EPA/600/R-14/052s
December 2013
THE ENVIRONMENTAL TECHNOLOGY VERIFICATION
PROGRAM
EPA
Baireiie
The Business of Innovation
TECHNOLOGY TYPE: AQUEOUS LEAD TESTING
APPLICATION:
LEAD ANALYSIS FOR DRINKING, WASTE, AND
ENVIRONMENTAL WATERS
TECHNOLOGY NAME: AND1000 Fluorimeter and LeadlOO Test Kit
COMPANY:
ADDRESS:
WEB SITE:
E-MAIL:
ANDalyze, Inc.
2109 S. Oak St., Suite 102
Champaign, IL 61820
http: //www.andalyze .com/
info@andalyze.com
PHONE: 217-328-0045
ETV Joint Verification Statement
The U.S. Environmental Protection Agency (EPA) has established the Environmental Technology Verification
(ETV) Program to facilitate the deployment of innovative or improved environmental technologies through
performance verification and dissemination of information. The goal of the ETV Program is to further
environmental protection by accelerating the acceptance and use of improved and cost-effective technologies.
ETV seeks to achieve this goal by providing high-quality, peer-reviewed data on technology performance to
those involved in the design, distribution, financing, permitting, purchase, and use of environmental
technologies. Information and ETV documents are available at www.epa.gov/etv.
ETV works in partnership with recognized standards and testing organizations, with stakeholder groups
(consisting of buyers, vendor organizations, and permitters), and with individual technology developers. The
program evaluates the performance of innovative technologies by developing test plans that are responsive to the
needs of stakeholders, conducting field and laboratory tests (as appropriate), collecting and analyzing data, and
preparing peer-reviewed reports. All evaluations are conducted in accordance with rigorous quality assurance
(QA) protocols to ensure that data of known and adequate quality are generated and that the results are
defensible.
The Advanced Monitoring Systems (AMS) Center, one of six verification centers under ETV, is operated by
Battelle in cooperation with EPA's National Risk Management Research Laboratory. The AMS Center evaluated
the performance of an aqueous lead (Pb) testing device for lead analysis in drinking, waste, and environmental
waters. This verification statement provides a summary of the test results for the ANDalyze, Inc. AND1000
fluorimeter and LeadlOO test kit (Lead 100/AND1000) for determining lead concentrations in environmental
water.
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VERIFICATION TEST DESCRIPTION
The verification testing of the Lead 100/AND1000 was conducted in two rounds: the first from July 10 to July 27,
2012, and the second from February 11 to March 13, 2013 at both various field sites and the Environmental
Treatability Laboratory (ETL) at Battelle's Main Campus in Columbus, OH. The verification test assessed the
performance of the Lead 100/AND 1000 relative to key verification parameters including accuracy, precision,
sample throughput, and ease of use. These performance parameters were evaluated using multiple variables that
challenged the Lead 100/AND1000's ability to detect Pb in a variety of aqueous matrices. The technology
evaluation was organized as four main tests. Each test evaluated the performance of Lead 100/AND 1000
operating under different laboratory and field conditions. The four tests were:
1. Initial demonstration of capability (IDC) and performance testing (PT) including determination of the limit of
detection (DLOD), determination of linear range (DLR), and determination of the effects of interferences
(DEI)
2. Testing accuracy and precision of the instrument for the analysis of finished drinking water samples (bottled
water, municipal drinking water, and treated groundwater)
3. Testing accuracy and precision of the instrument for the analysis of environmental water samples (surface
water and groundwater) and qualitative performance for the analysis of seawater
4. Testing accuracy and precision of the instrument for the analysis of wastewater effluent samples (municipal
wastewater effluent and metal finishing wastewater effluent)
The performance parameters were evaluated quantitatively using the statistical methods and qualitatively through
recorded observations. All samples analyzed with the Lead 100/AND 1000 were also collected, stored,
transferred, and analyzed in a certified laboratory using industry accepted analytical methods. All tests were
performed with the Lead 100/AND 1000 operating according to the vendor's recommended procedures as
described in the user's manuals and during training provided to the operator.
