U.S. EPA’s 2016 Recreational Waters Conference Session 3: Recreation Water Monitoring and Implementation Challenges/Successes


Wednesday, April 13
1:20 p.m.-2:50 p.m.

Session 3:

Recreation Water Monitoring and
Implementation Challenges/Successes

33

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U.S. EPA's 2016 Recreational Waters Conference

Monitoring Beaches Statewide in
Michigan for E. coli with qPCR (USEPA
Draft Method C)

Shannon Briggs, PhD

Michigan Department of Environmental Quality

Abstract

In 2015, Michigan initiated a statewide,
rapid beach testing program by providing
10 laboratories with $500,000 worth of qPCR-
related equipment. In collaboration with
Michigan State University (MSU) and the U.S.
Environmental Protection Agency (EPA), labo-
ratory personnel are being trained to use the
EPA's Draft Method C: Escherichia coli in Water
by TaqMan Quantitative Polymerase Chain
Reaction (qPCR). The training effort includes
developing manuals containing standard oper-
ating procedures that can be easily followed by
laboratory staff. Michigan's qPCR network of
16 labs is connected with the MiqPCR listserv
hosted by MSU. Beaches will be posted sooner
and reopened faster because test results will
be available the same day. Monitoring results
are posted on Michigan's BeachGuard website
at http://www.deq.state.mi.us/beach/. During
the transition to qPCR methods, beaches will
be monitored using both the culture and qPCR
methods so that correlations between the two
methods can be determined, allowing for future
derivation of water quality standards for the
new method.

Biosketch

Dr. Shannon Briggs is a toxicologist for
the Water Resources Division of the Michigan
Department of Environmental Quality (DEQ).
She received her bachelor of science degree in
animal science and her doctorate in pharma-
cology and toxicology from Michigan State
University. She is a member of a planning
team that will host the 2016 Great Lakes Beach
Conference in Marquette, Michigan, October
5-7, 2016. Dr. Briggs assists local health depart-
ments with state and federal grants for monitor-
ing beaches across the State of Michigan. She is
leading a water quality initiative of the DEQ to
provide rapid testing equipment and training
for 10 new laboratories that will test beaches
using the U.S. Environmental Protection
Agency's draft Method C (i.e., qPCR method
for E. coli). Dr. Briggs is an active member, past
president, and cofounder of the Great Lakes
Beach Association.

34

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Day One: Session 3

11,000 inland lakes
77,000 river miles T
1,200 public beaches ¦
4 Great Lakes	H

3,288 miles of coast
5.5 million acres of wetlands

4oinor5

MICHIGAN

yJlUililniiii,.,, 			i

¦ r.j J	sjiSteji

Monitoring Michigan Beaches Statewide
for E. coli with QPCR
(USEPA Draft Method C, June 2015)

Shannon Briggs
briaass4@michiaan.uov

Michigan rtepartment of
Fnvironmental Quality

Michigan Public Health Code and
Public Beaches

•	Monitoring is Voluntary for all 45 Health
Departments, however

•	County Ordinance can require testing

•	Health Officer has authority to test and close

•	Requires signs at all public beaches

•	Requires reporting if beach is tested

Beach Monitoring
"typical stats"

> 200 inland lake beaches monitored

•	200 Great Lakes beaches monitored

•	3.6% samples exceed WQS, n -186 samples

•	80% of beaches are open all season, n= 332

•	20% of beaches report action, n= 84 beaches

•	Most actions are 1 to 2 days

Path to qPCR for Beach Testing

QPCR Methods

Year

Beaches

Kary B. Mullis
invents PCR

1985

1986	—

oERA Ambient

Water Quality
Criteria for
Bacleiia - 1988

2000 BEACH Act

Path to qPCR for Beach Testing

QPCR Methods

Year

Beaches ]

Dr. Joan Rose at

2003

Monitor Beaches

Michigan State



with local, state &

University



federal funds

Water Fellows

2005

Identify Impaired

Lectures &



Beaches

Discussion





Microbial Source

2007

Beach Sanitary

Tracking (MST)



Survey Tool

35

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Path to qPCR for Beach Testing

Year

QPCR & Beaches j

2010

MST at Impaired Beaches

2011

Training Manual & Video for Beach



Testing with QPCR

2012

Public Meeting for MST Results



U.S. EPA Rec Water Quality Criteria



Includes Enterococci QCPR values

U.S. EPA's 2016 Recreational Waters Conference

QPCR Lab
at Lake St. Clair
Metropark Beach

$100,000 for equipment

Park renovated office to lab ($50,000)

bja

ML . <\ \
-



$500,000 for 10 New Labs

•	State of Michigan provided $500,000 to DEQ
for rapid beach testing equipment

•	Only health departments have authority to

test beaches

•	DEQ sent letters of invitation to 45 health
departments responsible for 83 counties

•	13 responses and description of lab capacity

Questions and Details

•	Commitment & Expectations in Memorandum
of Understanding between DEQ and HDs

•	10 Health Departments signed MOUs

•	MOU included equipment list with 50+ items
for each HD

•	$30l< for Training and Support from MSU

just added $28,000 more

O G Existing qPCR Labs
O 10 New qPCR I ahs

36

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£3

Day One: Session 3

You(B3

Performing the qPCR Assay
for Enterococcus

qPCR Training Video

uploaded on Nov 79. 7(111

and 2011 using a rapid method (quantitative polymerase chain reaction) to assess beach water quality at
sites in Orange and Los Angeles Counties Tltts video was produced to tram laboratory statt to execute

Path to qPCR for Beach Testing

1 Year

QPCR & Beaches

2014

10 New QPCR Labs (15 total)



U.S. EPA draft Method C



(QPCR - E. coli)

2015

Equipment Ordered & Delivered
Samples filtered & frozen
Train the Trainer for QPCR

Path to qPCR for Beach Testing

Year	QPCR & Beaches

2016 4-day Training on Draft Method C

Stockholm Water Prize awarded to
Dr. Joan Rose

37

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Jej*

U.S. EPA's 2016 Recreational Waters Conference

How would you rate your confidence in
your skills for each step of qPCR?

