Environmental Technology
Verification Test/QA plan
Drinking Water Systems Center
TEST/QA PLAN FOR THE MICROBIAL SEEDING CHALLENGE STUDY
OF THE DOW CHEMICAL COMPANY SFP-2880 ULTRAFILTRATION
MODULES FOLLOWING THE REQUIREMENTS OF THE EPA
MEMBRANE FILTRATION GUIDANCE MANUAL FOR LT2ESWTR
APPROVAL
Prepared by
NSF International
Under a Cooperative Agreement with
U.S. Environmental Protection Agency
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Contents
Abbreviations and Acronyms iv
Executive Summary v
1.0 Equipment Verification Testing Responsibilities 1
1.1 Verification Test Site 1
1.2 Roles and Responsibilities 1
1.2.1 NSF International 1
1.2.3 United States Environmental Protection Agency 2
2.0 Equipment Description 4
3.0 Experimental Design 5
3.1 Experimental Design 5
3.2 Challenge Organisms 5
3.3 Test Apparatus 5
3.4 Test Water Composition 6
3.5 Sanitizing the Test Rig 7
3.6 Module Conditioning 7
3.7 Membrane Integrity Tests 7
3.8 Microbial Challenge Test Procedure 7
4.0 Laboratory Operations Procedures 10
4.1 Introduction 10
4.2 Analytical Methods 10
4.2.1 Sample processing, and enumeration of MS-2 coliphages: 11
4.3 Analytical QA/QC Procedures 11
4.4 Sample Handling 12
4.5 Documentation 13
5.0 Quality Assurance Project Plan 14
5.1 Introduction 14
5.2 Quality Assurance Responsibilities 14
5.3 Data Quality Indicators 14
5.3.1 Representativeness 14
5.3.2. Accuracy 14
5.3.3 Precision 15
5.3.4 Statistical Uncertainty 16
5.3.5 Completeness 16
5.5 Data Validation and Reporting 17
5.5.1 Data Validation 17
5.5.2 Data Reporting 17
5.6 Testing Inspections 17
6.0 Data Management, Analysis, and Reporting 18
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6.1 Data Management and Analysis 18
6.2 Work Plan 18
6.3 Performance Reporting 18
6.4 Report of Equipment Testing 18
Tables
Table 4.1 Analytical Methods for Laboratory Analyses 7
in
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Abbreviations and Acronyms
ASTM American Society of Testing Materials
ATCC American Type Culture Collection
EC Degrees Celsius
CPU Colony Forming Units
cm Centimeter
DWS Drinking Water Systems
EPA U. S. Environmental Protection Agency
ETV Environmental Technology Verification
°F Degrees Fahrenheit
HPC Heterotrophic Plate Count
L Liter
LEVIS Laboratory Information Management System
mg Milligram
mL Milliliter
NaOH Sodium Hydroxide
ND Non-Detect
NIST National Institute of Standards and Technology
nm Nanometer
NSF NSF International (formerly known as National Sanitation Foundation)
NTU Nephelometric Turbidity Unit
PFU Plaque Forming Units
psig Pounds per Square Inch, Gauge
PSTP Product-Specific Test Plan
QA Quality Assurance
QC Quality Control
QA/QC Quality Assurance/Quality Control
QAPP Quality Assurance Project Plan
QMP Quality Management Plan
RPD Relative Percent Deviation
SOP Standard Operating Procedure
TDS Total Dissolved Solids
TSA Tryptic Soy Agar
TSB Tryptic Soy Broth
IV
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EXECUTIVE SUMMAR Y
This document is a Test / Quality Assurance Plan (TQAP) for the EPA/NSF Environmental
Technology Verification (ETV) Drinking Water Systems (DWS) Center. The purpose of this
document is to describe the TQAP for the verification of the Dow Chemical Company SFP-2880
ultrafiltration membrane module for removal of microbial contaminants per the requirements of
the Long Term 2 Enhanced Surface Water Treatment Rule (LT2ESWTR), as described in the
EPA Membrane Filtration Guidance Manual (MFGM). This TQAP describes the performance
evaluation test procedure and quality assurance/quality control procedures.
The Dow SFP-2880 ultrafiltration module will be challenged with the MS-2 coliphage virus and
endospores of Bacillus atrophaeus (ATCC 9372, deposited as Bacillus subtilis var. niger). The
endospores will function as a surrogate for Cryptosporidium parvum oocysts. One membrane
cartridge will also be challenged with live C. parvum oocysts to establish the surrogate
relationship between C. parvum and B. atrophaeus endospores.
The challenge protocol was adapted from the MFGM and the microbial seeding studies in the
ETV Protocol for Equipment Verification Testing for Physical Removal of Microbiological and
Paniculate Contaminants. This test plan only applies to microbial challenges. This verification
will not address long-term system performance over the life of the membrane, nor will it evaluate
cleaning of the membranes, nor any other maintenance and operation.
The experimental design conforms to the sample collection and test procedures for product-
specific testing as described in the MFGM. As the ETV Protocol is cited in the EPA's MFGM as
an acceptable approach for product-specific testing, the two documents are harmonized in their
respective requirements.
NSF International will perform all of the testing activities in their testing laboratory in Ann
Arbor, MI. Five membrane modules will be tested. The modules were selected by Dow from
different production runs. The membranes will be challenged at the flux specified by Dow. Each
module will be challenged for 30 minutes, with feed and filtrate samples collected for challenge
organism enumeration at start-up, 15 minutes, and 30 minutes. The modules will be operated at
the maximum specified flux of 70 gallons per square foot per day (gfd). The feed and filtrate
organism concentrations will be reported as logio numbers, and logio reductions will be
calculated.
