May 2011
Environmental Technology
Verification Report
ZEU INMUNOTEC
MICROCYSTIN TEST KIT:
MlCROCYSTEST
Prepared by
Battelle
Baireiie
77>o Business of Innovation
Under a cooperative agreement with
U.S. Environmental Protection Agency
ET1/ET1/ET1/
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May 2011
Environmental Technology Verification
Report
ETV Advanced Monitoring Systems Center
ZEU-lNMUNOTEC, S.L.
MICROCYSTIN TEST KIT:
MlCROCYSTEST
by
Ryan James, Anne Gregg, and Amy Dindal, Battelle
John McKernan, U.S. EPA
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Notice
The U.S. Environmental Protection Agency, through its Office of Research and Development,
funded and managed, or partially funded and collaborated in, the research described herein. It
has been subjected to the Agency's peer and administrative review. Any opinions expressed in
this report are those of the author(s) and do not necessarily reflect the views of the Agency,
therefore, no official endorsement should be inferred. Any mention of trade names or
commercial products does not constitute endorsement or recommendation for use.
11
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Foreword
The EPA is charged by Congress with protecting the nation's air, water, and land resources.
Under a mandate of national environmental laws, the Agency strives to formulate and implement
actions leading to a compatible balance between human activities and the ability of natural
systems to support and nurture life. To meet this mandate, the EPA's Office of Research and
Development provides data and science support that can be used to solve environmental
problems and to build the scientific knowledge base needed to manage our ecological resources
wisely, to understand how pollutants affect our health, and to prevent or reduce environmental
risks.
The Environmental Technology Verification (ETV) Program has been established by the EPA to
verify the performance characteristics of innovative environmental technology across all media
and to report this objective information to permitters, buyers, and users of the technology, thus
substantially accelerating the entrance of new environmental technologies into the marketplace.
Verification organizations oversee and report verification activities based on testing and quality
assurance protocols developed with input from major stakeholders and customer groups
associated with the technology area. ETV consists of six environmental technology centers.
Information about each of these centers can be found on the Internet at http://www.epa.gov/etv/.
Effective verifications of monitoring technologies are needed to assess environmental quality
and to supply cost and performance data to select the most appropriate technology for that
assessment. Under a cooperative agreement, Battelle has received EPA funding to plan,
coordinate, and conduct such verification tests for "Advanced Monitoring Systems for Air,
Water, and Soil" and report the results to the community at large. Information concerning this
specific environmental technology area can be found on the Internet at
http ://www. epa.gov/etv/centers/centerl .html.
in
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Acknowledgments
The authors wish to acknowledge the contribution of the many individuals that made this
verification testing possible. Quality assurance (QA) oversight was provided by Michelle
Henderson, U.S. EPA, and Zachary Willenberg, Battelle. We thank David Schumacher of the
Nebraska Department of Environmental Quality and Robert Waters from the New York, Suffolk
County Department of Health Services for their support of this verification by providing
recreational water samples, and also for peer review of the test/QA plan. We acknowledge the
support from Daniel Snow and David Cassada from the University of Nebraska Water Sciences
Laboratory for developing and validating the reference method in addition to analyzing the
samples for the verification. Finally, we want to thank Andrew Lincoff, U.S. EPA and Daniel
Snow of the University of Nebraska for their review of the test/QA plan and this verification
report.
IV
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Contents
Page
Foreword iii
Acknowledgments iv
List of Abbreviations ix
Chapter 1 Background 11
Chapter 2 Technology Description 12
Chapters Test Design and Procedures 14
3.1 Test Overview 14
3.2 Experimental Design 14
3.3 Test Procedures 15
3.3.1 QC Samples 15
3.3.2 PT Samples 15
3.3.2 RW Samples 16
Chapter 4 Quality Assurance/Quality Control 18
4.1 Reference Method Quality Control 18
4.2 Audits 20
4.2.1 Performance Evaluation Audit 20
4.2.2 Technical Systems Audit 21
4.2.3 Data Quality Audit 21
Chapters Statistical Methods 23
5.1 Accuracy 23
5.2 Linearity 23
5.3 Precision 24
5.4 Method Detection Limit 24
5.5 Inter-Kit Lot Reproducibility 24
5.6 Matrix Effects 24
Chapter 6 Test Results for the ZEU-INMUNOTEC MicroCystest Kit 25
6.1 ZEU-INMUNOTEC MicroCystest Kit Summary 25
6.2 Test Kit QC Samples 25
6.3 PT Samples 26
6.3.1 Accuracy 26
6.3.2 Precision 29
6.3.3 Linearity 31
6.3.4 Method Detection Limit 32
6.3.5 Inter-Kit Lot Reproducibility 33
6.3.6 Matrix Effect 33
6.4 RW Sample Results 36
6.5 Operational Factors 38
6.5.1 Ease of Use 39
6.5.2 Cost and Consumables 39
Chapter 7 Performance Summary for the ZEU-INMUNOTEC MicroCystest 40
Chapter 8 References 42
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APPENDIX A Reference Laboratory Method Detection Limit Memo 43
APPENDIXB ZEU-INMUNOTEC MicroCystest Raw Data 45
VI
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Tables
Table 1. Summary of Test Samples 16
Table 2. DQIs and Summary of Reference Method QC Results 19
Table 3. Summary of Reference Method CCV Percent Recoveries and Method Precision 20
Table 4. PEA Results: Analytical Comparison of Microcystin Standards 20
Table 5. PEA Results: Evaluation of Extracted Low Level Water Sample 21
Table 6. RB Sample Results for the ZEU-INMUNOTEC MicroCystest Kit 25
Table 7. Positive Control Sample Results for the MicroCystest 26
Table 8. ZEU-INMUNOTEC MicroCystest Sample Results and Reference Method Results for
LR 27
Table 9. ZEU-INMUNOTEC MicroCystest Sample Results and Reference Method Results for
LA 27
Table 10. ZEU-INMUNOTEC MicroCystest Sample Results and Reference Method Results for
RR 28
Table 11. ZEU-INMUNOTEC MicroCystest Precision Results 30
Table 12. Detection Limit Results for the ZEU-INMUNOTEC MicroCystest 32
Table 13. Inter-kit Lot Comparison of Kit Calibration Standards for the ZEU-INMUNOTEC
MicroCystest 33
Table 14. RW Matrix Interference Sample Results for the ZEU-INMUNOTEC MicroCystest. 34
Table 15. Chlorophyll-a Interferent Sample Results for the ZEU-INMUNOTEC MicroCystest35
Table 16. Statistical Comparisons between Interference Samples for the ZEU-INMUNOTEC
MicroCystest 36
Table 17. Recreational Water Sample Results for the ZEU-INMUNOTEC MicroCystest 37
Table 18. RW Lysing Extract Sample Results for the ZEU-INMUNOTEC MicroCystest 38
Table 19. ZEU-INMUNOTEC MicroCystest Performance Summary 40
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Figures
Figure 1. MicroCystest Microtiter Plate 13
Figure 2. Linearity for the ZEU-INMUNOTEC MicroCystest for LR 31
Figures. Linearity for the ZEU-INMUNOTEC MicroCystest for LA 31
Figure 4. Linearity for the ZEU-INMUNOTEC MicroCystest for RR 32
Vlll
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List of Abbreviations
ADQ audit of data quality
AMS Advanced Monitoring Systems
°C degrees Celsius
CCV continuing calibration verification
CR cross reactivity
CV coefficient of variation
DQI data quality indicator
DQO data quality objective
DI Deionized
EPA Environmental Protection Agency
ETV Environmental Technology Verification
LFM laboratory fortified matrix
LC-MS-MS liquid chromatography tandem mass spectrometry
LOQ Limit of quantification
MB method blank
MDL method detection limit
mg/L milligram per liter
mL Milliliter
nm Nanometer
NDEQ Nebraska Department of Environmental Quality
NRC National Research Council
NRMRL National Risk Management Research Laboratory
OD optical density
ppb parts per billion
%D percent different
PEA performance evaluation audit
PP protein phosphatases
PPIA Protein Phosphatase Inhibition Assay
pNPP p-nitrophenylphospate
pNP p-nitrophenol
PT performance test
QA quality assurance
QAO quality assurance officer
QC quality control
QMP quality management plan
%R percent recovery
r2 coefficient of determination
RB reagent blank
RW recreational water
RPD relative percent difference
RSD relative standard deviation
SD standard deviation
SOP standard operating procedure
IX
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SPE solid phase extraction
TFA trifluoroacetic acid
TQAP Test/Quality Assurance Plan
TSA technical systems audit
(ig/L microgram per liter
WSL Water Sciences Laboratory
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Chapter 1
Background
The U.S. Environmental Protection Agency (EPA) supports the Environmental Technology
Verification (ETV) Program to facilitate the deployment of innovative 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.