The first set of tests (IDC and PT) was entirely performed within Battelle's ETL. IDC required the detection of a
Pb spike of 25 parts per billion (ppb) in reagent grade water. DLOD was performed by measuring seven
replicates of Pb spiked at 10 ppb, five times the vendor-reported limit of detection (2 ppb). The DLR was carried
out by measuring the Lead 100/AND1000's ability to precisely and accurately measure seven samples with Pb
concentrations from 0 to 100 ppb. The samples were analyzed in triplicate and the coefficient of determination
was used to assess the linearity of the response of the instrument within this range. Finally, DEI was determined
using three synthetic water samples with differing characteristics: low total dissolved solids concentration
(LTDS), high total dissolved solids concentration (HTDS), and high iron (Fe) concentration and other dissolved
solids (HFe). Each of these synthetic water samples was split into required 100 mL subsamples and received a Pb
spike of 25 ppb and 50 ppb before Pb was measured in triplicate from each subsample by Lead 100/AND 1000.
Normal sample preparation procedures were followed for the LTDS and HTDS waters, and a special sample
preparation procedure for the removal of Fe interference was used to prepare the HFe sample for Pb analysis.
The next set of tests determined the accuracy and precision of the Lead 100/AND 1000 in recovery of Pb spikes in
finished drinking water. Three sets of samples were prepared with Pb spikes of 25 ppb: finished drinking water
samples collected from a water fountain (WF), bottled mineral water purchased from a local supermarket (Bottled
Water [BW]), and finished drinking water collected from the effluent of a local water treatment facility treating
groundwater (Finished Well Water [FWW]). All waters were analyzed without a Pb spike and in triplicate with a
Pb spike of 25 ppb.
The third series of tests aimed at determining the accuracy and precision of the Lead 100/AND 1000 in recovering
Pb spikes in environmental water samples. Three freshwater sources were sampled including water collected
from the reach of a freshwater river just outside and upstream of Columbus, OH (River Water [RiW]), samples
collected from a freshwater reservoir near the same location (Reservoir Water [ReW]), and raw groundwater
collected at the source of a drinking water treatment facility just outside Plain City, OH (Raw Well Water
[RWW]), which was collected from the source that feeds the facility from which the Finished Well Water was
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collected. In addition, one seawater sample was collected off the coast of West Palm Beach, FL (Seawater [SW])
to determine the accuracy and precision of the Lead 100/AND1000 in testing natural waters with high salinity.
All four environmental samples were analyzed with no Pb spike and in triplicate after the addition of a Pb spike to
25 ppb. Performance of tests on Seawater differ from the performance of tests on freshwater in that seawater was
diluted tenfold before being subjected to Lead 100/AND 1000 testing and results were analyzed qualitatively, not
quantitatively as with freshwater samples.
The final series of tests aimed at determining the accuracy and precision of the Lead 100/AND 1000 in recovering
Pb spikes in wastewater effluent samples. Three samples were analyzed during this series of tests: two effluent
samples collected from two separate traditional activated sludge treatment facilities treating domestic wastewater
in Columbus, OH (Municipal Wastewater Effluent #1 [MWWE#1] and Municipal Wastewater Effluent #2
[MWWE#1]) and a sample collected from the effluent of a metal finishing works (Metal Finishing Wastewater
Effluent [MFWWE]). The Metal Finishing Wastewater Effluent was collected from a facility conforming to 40
Code of Federal Regulations (CFR) 433 and/or 40 CFR 413 after all on-site pretreatment.
QA oversight of verification testing was provided by Battelle and EPA. Battelle QA staff conducted technical
systems audits of all laboratory testing and conducted a data quality audit of at least 10% of the test data. This
verification statement, the full report on which it is based, and the test/QA plan for this verification test are
available at http://www.epa.gov/nrmrl/std/etv/verifiedtechnologies.html#water.
