages
in the various effluents. Concentrations ranged
from 1 to 4.7 log PFU/L for F+ and from 1 to
4.67 for somatic phages in the effluents of three
WWTPs, with both types of coliphages detected
at similar concentrations per individual plant.
No coliphages were detected in one of the
plants throughout the course of the study. The
high variability observed in coliphage densities
between treated WWTP effluents highlights the
importance of these data when explaining the
dynamics of microbial contaminants in nearby
beaches. These results will be combined with
fate and transport information and relation-
ships to human pathogens and health data will
be explored to better understand the potential
advantages of coliphage as indicators of micro-
bial water quality.
Biosketch
Dr. Marirosa Molina is a research micro-
biologist with the Exposure Methods and
Measurement Division of the National Exposure
Research Laboratory located in Athens, Georgia.
Dr. Molina has a bachelor of science degree in
industrial microbiology from the University of
Puerto Rico-Mayagiiez, and a master of science
degree in microbiology and doctorate in ecology
from the University of Georgia. Her research
focuses on (1) assessing the fate and transport
of microbial contaminants, microbial source
tracking markers, and pathogens from point
and nonpoint sources in watersheds through
the application of field, laboratory, and model-
ing approaches; and (2) studying the response of
microbial communities to land use and climate
changes, including extreme events impacting
agricultural and urban landscapes. Dr. Molina
also has participated in the development and
evaluation of Virtual Beach, a software tool
designed to predict the concentration of fecal
indicator bacteria in surface waters using mete-
orological, hydrological, and physicochemical
parameters in an effort to provide nowcast capa-
bilities to beach managers and stakeholders.
103
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U.S. EPA's 2016 Recreational Waters Conference
SERA
Coliphage Levels
in the Effluents of Wastewater Treatment Plants
Discharging Nearby Great Lakes Beaches
MarirosaMolina1, Kelvin Wong2, Mike Cyterski1 , Richard Zepp1, and Gene Whelan1
'ITS Environmental Pretention Agency. ORD, Athens, CtA
2Uak Kidge institute for Science and bducation h ellow. Athens. (JA
The views expressed in this presentation are those of the authors and do not necessarily reflect
the views or policies of the U.S. Environmental Protection Agency
Introduction
Coliphages as alternative indicators
Understanding sources of contamination
critical
Objective
Waste Water Treatment Plant
coliphage contributions
F &T modeling = source
F+ or somatic col i phages
Is there a difference?
oEPA 201 5 Beach Study Sites
Michigan City Sanitary District, IN
Discharge: 12 MGD
Disinfection: Chlorine gas diffusion of tertiary effluent
Northeastern, OH
Discharge: 26 MOD
Disinfection by chlorination (Sodium
hypochlorite)
South Milwaukee and
Milwaukee South Shore, Wl
Discharee: 6 MGD
UV Disinfection
bysiem
Discharge: 90MGD
Disinfection by
Chlorination ot
secondary effluent
104
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Day Two: Session 6
oEPA
Sampling and Analysis
Samples collected
directly from WWTP
effluents
One sample /week
I4 weeks
- June - Sept 201 5
Analysis
- HFUF SAL
- DAL
Targets
F+ coliphages
Somatic coliphages
Aeration Tank
Primary
Rprlimentatinn
oEPA
Results
Both types of coliphages
were detected at similar
concentrations in the
effluents of the WWTP,
Northeastern OH had a
significantly higher
concentration relative to
the other three plants
No phages were detected
at Michigan City {which Is
in dose proximity to
Washington Hark) except
for one instance during
August 7D1S
uoiipnage Densities in waste v
i reatment riant trtiuents
Nortneastem u
li
oEPA
Results
FIB in WWTP Effluent*
Michigan City
Wnrthpayprn, flH
-jf
s s 5? 5r Sr
South Milwaukee
. 0 #»¦
%*
B«afiaaas
H 8 s a § 9 a s S
MitwdukccSuuLh Sl iutc
0
o n
mmm
o 5
/-V " .
sslllsssp
C 5 &¦ C! C I! C
SERA
Results
Correlation between Fl B and Coliphages Monitored In WWTP
Effluents
WWTP Plant
FIB
Coliphage
R7
£ coli
F+
0.09
Milwaukee South Shore
(MPN/100 ml)
Somatic
0.11
F+
0.42
South Milwaukee
Fecal Coliforms
Somatic
0.31
£ coli
h+
cum
Northeastern OH
(CFU/100 ml)
Somatic
0.01
* Michigan City did not have enough coliphage densities to develop a correlation
v>EPA
WWTP Characteristics
Northeastern
Ohio
South
Milwaukee
Milwaukee
South Shore
Michigan
Gty
Nearby Beach
Frigewater
Grant Park
Grant Park
Washington Park
Outfall
L. Eric
L Michigan
L. Michigan
Trail Creek
Design How
(MGD)
35
6
300
15
Average Flow
(MGD)
26
6
DO
12
Disinfection
chlori nation
(sodium
hypochlorite)
UV
rhlorination nf
secumJaiy effluenl
chlori nation of
tti lidiy effluent
FIB (method)
F mS
(Method 1603
modified m-Tec
fecal
Coliform
E. coli
(enzyme substrate.