ETV works in partnership with recognized testing organizations; with stakeholder groups
consisting of buyers, vendor organizations, and permitters; and with the full participation of
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 or 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 definition of ETV verification is to establish or prove the
truth of the performance of a technology under specific, pre-determined criteria or protocols and
a strong quality management system. The highest-quality data are assured through
implementation of the ETV Quality Management Plan. ETV does not endorse, certify, or
approve technologies.
The EPA's National Risk Management Research Laboratory (NRMRL) and its verification
organization partner, Battelle, operate the Advanced Monitoring Systems (AMS) Center under
the ETV program. The AMS Center recently evaluated the performance of the MicroCystest
Plate Kit offered by ZEU-INMUNOTEC.
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Chapter 2
Technology Description
This verification report provides results for the verification testing of the ZEU-INMUNOTEC
MicroCystest Plate Kit, based on information provided by the vendor. The information provided
below was not verified in this test.
The MicroCystest test is based on protein phosphatase inhibition assay (PPIA) and designed to
detect and quantify microcystins in water. The toxicity of microcystins is associated with the
inhibition of protein phosphatases (PP) 1 and 2A in the liver cells. MicroCystest is therefore
able to detect the potential toxicity caused by microcystins, as the kit measures the activity of the
PP2A enzyme in samples possibly contaminated with these toxins. PP2A is capable of
hydrolysing a chromogenic substrate like pNPP (p-nitrophenylphospate) to pNP (p-nitrophenol),
which can be detected at 405 nanometers (nm). Samples containing microcystins will inhibit the
enzyme proportionally to the amount of toxin contained in the sample. The test will respond to
all congeners of microcystins present in the sample (more than 80 congeners are known to exist).
The final concentration of microcystin can be calculated using a standard curve obtained from
the standards included in the kit, expressed as |J,g/L microcystin-LR equivalents.
The MicroCystest kit can be used with drinking water and recreational water samples. To
summarize, direct analysis of water (filtered or unfiltered) measures dissolved microcystins.
Then the filtered cellular residue is treated with methanol, trifluoroacetic acid (TFA), and Tween
20™ and centrifuged. The resulting solution is diluted and analyzed to measure the intracellular
microcystin and combined with the dissolved microcystin to determine the total microcystins.
The kits include ready-to-use standards and all reagents needed in the assay. A
spectrophotometer with 405 nm filter is required for results interpretation.
A maximum of 44 samples can be run with one 96-well kit; however if 11 or less samples are
required, each kit can be split a maximum of four times, because four individual vials of
phosphatase are provided. Each sample and standard is tested in duplicate and a standard curve
must be analyzed in every run.
The MicroCystest is shown in Figure 1 and measures 6.1 x 4.3 x 4.3 inches (15.5x 11.Ox 10.8
centimeters). The cost is $450 per 96-well plate kit. Other materials and equipment not
provided with the kits are pipettes, pipette tips, a photometer capable of reading at 405 nm, and
the supplies needed for filtering and lysing the sample.
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Figure 1. MicroCystest Plate Kit
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Chapter 3
Test Design and Procedures
3.1 Test Overview
This verification test was conducted according to procedures specified in the Test/Quality
Assurance Plan for Verification of Microcystin Test Kits1 (TQAP) and adhered to the quality
system defined in the ETV AMS Center Quality Management Plan (QMP)2. As indicated in the
test/QA plan, the testing conducted satisfied EPA QA Category III requirements. The test/QA
plan and/or this verification report were reviewed by:
• Andrew Lincoff, U. S. EPA
• Daniel Snow of the University of Nebraska
• Robert Waters, New York Suffolk County Department of Health Services.
Evaluating microcystin test kits was identified by the AMS Center stakeholders as a priority area
in 2005. With stakeholder input to the design, reference method selection, and submission of
recreational waters to be evaluated, the test assessed the performance of microcystin test kits
relative to key verification parameters including accuracy, precision, and method detection limit
(MDL). This verification test took place from July 26 through August 12, 2010. The reference
analysis was performed the week of August 16, 2010.
3.2 Experimental Design
The objective of this verification test was to evaluate the performance of the microcystin test kits
against a known concentration of each microcystin variant in ASTM International Type II
deionized (DI) water, as well as microcystin variants in unknown proportions from recreational
water (RW) samples. Battelle conducted this verification test with recreational samples provided
from the Nebraska Department of Environmental Quality (NDEQ), with the University of
Nebraska Water Sciences Laboratory (WSL) providing reference analyses. The technologies
were used to analyze a variety of water samples spiked with the variants microcystin-LR,
microcystin-LA, and microcystin-RR. Because none of the technologies tested can specify
between the different variants, the samples were spiked with individual variants. The
quantitative results from the ZEU microcystin test kit were compared to the results from the
reference method by calculating percent difference between the results. The reference method
for microcystin was based on direct injection liquid chromatography tandem mass spectrometry
(LC-MS-MS)3 for the determination of microcystins. To attain lower levels of detection, a
sample preparation method was developed by the WSL to extract the microcystins from the
water samples and concentrate the samples using solid phase extraction (SPE)4. The Zeu
MicroCystest kit provided a quantitative determination of microcystins by evaluating:
• Accuracy - comparison of test kit results (samples prepared in DI water) to results from a
reference method;
• Precision - repeatability of test kit results from three sample replicates analyzed in DI water,
matrix interference, and recreational water samples;
• Linearity - determination of whether or not the test kit response increases in direct proportion
to the known concentration of microcystin;
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• Method detection limit - the lowest quantity of toxin that can be distinguished from the
absence of that toxin (a blank value) at a 95% confidence level;
• Inter-kit lot reproducibility - determination of whether or not the test kit response is
significantly different between two different lots of calibration standards within the kits;
• Matrix interference - evaluation of the effect of natural recreational water matrices and
chlorophyll-a on the results of the test kits; and
• Operational and sustainability factors - such as general operation, data acquisition, setup, and
consumables.
Test kits were operated according to the vendor's instructions by a vendor-trained Battelle
technician. Water samples were tested according to the kit instructions, and in compliance with
the microcystin TQAP.
3.3 Test Procedures
The ability of each microcystin test kit to determine the concentration of microcystin was
challenged using quality control (QC) samples, performance test (PT) samples and RW samples.
These sample results were also compared to reference method results. Table 1 presents the test
samples analyzed during this verification test.
QC, PT, and RW samples were prepared by Battelle technical staff the day before testing began.