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1.0 Equipment Verification Testing Responsibilities
1.1 Verification Test Site
All testing will be performed at the NSF International Testing Laboratory in Ann Arbor, MI.
This laboratory is used for all of the testing activities for NSF certification of drinking water
treatment systems, and pool and spa treatment systems.
1.2 Roles and Responsibilities
1.2.1 NSF International
NSF International (NSF) is an independent, not-for-profit organization founded in 1944 for the
purpose of developing standards and providing third-party conformity assessment services
addressing the needs of governmental agencies, and manufacturers and consumers of products
and systems related to public health, safety, and environmental quality.
NSF entered into an agreement on October 1, 2000 with the U. S. Environmental Protection
Agency (EPA) to create a Drinking Water Systems (DWS) Center dedicated to technology
verifications. NSF manages an Environmental Technology Verification (ETV) Program within
the DWS Center for the purpose of providing independent performance evaluations of drinking
water technologies. Evaluations are conducted using protocols developed with stakeholder
involvement.
NSF will follow the procedures and adhere to the requirements of this TQAP, and will also
comply with the data quality requirements in the NSF Drinking Water Systems Center Quality
Management Plan (QMP).
The following are the roles and responsibilities of NSF staff involved with the verification
testing:
Mike Blumenstein:
• preparation of TQAP;
• provide logistical support, and schedule and coordinate activities in the testing
laboratory;
• co-manage, evaluate, and interpret data generated by the verification testing; and
• preparation of the first draft of verification reports and verification statements, and
revise these documents after each round of review.
Sal Aridi:
• direct and oversee the NSF Testing Laboratory staff as they perform the testing
activities as described in this document; and
• adhere to the QA requirements of this TQAP, associated NSF Standard Operating
Procedures (SOP), and the NSF International Laboratories Quality Assurance Manual.
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Rob Donofrio:
• direct and oversee the Microbiology Laboratory staff as they perform microbiological
analysis of samples as required by the TQAP; and
• adhere to the QA requirements of this TQAP, associated NSF SOPs, and the NSF
International Laboratories Quality Assurance Manual.
Kurt Kneen:
• direct and oversee the Chemistry Laboratory staff as they perform chemical analyses
as required by the TQAP; and
• adhere to the QA requirements of this TQAP, associated NSF SOP's, and the NSF
International Laboratories Quality Assurance Manual.
Joe Terrell:
• independent review of the TQAP to insure compliance with the requirements of the
NSF Drinking Water Systems Center QMP;
• a technical systems audit of the NSF laboratories involved with testing to confirm that
the product evaluation, sample management, and sample analyses follow the TQAP
and QMP; and
• reviews drafts of the verification reports as needed.
Bruce Bartley:
• co-preparation of TQAP;
• co-manage, evaluate, and interpret data generated by the verification testing;
• co-preparation of the first draft of verification reports and verification statements, and
revision of these documents after each round of review;
• designation of an internal technical/engineering reviewer of the TQAP and draft
report; and
• co-preparation of the draft and final verification statements.
1.2.3 United States Environmental Protection Agency
The EPA provides leadership in the nation's environmental science, research, education and
assessment efforts. The EPA works closely with other federal agencies, state and local
governments, and Native American tribes to develop and enforce regulations under existing
environmental laws. The agency is responsible for researching and setting national standards for
a variety of environmental programs and delegates to states and tribes responsible for issuing
permits, and monitoring and enforcing compliance. Where national standards are not met, the
EPA can issue sanctions and take other steps to assist the states and tribes in reaching the desired
levels of environmental quality. The Agency also works with industries and all levels of
government in a wide variety of voluntary pollution prevention programs and energy
conservation efforts.
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The following are specific EPA roles and responsibilities:
• QA oversight of NSF International;
• Technical review and QA oversight of TQAP;
• Direct the performance, at the EPA's discretion, of external technical systems audit(s)
during the verification testing;
• Review draft verification reports and statements; and
• Final report approval and clearance for signature by the EPA Laboratory Director.
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2.0 Equipment Description
The Dow SFP-2880 is a polyvinylidene fluoride (PVDF) hollow fiber ultrafiltration membrane
module. The module specifications and operating parameters are listed in Table 2-1. The SFP-
2880 is a pressure driven module, with the normal operating flow orientation from the outside to
the inside of the fibers.
The SFP-2880 is certified to NSF/ANSI Standard 61.
Table 2-1. SFP-2880 Specifications
Parameter
Dimensions:
Module outside diameter
Module length
Module volume
Nominal membrane pore size
Maximum membrane pore size
Average active membrane area (outer)
Operating Limits:
Filtrate flux range at 25°C
Flow range
Operating temperature range
Max. inlet module pressure
Max. transmembrane pressure (TMP)
Operating pH range
Max. NaOCl
Max. TSS
Max. Turbidity
Specification
8.9 inches (in) (225 millimeters, mm)
92.9 in (2360 mm)
10.3 gallons (gal) (39 liters, L)
0.03 (im
0.05 urn
829 square feet (ft2) (77 square meters, m2)
24-70 gallons per square foot per day (gfd) (40-120 L/m2/hr)
13.6-40.9 gallons per minute (gpm) (3.1-9.3 m3/hr)
34-104 Fahrenheit (°F) (1-40 Celcius, °C)
44 pounds per square inch (psi) (3.0 bar)
30 psi (2. 1 bar)
2-11
2,000 mg/L
lOOmg/L
300 NTU
Five modules will be tested. The modules were selected by Dow from five different production
runs. The module will not be tested in a pilot unit, but rather will be tested in a test rig
constructed by NSF. See Section 3.3 for more information about the test rig.