TECHNOLOGY DESCRIPTION
The following is a description of the technology, based on information provided by the technology
representative. The information provided below was not verified in this test.
The ANDalyze Lead 100/AND 1000 was designed to detect the presence of and measure the concentration of
soluble/bioavailable Pb in drinking water and environmental waters. Testing is intended to take place onsite at
the source of collection or in a temperature controlled facility without sample preservation. The test makes use of
two primary components: a handheld fluorimeter (AND1000) and a consumable test kit (LeadlOO) specific to
each metal or target (in the present case, Pb).
The AND 1000 fluorimeter is specifically designed to provide an interactive experience and allow testing, data
storage, and signal output without the use of a separate computational device. The fluorimeter has the capability
to analyze multiple targets with the appropriate test kit, though the sole target discussed here is aqueous Pb in
drinking water, wastewater effluent, and environmental waters. The AND 1000 fluorimeter enables field testing
to be performed in two steps. ANDalyze's catalytic deoxyribonucleic acid (DNA) sensors use a metal-specific
DNAzyme reaction that leads to an increase in fluorescence in the presence of a target contaminant substance
(i.e., aqueous Pb). The fluorescence of the reaction is measured by a fluorimeter to determine the concentration
of the target heavy metal and is reported in parts per billion (ppb). The product is a quantitative test that is
intended to detect metals in a linear range of 2 to 100 ppb — at and below EPA standards for drinking water. The
test is performed by injecting a buffered 1 mL water sample through the sensor and into the AND 1000
fluorimeter. This sample is then automatically analyzed and results are reported in less than 2 minutes. The
AND 1000 fluorimeter package includes the fluorimeter, USB to MINI-B cable, 100 uL fixed volume pipette and
tips, and pH test strips.
The second component is the LeadlOO test kit which is specific to Pb. The test kit provides all necessary
materials for in-field instrument calibration and sample testing. This kit contains the DNA sensors specific to Pb.
The kit is color coded to facilitate use and avoid measurement error. In addition, a product manual is provided
with step-by-step instructions including photographs. It should be noted that laboratory evaluation may require
additional supplies and standard laboratory glassware. Each kit includes 25 tests and/or calibrations, 25 sensor
bags with sensor and cuvette, 25 sample tubes (with buffer), 25 1 mL syringes, 25 disposable transfer pipettes, 8
mL analyte standard solution, instruction manuals, and material safety data sheets.
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VERIFICATION RESULTS
The verification of the Lead 100/AND1000 is summarized below.
Accuracy
Percent recovery was used for all Lead 100/AND 1000 observations to verify acceptability of on-site calibration
using control charts. Percent recovery was also used for IDC, ICC, DEI, finished drinking water testing,
environmental water testing, and wastewater effluent testing as acceptance criteria for observations, for which
percent recoveries between 75 and 125% were considered acceptable. Unacceptable recoveries were first
retested, then—if the recovery was still unacceptable—tested again after instrument recalibration, and finally
targeted in a root cause analysis (RCA).
No quality control (QC) issues were indicated on control charts for the 12 observations on IDC and ICC related
samples. However, retesting was performed four times and on-site recalibration was performed once during IDC
and ICC testing, and two IDC samples still did not meet criteria for percent recovery when compared to reference
concentrations. Several QC issues were indicated on control charts for the 28 observations corresponding to
DLOD and DLR. However, on-site recalibration was not required for DLOD and DLR testing and all samples
met criteria for percent recovery when compared to reference concentrations. One QC issue was indicated on
control charts for the 25 observations corresponding to DEI. Retesting was performed on four samples during
DEI, but on-site recalibration was not required for DEI as all samples met criteria for percent recovery when
compared to reference concentrations. Generally, recoveries during IDC, ICC, and DEI were considered
acceptable.
Two QC issues were indicated on control charts for the 19 observations corresponding to environmental samples.