7 T	

Pipetting Preparing Filtration DNIA Running DNA
Reagents	Extraction StepOne Analysis

Step in qPCR Analysis Plus

Path to qPCR for Beach Testing

Year

QPCR & Beaches

2017

Samples tested and reviewed with
previous 2 years

2018 Continue sampling

Present equivalent QPCR results to
USEPA and Local Health Officers

2019

Beach status determined by
QPCR methods
Molecular Source Track Training?

Path to qPCR for Beach Testing

Year

QPCR & Beaches

2016

Multi-lab Validation Study
2015 & 2016 Samples tested
Review Colilert & QPCR Results



jyj|


|

38

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Day One: Session 3

Report Data

DCa

BeacfiGuard' ,lon™' IAch^°*'9°'"omo t c°,»c,vo»,ort Incc I Ab°ut l*-¦

r quality campling roc ufe



Michigan Beaches

1??S Public Beaches

513 Private Beachec

Closures and Atfvlsorlco

No Current uosures or Acv.sor.es

QPCR is not the finish line

its just another tool in the tool box

Sleeping Bear Dunes Photo by Steve Keighly, Winner of the Instagram
Beach Photo Challenge for favorite beach to take a long walk.

Beach Sanitary Surveys

http://www2.epa.gov/beach-tech/beach-sanitary-surveys

Cf-S.I I	I **>«<*« I nto9«4>



Related lupus Technical Resources about Beaches

Beach Sanitary Surveys

On this page:

•	Bdikyiuund

•	Marinp Btmrh Unitary ^iirypy

•	other sanitary survey information

Background

A sanitary survey is a method of investigating the sources of fecal contamination to a water body. Sanitary

Beach Sanitary Surveys

Great Lakes Beach
Sanitary survey
User Manual

Canine Scent Tracking

Environmental Canine Services

I aura and Kama

39

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Remediation

landscaping
redesign slope of beach, groom beach

Great Lakes,

i Lanes fcteacn conference Adomonai into

Majcquette in October 5-7, 2016

Beach listservs
beachnet@great-lakes.net

MiQPCR@LIST.MSU.EDU
beachinfo@lists.epa.gov

40

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Day One: Session 3

Rapid Analyses of Water Quality at Five
Chicago Beaches, 2015

Abhilasha Shrestha

University of Illinois, School of Public Health

Abstract

In the summer of 2015, the Chicago Park
District (CPD) enhanced its beach monitoring
and notification through a pilot program of
rapid molecular testing of beach water. Water
samples were provided at approximately 8:30
a.m. 4 days per week to the University of Illinois
at Chicago School of Public Health (UIC SPH)
Water Research Laboratory. The results of the
rapid testing method, qPCR, were reported on
the same day by 1:00 p.m. The CPD used the
qPCR results to notify the public about mea-
sured bacterial concentrations. Previously, the
CPD posted notifications based on the most
probable number (MPN) of E. coli obtained from
overnight cultures.

Water samples from five Chicago beaches
were tested using the Enterococci qPCR. Similar
samples were set up for E. coli culture analy-
sis by a commercial laboratory on the same
days that UIC performed the qPCR test. The
CPD used the U.S. Environmental Protection
Agency's (EPA's) Beach Action Values (BAV) for
both the qPCR test results and the culture test.

Of the 270 qPCR tests, 23 exceeded EPA's
BAV, and of the 270 culture tests, 67 exceeded
the BAV. The results of E. coli culture test-
ing that became available on a given day (e.g.,
results that became available on a Thursday
from tests of beach water samples collected on
Wednesday) were frequently inconsistent with
the current qPCR results (from water samples
collected on Thursday). Our data suggest that
beach water notifications based on qPCR testing
presented a more accurate picture of same-day
water quality than the prior-day's culture test
results.

Biosketch

Ms. Abhilasha Shrestha is a doctoral stu-
dent in the Environmental and Occupational
Health Sciences Department at the University of
Illinois at Chicago School of Public Health (UIC
SPH). She earned her bachelor of science degree
in biology from the University of Minnesota-
Duluth and then worked as an aquatic toxicolo-
gist in a private laboratory in Minnesota for
more than 2 years. She completed her master's
degree from UIC SPH in 2013, focusing on
environmental and occupational health sci-
ences with a concentration in water quality and
health. Ms. Shrestha's research interests include
studying the use of different indicator targets/
genes for water quality assessment. In her dis-
sertation research, she is focusing on molecu-
lar methods for rapidly evaluating infectious
agents in surface water.

41

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U.S. EPA's 2016 Recreational Waters Conference

Rapid Analyses of Water Quality at Five Chicago Beaches, 2015

jjj |sa£
i im

Abhilasha Shrestha, PhD Student

Ira Heimler, Lathy Breitenbach, bamuel Uorevitch
U.S. EPA's Recreational Waters Conference
A|jiil 13,2016
University of Illinois at Chicago
School of Public I lealth
Chicago, IL

Overview

•	Introduction

•	Methods
v Beach Action Value (BAV)

•	Results

>	Data quality

>	£ coli culture results

>	Fntpracnrci qPCR results

>	One day delay In E. coll results, and associations with qPCR results

>	BAV exceeddiice after 0.5 iridi of idin

•	Conclusion

•	Future projects

•	Acknowledgment

O ******

Introduction

•	Chicago: 26 miles of public
beaches

•	~20 million visitors annually

•	Chicago Park District: 27 beaches

•	Point source discharges are rare

•	Monitoring: Culture-based
methods such as Colilert®

•	Prior-day culture poor
predictor of current conditions

•	2015: Pilot program with UIC

> 5 Chicago beaches

Methods

•	qPC R at I lit", lah, Tuesrlay-Fririay, May - August 30, 7015

•	Culture tests: Commercial laboratory, Colilert® method

•	1L samples, 2 transects each at 5 Chicago beaches (N-270)

•	Delivered at approximately 8:30 AM

•	Quantified for Enterococci DNA using the USEPA Method 1611 with
one modification

•	Results reported to the CPL1 on the same day by 1:00 PM

Beach Action Value (BAV)

Entcrococct culiurablc
-Ui&tbiki&lL...

I k. coli - culturable

(fresh)''	

I-Miniatcd I linos Rate
(NGI): 36 per 1,000
primary contact
rccrcaton

BAV
(Into n«r 100 ml.)