The module design allows for an optional reject line connection, but this port will be closed off
for the challenge tests. The modules will be operated in dead-end mode.
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3.0 Experimental Design
3.1 Experimental Design
The challenge protocol is adapted from the microbial seeding studies in the ETVProtocol for
Equipment Verification Testing for Physical Removal of Microbiological and Paniculate
Contaminants, and from the EPA MFGM. The ETV Protocol is cited in the MFGM as an
acceptable approach for product-specific testing. This test plan only applies to microbial
challenges. This verification will not evaluate cleaning of the membranes, nor any other
maintenance and operation.
3.2 Challenge Organisms
All five modules will be challenged with the MS-2 coliphage virus and endospores of Bacillus
atrophaeus (ATCC 9372, deposited as Bacillus subtilis var. niger). B. atrophaeus was selected
to function as a surrogate for Cryptosporidium parvum, due to the high cost and lack of
availability of suitable numbers of C. parvum for challenge testing. The strain of B. atrophaeus
to be used yields orange colonies with a distinctive morphology on trypicase soy agar (TSA), so
it can be distinguished from wild-type endospores that could be present as contamination. B.
atrophaeus endospores are ellipsoidal (football shaped), with an average diameter of 0.8 jim, and
an average length of 1.8 jim. In addition, one module will be challenged with live C. parvum
oocysts in order to experimentally confirm that B. atrophaeus is a suitable surrogate for C.
parvum. See Appendix A for further discussion regarding the use of Bacillus endospores as a
surrogate for Cryptosporidium.
The challenge organism suspensions will be injected into the feed water stream with the
following target concentrations in the feed water:
• MS-2 - 5xl05 plaque forming units per milliliter(PFU/mL);
• B. atrophaeus - IxlO7 colony forming units (CPU) per lOOmL; and
• C.parvum - 5xl05 oocysts per liter (L).
The MFGM calls for the maximum challenge concentration to be 6.5 logic above the organism's
detection limit (3.16x 106). The goal for the B. atrophaeus challenges is to be able to measure log
reductions greater than six. Based on previous testing experience and expected organism
recovery levels, it is necessary to set the target at approximately 0.5 logic above the 3.16xl06
CFU/100 mL limit to ensure that greater than IxlO6 CFU/100 mL will be measured in the feed
samples.
3.3 Test Apparatus
The modules will be tested in a test rig constructed specifically for these tests. The test rig
construction will conform to the requirements of the MFGM. See Figure 3-1 for a schematic
diagram of the test rig to be constructed for testing.
As stated in Section 2.0, the modules will be operated in dead-end mode.
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Deiorized Water Tank
Drain
Figure 3-1. Schematic diagram of the test rig to be used for verification testing.
The challenge organisms will be introduced into the feed water by intermittent injection during
the challenge tests. Injection and mixing of the organisms will follow the guidelines of the
MFGM. Specifically, the stock solution volume for injection will be between 0.5 and 2 percent
of the total test solution volume, a chemical metering pump that delivers a steady flow of the
challenge solution will be used, and the injection port will include a quill that extends into the
middle of the feed pipe.
Feed and filtrate grab samples will be collected from sample ports that also have quills extending
into the middle of the pipe, and the sample tap tips will be metal so they can be flame-sterilized
prior to sample collection. The feed sample tap will be located at least ten pipe diameters
downstream of the injection point, and the test rig will include an in-line static mixer in between
the injection and feed sample ports. The feed and filtrate sample ports will be located as close as
possible to the membrane modules.
3.4 Test Water Composition
Local tap water treated by carbon filtration, reverse osmosis, ultraviolet disinfection, and
deionization will be used as the base water for the tests. The base water has the following quality
control (QC) requirements for use in the NSF testing laboratory:
• Conductivity < 2 microsiemens (|lS) per centimeter (cm) at 25°C;
• Total organic carbon < 100 micrograms (|lg) per L;
• Total chlorine < 0.05 milligrams (mg) per L; and
• Heterotrophic bacteria plate count < 100 CFU/mL.
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Of the above parameters, only total chlorine will be measured specifically for this verification.
The other parameters are measured periodically by NSF as part of the internal quality assurance
(QA)/QC program for test water quality.
If necessary, the water will be treated by further mechanical filtration to reduce the concentration
of suspended solids to as low as possible.
A water supply tank will filled with the base water, and sodium bicarbonate will be added in
sufficient quantity to provide alkalinity at a target of 100 ± 10 mg/L as calcium carbonate. The
pH will then be adjusted as necessary with hydrochloric acid or sodium hydroxide to reach the
target range of 7.5 ± 0.5.
Feed water samples will be collected prior to each challenge period for analysis of total chlorine,
alkalinity, pH, temperature, total dissolved solids, total organic carbon, and turbidity. These
samples will be collected prior to injection of the challenge organism.
3.5 Sanitizing the Test Rig
Prior to initiation of testing, and during each module changeout, the test rig will be sanitized
using a bleach solution at an appropriate CT. Deionized water shall be used for the sanitization
procedure.
3.6 Module Conditioning
Prior to testing, the modules will be conditioned following a procedure supplied by Dow.
Immediately prior to testing, each module will also be backflushed per Dow's specifications.