The test samples were prepared in glass volumetric flasks and stored in amber glass vials at 4 °C
± 3 °C until use. The reference samples that were prepared from the test solutions were stored in
amber glass bottles at < -10°C until analysis approximately two weeks later. Replicate samples
for the test kits were taken from the same sample bottle. The QC, PT, and RW samples were
prepared blindly for the operator by coding the sample labels to ensure the results were not
influenced by the operator's knowledge of the sample concentration and variant.
Because the reference method is specific to individual microcystins, PT samples for each of the
three different variants were combined into a volumetric flask and brought up to a known
volume with DI water before being sent to the reference laboratory. Then the calculated dilution
factor was applied to the reference method result to determine the PT sample concentration of
each variant. The RW samples were sent for reference analysis without dilution.
3.3.1 QC Samples
Reagent blank (RB) samples were prepared from DI water and exposed to identical handling and
analysis procedures as other prepared samples, including the addition of all reagents. These
samples were used to ensure that no sources of contamination were introduced in the sample
handling and analysis procedures. At least 10% of all the prepared samples were RBs.
3.3.2 PT Samples
PT samples were used to verify the accuracy, precision, linearity, MDL, and inter-kit lot
reproducibility of the test kits. All PT samples were prepared at Battelle using DI water as the
water source. PT samples were individually spiked with microcystin-LR, microcystin-LA, and
microcystin-RR and analyzed in triplicate. The concentration levels were 0.10 (LR only), 0.50,
1.0, 2.0, and 4.0 (RR only) ppb to evaluate the full dynamic range of the test kits for these
variants. The cross-reactivity (CR) of the response of the variants caused the variants to be
analyzed at various concentration levels. EPA Guidelines5 were followed to estimate the MDL
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of the quantitative test kits. In doing so, a solution with a concentration five times the vendor's
reported detection limit for each variant was used. A minimum of seven replicate analyses of
this solution were made individually for each variant to obtain precision data with which to
determine the MDL.
Table 1. Summary of Test Samples
Type of Sample
QC Samples- Laboratory
Reagent Blank (RB)
PT Samples - DI Water
PT Samples - RW Matrix
Interference Samples:
RW sample and tenfold
dilution
PT Samples -
Chlorophyll-a Matrix
Interference Samples:
Chlorophyll-a sample and
tenfold dilution
PT Samples - Inter-kit lot
reproducibility
RW Samples- Through
freeze-thaw lysing
procedure
RW Samples- vendor
recommended lysing
procedure
Microcystin
Variant
none
LR
LA
RR
LR
LA
RR
LR
LA
RR
LR
LA
RR
Microcystin
Concentration
(ppb)
0
0.10,0.50,1.0,
2.0
0.50,1.0,2.0
0.50, 1.0, 2.0,
4.0
5 times the vendor
stated MDL
5 times the vendor
stated MDL
5 times the vendor
stated MDL
2.0*
2.0*
2.0*
2.0*
2.0*
2.0*
Replicates
3
3
3
3
7
7
7
3
3
3
3
3
3
Total Number
of Samples per
Test Kit
10% of total test
samples, 2
12
9
12
7
7
7
6
6
6
6
6
6
A second set of vendor provided calibration standards from a different lot
analyzed following the vendor's procedure
Unknown
Unknown
three samples >20,
three samples >10,
three samples ND
three samples at
unknown
concentrations
3
3
27
9
""concentration that is within the calibration range of the test kit
Additional performance testing was conducted to verify the impact of possible matrix
interferences. Two types of possible matrix interferences, RW water and chlorophyll-a, were
tested. Testing was performed using a RW sample with a low level of native microcystin
concentration (based on information from NDEQ). This RW sample was serially diluted by a
factor of 10 with DI water to provide a less concentrated level of the RW matrix. Then both the
original RW sample and diluted RW samples were fortified with 2.0 ppb of microcystin LR, LA,
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or RR. The spike level chosen was dependent on the detection range of the kit. The test kit
results in each of the matrices were compared to determine the impact of the matrix
concentration on the test kit results. In addition, the results from the matrix samples were
compared with the PT sample in DI water of the same microcystin concentration.
To evaluate the effect of chlorophyll-a as a possible interference, the test was designed for a DI
water sample fortified with 10 milligram/Liter (mg/L) of chlorophyll-a (Sigma Aldrich, Cat #
C5753-5MG chlorophyll-a from spinach) to be prepared by adding a known amount of
chlorophyll-a into a volumetric flask and diluting to volume. Because ZEU-INMUNOTEC
recommends their specific lysing procedure, the chlorophyll-a interference sample preparation
was modified. For this test kit, the chlorophyll-a was spiked into the filter extraction solvent as
if the water sample had already been filtered and the filter had already been extracted by the
solvent. Then the samples went through the rest of the procedure. The solvent samples were
spiked at 10 and 1.0 mg/L chlorophyll-a. Then each of these concentration levels was fortified
with 2.0 ppb of microcystin-LR, LA, or RR. The test kit results in each of the matrices were
compared to determine the impact of the chlorophyll-a.
Lastly, the calibration standards provided with the microcystin test kits from different lots could
cause variability in the results across test kits. Therefore, two separate lots of calibration
standards were analyzed using the kits and compared to determine the inter-kit lot
reproducibility.
3.3.3 RWSamples
RW samples were obtained from lakes in and around Lincoln, Nebraska to assess kit
performance in recreational waters. The procedure for collecting and preparing the samples for
verification testing and reference analysis is described in the NDEQ standard operating
procedure for microcystin analysis (SOP# SWS-2320.1A)6. In summary, staff from NDEQ
collected the water samples from lakes where there is a potential for human exposure to
microcystins. The RW samples were collected in brown plastic bottles with head space
remaining and returned to the laboratory where they were frozen and thawed three times to lyse
the cyanobacteria and free the microcystin into solution, making it available for analysis. Then
the samples were split for verification testing and reference analysis. Using analytical data
generated by NDEQ, samples used for ETV testing were selected from lakes that had both
detectable and not-detectable microcystin concentrations. Because not all possible variants are
monitored by the reference method, there could be a discrepancy between the test kit results and
the total microcystin determined by the reference method.
As previously discussed in Chapter 2, the MicroCystest contains a specific lysing procedure to
analyze for microcystin. For this test kit, three of the RW samples were split before the fireeze-
thaw process to compare the results using the two lysing procedures. The MicroCystest was
used to analyze the three RW samples with and without the freeze-thaw lysing. One of the three
RW sample extracts from the ZEU-INMUNOTEC lysing procedure was analyzed by the
reference method to compare the lysing process and the entire test procedure.
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Chapter 4
Quality Assurance/Quality Control
QA/QC procedures were performed in accordance with the AMS Center QMP and the TQAP for
this verification test. QA level III, Applied Research was specified for this test by the EPA
Project Officer. These procedures and results are described in the following subchapters.
4.1 Reference Method Quality Control
To ensure that this verification test provided suitable data for a robust evaluation of performance,
a variety of data quality objectives (DQOs) were established for this test. The DQOs indicated
the minimum quality of data required to meet the objectives of the verification test. The DQOs
were quantitatively defined in terms of specific data quality indicators (DQIs) and their
acceptance criteria. The quality of the reference method measurements were assured by
adherence to these DQI criteria and the requirements of the reference methods, including the
calibration and QA/QC requirements of the method. Blank samples were required to generate
results below the detection limit and the Laboratory Fortified Matrix (LFM), duplicate, and
Performance Evaluation Audit (PEA) sample results were required to be within 30% of the
expected results. Continuing calibration verification (CCV) standards were required to be within
20% of the expected result. Battelle visited the reference laboratory prior to initiation of the
reference analysis and audited the data package provided by the reference laboratory following
analysis. More details about the audits are provided in Section 4.2. Table 2 presents these DQIs
and the reference method QC sample results. A total of 22 samples were analyzed by the
reference method; 17 were extracted prior to analysis, and five were analyzed by direct injection.