5M method 9223
B)
F. cnli
(Method 1603
modified m-Tec)
Correlation
Low
High
Low
N/A
*>EPA
Results
Solar Irradiation Experiment
Phage photoinactivation could be
described by first order kinetic
expressions.
* Heray* of up to «pv*>n order* of
magnitude were observed.
Surrogace somatic phage exhibited
the largest decay > F+RNA/Somatic
phage Communities > Surrogate F +
KNA
Process Relationships for Evaluating the Role of Light-induced Inactivation of Coliphages at Selected
Beaches and Nearby Tributaries of the Great Lakes. Richard G. Zcpp1, Mamosa Molina1, Mike Cytcuki1, Gene
Whelan1, RaibirParmar1, Kelvin Wong2, Brad Acrev3, and Kama Ueorgacopoulos3
105
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oEPA
Summary of Results
Both types of coliphages were detected at similar concentrations.
No correlation was identified between coliphages and Ecoh in any of the
WWTP effluents rested However, a significant correlation was ohservert
between fecal coliforms and both types of coliphages in one of the treatment
plants.
Disinfection treatment, treatment capacity??
U.S. EPA's 2016 Recreational Waters Conference
oEPA
Final Considerations
The potential impact of the WWTP on adjacent beaches based on the phage
effluent concentrations is as follows:
- Edgewater > Grant Park > Washington
- For FIB : Grant Park^Edgewater>'Washington
- Basically, no phages were detected in the effluent of Michigan City WWTR
therefore, any phages detected at Washington Park arc likely coming from
other sources in the watershed.
&EPA Next steps
Factors affecting fate and transport
- Effect of sunlight irradiation
- Interaction with organic matter
* Predictive vs. process modeling approaches
TtotksS
106
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Day Two: Session 6
Advances in Measurements and Indicators for
Determining Shellfish Growing Area Classifica-
tions Adjacent to Wastewater Treatment Plant
Outfalls
Yaping Ao
U.S. Food and Drug Administration
Abstract
Since 1987, the U.S. Food and Drug
Administration (FDA) has recommended at
training courses and other venues the use of a
1000:1 dilution as the minimum level of dilu-
tion needed around a wastewater treatment
plant (WWTP) outfall to mitigate the impact of
viruses. In 1995, this estimated level of necessary
dilution was further calculated and explained
by FDA using assumptions based on the most
relevant scientific literature available at that
time. Since then major advances in the detection
and enumeration of norovirus in wastewater
and shellfish have been made, and advances in
fluorometer technologies have enabled more
sophisticated and accurate hydrographic dye
study methods. Using these advances, FDA has
conducted hydrographic dye dilution studies
within estuaries of various geographic locations
and conditions. Shellfish sentinels placed at
various dilutions were tested for enteric viruses
and male-specific coliphage. This has afforded
FDA, for the first time, with a means to directly
determine the viral risk posed by WWTP efflu-
ent on shellfish resources. The results of these
studies provided the scientific basis behind FDA's
dilution guidance that was recently adopted
into the National Shellfish Sanitation Program.
The results also have proven valuable for a joint
United States/Canada quantitative norovirus risk
assessment for molluscan shellfish as well as cali-
bration and validation of hydrodynamic models
of WWTP discharges to growing areas currently
being developed by FDA. The data collected
might additionally support future forecasting
models used to predict the sanitary impacts to
growing areas attributed to forecasted storm-
related events.
Biosketch
Ms. Yaping Ao is a visiting associate
with the U.S. Food and Drug Administration,
Center for Food Safety and Applied Nutrition
in College Park, Maryland. She serves as a lead
modeler in the application of computer fate and
transport models to assess pollution source
impacts to shellfish growing areas. She assisted
with the development of dilution models to
support a joint U.S.-Canada norovirus risk
assessment and has provided training on and
led specialized field and hydrographic studies
to identify and assess pollution sources in the
environment. Ms. Ao's expertise includes sup-
porting the development of guidance for irriga-
tion water use for produce safety. She received
her master of science degree in civil engineering
from Marquette University in Wisconsin and
her bachelor of science degree in environmental
engineering from the Chengdu University of
Technology, China.