3.7 Membrane Integrity Tests
Before and after each challenge test, each module will undergo a 20-minute pressure decay test to
satisfy the non-destructive performance test requirement in Section 3.6 of the MFGM. The test
procedure will follow ASTM D6908-03 Standard Practice for Integrity Testing of Water
Filtration Membrane Systems. The water will be drained from the feed side of the membrane,
but not the filtrate side. Air pressure will be applied to the feed side to measure the decay rate.
The applied pressure will be measured every minute to chart the pressure decay.
The baseline pressure decay of the test rig will also be measured over 20 minutes and recorded
prior to installation of each module. Then, the initial applied pressure for that module's pre-
challenge and post-challenge pressure decay tests will be greater than or equal to 20 psig plus the
total baseline decay value measured over 20 minutes. This applied pressure will meet the applied
pressure resolution requirement of Section 4.2.1 of the MFGM.
3.8 Microbial Challenge Test Procedure
Each of the five SFP-2880 modules submitted for testing will be challenged individually. The
test rig will be sanitized with a bleach solution before the start of testing, and as part of the
changeout procedure for each module. The target flux for membrane operation will be Dow's
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maximum recommended value of 70 gfd at 25 °C, which equals a flow rate of approximately 40
gpm.
Separate challenge tests will be conducted for each challenge organism, so each module will be
tested twice over the course of the testing activities, and one module will be tested a third time
with C. parvum. The module chosen for the C. parvum challenge will be the one with the
highest filtrate counts from the Bacillus endospores challenge. The testing laboratory expects to
run both the MS-2 and Bacillus challenges in the same day, so that one module is tested per day.
The modules will be "brand new" when challenged. There will be no seasoning period, other
than that specified by Dow to sufficiently rinse out the membrane preservative and wet the
membranes.
Each membrane will be individually plumbed to the test rig after the rig has been sanitized and
rinsed. If it is the first time the module is installed, it will be flushed per Dow's flushing and
conditioning procedure. If the module has already been tested once (or twice in the case of the
module for the C. parvum test), the module will only be forward flushed at 40 gpm for one
minute. Immediately prior to testing, each module will be backflushed for one minute at a flow
rate of 40 gpm.
The next step will be the pre-challenge pressure decay test. See Section 3.7 for the pressure
decay test procedure. After the pressure decay test is complete, the test water feed (minus
challenge organism injection) will be resumed at 40 gpm. After an additional minute of
membrane flushing, a negative control filtrate flush sample will be collected for challenge
organism enumeration. During this flush, also collect an additional filtrate sample to serve as the
matrix spike sample, and adjust the flow rate and feed/filtrate pressures as necessary, to prepare
for the challenge test.
Each challenge test will be approximately 35 minutes in length. As discussed in Section 3.3, the
challenge organisms will be intermittently injected into the feed stream prior to, and during
sample collection. Sections 3.10.2, 3.10.4, and 3.12.4 of the MFGM describe the requirements
for the challenge test sampling plan. The MFGM requires that feed and filtrate samples not be
collected until at least three hold-up volumes of water containing the challenge organism have
passed through the membrane, to allow for establishment of equilibrium (equilibrium volume).
The hold-up volume is defined as the "unfiltered test solution volume that would remain in the
system on the feed side of the membrane at the end of the test." Dow's specification sheet for the
SFP-2880 gives the module volume as 10.3 gal. It is assumed that this volume is the total water
holding volume of the module, not just the volume of the feed side of the membranes. As such,
its use as the hold-up volume will add a safety factor to the hold up volume calculation.
The MFGM also specifies that the challenge organisms are injected at least 10 pipe diameters
upstream of the feed sample tap, and that the feed sample tap should be as close as possible to the
modules. The inlet and outlet fittings on the SFP-2880 module are 2 in (DN50), so the pipe to be
used for the test rig will also be 2 inches in diameter. Therefore, the injection point must be at
least 20 in upstream of the feed sample tap. The test rig has not yet been constructed as of this
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writing, so the test plan will speculate here about the expected hold-up volume of the test rig.
The injection point will be at most 36 in from the feed sample tap. Thirty-six inches of 2-inch
diameter pipe has a volume of 113 in3, which translates into 0.49 gal. The maximum expected
pipe volume plus the module volume gives a hold-up volume of approximately 10.8 gal, which
will be rounded up to 11 gal here for simplicity. If the hold-up volume is 11 gal, then the
equilibrium volume is 33 gal. The challenge flow rate will be approximately 40 gpm, so the
challenge organisms will be injected for 1 minute prior to sampling to meet the requirement of
passing the equilibrium volume.
The challenge organism will be injected into the feed stream at start-up, after 15 minutes of
operation, and after 30 minutes of operation. After 1 minute of injection, grab samples will be
collected from the feed and filtrate sample taps. The sample taps will be flame sterilized prior to
sample collection. Also, at least 100 mL will be collected and discarded prior to sample
collection to flush the taps. After sample collection is complete, injection will be turned off and
clean water will be pumped through the modules until the next sampling point.
Log reduction values (LRV) will be calculated for each set of feed and filtrate samples.