23 > cfu

jc

K*timatcd Illness Rate
(NCI): 32 per 1,000
primary contact
retreator*

BAV
(iHitipw 100 ml.)

60 cfti

iwcfa

Eruavcoccus spp

qPCR (fresh and mnnne)r	1,000 cce	I	640 c

1,000 ccc	I	

"	uM-auu«d UM1151 epa MmIumI il00(U v f ;>.\ 3002a) m MMb« wplvalMt im^kkM

cultivable euterocotci

6 E coli	iuius EPA Method 1403 (U S EPA 2002b), ot my otlut  Nine standard curve runs, each in triplicate, initially and every two weeks
v R4= 0.9957 (high accuracy)

1 Parameter



Standard

05% lower

05% upper 1



Mean

deviation

bound

bound

Slope

3.4945

0.0202

3.5345

3.4545

Intercept

38.2229

0.061

38.1122

38.3535

qPCR Precision/ Calibrators & sample processing controls (SPC)
> 55 calibrators

Sample processing control

23.00

026

1.11*

Enterococci cells



053

2.05*

qPCR Inhibition.

>- Of the 510 total beach samples, only two (0.37 %) exceeded the 3 CT unit
offset; other two (0.37%) had offsets in the 2-3 cycle range

42

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Day One: Session 3

E. coli culture (MPN/lOOmL geomean)

Enterococci qPCR (CCE/lOOmL geomean



383.1 (14.9 - >2420.0) 33(61.1) 14(25.9) 7(ia-°)



171 f, (1.(- ->9490 n) 16(^3.3) X13") '(3 7)
167.8 CS-fi ¦ >2420.0) 46(85-2) 7(13-0) 1(1.9)

i86.y (2.8 - iuzu./) 4i(y/.8) 11(20.4) i(i.y)

w*1	

057.8 (6.0-01900) 37(68-0) 13(24-1) 4(7-4)

[All beaches



\ \ \ \ \ \

D*li

B««dl — L	— RB —	^rt Note: 03 values were truncated at 24G0



liRilRIHSinBiBBil

Montrose

447-9

C27.1-569b.oj

49190-7)

5(9-3)

O.V'SHcc*

240.9

(14.2-1O77.G)

49(90-7)

5 (9-3)

SnnHl Shnro

3°6 0

0" 6-3136 s)

61 (91 1)

3(6 Q

Rainbow

501-9

(23.3-10385.8)

49(90.7)

5 (9-3)

(ATI hpartip«j

4&U-1

9*94

(2u.6-38m-3)
(10 5-1038S.8)

BBBHBH

5(9-3)

Time series graphs of daily measures of culture and qPCR

I -



Beach

Pearson's R

p-value

Montrose

0.78

<0001

6}rd Street

078

<¦0001

South Shore

060

<0001

Rainbow

0.29

0.035

calumet

O.tffl

<0001

One day delay in E. coli results, and associations with qPCR results

Beach management decisions based on today's qPCR results and the E. coli
results from yesterday's water sample were not associated, with the
exception of 63rd Street beach.

1 Pr ior day culture results frequently lead to the erroneous decisions when
rnmparpd tn the same day qPCR results as the gnld standard.

H¦¦¦¦¦¦¦¦J





| i 3 4

1 9

39 48

1 10

42 52

| ORl

44 fo.13,15.55)









BFr'^

347

ao a6 46



^3 30 53

nil nt»»(mn J »(.)















2 s /



16 29 45



10 34 52



OR O.T3 fO.13.4.17)

BAV exceedance after 0.5 inch of rain

•	Odds of exceeding either the E. coli culture MPN BAV or the
Enterococci qPCR CCE BAV were increased

•	Enterococci qPC'K: Odds ratio 4 2b (l.b9 - 11.43)

•	E. coli Culture: Odds ratio 1.90 (0.85 - 4.24)



CCE <1,000

CCE> 1,000

Total

-0 0 inches past 24 hours

224 (93.3%)

16 (6.7%)

240 (100%)

i0.5 inches past 24 hours

23 (76.7%)

7 (23.3%)

30(100%)

Total

247

23

270





MPN<235

MPNi 235

Total

<0 5 inches past 24 hours

184 (7fi 7)

SB (23.3%)

?4O(10O%)

*0.5 Inches past 24 hours

19 (63.3%)

11 (36.7%)

30(100%)

Total

203

67

270

Conclusions

•	Accurate, precise qPCR results can be available by 1.00 PM.

•	Daily qPCR CCt values resulted In BAV exceedance less frequently
than the E. coli culture results (8.5% vs 24.8% of samples).

•	Inhibition of the qPCR reaction was rare (<1% of samples).

•	Results of £ coli testing (from prior day water samples) were not
consistently related to qPCR results.

•	Beach management decisions should be based on same-day
rather than prior-day information.

•	Hpavy precipitation tends to increase Fntprnrocci qPCR CCF
results significantly, and to a lesser degree, £ coli MPN.

43

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Jej*

U.S. EPA's 2016 Recreational Waters Conference





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>

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oc

<

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Q.
HI



Future Projects

•	Archived filters

>	Evaluate the concentration of a human-specific molecular
target like HF 183.

•	Summer 2016

>• qPCR testing expanded to additional beaches, particularly
those that tend to have relatively frequent BAV exceedance
based on E. coli culture results.

v 9 beaches, 5 days a week, Wednesday- Sunday

>	Goal; Earlier sample collection and results by noon.

THANK YOU

Acknowledgement

funding for this project was provided by the Chicago Park District.