107
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U.S. EPA's 2016 Recreational Waters Conference
U.S. Food and Drug Administration
Protecting and Promoting Your Health
Advances in Measurements and Indicators for
Determining Shellfish Growing Area
Classifications Adjacent to Wastewater
Treatment Plant Outfalls
Yapinq Ao1. Gregory Goblick', Steven TidwelP, Eric Tate1, Julie Anbarchian',
Jacqueline Woods2, William Burkhardt, III2, and Kevin R. Calcl2
'Office of Food Safety, Center for Food Safety and Applied Nutrition,
US FDA, College Park, MD 20740
2Gulf Coast Seafood Laboratory, Center for Food Safety and Applied Nutrition,
US FDA, Dauphin Island. AL 36528
le in format ion contained in trvs presentation e unpublished The data, the presentation slides, or portions or the presentation sfx»s may
National Shellfish Sanitation Program MO Section II
Chapter IV - Shellfish Growing Areas
@.03 - Growing Ana Classification
F, - Prohibited Classification
(5) - Wastewater Discharges
0,\) An area classified as prohibited shall be established
adjacent to each sewage treatment plant outfall or any
other point source outfall of public health significance
Disclaimer Some of trie intormaton
) this presentation is unpuDisheo Theoata, the presentation slides, or portions of the presentation sltoes may
Why are Enteric Viruses a Concern?
Viruses have a longer survival time than the coliform bacteria group
Viruses are more resistant to disinfection and may be found in treated
WWTP effluent
Build-up of virus levels can occur in poorly flushed estuaries
Shellfish can bio-accumulate viruses >50 fold overlying waters
Bacterial indicators do not adequately index viral risk posed by WWTP
effluents - especially for conventional WWTP treatment
YoC^g|00ffrt rt.
Tim data. Uie presentation slides, or portions of the presentation slides rnay
Advances in Indicators
Male-Specific Coliphaae:
¦ Virus which infect coliform bacteria
¦ Culturable using rapid, inexpensive techniques
¦ Chlorine resistant
¦ More resistant to environmental stresses than fecal coliforms
Good indicator of:
¦ Raw or treated municipal sewage
¦ exfiltration from a sewage collection system
¦ overflows or spills from a collection system
¦ viral impact on shellfish from the above sources
50 PFU/100 g (shellfish) - has been adopted by the NSSP - re-
opening after sewage contamination events from WWTPs
100 PFU/100 g (shellfish) - level at which outbreaks in EU shellfish at
market begin to occur
Advances in Measurements/Tools
Submersible Fluorometers:
High sensitivity
Less expensive
Small and light weight
No calibrations - regressions for dye
No false positives from air or carryover
Can use in moored mode
Can use In plume tracking mode
Several parameters possible
Advances in Analytical Tools
Hydrodynamic and Transport Models:
¦ Models were calibrated and validated against dye studies and
were used to evaluate wet weather driven pollution events
¦ Models allow different conditions and factors to be assessed
that were not captured during dye study
¦ Can be used for comparing factors to determine worst case and
useful for management and classification of growing areas
lunpubtshed Thedeta, toe presentation glides,orportions ot tin presentation shdesmey
108
-------
Day Two: Session 6
FDA studies
In recent years FDA has conducted >50 hydrographic dye dilution studies
Since 2007, FDA has conducted combined dilution/shellfish meat bio-accumulation
studies:
¦ Gulf Coast - AL", MS*
¦ Atlantic Coast - VA
¦ West Coast - OR, WA. CAA
¦ East Coast - ME*, NH", MA", Rl", CT
** Seasonal data
** Starting to add to seasonal data
* WWTPonly
A Recently conducted - not included
216 samples determined dilution and analyzed shellfish for Male-Specific Coliphage
(MSC) and 161 of these for Human NoroVirus (HuNoV)
Samples collected collaborative effort with States and Industry
¦ Many additional samples provided by Spinney Creek Shellfish
¦ On-going studies/samples with: CT, NH, WA
Shellfish species: pacific and eastern oysters, hard and soft-shell clams, mussels
FDA Field Studies
Example: Typical Technical Assistance & Research Study
WWTP Disinfection technologies: conventional chlorination, UV, Membrane
in mis presentation s unpublished Theaeta, tne presentation slides, or portions of tne presentation slides may
Disclaimer. Some ot the intormaton contained in tms presentation/sunpubtshed Theoata, the presentation sudes, or portions of tne presentation sloes may
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in mis presentation is unpublished The data,
the presentation skies, or portions of the
presentation slides may not be further
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In the Royal and Cousins River for
Approximately 6 days. This indicates that
once a pollutant is released it wll remain in the
Rivers for at least 6 days
A ,a ,i% i>a1. <
5/24 5'25 5/26 5/27 5/28 5/29 5/30 5/31
Contmous Headings SuBmerisBle Huorometer
o stMtiy sat# W. i iflai Day p«k in
-V- steaay star# 1/2 liaal uayAvofage
- siMrty state 1/7 Ttrtal Day I rui Tirtm Concentration
50 Meter Buffer Plume Traekino FHiorometer (Wei Labs)
Yarmouth, ME - Dye Study Results
Concentration of 5-Point Moving Average on May 24-26, 2010 (Accumulated[
0.6!6, M6iS
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26280-13140
Some ottne information contameci m tnispresemi!:.-
« unpublished The data, the presentation slides, or portions ot h
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109
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Yarmouth, ME - Empirical Model Results
Concentration of 5-Pomt Moving Average on May 24-26, 2010 (Accumulated.