The test procedure can be summarized as follows:
1. Sanitize the test rig with deionized water spiked with an appropriate amount of bleach.
2. Install and condition the module, or flush for one minute.
3. Backflush module at 40 gpm for 60 seconds.
4. Conduct the pre-challenge pressure decay test.
5. Conduct the microbial challenge test
a. Flush the module for 1 minute, then collect the filtrate flush and matrix spike
samples.
b. Adjust the flow and pressure if needed.
c. Collect feed samples for the water quality analyses.
d. Begin injection of the challenge organism suspension.
e. Inject the challenge organism for at least one minute, then collect the required
volumes of feed, then filtrate for microbial analysis. Flame sterilize the sample
taps prior to sample collection. Flush the sample taps with at least 100 mL prior
to beginning sample collection.
f. After sample collection is complete, turn off injection.
g. Operate the module using the feed water minus the challenge injection until the
next sampling point.
h. Repeat steps d through g after 15 minutes and 30 minutes of elapsed module
operation time.
6. Conduct the post-challenge pressure decay test.
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4.0 Laboratory Operations Procedures
4.1 Introduction
This TQAP specifies procedures that will be used to ensure accurate documentation of
UF module performance. Careful adherence to these procedures and to the analytical
procedures will result in verifiable performance data.
4.2 Analytical Methods
A list of laboratory analytical methods for all parameters but MS-2 enumeration can be found in
Table 4.1. The analytical method for MS-2 is explained below the table.
Table 4.1 Analytical Methods for Laboratory Analyses
hd
re
"*
Alkalinity (total)
pH
TDS
Total Chlorine
Turbidity
MS-2
B. atrophaeus Endospores
C. parvum oocysts
2
o
c.
SM 2320B(2)
SM 4500-H+ B
SM 2540 C
SM 4500-C1 G
SM2130B
see below
SM9218(5)
EPA 1623
f?
•a
HH °
3 | %
ff Cf« *3
5mg/L
5mg/L
0.05 mg/L
0.1 NTU(4)
1 PFU/mL
1 CFU/lOOmL
1 oocyst/L
9£
X*
0 °
3 3
^ VI
90-110
±0.1
units
90-110
90-110
95-105
NA
NA
NA
r
9 *
5?
d %.
P O
O^ B
<13
<10
< 10
<10
NA
NA
NA
H
o
C.
cr«
H
§'
re
14 days
(3)
7 days
(3)
(3)
8 hours
30 hours
72 hours
O
o
B C«
s-1
B u
re &
i re
1 L plastic
NA
1 L plastic
NA
NA
125mL
plastic
125mL
plastic for
feed, 1 L
plastic for
filtrate
1 L plastic(6)
hd
B
5 $
8. 3
B re"
none
none
none
none
none
polysorbate 20
(Tween), store
at 3 + 2 °C
polysorbate 20
(Tween), store
at 3 + 2 °C
polysorbate 20
(Tween), store
at 3 + 2 °C
(1) RPD = Relative Percent Deviation
(2) SM = Standard Methods
(3) Immediate analysis required
(4) NTU = Nephelometric Turbidity Unit
(5) Trypticase soy agar (ISA) will be substituted for nutrient agar in SM 9218 so that the challenge endospores could be
distinguished from wild-type endospores. ISA gives orange colonies with a distinctive morphology.
(6) For the required triplicate analyses, plus backup samples, the Microbiology Laboratory will need six 1 L bottles of filtrate at
each sample point. Two 1 L bottles of the feed will be needed. The feed and filtrate samples should be collected into a single
sterile container, and the 1 L bottles filled from these volumes.
The following are the analytical instruments to be used for the water quality measurements:
10
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Alkalinity - SmartChem Discrete Analyzer;
pH - Orion EA 940 pH/ISE meter;
Temperature - Fluke 51 II digital thermometer;
Total Chlorine - Hach DR/2800 spectrophotometer using AccuVac vials; and
Turbidity - Hach 21 OOP turbidimeter.
4.2.1 Sample processing, and enumeration of MS-2 coliphages:
One milliliter volumes of the feed samples will be serially diluted for enumeration. One
milliliter volumes of the filtrate samples will be both enumerated directly and serially diluted for
enumeration. The one mL volumes are added to tubes containing the host E. coli in tryptic soy
broth (TSB). The tube is vortexed for 30 seconds, and then 4 mL of molten, tempered 1% tryptic
soy agar (TSA) is added to the tube. This mixture is then poured over a TSA plate, and the plate
is incubated at 35 °C for 18-24 hours. The viral plaques will be counted using a Quebec Colony
Counter.
4.3 Analytical QA/QC Procedures
Accuracy and precision of sample analyses shall be ensured through the following measures:
• Alkalinity - A certified QC sample is analyzed each day. The acceptable recovery limit is
that specified with the sample.
• pH - Three-point calibration (4, 7, 10) of the pH meter used to give the reportable data
shall be conducted daily using traceable buffers. The accuracy of the calibration shall be
checked daily with a pH 8.00 buffer. The pH reading for the buffer shall be within 10%
of its true value. The precision of the meter shall be checked daily using duplicate
synthetic drinking water samples. The RPD of the duplicate samples shall be less than
10%.
• TDS - A QC sample is analyzed with each sample batch. The percent recovery must be
within 10%, or the QC sample manufacturer's specified limits. Also, one blank (empty
evaporating dish) is run with each batch, and must be within 0.5 mg of original weight.
Ten percent of samples are analyzed in duplicate, and should agree with 5% of average
weight (10% RPD).
• Temperature - The thermometer used to give the reportable data shall have a scale
marked for every 0.1°C. The thermometer is calibrated yearly using a Hart Scientific Dry
Well Calibrator Model 9105.