, V)

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g JO I

2 ^ Z h

5 Tia J

E

I gou«zm
Z Z U kiJ u 3
ilKOIlAL



KEEP
CALM

AND

RUN
qPCR

44

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S3

Day One: Session 3

Application of Rapid qPCR-Based Tests for
Enterococci (Method 1611) in Hawaiian Coastal
Waters

Marek Kirs, PhD

University of Hawaii

Abstract

To evaluate the applicability of the U.S.
Environmental Protection Agency's (EPS's)
enterococci qPCR method 1611 for beach waters
of Hawaii, a total of 127 water samples were
collected from 12 beaches on Oahu over a
10-month period. The samples were analyzed
using EPA methods for Enterolert®, 1600, and
1611. Clostridium perfringens, human-associated
Bacteroides, and human polyomaviruses
also were enumerated. Concentrations of
enterococci and C. perfringens varied from <
10 to 389 colony-forming units (CFU) 100ml-l
(Enterolert®), from < 1 to > 151 CFU 100ml-l
(1600), and from < 1 to 96 CFU 100ml-l (mCP).
Four samples (3.1%) analyzed using Enterolert,
and two samples (1.6%) using method 1600
exceeded the EPA-recommended statistical
threshold value (STV) of 130 CFU 100ml-l, while
C. perfringens concentrations exceeded 50 CFU
100ml-l in a single sample (0.8%), indicating
generally good water quality at the beaches
studied. In the samples exceeding the STV,
human-associated Bacteroides was detected in
a single sample, while human polyomaviruses
were not detected. Importantly, 88 samples
(69.3%) tested using method 1611 could not
be quantified because of the PCR inference.
After those samples were diluted in molecular
grade water (1:10), the majority of the samples
(85 samples, 66.9%) remained compromised
by the PCR inference. In contrast, for an addi-
tional set of monthly samples (n=39) collected
at three sites from the brackish Ala Wai Canal,
only a single sample was compromised (2.5%).
Although good agreement existed between the
methods for enterococci when samples were not

compromised, our data indicate serious short-
comings for the recommended qPCR method
1611 for enterococci enumeration for Hawaiian
beaches. New technology that alleviates inhibi-
tion issues for qPCR is being evaluated.

Biosketch

Dr. Marek Kirs is an assistant researcher
at the Water Resources Research Center of the
University of Hawaii. He received his bach-
elor of science degree from Tartu University in
Estonia, his master of science degree from the
University of Edinburgh in the UK, and his
doctorate from the University of Rhode Island.
He also has completed postdoctoral training at
the University of North Carolina at Chapel Hill.
More recently, Dr. Kirs worked at the Cawthron
Institute in New Zealand, where he was
involved in establishing microbial source track-
ing services and lead microbial water quality
research and consultancy projects. His research
focuses on a wide range of microbial water
quality and related public health issues.

45

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U.S. EPA's 2016 Recreational Waters Conference

Application of rapid qPCR-based
tests for enterococci (Method 1611)
in Hawaiian coastal waters

Marek Kirs, Denene Blackwood, Rachel Noble,
Philip Moravcik
April 13, 201G

U.S. EPA's 2016 Recreational Water Conference, New Orleans

INIVCRS1TY ||,||
-------
Water quality

Enterococci

Method 1600(mEI)

Water quality

Enterococci

Enterolerf l600(mEl)

Water quality

Clostridium perfringens

Water quality

Human-associated Bacteroides; human polyomaviruses

Water quality

Summary

• Fnterocncri and C pprfringpns indicated good water quality
on the beaches studied

• 3.1% of the samples exceeded STV for enterococci by Enterolert®

• 1.6% of the samples exceeded STV for enterococci by method 1600

• Only a single sample exceeded both , the STV for enterococci and

threshold level for C. perfringens
• Human sewage was not conclusively identified as the contamination
source in any of Ihe coastal samples based on the markers

Rapid Method Application (1611)

PCR Inference (inhibition)

PCR inference can be caused by:

• Mechanical blocking of the enzyme, template

• Physical and chem. modification of the enzyme, template

• Binding and chelating of other chemicals necessary in PCR

• Other ..(see Schraderetal, 2012, J. Appl. Microbiology 113:1014-1026)

PCR inference results in:

• Severe underestimate of bacterial concentrations

• False negatives

-------
U.S. EPA's 2016 Recreational Waters Conference

Rapid Method (1611)

PCR Inference (inhibition)
PCR inference was measured:

23 3 cycles)

Rapid Method (1611)

PCR Inference
27 3

63.7 , m 100.0
63 6 tjj Tin Total of 69.3% samples

compromised

& ¦v/
Sc. - 81.8

8LS

''' .

% compromised samples0 45.5" 81.° 455
(ACtsketa >3.3 cycles)

0% samples extraaed using
commercial kit compromised
(AC^iO.61)

(mixed cellulose filters)

Rapid Method (1611)

PCR Inference 1:10 diluted

'\ . -27.3
bi. 7 100.0

03 G it,. 72.7

- '

% compromised samples

(AC^ 23 3 cVcles)

67.7% samples
still compromisec

27.3 81.8

Rapid Method (1611)

Ala Wai

Site

Description

Salinity

Compromised

(PPt)

(%i

Coastal

34.9

7G.9

Canal

27.6

7.7

Canal

23.4

Stream

6.8

¦ — - _

Rapid Method (1611*)

Comparisons with cultivation based methods (combined set)

1A-0 90

R2=0M7

i!:

jB* '

to- 10>
Cnterolert

• *'

! *

n=32
IA=0.884
R*=o a?o

Rapid Method (1611*)

Beach management decisions (combined set)

Method 1600

Open

tnterolert*

Clnsp

Open

Method 1611

open

Enterolert®

Close-

Open

Method 1611

open

Method
1600

Open

maam

-------
Day One: Session 3

Rapid Method (1611)

Summary

Good water quality of the beaches sampled

PCR inhibitors can compromise application of
rapid qPCR based methods in Hawaiian coastal waters

There was good agreement between enterococci concentration
estimates as well as beach management decisions based on
all three methods

Rapid accurate methods are highly desired in HI
(number of beach goers, distances, impact)

Rapid Method (1611)

Future plans

A study funded by the Sea Grant College Program/NOAA:

1) identify cause,

2)tmuhleshnnt, arid

3)secondary assay needed

Coral sand?