Retesting was performed on four samples during environmental sample testing and on-site recalibration was
required and a RCA was performed for raw well water samples suspected of containing high levels of Fe that
interfered with the test. Nevertheless, five observations did not meet criteria for percent recovery when compared
to reference concentrations. Two QC issues were indicated on control charts for the 17 observations
corresponding to drinking water samples. Retesting was performed on three samples during drinking water
sample testing, and on-site recalibration was required twice during drinking water testing. During testing, BW
did not appear to meet QC criteria and a RCA was performed. It was determined that these observations were in
fact within QC criteria once reference data were received. Nevertheless, four observations did not meet criteria
for percent recovery when compared to reference concentrations. No QC issues were indicated on control charts
for the 15 observations corresponding to wastewater effluent samples. Retesting was performed on two samples
during wastewater effluent testing, and on-site recalibration was required once. Nevertheless, five observations
did not meet criteria for percent recovery when compared to reference concentrations. More unacceptable
recoveries were observed during drinking water, environmental water, and wastewater effluent testing than in the
previous IDC and PT testing; however, recoveries were still generally considered acceptable based on the
complexity of those matrices.
Precision
Mean observation value, standard deviation (SD), and coefficient of variation (CV) were determined for all 23
samples analyzed in triplicate. Standard deviations ranged from 0.58 to 11.14, corresponding to coefficients of
variation ranging from 3% to 19%.
Linearity of Response
Observations from DLR sample testing were plotted against concentrations from reference analysis. Linearity
was calculated in terms of slope (0.8841), intercept (-0.8418), and the square of the correlation coefficient (r2)
(0.9927). Results from the Lead 100/AND 1000 were generally in good agreement with results from the reference
analysis, as indicated by a slope close to 1 and a coefficient of determination close to 1.
Limit of Detection
DLOD testing included statistical analyses in accordance with 40 CFR Part 136. The limit of detection of the
Lead 100/AND 1000 was calculated to be 1.534 ug/L Pb, which is below the vendor's estimated detection limit.
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Qualitative Results for Seawater Samples
Precision was also calculated for Seawater samples to assist in qualitative analysis. As expected, the coefficients
of variation for Seawater observations were significantly higher than those associated with any other test owing to
the high salinity of the matrix. However, observations aligned with reference concentrations (i.e., no observations
for samples prepared with 20 ug/L Pb were greater than any observations for samples prepared with 40 ug/L Pb).
This demonstrates that the Lead 100/AND1000 is capable of indicating whether seawater has a high or low
concentration of Pb, despite its low precision when analyzing seawater samples.
Operational Factors
In general, the ease of use of the Lead 100/AND 1000 was high. In several cases, the rechargeable battery
provided with the AND1000 lost power after less than 8 hours. In one instance, the instrument displayed a screen
that was foreign to the user making the instrument unusable; however, simply turning the instrument off and
rebooting retuned the AND 1000 to normal working order. The Lead 100 test kits generate a significant amount of
solid waste if many tests are performed. Under remote/austere sampling conditions in which waste minimization
is essential, this should be taken into account by the user. The instrument cannot be used to determine particulate
Pb. Additionally, samples are intended to be unpreserved for analysis by the technology and the pH of the
samples is an important parameter to measure to ensure that the pH is within the specifications for accurate
analysis.
Signed by Spencer Pugh
1/14/14
Spencer Pugh
General Manager
Energy and Environment Business Unit
Battelle
Date
Signed by Cynthia Sonich-Mullin 1/23/14
Cynthia Sonich-Mullin Date
Director
National Risk Management Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
NOTICE: ETV verifications are based on an evaluation of technology performance under specific,
predetermined criteria and the appropriate quality assurance procedures. EPA and Battelle make no
expressed or implied warranties as to the performance of the technology and do not certify that a technology
will always operate as verified. The end user is solely responsible for complying with any and all applicable
federal, state, and local requirements. Mention of commercial product names does not imply endorsement.
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