U.S. EPA's 2016 Recreational Waters Conference
- Original 2010 Study Clam Sampling Station
(sj) - S1 offset to north shore Clam Sampling Site
(sb) - SB Upper 2011 Line (2012 station)
@) - Upper flats 300 yards from landing (2012 station)
(so) - Above outfall and below marina (201? station)
($e) - Adjacent to outfall on northern shore (2012 station)
Sampling I lata
Disclaimer Some of the Intormatori contained in Hits presentation e unpublished Vmdata, the presentation slides or portions ol the presentation slides rnay
not be turner disseminated
MSC and FC in Effluent versus Instantaneous Flow Rate
Yarmouth WWTP - April 2012 Wet Weather Study
12 3 4
Instantaneous Flow Rate (MGD)
Disclaimer. Same ot the intormaton contained in this presentation is unpublished Thedata, Vie presentation slides, or portions oUbe presentation slides may
not tie further nissemmatea
Hydrodynamic Model Simulation
Determine 1000:1 Dilution under wet weather
I SMWiCagei
Track Data
Cooc#ntratkm ppb
disclaimer Stirhft ol the In formation contained n this presentation
s unpublished i negate. the presentation Hides, or portions of the
protxntotion otdoo may not bo turthor disseminated '
110
-------
Day Two: Session 6
anoiitsn » so wui lugg
10000 -
:Vi 1 :
,00
..
** 4$
*
*> ^
"
+ ~
Dilution in Receiving Water
A Virginia (non-detect)
v Maine
v Maine (non-deled)
O Washington
New Hampswre
u New nampsnire (non-oetect)
Connecticut (non-oetect)
vnkaiKJ in i/«a pnaviiMivii ts uiifjuoimiKiJ TrmOab, v*t iHv&atiaUuii snues, vi puiliuim uf iik fimtmnueun sSUes
Mean Male-3(jecific Culipliaye ill 31 ic llfish v. a. Mean Dilutiui)
(All data - Removing "Malfunction", "Primary", and "Membrane")
i
>.
4-
Mean Dilution
| Mean MSC during "Normal" WWTP operation
- Cegrecon at mean MSC ((?' - ©0091)
Keyccssiwi uf oil M3C (It' - 0.4033)
Virginia (non-doled)
in mis fxumnilaHijii Ki uiiiMiiHsiiaJ. T/inUaKJ, Uk pnfsotHttlfoi sMts, in fiuiifijini v! u*t piazamn
Conclusion
¦ These new advances in tools has enabled FDA to develop
Dilution Guidance on how to size a prohibited buffer zone
protective of viral impacts
¦ When WWTPs operating within "normal" conditions results fall
below MSC end-point within the 1000:1 recommended dilution
¦ 1000:1 does not appear to be applicable for:
¦ When WWTPs "malfunction" frequently - such as bypass
primary or secondary or change operations frequently
causing degradation of effluent
¦ Unconventional treatment technologies that have not been
validated -eg some membrane technologies
¦ WWTPs with only primaiy treatment
Disclaimer, some of the mtormaton contained in this presentations unpublished Ihedata, the presentation sities, or portions of trie presentation slides may
not be further disseminated.
Uses of MSC
¦ To characterize Ihe performance ofWasle Water
Treatment Plants (WWTP).
¦ To characterize the shellfish growing area catchment,
the pollution sources and strength.
¦ To classify shellfish harvest areas that are adjacent to
WWTPs.
¦ To manage sewage spill events and to determine when
shellfish can be safely harvested again after the event.
¦ To manage shellfish relaying operations where shellfish
are moved to clean environmental waters for long term
cleansing.
¦ To assess viral illness events associated with shellfish.