Acknowledgements

Contributors:

Dr. Roger Fujioka

Dr. Valerie lody Harwnnd

Dr. Mayee Wong

Ms. Martina Frycova

Clean Water Branch {HI DOH)

Funding:

National Institute of Water Resources (USGS) and
start up

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U.S. EPA's 2016 Recreational Waters Conference

Multi-Laboratory Survey of U.S. EPA
Enterococci qPCR Methods Acceptability for
Analyses of U.S. Coastal and Inland Waters

Richard Haugland, PhD

U.S. Environmental Protection Agency, Office of Research and Development

Abstract

The U.S. Environmental Protection Agency
(EPA) offers two similar quantitative poly-
merase chain reaction (qPCR) methods, method
1611 and method 1609, for the rapid estimation
of enterococci fecal indicator bacteria densities
in recreational surface waters. Water quality
monitoring results from either of these methods
can be compared with 2012 EPA Recreational
Water Quality Criteria (RWQC) values for
site-specific notification programs if the meth-
ods are demonstrated to meet performance
acceptability guidelines at the site. Current
site acceptability guidelines that are available
from EPA recommend a maximum frequency
of 10% of samples that can exhibit excessive
sample matrix interference to the EPA methods
as assessed by results and acceptance criteria of
the sample processing and/or amplification con-
trol assays prescribed in the methods. Here we
report the results of a multi-laboratory survey
of 22 different marine, Great Lakes, inland lake,
and river or stream sites from across the U.S. for
their potential acceptability in implementing
methods 1611 and 1609 based on these guide-
lines. Combined laboratory results from 20 and
16 of these sites were found to meet the guide-
lines using methods 1609 and 1611, respectively.
The benefits of augmenting the control assay
results with qPCR analysis estimates of recover-
ies of target sequences from enterococci that are
spiked into the test samples also are presented.
Results from the analyses in this study indi-
cated that the recommended protocol in method
1609 provided the greatest assurance (>98%) of
preventing excessively underestimated entero-
cocci densities (< 50% recovery) caused by

matrix interference in samples meeting control
assay results acceptance criteria.

Biosketch

Dr. Richard Haugland is a microbiologist
in the Environmental Methods & Measurements
Division of the National Exposure Research
Laboratory. He received his bachelor of science
degree in biology from Muskingum College,
Ohio, and his doctorate in developmental
biology from the Ohio State University. His
past research has addressed diverse prob-
lems including biodegradation of hazardous
chemicals in the environment, assessment of the
microbiological quality of indoor environments,
detection of biothreat agents for homeland
defense, and most recently, monitoring ambient
water quality using bacterial indicators of fecal
pollution. Since joining the U.S. Environmental
Protection Agency (EPA) in 1991, Dr. Haugland
has authored or coauthored more than 60 pub-
lications and has received a number of awards
for his work, including the EPA bronze and gold
medals.

-------
Day One: Session 3

Multi-laboratory survey of U.S. EPA
enterococci qPCR methods
acceptability for analyses of U.S.
coastal and inland waters

RichardHaugland, Shawn Siefring, Manju Varma Kevin H
Oshima, Mono Sh'aganesait, YipingCao, Mcir cktn Raith,
John Griffith, Stephen B. Wetsberg, Rachel T. Noble, A.
I)i>t>ene. Blackwood, Julie K inzelman, Tamam A nan eva,
RcbeccaN. Bushon, Erin A. Stelzor, Valaric J. Norwood,
Kuirinu V. Ov/xlon, Chiitlupher Siiiigulliunu

SEFW,

Study background

• QPCR methods can provide rapid (same day)
estimates of fecal indicator bacteria (FIB) densities in
recreational waters.

• Enterococci FIB densities determined by qPCR have
been found in a series of epidemiological studies.
(U.S.EPA NEEAR studies and others) to correlate with
bather gastrointestinal illness rates.

• Based on these observations, qPCR density values for
enterococci are provided In the U.S.EPA (EPA), 2012
Recreational Water Quality Criteria (RWQC).

oEPA

Study background

~ 2012 RWQC further indicates that: "overall testing of the
qPCR method with different types of ambient waters, and by
different laboratories, remains limited and (EPA) anticipates that
there may he situations at some locations where the performance
of the qPCR method may be inconsistent'.

~ For this reason, the RWQC suggests that: "states
evaluate the qPCR method with respect to laboratory
performance and sample interference in their prospective waters
prior to developing new or revised standards relying on this
method

SEPA

Study background

~ EPA has provided guidelines for determining acceptability of
qPCR method performance at prospective sites based on
Ihe peicenlaye of samples passing Ihe uonLiol assay
acceptance criteria specified in the EPA methods:
(http://www2.epa.gov/cwa methods/other clean water act
test-methods-microbioloaicalV

* EPA offers two methods (Method 1611 and Method 1609)
that can be evaluated at prospective sites for their ability to
meet these performance acceptability guidelines.

SERA

Study objectives

~ Use the two EPA methods and different EPA-recommended
or alternative method permutations to:

1. Determine the percentage of samples passing EPA Method
specified and alternative control analysis acceptance criteria
from a variety of different water body types based on analyses
of shaied samples by multiple labs.

2. Evaluate the reliability of the controls in identifying accurate
sample analyses based on estimated recoveries of target gene
sequences from spiked enterococci in these water sample
matrices.

Site Water body type

River or Stream (RS) Racine, W

White Sands Beach
Fischer Park Beach

Quarry I alreParlr

Cabrillo teach
Dohenv Beath

Study sites

Buckeye lake, Ohio
Lake Carroll, Florida
Browns Lake. Wisconsin

Kwer or stream (Kbj Near mouth, uncmnati, umo
River or Stream (RS) Riverfront Park.Tamra. Florida

Inland Loire (IL) Buckeye Lolic, Ohio

interna lake (il)

Inland Lake(lL)

Inland Lake (IL)

Inland I al» (II)
i*eat Lakes |t.ij

Atlantic Ocean (AO) Wilmington North Caroline

-------
U.S. EPA's 2016 Recreational Waters Conference

S-EPA

Study design

I ahoratnry 1* 11S FPft. National Fvprmirp R«parrh I ahnratnry. finrinnati. f»H
Laboratory 2: Southern California Coastal Wotcr Research Project, Costa Mesa, CA
LoUuioloi v 3. Cily i>r Rounc Heallli Dcyoi liuciil, Racine, Wl
LaDoratory 4: uepartment of biology, university of bouth Honda, lampa. n
Laboratory 5: U.S. Geological Survey. Columbus, OH

lahorafmvfi'lnrtitiitpnf Marinp Sripnr« I lniwpr<:itvnf Nnrth farnlina at Thanfl Hill MnrphpaH fitv Nf