One sample duplicate was processed with the 17 extracted samples to assess the DQI. No
sample duplicate was included for samples analyzed via direct injection.
The calibration of the LC-MS/MS method was verified by the analysis of a CCV at a minimum
of every 10 samples. All of the calibration standards were used as CCVs and were interspersed
throughout the run every five samples. The percent recoveries (%R) of CCVs were calculated
from the following equation:
C
o/0^ = _Lx100 (1)
s
where Cs is the measured concentration of the CCV and, s is the spiked concentration. If the
CCV analysis differed by more than 20% from the true value of the standard (i.e., %R values
outside of the acceptance window of 80 to 120%), the instrument was recalibrated before
continuing the analysis. As shown in Table 3, all reference CCV analyses were within the
required range.
Spiked samples were analyzed to assess the efficiency of the extraction method. There was a
LFM spike performed every 20 samples and this was assessed by calculating the spike percent
recovery (%Rs) as below.
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%Rs = •
C-C
•xlOO
(2)
Csis the measured concentration of the spiked sample, C is the measured concentration of the
unspiked samples, and s is the spiked concentration. The spike %R was required to be within
30% of the spiked amount. The two LFM sample results were within this range for all three of
the variants.
The relative percent difference (RPD) of the duplicate sample analysis was calculated from the
following equation.
RPD = -
\C-CL
(C + CD)I2
-xlOO
(3)
C is the concentration of the sample analysis, and CD is the concentration of the duplicate sample
analysis. If the RPD was greater than 30%, then the extraction method and the analytical
methods were investigated. Reference method CCV RPD results are provided in Table 2.
Reference method precision of laboratory samples was not determined because the duplicate
extraction was performed on the reagent blank sample.
Table 2. DQIs and Summary of Reference Method QC Results
DQI
Performance
Evaluation Audit
(PEA)
Method
contamination
check
Method
Calibration
Check
Method precision
Method accuracy
Method of Assessment
(Frequency)
PEA Samples (Once before testing
begins)
Method Blank (MB) (Once every
20 samples)
Continuing Calibration Verification
(CCV) (Once every 5 samples)
Laboratory Duplicates (Once
every 20 samples)
Laboratory Fortified Matrix (LFM)
Spikes (Once every 20 samples)
Acceptance
Criteria for
Microcystins
70% - 130%
Recovery
< Lowest
Calibration
Standard
80% - 120%
Recovery
< 30% Difference
70% - 130%
Recovery
Results
See Tables 4 and 5 in Section
4.2.1
ND for all three variants
See Table 3
See Table 3
LRM1
93% LR
79% LA
97% RR
LRM2
103% LR
105% LA
88% RR
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Table 3. Summary of Reference Method CCV Percent Recoveries and Method Precision
CCV Cone.
(ppb)
10
30
30
60
60
75
Variant % Recovery
LR LA RR
99.5
109
96.5
97.6
103
98.7
98.2
104
97.1
94.2
109
91.8
96.1
112
98.7
93.5
108
101
Variant RPD
LR LA RR
NA
12%
5%
NA
NA
7%
14%
NA
NA
13%
14%
NA
4.2 Audits
Three types of audits were performed during the verification test: a performance evaluation audit
(PEA), a technical systems audit (TSA) of the verification test procedures, and an audit of data
quality (ADQ). Audit procedures are described further below.
4.2.1 Performance Evaluation Audit
A PEA was conducted to assess the quality of the reference measurements made in this
verification test. National Institute of Standards and Technology traceable standards of
microcystin are not available; however, the Canadian National Research Council (NRC) offers
standards that have gone through the most validation of any commercially available standards
and were recognized by the vendors and stakeholders reviewing the TQAP as the most reliable
standards. The microcystin-LA variant was not available through the Canadian NRC and
therefore was obtained from Abraxis. The approach of using the microcystin-LA variant
standard from Abraxis was approved by all participating vendors prior to use. The standards
obtained from both sources were prepared at 50 ppb in DI water and sent blindly to the reference
laboratory for analysis. These PEA samples were analyzed directly (i.e., without additional
preparation) and were in the mid-level of the calibration range of the reference method. The
stock solutions used to prepare the calibration standards by the reference laboratory were
prepared by dissolving neat standards (not solutions) obtained from EMD Biosciences
(microcystin-LR), Sigma Aldrich (microcystin-LA), and ENZO Life Sciences (microcystin-RR).
The results from the analyses are presented in Table 4.
Table 4. PEA Results: Analytical Comparison of Microcystin Standards
Standard
Source
NRC
Canada
Abraxis
#of
Replicates
2
8
2
8
Analysis
Date
27-May
9-Jun
27-May
9-Jun
MC-LR
(% Recovery)
150% ±3%
135% ±7%
129% ± 2%
121% ±6%
MC-LA
(% Recovery)
Not available
Not available
86% ± 2%
86% ±5%
MC-RR
(% Recovery)
192% ± 1%
194% ±12%
1440/0 ± Oo/o
153% ±10%
Shading indicates results outside acceptable 30% tolerance based on TQAP
The recoveries of the NRC and Abraxis standards revealed that the reference laboratory method,
using the standards from alternate sources, were outside the acceptance range of ±30%. It was
then discussed with the stakeholders, and accepted by the vendors and the EPA Project Officer,
that the reference laboratory use the two available NRC standards (LR and RR) as well as LA
20
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from Abraxis for preparing the reference method calibration solutions. It is not a common
practice for calibration standards and test solutions to be generated from the same source.
However, since the objective was to generate comparable vendor and reference data, it was
deemed necessary and appropriate for this verification test due to the difficulties in obtaining
certified microcystin standards.
To achieve the low detection limits required to analyze the test samples, an SPE extraction
method was also developed and used by the reference laboratory for samples expected to be
below 5.0 ppb. The MDL of this method was determined from extraction and analysis of eight
solutions of LR, LA, and RR at 0.38 ppb. The reference method MDLs for LR, LA, and RR
were determined to be 0.10 ppb, 0.14 ppb, and 0.13 ppb, respectively. Appendix A is the memo
from the reference laboratory presenting the MDL data.
A second PEA was performed to evaluate the extraction method efficiency and the analytical
method at a lower concentration relevant for this verification test. Battelle provided WSL with a
blind spiked DI sample at 0.25 ppb that was extracted in triplicate. The results from the second
PEA are presented in Table 5.
TableS. PEA Results: Evaluation of Extracted Low Level Water Sample
0.25 ppb Spiked
Sample
Replicate 1
Replicate 2
Replicate 3
Average
Standard Deviation
LR
Cone. %
(ppb) Recovery
0.23 92%
0.25 100%
0.23 92%
0.24 95%
0.01 5%
LA
Cone. %
(ppb) Recovery
0.21 84%
0.23 92%
0.22 88%
0.22 88%
0.01 4%
RR
Cone. %
(ppb) Recovery
0.24 96%
0.22 88%
0.26 100%
0.24 96%
0.02 8%
4.2.2 Technical Systems Audit
Battelle's Quality Assurance Officer (QAO) conducted a TSA to ensure that the verification test
was being conducted in accordance with the TQAP and the AMS Center QMP. As part of the
TSA, test procedures were compared to those specified in the TQAP, and data acquisition and
handling procedures as well as the reference method procedures were reviewed. Two
observations on storage of test records and sample handling and custody were documented and
submitted to the Battelle Verification Test Coordinator for response. The observations from the
TSA were addressed and documented as necessary. The conclusion of the TSA was that
verification testing was performed according to the TQAP. TSA records are permanently stored
with the QAO.