• Total chlorine - The calibration of the chlorine meter shall be checked daily using a DI
water sample (blank), and three QC standards. The measured QC standard values shall
be within 10% of their true values. The precision of the meter shall be checked daily by
duplicate analysis of synthetic drinking water samples. The RPD of the duplicate samples
shall be less than 10%.
• Turbidity - The turbidimeter shall be calibrated as needed according to the
manufacturer's instructions with formazin standards. Accuracy shall be checked daily
11
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with a secondary Gelex standard. The calibration check shall give readings within 5% of
the true value. The precision of the meter shall be checked daily by duplicate analysis of
synthetic drinking water samples. The RPD of the duplicate samples shall be less than
10%.
• Sample processing and enumeration of MS-2
o Samples will be stored in the dark at 3 ± 2 °C until analyzed.
o All samples will be analyzed in triplicate.
o All batches of media will be checked for sterility and for positive growth
response.
o Membrane filters and dilution water will also be checked for sterility.
o Cultures will be checked for purity.
• Sample processing and enumeration of B. subtilis endospores.
o Samples will be stored in the dark at 3 ± 2 °C until analyzed.
o All samples will be analyzed in triplicate.
o All batches of media will be checked for sterility and for positive growth
response.
o Membrane filters and dilution water will also be checked for sterility.
• Sample processing and enumeration of C. parvum
o Samples will be stored in the dark at 3 ± 2 °C until analyzed.
o All samples will be analyzed in triplicate.
o A matrix spike (MS) sample will be processed and enumerated with every set of
samples. The percent recovery of the oocysts will be measured using the equation
in Section 5.3.2. The upper and lower control limits for percent recovery are
defined as the mean percent recovery from the last 20 recovery analyses ± 3
standard deviations. The NSF Microbiology Laboratory's current percent
recovery control limits are 53% to 135%. The mean recovery is 94.3%. New
control limits are calculated after every 10 recovery analyses. The matrix spike is
also used as the positive antigen control for the Crypto-a-glo™ antibody.
Please note that NSF analyzes many samples for these parameters every day. The samples for
alkalinity and TDS will be included in larger sample batches. Duplicate sample analysis
requirements apply to the whole batch, so NSF may not perform duplicate analysis on 10% of
samples from this test.
4.4 Sample Handling
All samples not immediately analyzed will be labeled with unique identification numbers. These
identification numbers will be entered into the NSF Laboratory Information Management System
(LDVIS), and will appear on the NSF lab reports for the tests. All challenge organism samples
will be stored in the dark at 3 ± 2 °C until processed for analysis.
Chlorine, pH, turbidity will be measured immediately after sample collection.
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4.5 Documentation
All laboratory activities will be thoroughly documented using lab bench sheets and NSF LIMS
laboratory reports.
NSF will be responsible for maintaining all documentation. Lab bench sheets will be used to
record all water treatment equipment operating data. Each page will be labeled with the project
name and number. Errors will have one line drawn through them and this line will be initialed
and dated.
Any deviations from the approved final TQAP will be thoroughly documented at the time of
inspection and in the verification report.
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5.0 Quality Assurance Project Plan
5.1 Introduction
The Quality Assurance Project Plan (QAPP) for this verification test specifies procedures that
will be used to ensure data quality and integrity. Careful adherence to these procedures will
ensure that data generated from the verification testing will provide sound analytical results that
can serve as the basis for the performance verification.
This section outlines steps that will be taken by NSF to ensure that data resulting from
verification testing is of known quality and that a sufficient number of critical measurements are
taken.
5.2 Quality Assurance Responsibilities
A number of individuals will be responsible for test equipment operation, sampling, and analysis
QA/QC throughout the verification testing. Primary responsibility for ensuring that these
activities comply with the QA/QC requirements of this TQAP rests with the supervisors of the
individual NSF laboratories.
NSF QA/QC staff will review the raw data records for compliance with QA/QC requirements.
NSF ETV staff will check 100% of the raw data records against the reported results in the LEVIS
reports.
5.3 Data Quality Indicators
The data obtained during the verification testing must be of sound quality for conclusions to be
drawn on the treatment equipment. For all verification activities, data quality parameters must be
established based on the proposed end uses of the data. These parameters include five indicators
of data quality: representativeness, accuracy, precision, statistical uncertainty, and completeness.
5.3.1 Representativeness
Representativeness refers to the degree to which the data accurately and precisely represent the
conditions or characteristics of the parameter represented by the data, or the expected
performance of the RO system under normal use conditions. Representativeness will be ensured
by executing consistent sample collection protocols, including timing of sample collection,
sampling procedures, and sample preservation. Representativeness will also be ensured by using
each analytical method at its optimum capability to provide the most accurate and precise
measurements possible.
5.3.2. Accuracy
Accuracy is a measure of the deviation of the analytical value from the true value. Since true
values for samples can never be known, accuracy measurements are made through analysis of
certified standards or QC samples of a known quantity.
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Accuracy will be maintained through the following items:
• Maintaining consistent sample collection procedures, including sample locations, timing
of sample collection, and sampling procedures;
• Calibrated instruments; and
• Laboratory control samples (e.g., method blanks, duplicates, matrix spikes, matrix spike
duplicates, and performance evaluation samples).
Recoveries for spiked samples will be calculated in the following manner:
100*(SSR-SR)
Percent Recovery = —
xj^i
where: SSR = spiked sample result
SR = sample result
SA = spike amount added
Recoveries for laboratory control samples are calculated as follows:
100 * (Found Concentration)
Percent Recovery =
True Concentration
For acceptable analytical accuracy, the recoveries must be within control limits. The NSF
laboratory's minimum acceptable accuracy for each parameter is listed in Table 4.1.