^m (i i rn a in ens. l- i

sx CZ] h q m Q Q

From "Millb-laboratory survey of qPCR enterococc* analysis method performance in U.S. coastal and inland surface
waieisHauoland."byR. A. Hauoland.S-Sefrmo M Vaima. KH Gshima.M. Si/aoanesan. Y. Cao M Ralh J Griffith. S.B.
WfeishfirO.RT Nnhlfi.AD RhrVwnnd..1 Krophian.T Anan'pwa.RN Fkishfm V.I Hsrwnnd.KV Rnrrlnn anilC
Rinigalliann. Tfllfi../ Micratonl Method* fflp|> 1U-I?5

5-EPA

Sample analysis methods and

permutations

Method

PCR Master

Extract Dilution

Calculation

Mix Reagent

Analyses

Models

EPA Method

Universal

.Sx-diluted extracts

Delta-Delta Ct

1611

Master Mix

(recommended in

Method], undiluted extract

Method) & Delta Ct

data collected but not

recommended in Method

EPA Method

Environmental

Undiluted extracts

Delta-Delta Ct

1609

Master Mix

(recommended in Method)

(recommended In

& 5x-dlluted extracts

Method) & Delta Ct

(optional in Method)

—

SEPA Matrix interference control

analyses & acceptance criteria

Control Analysis

Acceptance Criterion

Reference

Salmon DNA sample
processing control (SPC)
assay

lebl bdtnple Cl within'3
units of positive control
samples

EPA Melhudb 1611
&1G09

Competitive Internal
Amplification Control
(IAC) assay

test sample Ct within 1.5
units of negative control
samples

EPA Method 1600
& updated Method
1611

Enterococcus assay Ct
shift across undiluted - 5x
sample extract dilutions

test sample Ct shift within
2.32 ±1 units

Caoctal., 2012*

HI *J. flppl. Microbiol. 1tl 66-7?

SEPA

Spike recovery estimations

• Spiked test matrix (SIM) samples: ~1UA4 t. taecalis (hnt) cells
added to filters containing water sample retentates.

• Spiked control matrix (SCM) samples: same number of Hnt cells
added to clean filters.

• Ratios ot total hnt target sequences recovered from SIM/SCM
samples calculated by Delta & Delta-Delta Ct formulas:

• ACt ratio = AI~A(-(a - c))

• AACt ratio = AFA(-((a - b) - (c - d)))

• where AF = amplification factor (amp efficiency + 1), a - mean STM
sample Ent Ct, b = mean STM sample SPC Ct, c = mean SCM
sample hnt Ct. d = mean S( ;m sample SPC: C :t

- Ratios converted to STM/SCM recovery percentages

• The same analyses and calculations weie perluimed fur
corresponding unspiked samples and recoveries subtracted from
the spiked sample recoveries to determine net spike recoveries.

• Net recoveries within 50 200% were considered as acceptable

SERA

Overall results

Method

Total analyses
(sample extract , .

dilution) |N>

percent of

analycec

passing Sft &
IAC control
assay cntena

Percent of

analyses patting
Enterococcus
assay Cl shift
criterion (Cao et

dl>

Percent of act Percent of flflCt

net recovery net recovery
analyses within analyses within

50-200% 50-200%
recovery ranee recovery range
STM/SCM STM/SCM

1611 (lx)

Not determined

)t determined 8/%

84%*

xbll (SX) II8 34%

* Peicenlaye leUuteii by a yiuup of sample analyses Dial would nul meet current QC triletia

&EFA

Summary of site acceptability
analyses based on current
control assay criteria

Method
(extract
dilution)

Sample
analysis
acceptability
criterion

Sites passiny
EPA guidelines
(> 90% earn pie
analyses pass
criteria)

Sites passiny ui
approaching EPA
Guidelines (5 80%
sample analyses
pass criteria)

Method 1609
(undiluted)

SPC and IAC
assay controls

14/22(64%)

18/22 (82%)

Method 1609
(undiluted)

Enterococcus
assay Ct shift

13/22(59%)

17/22 (77%)

Method 1H09
(5x-diiuted)

vSK: and IAC
assay controls

A) m («1%)

¦/?!/? (100%)

Method 1611
(5x-diluted)

SPC and IAC
assay controls

16/22(73%)

21/22 (95%)

¦a

-------
Day One: Session 3

SEPA

Great Lakes,

Lake Michigan site

y North Beach, Racine, Wisconsin

E9Aft*" M

/ mai.oI »« * Analyses % ACT net % MCT net
Method ... labs ' . nutans rprrwcrv rpriMMV

(extract doi Passings & Cnterococcu' anal » analye"

MjZ.,1 Analvses :Z. lACcontrol

a«avrntwia ^

1609 (IX) S9 1 « O » O » M

1609 (5x) 93 O ®

KllM << « O

¦a

o Passes CPA site guide# nes (s90%)

O Approochoc EPA cito guidolinoc (80 00%), furthor anatyooc warrantod?
© FailsFPA site (jiiflfJinpc; (< Sfl1*.)

AEPA

Pacific Ocean sites

4o.~v

Newport Dunes Beach, Newport, California

Analyses

% Analyses

% ALi net % MCI net

dilution) Anal''ses

analyse,,

|M*>imS SPC a

ial contra

RltMWlYriK

analvw analyw

1C09 (lx) 32

76 O

09 O

1«W M 40

« O

1611 (5x1 50

86 O

90 84

Doheny Beach, Dana Point, California

(attract Tl*al
dilution) Aria v™'

analyses

% Analyses
Dassine SPC &

IAC control

assay Ct shift

analyses analyses
within 50- within 50-
200% 20O9S

1609 (lx) 31

ioo m

90 100

1609 (bXJ 3b

1U0 Q }

9/ 9/

1#11 (SX) 34

100 ©

AEPA

Environmental Protection

Atlantic Ocean sites

, Jockey

s Ridge Beach, Outer Banks, North Carolina

(ora

% Analyses
Total ' na«ine SPC ft
Analyse) ^ lACcontrol

tnterococcus analyses 'analyses

dilution)

0 " analyses a ' Uj™,j0

assay Q shift within 50- within 50-

1609 (lx)

20 2 100

95 © 85 100

16U9 (bx|

24 I 1UU

92 1U0

1S11 (SX)