4.2.3 Data Quality Audit
Two ADQs were performed for this verification test. The first was for the data collected on the
first day of testing and the second was on the complete data package generated during verification
test preparation and execution. During the audits, test kit data were reviewed and verified for
completeness, accuracy and traceability.
Because the EPA Project Officer designated this as an EPA Category III verification test, at least
10% of the data acquired were audited. The QAO traced the data from the initial acquisition,
21
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through reduction and statistical analysis, to final reporting to ensure the integrity of the reported
results. All calculations performed on the data undergoing the audit were checked.
Observations and findings (mostly related to test record documentation) were reported and
submitted to the Battelle Verification Test Coordinator after the TSA and all observations were
addressed prior to the submission of this final report.
22
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Chapter 5
Statistical Methods
The statistical methods used to evaluate the quantitative performance factors listed in Section 3.2
are presented in this chapter. Qualitative observations were also used to evaluate verification test
data.
The microcystin test kits being verified report total microcystin and are also calibrated against
microcystin-LR. Because of this, the kit data were converted from microcystin-LR equivalents
to compare the test kit results to the reference method results for all PT samples. Using cross
reactivity data provided by each vendor (specific to each test kit), the microcystin-LR
equivalents were converted to microcystin concentration by variant as follows:
byvariant
"LRequiv ,.,
CR ^ ^
where CiRequiv is the test kit result in equivalents of microcystin-LR and CR is the mass-based
cross reactivity of the variant.7
For the RW samples, each variant identified through analysis by the reference method was
converted to LR-equivalents, and added together to calculate the total microcystins. The total
microcystin-LR equivalents from the RW reference analyses were compared to the total
microcystin results from the test kits. Because not all possible variants are monitored by the
reference method, there could be a discrepancy between the test kit results and the total
microcystin determined by the reference method.
5.1 Accuracy
Accuracy of the test kits verified was assessed relative to the results obtained from the
reference analyses. The results for each set of analyses were expressed in terms of a percent
difference (%D) as calculated from the following equation:
C -C
%D = — x 100 (5)
CR
where CT is the microcystin-LR equivalent results from the test kits being verified and CR is the
concentration as determined by the reference method.
5.2 Linearity
Linearity was determined by linear regression with the toxin concentration measured by the
reference method as the independent variable, and the test kit result being verified as the
dependent variable. Linearity was expressed in terms of the slope, intercept, and the coefficient
of determination (r2). In addition, plots of the observed and predicted concentration values were
constructed to depict the linearity for each variant of microcystin being tested.
23
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5.3 Precision
The standard deviation (S) of the results for the replicate samples were calculated and used as a
measure of test kit precision at each concentration. S was calculated from the following
equation:
s =
1
:-C)
(6)
where n is the number of replicate samples, Ck is the concentration measure for the kth sample,
and C is the average concentration of the replicate samples. The kit precision at each
concentration was reported in terms of the relative standard deviation (RSD) presented below.
RSD =
(7)
5.4 Method Detection Limit
MDL was determined by seven replicate analyses of a fortified sample with the toxin
concentration of five times the vendor's estimated detection limit. The MDL was calculated
from the following equation:
MDL = txS (8)
where / is the Student's value for a 95% confidence level, and S is the standard deviation of the
replicate samples.
5.5 Inter-Kit Lot Reproducibility
Inter-kit lot reproducibility was assessed by calculating the RPD (Equation 3) between optical
density (OD) results that are given to compare between the lots of calibration standards.
5.6 Matrix Effects
Matrix interference effects also were assessed by using a t-test to compare the microcystin test
kit results generated from samples made by spiking undiluted and diluted interference matrices
with the PT sample results at the same spiked concentration (either 2 or 4 ppb spike
concentration). Each paired t-test was performed using the replicate data from each type of
sample. The null hypothesis is that there is no difference between the two sets of data.
Therefore, the resulting probability (p)-value gives the likelihood of observing a difference as
large as is seen in the data, or a larger difference, if the null hypothesis were true. Therefore, at
the 95% confidence interval, p-values less than 0.05 will indicate there is evidence against the
null hypothesis being true and therefore a significant difference between the two sets of data.
Since the number of replicates was predetermined by the test kit instructions and TQAP, power
and sample size calculations were not conducted for this assessment.
24
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Chapter 6
Test Results for the ZEU-INMUNOTEC MicroCystest Kit
The following sections provide the results of the quantitative and qualitative evaluations of this
verification test for the ZEU-INMUNOTEC MicroCystest Kit.
6.1 ZEU-INMUNOTEC MicroCystest Kit Summary
The MicroCystest requires that each standard and sample be analyzed in duplicate and then the
raw data output from the plate reader software reports a mean concentration of the duplicate
analyses. Therefore, a sample indicated in Table 1 to have three replicates corresponded to six
wells being filled as part of the MicroCystest. Each MicroCystest plate contains four calibration
solutions. Following the analysis method, the plate reader measures the absorbance of the wells
containing the calibration solutions at a wavelength of 405 nm and the calibration curve is
generated based on the OD of each well. These results are plotted against concentrations using a
vendor-provided spreadsheet that generated a four parameter curve to quantify the rest of the
samples.
If the MicroCystest determined a result to be either above or below the calibration range, an "out
of range" result was indicated and the sample was either diluted into the linear range or reported
as being less than the limit of quantification (
-------
Other quality control samples of the MicroCystest include calibration standards; however, this
test kit does not routinely include positive or negative controls. Positive controls were received
from the vendor for the training before testing began. Even though a positive control is not
specified by the vendor, the technician analyzed a 0.70 ppb positive control at the end of each
MicroCystest plate to ensure the proper technique was used by the technician. At least one
positive control was analyzed at the end of each plate and in some instances when space allowed,
additional positive controls were analyzed. As shown in Table 7, the percent recovery results
ranged from 78% to 113% recovery. All but two coefficients of variation (CV) results were
below 10%. The exceptions were Plate 1 (30%) and Plate 6 (12%). Since no acceptance criteria
were provided by the vendor, no results were rejected or rerun based on these data.
Table 7. Positive
Control
Sample Results for the
MicroCystest
Positive Control ID Plate Mean Concentration (ppb) CV (%)
1
2a
2b
3
4
5a
5b
6
1
2
2
3
4
5
5
6
0.55
0.65
0.75
0.62
0.79
0.66
0.60
0.56
30
7.3
1.2
3.8
2.6
0.80
4.5
12
Percent Recovery (%)
78%
93%
110%
89%
110%
95%
86%
81%
6.3 PT Samples
Tables 8, 9, and 10 present the results for the PT samples for the three variants of microcystin
used during this verification test. In addition, the tables present the sample concentration
corrected for the microcystin inhibitory ability, the reference method results and the accuracy
results by variant for the PT samples prepared in DI water. All samples were analyzed in
triplicate.
6.3.1 Accuracy
Tables 8, 9, and 10 also present the accuracy results for the MicroCystest, expressed as %D
when calculated with the theoretical spike concentration and the reference method concentration.
As shown in Equation 5 (Section 5.1), the reference method value was used for calculation of
accuracy.