The accuracy of the benchtop chlorine, pH, and turbidity meters will be checked daily during the
calibration procedures using certified check standards. For samples analyzed in batches
(alkalinity and TDS), certified QC samples will be run with each batch.
5.3.3 Precision
Precision refers to the degree of mutual agreement among individual measurements and provides
an estimate of random error. Precision will be measured through duplicate sample analysis. One
sample per batch will be analyzed in duplicate for the TDS and alkalinity analyses. To check the
precision of the benchtop chlorine, pH, and turbidity meters, duplicate synthetic drinking water
samples will be analyzed daily. Precision of the duplicate analyses will be measured by use of
the following equation to calculate RPD:
RPD =
X200
where:
Sl = sample analysis result; and
^2 = sample duplicate analysis result.
Acceptable RPD values for each parameter are given in Table 4.1.
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5.3.4 Statistical Uncertainty
Statistical uncertainty of the triplicate challenge organism counts will be evaluated using
Microsoft® Excel 2003 to calculate the 95% confidence intervals. The following formula will be
employed for confidence interval calculation:
confidence interval = X ± 11_- (S/-Jnj
where:
X is the sample mean;
S is the sample standard deviation;
n is the number independent measures included in the data set;
t is the Student's t distribution value with n-1 degrees of freedom; and
V is the significance level, defined for 95% confidence as: 1 - 0.95 = 0.05.
5.3.5 Completeness
Completeness refers to the amount of data collected from a measurement process compared to
the amount that was expected to be obtained. Completeness refers to the proportion of valid,
acceptable data generated using each method. This portion of the required data for the selected
test plan will be reported at the conclusion of each testing period.
The completeness objective for data generated during verification testing is based on the number
of samples collected and analyzed for each parameter and/or method. The following chart
illustrates the completeness objectives for performance parameter and/or method based on the
sample frequency:
Number of Samples per
Parameter and/or Method
0-10
11-50
>50
Percent
Completeness
80%
90%
95%
Completeness is defined as follows for all measurements:
%C = (V/T)X100
where:
%C = percent completeness;
V = number of measurements judged valid;
T = total number of measurements.
Retesting may be required if the completeness objectives are not met.
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The following are examples of instances that might cause a sample analyses to be incomplete:
• Instrument failure;
• Calibration requirement not being met; or
• Elevated analyte levels in the method blank.
5.4 Data Validation and Reporting
To maintain good data quality, specific procedures will be followed during data validation, and
reporting. These procedures are detailed below.
5.4.1 Data Validation
For the analytical data:
• NSF ETV staff will review calculations and inspect laboratory logbooks and data sheets
to verify accuracy of data recording and sampling;
• The NSF QA/QC department will verify that all instrument systems are in control and
that QA objectives for accuracy, precision, and method detection limits have been met;
and
• NSF QA and ETV staff will review the raw data records for compliance with QC
requirements and check 100% of the data against the reported results from the LEVIS
reports.
Should QC data be outside of control limits, the analytical laboratory supervisor will investigate
the cause of the problem, and discussion of the problem will be included in the final report.
Depending on the severity of the problem, the data in question may be flagged, or not reported.
5.4.2 Data Reporting
The data to be reported will be the feed and treated water microorganism counts, log reductions,
and the water chemistry data. The QC data, such as calibrations, blanks and reference samples
will be not be reported, but will be kept on file at NSF.
5.5 Testing Inspections
NSF QA staff will conduct an audit of the laboratory during testing to ensure compliance with
the test procedures and requirements of this TQAP. The results of all audits will be reported to
the NSF ETV staff. Throughout testing, ETV staff will carry out random spot inspections. Any
variances will be reported to NSF QA staff.
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6.0 Data Management, Analysis, and Reporting
6.1 Data Management and Analysis
All operational and analytical data will be gathered and included in the Final ETV Report. The
data will consist of results of analyses and measurements and QA/QC reports.
The data management system for this verification involves the use of the NSF LEVIS computer
system, spreadsheet software and manual recording of system operating parameters.
6.2 Work Plan
The following is the work plan for data management:
• Laboratory personnel will record equipment operation, water quality and analytical data
by hand on bench sheets.
• All bench sheet entries will be made in water-insoluble ink.
• All corrections on the bench sheets will be made by placing one line through the
erroneous information. Any corrections will be dated and initialed by the lab personnel
making the correction.
• Pertinent information from the bench sheets will be entered into the LEVIS system. When
the test is complete, a preliminary report will be generated. The preliminary report will
be reviewed by the manager of any laboratory that entered data. Once the preliminary
report is approved, a final laboratory report will be generated and given to ETV staff.
The database for this verification testing program will be set up in the form of custom-designed
spreadsheets. Pertinent data from the LEVIS reports will be entered into the appropriate
spreadsheets. All recorded calculations will also be checked at this time. Following data entry,
the spreadsheet will be printed out and the printout checked against the LEVIS report.
6.3 Performance Reporting
Microorganism removal by the UF module will be evaluated through log reduction calculations.
All challenge organism samples will be analyzed in triplicate, so the geometric mean of each
triplicate set of results will be used for the calculations.
6.4 Report of Equipment Testing
The report will be issued in draft form for review prior to final publication. The reports will be
prepared by NSF and will consist of the following:
• Introduction;
• Description and Identification of Product Tested;
• Procedures and Methods Used in Testing;
• Results and Discussion, including QA/QC discussion; and
• References;
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This report will be prepared using Microsoft Word® 2003.