Disclaimer: Some ot theintormaton contained m this presentation is unpublished Ihedata. foe presentation slides or portions of the presentation slides may
no? be further disseminated
A zkn o V-'J y d i\ ysnafj i
Treatmwt Plant BBP|
Spinney Creek Shellfish
Maine Department of Marine Resources
Joint Canada-U.S. Risk Assessment working group members
hDA employees that have assisted on field studies
DHI
111
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U.S. EPA's 2016 Recreational Waters Conference
Coliphages as Indicators of Gastrointestinal Ill-
ness in Recreational Waters: A Pooled Analysis
of Six Prospective Marine Beach Cohorts
Jack Colford, Jr., MD, PhD
University of CaliforniaBerkeley
Abstract
Background: Coliphages have been pro-
posed as potential indicators of fecal contamina-
tion of marine recreational waters because they
might be able to predict the presence of viruses
better than fecal indicator bacteria. We esti-
mated the association between coliphages and
gastrointestinal illness.
Methods: We pooled data from six pro-
spective cohort studies conducted from 2003 to
2009 that enrolled beachgoers in the summer
at coastal beaches in Alabama, California, and
Rhode Island. Studies collected water samples
and recorded incidents of gastrointestinal ill-
ness within 10 days of the beach visit. Samples
were tested for male-specific and somatic
coliphage using U.S. Environmental Protection
Agency (EPA) methods 1601 and 1602. We
estimated cumulative incidence ratios (CIRs) for
the association between gastrointestinal illness
and coliphage when human fecal pollution was
suspected to be present ("high-risk conditions")
and not present ("low-risk conditions").
Results: Under high-risk conditions, a
1-loglO increase in male-specific coliphage levels
was associated with a CIR of 1.30 (95% CI 0.94,
1.81) (EPA 1601; n=6 beaches) and 2.20 (95%
CI 1.30, 3.71) (EPA 1602; n=2 beaches); under
low-risk conditions the CIRs were 0.83 (95%
0.70, 1.00) (EPA 1601) and 0.71 (95% 0.19, 2.72)
(EPA 1602). The CIRs for a 1-loglO increase in
somatic coliphage (EPA 1602) were 1.27 (95% CI
0.92, 1.76) under high-risk conditions and 0.98
(95% 0.82, 1.16) under low-risk conditions (n=2
beaches).
Conclusion: Coliphage was associated with
increased gastrointestinal illness risk at beaches
with suspected human fecal pollution.
Note: This abstract does not represent EPA
policy.
Biosketch
Dr. Jack Colford is a professor of epide-
miology at the University of California (UC),
Berkeley School of Public Health. He trained at
Johns Hopkins University (doctorate in medi-
cine), Stanford University (chief medical resi-
dent), UC San Francisco (residency in internal
medicine and fellowships in infectious diseases
and HIV/AIDS), and UC Berkeley (doctorate in
epidemiology). Dr. Colford has served as the
principal investigator for numerous random-
ized controlled trials and observational stud-
ies evaluating the impact of water, sanitation,
and hygiene interventions in India, Bolivia,
Guatemala, Bangladesh, Kenya, Mexico, and
the United States. His research has been sup-
ported by the National Institutes of Health,
Centers for Disease Control and Prevention,
U.S. Environmental Protection Agency, and
Gates Foundation. He teaches courses each year
at UC Berkeley on epidemiologic methods, the
design of randomized controlled trials, and
impact evaluation for health professionals.
112
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f*"!
Day Two: Session 6
J
iL2a
EPA's Development of Recreational Water
Quality Criteria for Coliphage: Updates and
Experts Coliphage Workshop Overview
Sharon Nappier, PhD
U.S. Environmental Protection Agency
Abstract
Recreational Water Quality Criteria
(RWQC) are recommendations intended to be
used by states in adopting water quality stan-
dards (WQSs) to protect the designated use of
primary contact recreation. WQSs are then used
to develop point source permits, to identify
impaired waters, and for beach notifications.
Historically, RWQC recommendations have
been based on fecal indicator bacteria E. coli and
enterococci. The U.S. Environmental Protection
Agency (EPA) is now evaluating coliphage, a
viral indicator, to help prevent viral associ-
ated illnesses. EPA recently held an experts
workshop to engage a group of internationally
recognized experts on the state of the science of
coliphage and their usefulness as a viral indica-
tor for the protection of public health. Topics for
discussion included the need for a viral indica-
tor, coliphage as a predictor of gastrointestinal
illnesses, coliphage as an indicator of waste-
water treatment performance, male-specific
vs. somatic coliphage, a systematic literature
review of viral densities, and data gaps and
future research. EPA will provide an overview
of the recent Experts Coliphage Workshop and
an update on the development of RWQC for
coliphage.