24 2 100 ©

- South Naes Head Beach, Outer Banks, North Carolina

Method

iotal .s passing iKL &
Analyses s lACcontrol
"" v*a assay criteria

% Analyses KACTnet KMCTnet

1009 (lx)

21 2 B1 ©

80 © 07

1G09 (5x)
Ittl (Ex)

100 ©
2 100 ©

02 100

Midwest inland lake sites

Tischer Park Beach, Browns Lake, Wisconsin

1609 (lx) 33 2 91 C J 85 O 70

1609 (bXj 21 2 1U0 © ill

mi 1S*1 21 2 100 O "

Quarry Lake Park Beach, Racine, Wisconsin

« Analyses % ACT ne

sum ""i its I-*"-* s"™ s™

Analyses ® IAC control . . ^

SEPA

Midwest inland lake sites

Rrnnk«: Rpath, Riirkpyp lakp, fpntral Ohin

MAid % Analyses %ACTnet «AACTne

»ni if Mwcn zmz nzir

(extract ""S lACcontrol 0rQ"""' ^T.00'

Uiluliuii) alidpo assay Ctshrft within 50- within 50-

cntenon 20U%

Crystal Beach, Buckeye Lake, Central Ohio

-------
S-ER<\ Spike Recovery Estimates

Samples passing SPC & IAC criteria Samples failing SPC & IAC criteria

> MSJ!

U.S. EPA's 2016 Recreational Waters Conference

SERA

Summary

uslnq data from all labs, 20 out of 22 of the sights passed the current EPA site
acceptability guidelines based on Method 1609 analyses of either undiluted or
diluted sample extracts 1fi sites passed hased Method 1fi11 analyses nf diluted
sample exliads.

Ayieemenl un bile acceptability by different labs was 76% (seveidl facluis may be
involved in the differences)

The current controls were generally, but not always, accurate in predicting
acceptable (50-200%) spike recoveries.

Enterococcus assay ct shift and spike recovery results from delta ct analyses
suggested that some of the samples (e.g. Buckeye Lake) interfered with the
analyses.

Delta delta Ct analyses suggested that use of SPC assay results in the calculation
model was effedive ill adjusliny reuuvety estimates lu the acceptable lanye in
many of these interfering samples.

Method 1609 vwth undiluted extracts passed the control assay criteria at a lower
rate but. when outside the accepted spike recovery range, the delta delta Ct
estimates from these analyses were nearly always high rather than low.

Method 1609 (and 1611) with diluted extracts passed the CPA control assay
criteria at a higher rate but delta delta Ct recovery estimates were below 50% in a

higher number of sample analyses passing these controls.

oEPA Possible future guidelines for site

Environment! Protection evaluations (based on this study)

Use Method 1609 with undiluted extracts

Current EPA guidelines
are met:

AACt Spike recoveries

> sn% a < ?nn%-

Current EPA guidelines
are met (& AACt spike
recoveries > 50%):

yes

Determine spike recoveries

Use Method 1609 (or 1611) with diluted extracts
yes no

li I

Acceptable Site Site not acceptable

-------
S3

Day One: Session 3

Towards Field-Portable Instrumentation for
Real-Time Water Quality Monitoring Using
Digital Droplet PCR

Kevan Yamahara, PhD

Monterey Bay Aquarium Research Institute

Abstract

The release of the 2012 Recreational Water
Quality Criteria allows beach managers to
utilize quantitative PCR (qPCR) for routine
water quality monitoring. While methods used
to assess water quality have advanced, tech-
niques for automating the process have lagged;
few technologies exist that fully automate the
water quality monitoring process from sample
collection to delivery of quantitative results.
The Environmental Sample Processor (ESP) is
one tool that may enable researchers and beach
managers to monitor beach water quality in an
autonomous manner. Current development of
the ESP system is designed to allow for in-situ
sample collection, sample lysis, and continuous
flow digital droplet PCR (ddPCR) to quantify
the Enterococci 23rDNA gene and other source
tracking targets. Processes performed using the
new ESP system, including sample collection,
DNA extraction, and ddPCR quantification, are
shown to be equivalent to traditional laboratory
methods using real-time qPCR for quantifica-
tion of enterococci. Quantification of enterococci
gDNA by the continuous flow ddPCR instru-
ment developed during the course of this proj-
ect is positively correlated with quantifications
using the BioRad ddPCR instrument (slope =
0.72, R2 = 0.99, p=0.0001). The evolving ESP/
ddPCR technology may provide a new plat-
form for conducting water quality monitoring
tests that can be packaged in a portable, field-
deployable unit, reducing sample handling and
complex assay standardization associated with
traditional qPCR.

Biosketch

Dr. Kevan Yamahara is a research special-
ist at the Monterey Bay Aquarium Research
Institute (MBARI) in Moss Landing, California.
He earned his doctorate in environmental
engineering and science at Stanford University,
where his dissertation focused on the fate and
transport of fecal indicators and pathogens in
California beach sands. At MBARI, he focuses
on developing new technologies for biologi-
cal monitoring of the marine environment.
Dr. Yamahara is currently developing field-
portable instrumentation for monitoring fecal
indicators and source-tracking markers and
autonomous vehicle instrumentation to detect
environmental DNA of marine phytoplankton
and vertebrates.

-------
S3

U.S. EPA's 2016 Recreational Waters Conference

at
<

<
a

Towards Field-
Portable
Instrumentation for
Real-Time Water
Quality Monitoring

Kevan Yamahara, Andrew Hatch, Joshua
Steele, Cody Youngbull, John Griffith,
Christopher Scholin

WARNING
BEACH CLOStD

Santa Cruz Wharf, CA, Deployment

~ P. australis ~ P.multicorioe ~ p muHiteriesMelics
O Pseudo-nltzschla spp # P a

Yamahara etal. 2015. Lett Appl Microbiol

Proof of Concept

0E5P

! Sample
Collection

1 1

—1 1—

§ 1

jk-

1 1

Yarr

1 ¦ '

ahara etal. 2015. Lett Appl Microbiol

Sample
Collection

5-nn*

.uu

Data
Uploaded

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IU.UU

Outline

_| WARNINb

¦ BEACH CLOSFR

arngmmm

¦: : : .

• Environmental Sample
Processor (ESP)

tT•*' isf- ' "•

k a*.-;.. .