NSF ETV staff will prepare the first draft of the Verification Report and Verification Statement.
These documents will be reviewed by the NSF QA officer, and then will be sent to an outside
technical advisor for review. NSF will also send the draft documents to the EPA for review
concurrent with the technical advisor review.
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Appendix A
Bacillus Endospores as a Surrogate for C parvum Oocysts
The EPA LT2ESWTR allows the use of a surrogate for C. parvum, provided the surrogate is
conservative. The EPA MFGM specifically discusses Bacillus subtilis as a surrogate, but states
"Because there is limited data currently available regarding the use of Bacillus subtilis in
membrane challenge studies, a characterization of this organism would be necessary to determine
whether it could be used as a Cryptosporidium surrogate..." The MFGM also states "Based on
the size.. .Bacillus subtilis could potentially be considered a conservative surrogate.. .pending a
comparison of other characteristics (e.g., shape, surface charge, etc.)..."
1. Organism Size and Shape
C. parvum is spherical in shape, while Bacillus endospores are ellipsoidal in shape (football
shaped). C. parvum has a diameter of 4-6 jim. Bacillus endospores are approximately 0.8 jim in
diameter, and 1.8 jim in length. Therefore, Bacillus endospores are a conservative surrogate for
C. parvum, no matter what the orientation of the endospore is when it impacts the test membrane.
Baltus et. al. (2008) studied membrane rejection of bacteria and viruses with different length vs.
diameter aspect ratios. They theorized, based on a transport model for rod-shaped particles, that
rejection would improve as the aspect ratio (length vs. diameter) increased for a fixed particle
volume. However, their experimental results contradicted this, with similar rejection rates for
particles with a range of aspect ratios. The model assumed that particles would impact the
membrane with equal frequency for all particle orientations. They theorize that instead, an end-
on orientation was favored for transport of the particles in the water stream. They concluded that
microorganism removal by membranes could be conservatively estimated using only the rod
diameter in transport models. These findings add an additional safety factor to using Bacillus
endospores as a surrogate for C. parvum.
2. Electrophoretic Mobility and Isoelectric Point
A suitable surrogate should have a surface charge similar to C. parvum, as measured through the
isoelectric point and electrophoretic mobility (EPM). The isoelectric point is the pH at which the
particle has a neutral surface charge in an aqueous environment. Below this point the particle has
a net positive charge, above it a net negative charge. Many studies have pegged the isoelectric
point of C. parvum between pH values of 2 and 4, thus it would have a negative surface charge in
the neutral pH range. The isoelectric point can be found by measuring the EPM of the particle at
various pH values. The pH where the EPM is zero is classified as the isoelectric point.
Lytle et. al. (2002) measured the EPM of both C. parvum and B. subtilis endospores in solutions
of increasing buffer concentration (0.915 millimolar, mM, 9.15 mM, and 91.5 mM KH^PO/i).
They found that increasing the buffer concentration also increases the EPM toward a positive
value. The buffer concentration of the test water for the Siemens tests was approximately 1 mM.
Therefore, the 0.915 mM data from this study should be the most accurate representation of the
C. parvum and B. subtilis EPM for the ETV tests. In 0.915 mM solutions at pH values between 7
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and 8, they observed EPM of approximately -2.2 to -2.6 jim cm V"1 s"1 for C. parvum, and -1.9 to
-2.2 |im cm vV1 for B. subtilis. For B. subtilis, the researchers did not measure an isoelectric
point at any buffer concentration. For C. parvum, they did find an isoelectric point at a pH
around 2.5, but only for the 9.15 mM solution. For both organisms, the 0.915 mM solution
generally gave lower (more negative) EPM values than the solutions with higher buffering
capacity.
3. Aggregation
The NSF Microbiology Laboratory microscopically examined a sample of the B. atrophaeus
stock solutions purchased for the tests. The sample was suspended in sterile, buffered, deionized
water and stirred at moderate speed for 15 minutes. The estimated cell density was IxlO9
CFU/100 mL, which is approximately 100 times higher than the suspensions injected into the
pilot units to challenge the UF membranes. Figure 1 is a photograph of the B. atrophaeus
endospores in the sample. The magnification is lOOOx oil immersion with differential
interference contrast microscopy. No evidence of endospore aggregation was found.
Figure B-l. Mono-dispersed B. atrophaeus endospores used for challenge tests.
References
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Baltus, R. E., A. R. Badireddy, W. Xu, and S. Chellam (2009). Analysis of Configurational
Effects on Hindered Convection of Nonspherical Bacteria and Viruses across Microfiltration
Membranes. Industrial and Engineering Chemistry Research. In press.
Brush, C. F., M. F. Walter, L. J. Anguish, and W. C. Ghiorse (1998). Influence of Pretreatment
and Experimental Conditions on Electrophoretic Mobility and Hydrophobicity of
Cryptosporidium parvum Oocysts. Applied and Environmental Microbiology. 64: 4439-4445.
Butkus, M. A., J. T. Bays, and M. P. Labare (2003). Influence of Surface Characteristics on the
Stability of Cryptosporidium parvum Oocysts. Applied and Environmental Microbiology. 69:
3819-3825.
Lytle, D. A., C. H. Johnson, and E. W. Rice (2002). A Systematic Comparison of the
Electrokinetic Properties of Environmentally Important Microorganisms in Water. Colloids and
Surfaces B: Biointerfaces. 24: 91 -101.
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Appendix B
Dow Flushing Procedure
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