Biosketch
Dr. Sharon Nappier specializes in environ-
mental microbiology and quantitative microbial
risk assessment and has more than 13 years of
national and international experience work-
ing on foodborne and waterborne diseases;
microbial method development and evaluation;
program and contract management; national
water policy development; and science com-
munication. Dr. Nappier received her master's
degree from the University of North Carolina
at Chapel Hill in environmental sciences and
engineering and her PhD from the Johns
Hopkins Bloomberg School of Public Health in
environmental health engineering. She has been
working at the U.S. Environmental Protection
Agency for the past 6 years. Her major projects
include chairing the 2012 Recreational Water
Quality Criteria workgroup; leading efforts to
develop recreational water quality criteria for
coliphage; and assessing the microbial risks
associated with direct potable reuse. Since 2011,
Dr. Nappier also has served as a professorial
lecturer at The George Washington University's
School of Public Health, teaching applied envi-
ronmental health microbiology.
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U.S. EPA's 2016 Recreational Waters Conference
Recreational Water Quality Criteria for Coliphage;
Updates and Experts Workshop Overview
Sharon P Nappicr, MSPH, PhD
Office of Water, Office of Science and Technology
US Environmental Protection Agency
April 14, 2016
Outline
Recreational Water Quality Criteria
Experts Workshop
Next Steps
Clean Water Act (CWA)
Goal: Restore and maintain oceans, watersheds, and
their aquatic ecosystems to protect human health,
support economic and recreational activities, and
provide healthy habitat for fish, plants and wildlife.
Establishes basic structure for state water quality
standards, including regulation of pollutant discharge
into the waters of the United States.
Recreational Water Quality Criteria (RWQC)
Intended to be used by states adopting water quality standards
to protect the designated use of primary contact recreation.
BEACH ACT (CWA 304(a)(9)(B)) requires EPA to review coastal
RWQC every five years (next review: 2017)
RWQC recommendations:
Prevent illness
By preventing fecal contamination and/or pathogens from entering surface
waters
Point source permits (NPDES permits)
Identify impaired waters
303(d) Listing, Total Maximum Daily Loads (TMDLs)
Identify potentially hazardous conditions
. Beach notifications
Conceptual Model
rz
Fecally-Associated Pathoger
s in Fresh and Marine Waters
1
1
1
1 1
Noti-poinl n
n off | | Wastewater discharge
1
CSOs/SSOs | | Septic systems
i
1
1
I
Media
Route
Receptors
Endpoints
Freshwater (inland
rivers and lakes)
Coastal marine waters
(including Great Lakes)
Adults and children recreating in
fresh and marine waters
Gastrointestinal
1 Respiratory J [ Dermal 1 J Far j
! _ illness 1 I irritation J 1 liifection_ 1
2012 Recreational Water Quality Criteria
The 2012 RWQC for primary contact recreation are associated with
bacterial indicators of fecal contamination.
Highlights:
Indicators:
Enterococci (marine and freshwater) and £ coli (freshwater)
Specified magnitude, duration (30 day), and frequency
Two sets of recommended criteria, each corresponds to a
different illness rate
Includes supplemental tools
qPCR method for same-day notification
Beach action values for precautionary notification
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Day Two: Session 6
Current Status
To prevent illness
Bacterial pathogens targeted through bacterial indicators
Historically bacteria were thought to cause majority of illnesses
Wastewater treatment improvements and permits based on bacterial indicators
effectively control bacterial pathogens
QMRA, epidemiological, and microbial water quality studies indicate viruses cause
majority of swimming-associated illnesses in human-impacted waters
Current treatments, indicators, and permits do not specifically target viruses
Thus, viruses enter surface waters from treated & untreated human sources
To identify impaired waters or potentially hazardous
conditions
Culturable bacterial indicators used
Effective at predicting bacterial impairments of water quality
Epi studies indicate they may not always be predictive of viral illnesses
Coliphage - a viral indicator
In use since the 1970's:
EPA: Ground Water Rule recommended coliphage to
detect and/or quantify viral indicators in ground water
ISSC/FDA: Evaluating the use of male-specific coliphage
for shellfish bed closure decisions
NWRI: Framework for Direct Potable Reuse recommends
coliphage be used as a surrogate for evaluating virus
removal in reuse configurations
Coliphage-a viral indicator
Recreational Water Quality Criteria - Coliphage
Coliphage advantages:
F+ coliphage . j
Of fecal origin/highly concentrated in sewage
Infect via cell wall
Physically similar to enteric viruses of concern
hRb
Similar persistence patterns to enteric viruses of concern
To treatment and to environmental insults
No appreciable re-growth in ambient waters
Somatic
/ \ ^^^^^aftcoliphage
| j Infect via F-pili
Non-pathogenic
Indicators rather than pathogenic viruses:
Male-Specific (F+) Coliphage Somatic
and Host Coliphage and Host
Currently not feasible to assess all pathogenic viruses due to
methodological and time constraints
Recreational Water Quality Criteria - Coliphage
Prevent viral illness V
Coliphage-based discharge permits can prevent viruses
entering source waters, thus preventing viral illnesses
Identify impaired waters or potentially hazardous
conditions!/
Epidemiological studies indicate coliphage may provide a
tool to better protect from viruses
Coliphage Experts Workshop: March 1-2, 2016
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U.S. EPA's 2016 Recreational Waters Conference
Coliphage Experts Workshop
Purpose: Have internationally recognized experts engage on the
topic of how best to protect public health from viral
contamination of water given currently available information.