26
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Table 8. ZEU-INMUNOTEC MicroCystest Sample Results and Reference Method Results
forLR
Sample
Description
0.10LR
Avg ± SD
0.50 LR
Avg ± SD
1.0 LR
Avg ± SD
2.0 LR
Avg ± SD
Kit Results: LR
Equivalents
(ppb)
-------
Table 10. ZEU-INMUNOTEC MicroCystest Sample Results and Reference Method
Results for RR
Sample
Description
0.50 RR
Avg ± SD
1.0 RR
Avg ± SD
2.0 RR
Avg ± SD
4.0 RR
Avg ± SD
Kit Results: LR
Equivalents
(ppb)
0.45
0.78
0.61
0.61 ±0.17
1.7
1.7
1.7
1.7 ±0.10
2.0
2.2
2.2
2.1 ±0.10
2.4
2.4
2.3
2.4 ±0.10
Accuracy by LR
Equivalents for
Theoretical
Concentration (%
Difference)
-11%
56%
21%
22% ±33%
70%
72%
65%
69% ±4%
-1%
8%
9%
5% ±5%
-40%
-41%
-42%
-41%±1%
Accuracy by LR
Equivalents for
Reference
Concentration (%
Difference)
17%
110%
59%
61% ±44%
220%
220%
210%
2 10% ±7%
24%
35%
36%
32% ±6%
-24%
-26%
-27%
'-26%±1%
Reference
Concentration
(ppb)
0.38
0.54
1.6
3.2
For the LR spiked samples, the reference method results ranged from 0% to 17% less than the
target concentration. For LR, the percent difference ranged from 20% to 280%, with overall
average percent difference values ranging from 41% to 260% between the MicroCystest and the
reference method. For the 0.10 ppb samples, only two of the three replicate MicroCystest
samples were detectable. These results were just above the LOQ and the %D was 230% and
280%, corresponding to an absolute maximum difference from the reference concentration of
0.28 ppb. For the 0.50 ppb samples, the %D ranged from 20% to 55%, but the absolute
difference from the reference concentration was no more than 0.23 ppb. For the 1.0 ppb
samples, the %D ranged from 85% to 110%, corresponding to a maximum absolute difference
from the reference concentration was 0.87 ppb. Similarly, for the 2.0 ppb samples, the %D
ranged from 35% to 48% and the maximum absolute difference from the reference concentration
was 0.92 ppb. For LR, the %D when compared to the theoretical spike concentration ranged
from 1% to 280% with the overall average %D values ranging from 18% to 230%.
For the LA spiked samples, the reference method results were approximately 15% to 33% lower
than the spike value. For LA, the percent difference ranged from 43% to 110%, with overall
average percent difference values ranging from 50% to 100%. For the 0.50 ppb samples, the %D
ranged from 43% to 58%, but the absolute difference from the reference concentration was no
more than 0.23 ppb. For the 1.0 ppb samples, the %D ranged from 97% to 110%, corresponding
to a maximum absolute difference from the reference concentration was 0.79 ppb. Finally, for
the 2.0 ppb samples, the %D ranged from 47% to 54% and the maximum absolute difference
from the reference concentration was 0.92 ppb. For LA, the %D when compared to the
28
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theoretical spike concentration ranged from 14% to 49% with the overall average %D values
ranging from 18% to 43%.
For the RR spiked samples, the reference method results were approximately 20% 46% lower
than the spike value. For RR, the percent difference ranged from -27% to 220%, with overall
average percent difference values ranging from -26% to 210%. For the 0.50 ppb samples, the
%D ranged from 17% to 110%, corresponding to an absolute maximum difference from the
reference concentration of 0.40 ppb. For the 1.0 ppb samples, the %D ranged from 210% to
220% and the maximum absolute difference from the reference concentration was 1.2 ppb. For
the 2.0 ppb samples, the %D ranged from 24% to 36% corresponding to a maximum absolute
difference from the reference concentration of 0.58 ppb. For the 4.0 ppb samples, the %D
ranged from -24% to -27%, corresponding to a maximum absolute difference from the reference
concentration of 0.86 ppb. For RR, the %D when compared to the theoretical spike
concentration ranged from -42% to 72% with the overall average %D values ranging from -41%
to 69%.
6.3.2 Precision
Precision results for the MicroCystest are presented in Table 11. The RSD was determined as a
percentage according to Equation 7 (Section 5.3) for all DI water, matrix interference and
recreational water samples. The RSDs ranged from 1% to 13% for the LR variant (mean 5.2%)
For LA, the RSDs ranged from 1% to 10% (mean 4.2%) and from 1% to 27% for the RR variant
(mean 6.8%); however, seven of the eight sample sets had RSDs lower than 15%. The precision
for the RW samples ranged from 1% to 6% (mean 3.9%). The overall average of all RSDs was
5%, with a minimum of 1% and a maximum of 27%.
29
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Table 11. ZEU-INMUNOTEC MicroCystest Precision Results
Variant
LR
LA
RR
Unknown
Sample Concentration in DI
O.lOppb
0.50 ppb
1.0 ppb
2.0 ppb
2.0 ppb LR in 1.0 mg/L Chlorophyll -a DI
2.0 ppb LR in 10 mg/L Chlorophyll-a DI
2.0 ppb LR in lOx dilution of RW Matrix
2.0 ppb LR in RW Matrix
0.50 ppb
1.0 ppb
2.0 ppb
2.0 ppb LA in 1.0 mg/L Chlorophyll-a DI
2.0 ppb LA in 10 mg/L Chlorophyll -a DI
2.0 ppb LA in lOx dilution of RW Matrix
2.0 ppb LA in RW Matrix
0.50 ppb
1.0 ppb
2.0 ppb
4.0 ppb
2.0 ppb RR in 1.0 mg/L Chlorophyll-a DI
2.0 ppb RR in 10 mg/L Chlorophyll-a DI
2.0 ppb RR in lOx dilution of RW Matrix
2.0 ppb RR in RW Matrix
RW 1 (20x dilution)
RW 2 (20x dilution)
RW 3 (20x dilution)
RW 4 (4x dilution)
RW 5 (4x dilution)
RW 6 (2x dilution)
RW7
RW8
RW9
Precision (%RSD)
12%
13%
6%
5%
2%
4%
1%
1%
5%
4%
2%
4%
1%
10%
3%
27%
2%
5%
2%
2%
15%
1%
6%
2%
3%
4%
3%
1%
6%
NA
6%
6%
NA - Result was less than the LOQ so no calculation of RSD
30
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6.3.3 Linearity
The linearity of the MicroCystest measurements was assessed by performing a linear regression
of the MicroCystest kit results against the reference method results for the four PT samples
ranging from 0.10 ppb to 2.0 ppb of microcystin LR in DI water, three PT samples ranging from
0.50 ppb to 2.0 ppb for microcystin LA, and four PT samples ranging from 0.50 ppb to 4.0 ppb
RR in DI water. Figures 2, 3, and 4 present the results of the linear regressions for LR, LA, and
RR respectively. The slope, intercept, and coefficient of determination (r2) for each regression
equation are shown on the charts. The linear regressions compared to the reference method
results had coefficients of determination of 0.95for LR, 0.95 for LA, and 0.63 for RR. In
general, LR looked to generate data that was reasonably linear while LA and RR seem to
indicate a log-normal relationship between the reference method concentration and the
MicroCystest measurements.
&
a.
a.
-------
3.5
3
01
M
1
0.5
0
y = 0.4765X + 1.013
R2 = 0.6271
0 0.5 1 1.5 2 2.5
Reference Cone, (ppb)
3.5
Figure 4. Linearity for the ZEU-INMUNOTEC MicroCystest for RR
6.3.4 Method Detection Limit
The MDL was assessed by analyzing at least seven replicates of a sample spiked at
approximately five times the vendor-stated detection limit for the microcystin test kit (which was
0.25 ppb). Table 12 lists the replicate results; the %CV of the duplicate MicroCystest analysis for
each replicate, the standard deviations for the replicate results, and shows the calculated MDLs
for the three variants. The calculated MDL values were 0.24, 0.17, and 0.61 ppb for LR, LA, and
RR respectively. The increased variability in the RR results was unexpected because the sample
bottle, plate, analysis method, and operator were the same. It is possible that the increased MDL
was due to an operational issue.