• Proof of concept study
for water quality
monitoring

• New sensor
development

2nd Generation Environmental Sample
Processor (ESP)

Collection

Concentration

Extraction

Detection

-------
Day One: Session 3

Proof of Concept

¦ I1"1 1 1

Quantification of BOTH fecal indicators and harmful
algae from the same sample

Sample to Results in ~ 4 hours

I imitations nf si7P and portability

The Next Conceptual Idea

\-Vy * r '*

wf|'i 1

Instrument to survey a number of locations and
to determine "hot spots"

The Next Conceptual Idea

Instrument that allows for tracking sources of pollution

Instrument Design Criteria

GD,

Samplp f nllprtinn/

Processing

£3 u SMJ

MP
¦

Analyte Detection

V;.-.

^ I ¦ #

• Tracking sources of contamination requires mobility

- Engineering design for a hand-carry instrument

- Modular design - separate sample collection and detection

Sample Collection/Processing

Sample Collection/ ¦ MP::
Processing

SSE JH, %

:l r

r 1 k ,J'

3rd Generation ESP Solution

Rotating Section
^Tump Heads ^aste x Analytical Modules
and Ambient Valves

Filter housing

seawaierour

Same engineering concepts, different form factor

-------
U.S. EPA's 2016 Recreational Waters Conference

Prototype 3rd Generation ESP

3rrl Generation (3G) ESP technology

- Sample Collection and Processing

• Preservation and In-situ Lysis

- Digital PCR (ddPCR) or Surface Plasmon Resonance (SPR)

ESP DNA Extraction Comparison

>
3

iff

O ,0

qPCR ddPCR qPCR

8ead-beating Lysis esp I

ddPCR
s Method

Analyte Detection

ASU Droplet Digital PCR Module

Partition a normal PCR reaction with many
DNA templates into many individual PCR reactions

Digital readout of positive and negative reactions
provides an absolute quantification

10 110 1
110 0 10
0 0 1 0 0 1
0 10 0 10
0 10 111

Droplet Digital PCR Module

Partition a normal PCR reaction with many
DNA templates into many individual PCR reactions

Diyiial readout of positive and negative reatlioitb
provide an absolute quantification

10 110 1
1 10 0 10
0 0 1 0 0 1
0 10 0 10
0 10 111

Potitlv* fiw tiniK

Nrgtm* Rwilom

Partitioning to 1-nL Reactions Racetrack

Thermotytler

Digital Positive & Negative
Droplets

ddPCR Quantification of Enterococcus

3 10* -

r - 0.99

oearson

p = 0.0001

o ,

U 10

£ io21

a! 10' -

\ /

ul
<£

10°

BioRad ddPCR Concentration (Copics/uL)

-------
ddPCR Quantification of HF183

Wo»=°-97
p = 0.002

Qubit Fluorometric Concentration (Copies/uL)

Conclusions and Next Steps

The challenge of portable biological sensors for
water quality monitoring ib surriple acquisition
and processing for downstream analyses

Modular designs may allow for greater flexibility
for detecting/quantifying intended targets (e.g.
ddPCR tor DNA, cELISA for toxins)

- New analytical detection methods are being
developed all the time

Field sampling trials will begin later this year in
2016

Day One: Session 3

Quantification of Environmental
Samples Using ESP Methods

Entorocoeci Concentration (Copfo/uL)

Sample l

Acknowledgements

Sesp

Chris Seholin
James Birrh
Douglas Pargetr
Scull Jeiiben
Drent Roman
Chris Preston
Roman Marin
Rill Ussier
MBARI Technicians

the David

Luc!lcPackard

FOUNDATION

MCll' School of Earth
rw & Space Extlohation

Cody Youngbull
Andrew Hatch
Andrew Larson
Tathagata Ray
Kelly Lmtecum
Greg Wells

Steve welsberg
John Giiffilli
Joshua Steele
Blythc Layton

&, , V MBARI sjS MOORE

M-mtwey Bay Aquarium 1 v ,VV-,\L'

:h Institute

FOUNDATION

-------
J.

U.S. EPA's 2016 Recreational Waters Conference

Question & Answer Session
Question 1

(Unknown): How long does it take for a digital droplet?

Answer 1

Kevan Yamahara: It's about the same time as for the qPCR [quantitative polymerase chain
reaction] system; we could reduce the number of cycles so we are looking into that.

Comment 1

(Unknown): Rumor was that it takes 5 hours for results with digital qPCR.

Comment 1 (follow-up)

Kevan Yamahara: No, it is probably less than an hour.

Question 2

(Unknown): How do you keep the integrity of the sample once you launch it? When the sample goes
from point A to point B, how do you make sure the second site doesn't have the carryover from the first
site?

Answer 2

Kevan Yamahara: We have looked at how to flush the system out. We let it sit for 15 to 20 min-
utes, then flush it with a solution, and are working on a handoff system between cartridge
handling (based on bleach or other solution).

Answer 2 (follow-up)

John Griffith: We work closely with EPA. It's not ready for prime time, but in the upcoming
year it will be comparable to regular qPCR. We'll communicate with EPA as usual.

Question 3

Steve Weisberg: For Shannon Briggs. I find this session to be gratifying. I took a look back at prior
beach conferences. I looked back at the needs back then, then how we started developing the newer
technologies to respond to those needs, then how we started getting more specific, then getting into
application and learning from the challenges. It is great to see the transition from concept and method-
ology to the application. But, what is next? You put effort and resources into training these laboratories
in qPCR, but who is watching you? Shannon, you invested a lot in this equipment, and it could be
replaced in a few years. Was this a good time to make the investment?

Answer 3

Shannon Briggs: Yes things have evolved. The certification process has changed. We're not
near drinking water yet; we discussed this last night. The site-specific document that came
out in 2014 is a bit of a guidance that proves we are doing something right. But it's a day-by-
day thing. Kevan's stuff looks very promising. This thing landed on us by chance—the con-
nection started because of a public meeting. But, yes, I have 5 years to make it work.

Question 4

Suzanne Young: For the extraction methods for DNA, is everyone using kits?

Answer 4

Abhilasha Shrestha: It was a crude extraction for us.

Answer 4 (follow-up)

Kevan Yamahara: Ours was crude with a DNA sequence. We used a gene extraction kit.

-------