Specific Goals:
Obtain input on science questions from experts in fields of
environmental microbiology, microbial risk assessment, and
environmental epidemiology.
Gather scientific insight to determine the best coliphage type
(male-specific and/or somatic) for use in CWA 304(a) criteria.
Identify situations where these coliphage types may be most useful for
preventing illnesses and identifying impaired waters
Identify research needs that can be addressed by 2017.
Coliphage Experts Workshop - Experts
Name
1 Affiliation
Nicholas Ashbolt
U niversity of AJ berta
William Burkhardt
U.S. Food and Drug Administration
Kevin Calci
U.S. Food and Drug Administration
Jack Colford
University of California, Berkeley
John Griffith
Southern California Coastal Water Research Project
Vincent Hill
Centers for Disease Control and Prevention
Juan Jofre
University of Barcelona, Spain
Sanitation Districts of Los Angeles County
Rachel Noble
University of North Carolina, Chapel Hill
Joan Rose
Michigan State University
Mark Sobsey
University of North Carolina, Chapel Hill
Timothy Wade
U.S. Environmental Protection Agency
Coliphage Experts Workshop Scope
Focused on recreational risks associated with fecal
contamination
Other risks not considered: sunburns, shark attacks, etc.
« Focused on science aspects of criteria development
Minimized policy and implementation discussions
Coliphage Experts Workshop - Topic Areas
1. Need for a Viral Indicator
2. Coliphage as a Predictor of Gastrointestinal Illness
3. Coliphage as an Indicator of WWTP Performance
4. Male-specific vs Somatic Coliphage
5. Systematic Literature Review of Viral Densities
Coliphage Experts Workshop - Meeting Format
Experts assigned a topic with associated charge questions
Experts provided written responses to charge questions to
EPA prior to Workshop
Responses compiled and provided to all experts prior to
Workshop
Each expert gave 10-15 min presentation, based on their
answers to charge questions
Group collectively discussed charge questions
Group captured main points in discussion summary
Coliphage Experts Workshop - Highlights (1)
Topic 1: Need for a Viral Indicator
Individual experts agreed that viruses are a source of illness in
recreational water exposures.
Viruses can enter surface waters via WWTP effluent.
Especially during wet weather and when WWTPs exceed design flows.
Coliphages are more similar to human pathogenic viruses compared to
the traditional fecal indicator bacteria (FIB).
Mimic human pathogenic viruses.
Coliphages have demonstrated value added for managing risks and are
used full-scale to address WWTP water quality and related applications.
Ex: NC reclaimed water, Ground Water Rule, and by FDA for reopening
shellfish harvesting areas after catastrophic spills.
Coliphage methods are available, inexpensive, and could be developed
into easy-to-use commercial kits.
Faster methods (less than 8 hours) are available.
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Day Two: Session 6
Coliphage Experts Workshop - Highlights (2)
Topic 2: Predictor of Gl Illness
Future epidemiological studies should specifically include
coliphages as measured indicators.
Topic 3: Indicator of WWTP performance
Coliphages are consistently present in municipal sewage, and
provide a baseline for looking at different WWTP processes under
varied conditions.
Some experts indicated the literature suggests coliphage and human
viruses have more similar log-reductions during wastewater
treatment, compared to traditional FIB.
Coliphage Experts Workshop Highlights (3)
Topic 4: Male-specific vs Somatic Coliphages
Opinions ranged on whether somatic, male-specific coliphage, or both
would be better for various applications.
Evidence for both showing relationship to Gl illness.
Male specific coliphage behave more similarly to RNA viruses under some
conditions and are currently used successfully by J-DA/ISSC.
Somatic may persist longer than male-specific coliphage and may be present
in greater concentrations in raw sewage.