Table 12. Detection Limit Results for the ZEU-INMUNOTEC MicroCystest
Variant
Sample
Concentration
(ppb)
.3
o
.J
o
.J
.3
.3
o
.J
o
.J
Standard
Deviation
t (n=7)
MDL
LR
Mean
Cone, (ppb)
2.1
2.3
2.4
2.4
2.2
2.2
2.1
0.13
1.9
0.24
%CV
2.6
1.7
1.1
1.2
7.7
4.3
1.5
LA
Mean Cone.
(ppb LR
Equivalents)
1.9
1.9
1.8
1.7
1.9
1.9
1.9
0.09
1.9
0.17
%CV
8.2
3.0
1.9
17
0.70
3.1
1.2
RR
Mean Cone.
(ppb LR
Equivalents)
1.6
1.4
1.5
1.9
2.1
2.2
1.9
0.31
1.9
0.61
%CV
20
2.5
5.0
18
0.20
4.1
12
32
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6.3.5 Inter-Kit Lot Reproducibility
Two sets of kit calibration standards were analyzed on the sample plate to determine whether or
not the calibration standards from different lots were similar. The data are presented in Table 13.
The OD values were compared by calculation of the RPD between each pair of OD
measurements. All RPDs except one were less than 9% with the highest RPD value at 25%.
Table 13. Inter-kit Lot Comparison of Kit Calibration Standards for the ZEU-
INMUNOTEC MicroCystest
Standard (ppb)
0.25
0.50
1.0
2.5
OD Values
Set A
1.63
1.37
1.13
1.16
0.826
0.696
0.355
0.375
(ppb)
SetB
1.56
1.50
1.19
1.20
0.641
0.699
0.344
0.351
RPD
4%
9%
5%
3%
25%
0%
3%
7%
6.3.6 Matrix Effect
Matrix interference effects were assessed by using a t-test to compare the MicroCystest results
generated from samples made by spiking undiluted and diluted interference matrices with the PT
sample results at the same concentration. The two possible interfering matrices included a RW
sample both undiluted and after undergoing a tenfold dilution and chlorophyll-a at 10 mg/L and
1.0 mg/L. Tables 14 and 15 provide the MicroCystest sample results for the RW matrix
interference samples and chlorophyll-a interference samples, respectively, including the average
and SD for each sample. Because this comparison is made to evaluate only the impact of the
matrix on the sample result, LR equivalents are used.
Each paired t-test was performed using the replicate data from each type of sample. The null
hypothesis is that there is no difference between the two sets of data. The resulting probability
(p)-value gives the likelihood of observing a difference as large as is seen in the data, or a larger
difference, if the null hypothesis were true. Therefore, at the 95% confidence interval, p-values
less than 0.05 will indicate there is evidence against the null hypothesis being true, and therefore
a significant difference between the two sets of data exists.
Table 16 summarizes the results of a paired t-test for both sets of interference data by showing
the p-values associated with each of the applicable comparisons across both types of possible
interfering matrices. Across both the chlorophyll-a and RW results, none of the 18 comparisons
were determined to be statistically different. Therefore, the interferences tested during this
verification test did not affect the performance of the MicroCystest. There are p-values from 18
tests reported in Table 16 and none them is smaller than 0.05. At a significance level of 5%, we
would expect one test out of every 20 to have a p-value below 0.05 just by chance, even if the
null hypothesis were true in each case. A formal multiple comparisons adjustment is not needed
here because a performance standard is not being evaluated as this is more of an exploratory test
33
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to determine if there is any difference caused by the matrix. However, a conservative Bonferroni
correction, for example, would set the p-value associated with a significant result at 0.05 divided
by 18, corresponding to a p-value of 0.0028 for the individual tests.
Table 14. RW Matrix Interference Sample Results for the ZEU-INMUNOTEC
MicroCystest
Variant
Unknown
LR
LA
RR
Sample Description
Unspiked RW Matrix
(RW9)
2.0ppbLRinDI
2.0 ppb LR in tenfold
dilution of RW Matrix
2.0 ppb LR in RW Matrix
2.0 ppb LA in DI
2.0 ppb LA in tenfold
dilution of RW Matrix
2.0 ppb LA in RW Matrix
2.0ppbRRinDI
2.0 ppb RR in tenfold
dilution of RW Matrix
2.0 ppb RR in RW Matrix
Mean Kit Results: LR Average Result
Equivalents (ppb) (ppb)
0.69 0.72
0.77
0.69
2.8 2.7
2.7
2.6
2.7 2.7
2.7
2.7
2.7 2.7
2.7
2.7
2.5 2.6
2.6
2.6
2.5 2.4
2.5
2.1
2.6 2.6
2.6
2.7
2.0 2.1
2.2
2.2
2.2 2.2
2.3
2.2
2.4 2.5
2.7
2.4
SD
0.05
0.10
0
0.10
0.10
0.20
0.10
0.10
0.10
0.20
34
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Table 15. Chlorophyll-a Interferent Sample Results for the ZEU-INMUNOTEC
MicroCystest
Variant
LR
LA
RR
Sample Description
2.0ppbLRinDI
2.0ppbLRinl.Omg/L
Chlorophyll-a DI
2.0 ppb LR in 10 mg/L
Chlorophyll-a DI
2.0 ppb LA in DI
2.0 ppb LA in 1.0 mg/L
Chlorophyll-a DI
2.0 ppb LA in 10 mg/L
Chlorophyll-a DI
2.0ppbRRinDI
2.0 ppb RR in 1.0 mg/L
Chlorophyll-a DI
2.0 ppb RR in 10 mg/L
Chlorophyll-a DI
Mean Kit Results: LR Average Result
Equivalents (ppb) (ppb)
2.8 2.7
2.7
2.6
2.6 2.6
2.5
2.6
2.6 2.7
2.6
2.8
2.5 2.6
2.6
2.6
2.6 2.5
2.5
2.4
2.5 2.5
2.4
2.5
2.0 2.1
2.2
2.2
2.0 2.0
2.0
2.0
1.9 2.1
2.0
2.5
SD
0.10
0.10
0.10
0.10
0.10
0
0.10
0
0.30
35
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Table 16. Statistical Comparisons between Interference Samples for the ZEU-
INMUNOTEC MicroCystest
Description of Comparison
2.0 ppb in DI compared with 2.0 ppb in
tenfold dilution of RW
2.0 ppb in DI compared with 2.0 ppb in
undiluted RW
2.0 ppb in undiluted RW compared with
tenfold dilution of RW
2.0 ppb in DI compared with 2.0 ppb in 1.0
mg/L Chlorophyll-a DI
2.0 ppb in DI compared with 2.0 ppb in 10
mg/L Chlorophyll-a DI
2.0 ppb in 1.0 mg/L Chlorophyll-a DI
compared with 2.0 ppb in 10 mg/L
Chlorophyll-a DI
p-value (D-different, ND-not different)
LR
0.962 (ND)
0.890 (ND)
0.526 (ND)
0.170(ND)
0.729 (ND)
0.300 (ND)
LA
0.369 (ND)
0.258 (ND)
0.308 (ND)
0.617 (ND)
0.132(ND)
0.490 (ND)
RR
0.137(ND)
0.03 1(ND)
0.073 (ND)
0.218 (ND)
0.848 (ND)
0.505 (ND)
6.4 RW Sample Results
Table 17 presents the RW results for the MicroCystest and the reference analysis. The
concentrations were determined by the reference method for only three of the approximately 80
variants that are naturally occurring in recreational waters. The total microcystins measured by
the MicroCystest may have other variants present that would not have been detected by the
reference method. Therefore, testing included only a qualitative comparison between the
MicroCystest and the reference method results. In general, the samples that were determined to
have higher total concentrations by the MicroCystest had higher total concentrations with the
reference method as well. All of the MicroCystest total microcystin results were greater than the
reference method results, which was limited to quantifying three of the -80 known variants.