Hosts are available that can detect both.
Topic 5: Review of Viral Densities
Individual experts supported how the systematic analysis was structured
and conducted.
Coliphage Experts Workshop - Products
Presentations:
2016 UNC Water Microbiology Conference (May 2016)
Publications:
Fact-sheet (summer 2016)
Peer-reviewed Proceedings Report (winter 2017)
Status and Timeline
I date
I milestone
04/17/2015
Review of Coliphages as Possible Viral Indicators of Fecal
Contamination for Ambient Water Quality
10/15/2015
Stakeholder Webinar
03/01/2016
Coliphage Expert Workshop
fact sheet (summer 2016) and proceedings (winter 2017)
2016
Listening Sessions/Webinars
Conferences (New Orleans and Chapel Hill)
States
Other stakeholders (industry/environmental groups)
summer 2016
Analytical method multi-lab validation
late 2017
Draft Criteria released for public review
Questions?
Contact:
Sharon Nappier
Nappier.Sharon(5>epa.gov
(202)566-0740
wafer? ar& Tn&
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U.S. EPA's 2016 Recreational Waters Conference
Question & Answer Session
Question 1
Mark Sobsey: I'm glad to see the interest in viruses. I have a comment to Asja [Korajkic]. The EPA
study involved three methods. There are other methods, why weren't some of them included? MPN
[most probable number] 1601 is easy to do and less expensive, but it takes longer because there are
two steps. There is membrane filtration and an adsorption dilution method. If you took that method
and stuck it in a dilution and waited, I think the dilution would be more successful. You could then do
another assay. If you revisit, you should consider looking at this.
Answer 1
Asja Korajkic: Thanks for developing all those methods. When we looked between methods
1601 and 1602, we wanted to take the standard methods. So, it added 1 day for 1601, which is
why we didn't use it. It was because of the logistics. All your points are great, and we have
a system for alternative methods. This is to get the ball rolling. These aren't the end-all-be-
all. I'd love to try to modify some other methods, especially to get results in a shorter time
frame: 16 hours or so. All of these methods were initially developed for groundwater and
we're trying to get them suitable for surface water. Dilution might improve it, but we opted
to go for simplified versions of the methods first.
Comment 1
Mark Sobsey: I think there is a future for reducing the time for results. There is an opportu-
nity for revisions of these methods.
Question 2
Dan Shapely: Regarding wastewater treatment, in New York there has been some analysis and push
for new standards. Our plants, with holding time, can't meet enterococcus standard but can meet E.
coli standard. They require smaller holding tanks than other states require. Have there been any studies
about holding times in wastewater treatment with coliphages or viral indicators?
Answer 2
Marirosa Molina: When we talked to WWTP [wastewater treatment plant] operators, they
were very helpful and cooperative. It was interesting that Michigan City has a specific res-
ervoir for exposure to chlorine; it goes above the minimum of 15 minutes. That has some-
thing to do with it. We also found no coliphage at the plant that never exceeded their E. coli
standard.
Answer 2 (follow-up)
Sharon Nappier: Free chlorine is different though, so it depends what they use for
disinfection.
Question 3
Ali Boehm: For your low versus high risk, did you categorize sources? Why isn't there a relationship
with low-risk beaches?
Answer 3
Jack Colford: Yes, we did parse based on times of day. I don't know about lack of relationship.
Maybe there was not enough evidence yet.
Answer 3 (follow-up)
Steve Weisberg: Phages are not as abundant in the environment as fecal indicators. So, you see
a lot more minimums in the study. That is one reason for low numbers in low-risk condi-
tions, there are lots of nondetects.
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Day Two: Session 6
Answer 3 (follow-up)
Sharon Nappier: All those samples were under 100 mL [milliliters]. Future studies for EPA
will sample using 2 L [liters]. I recommend we include the larger volumes so there is a better
chance of catching coliphage
Question 4
Phil Scanlan: I'm here to try to share what we did in New Jersey. Many states have told me they want
to cut the amount of pollution at their beaches in half by 2020. In 2013, the average number ofexceed-
ances was 10 to 12 percent of beaches. If we change to coliphage, how does that change exceedance s?
What will the exceedance be then?
Answer 4
Steve Weisberg: That is a fair question but it is complicated. It depends on the threshold.
Question 5
Linda Pechacek: When you discussed the results of your study, you said there were two possible fac-
tors: disinfection time and flow. And, one plant was operating near capacity and one was operating far
below its permitted flow. Could there be a third factor, like tertiary treatment in Michigan City?
Answer 5
Marirosa Molina: Yes, there could be. They do have tertiary treatment as well. There are a
number of factors affecting things.
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