However, the results of the MicroCystest were usually within a factor of four of the reference
method, indicating that the LR, LA, and RR variants make up a considerable proportion of the
microcystins that are measurable by the MicroCystest.
RW 6, 7, and 8 were lysed using the freeze thaw method used with the other RW samples. In
addition, an aliquot of each was removed before the lysing to follow the procedure in the
MicroCystest Kit. The dissolved (unfiltered RW 6), filtrate (filtered RW 6), and intracellular
partitions (solid algae collected on filter to be lysed) of the RW samples were analyzed by the
Microcystest and the intracellular portion of RW 6 was also analyzed by the reference method.
The results of these samples are presented in Table 18.
Because RW6 sample had detectable levels of microcystin in each sample fraction (dissolved,
filtrate, and intracellular), the RW 6 sample illustrated the use of the Zeu lysing method. The
RW 6 dissolved and liquid filtrate results should represent the same amount of dissolved
microcystin as one is just the filtered form of the other. The results were consistent as the RW 6
dissolved and filtrate fractions produced concentrations of 0.31 and 0.35 ppb, respectively. The
RW intracellular represented the microcystins bound in the algae cells. The result from the
MicroCystest was 2.0 ppb. Lastly, the RW 6 freeze-thaw result of 2.6 ppb represented the total
amount to microcystin released from the algal cells during the freeze-thaw cycling in
36
-------
combination with the already dissolved microcystin. As would be expected, the freeze-thaw
result was similar to the total result of the MicroCystest RW 6 dissolved result of 0.31 ppb and
the MicroCystest RW 6 intracellular result of 2.0 ppb for a total microcystin concentration of
approximately 2.4 ppb. The total determined by the reference method (using freeze-thaw lysing)
was 2.0 ppb and the total determined by the reference method (using the MicroCystest lysing
method) was 1.0 ppb. Therefore, when comparing the MicroCystest results, the lysing procedure
produced very similar results, but with the reference method the results were different by 1.0
ppb.
Table 17. Recreational Water Sample Results for the ZEU-INMUNOTEC MicroCystest
Sample
Description
RW 1 (20x
dilution)
RW 2 (20x
dilution)
RW 3 (20x
dilution)
RW 4 (4x dilution)
RW 5 (4x dilution)
RW 6 freeze thaw
(2x dilution)
RW 7 freeze thaw
RW 8- freeze thaw
RW9(RW
Matrix)
Test Kit Re suits (ppb)
Kit Re suits:
LR
Equivalents
(ppb)
2.0
2.1
2.1
21
2.0
1.9
2.1
2.0
1.3
1.2
1.2
1.6
1.5
1.6
12
1.4
-------
Table 18. RW Lysing Extract Sample Results for the ZEU-INMUNOTEC MicroCystest
Sample
Description
RW6
dissolved
RW6
filtrate
RW6
intracellular
RW 6 freeze
thaw (2x
dilution)
RW7
dissolved
RW7
filtrate
RW7
intracellular
RW 7 freeze
thaw
RW8
dissolved
RW8
filtrate
RW8
intracellular
RW8-
freeze thaw
Test Kit Results (ppb)
Kit Results:
LR
Equivalents
(ppb)
0.33
0.28
-------
6.5 Operational Factors
During testing activities, the technical operators were instructed to fill out an Ease of Use
Questionnaire. This section summarizes these observations as well as other operational
considerations about the technology.
6.5.1 Ease of Use
The test kit operator reported that the MicroCystest was easy to use. The brochure explains the
extraction and analyses procedure clearly. Solution preparation involves hydrating the
phosphatase buffer and gently shaking the solution for an hour. The procedure includes one 30-
minute incubation period at 37 °C. Previous knowledge or training on the use of micro-pipettes
and or multi-channel pipettes with 96-well plates is recommended for consistent readings. The
Battelle operator trained by the vendor had experience with pipetting and 10+ years of laboratory
experience. A spectrophotometer plate reader is necessary for obtaining the spectrophotometric
readings at 405 nm that are then analyzed using any commercial plate reading evaluation
program (a four-parameter plate reading program is recommended by the vendor). The lysing
procedure was not included in the ease of use evaluation of the test kit.
6.5.2 Cost and Consumables
Once the analysis is complete, the remaining solutions and well contents were disposed of
according to local regulations.
The kit has a 6-month shelf life as received, and should be stored at 4 - 8 °C. Of the 96-wells on
one plate, eight are needed for calibration samples. The remaining 88 are for sample analyses
that are performed in duplicate (44 total samples). Other equipment and consumables not
included in the kit are pipettes, pipette tips, DI water, a photometer capable of reading at 405
nanometers, and the supplies needed for filtering and lysing of the sample. The price for the
MicroCystest at the time of the verification was $450 per 96-well plate kit.
39
-------
Chapter 7
Performance Summary for the ZEU-INMUNOTEC MicroCystest
The verification of the ZEU-INMUNOTEC MicroCystest is summarized by the parameters
described in Table 19.
Table 19. ZEU-INMUNOTEC MicroCystest Performance Summary
Verification Parameters
LR
LA
RR
Accuracy (ppb, range of %Difference)
0.10
0.50
1.0
2.0
4.0
Precision (range of %RSD)
Precision (RW samples)
Linearity (y=)
Method Detection Limit (ppb)
230% and 280%
20% - 55%
85% -110%
35o/0 . 48%
1% - 13%
43% - 58%
97% -110%
47o/0 . 5407,,
1% - 10%
17% - 105%
2 10% -220%
24% - 36%
-27% to -24%
1% - 27%
I%to6%
1.4x + 0.23
r2 = 0.95
0.24
1.4x + 0.21
r2 = 0.95
0.17
0.48x+1.0
r2 = 0.63
0.61
Inter-kit lot reproducibility. Calibration standards from two different lots were measured and
all of the RPDs except one were less than 9% with the highest RPD value at 25%.
Matrix Interference. Matrix interference effects were assessed by using a t-test to compare
results from samples made by spiking undiluted and diluted interference matrices with the PT
sample results at 2.0 ppb spiked concentration. Across both the chlorophyll-a and RW results,
none of the 18 comparisons were determined to be statistically different (at low power), and
therefore, the interferences tested during this verification did not affect the performance of the
MicroCystest.
Recreational Water (RW). In general, the samples that were determined to have higher total
concentrations by the MicroCystest also had higher total concentrations as determined by the
reference method. All of the MicroCystest total microcystin results were greater than the
reference method results, which were consistent with the likelihood that all of the microcystins
were not being measured by the reference method that is limited to measuring three variants.
However, the results of the MicroCystest were usually within a factor of three or four of the
reference method, indicating that the LR, LA, and RR variants are common in the RW samples
tested, making up more than a quarter of the microcystins measurable by the MicroCystest.
In addition to the freeze-thaw method of lysing algae cells to release microcystins, a Zeu-specific
lysing technique was verified. Three RW samples were analyzed using both lysing approaches
and the results reported.
Operational Factors. The test kit operator reported that the MicroCystest was easy to use. The
brochure explains the extraction and analyses procedure clearly. Solution preparation involves
hydrating the phosphatase buffer and gently shaking the solution for an hour. The procedure
40
-------
includes one 30-minute incubation period at 37 °C. Previous knowledge or training on the use of
micro-pipettes and or multi-channel pipettes with 96-well plates is recommended for consistent
readings. The Battelle operator trained by the vendor had experience with pipetting and 10+
years of laboratory experience. A spectrophotometer plate reader is necessary for obtaining the
spectrophotometric readings at 405 nm that are then analyzed using a commercial plate reading
evaluation program. The lysing procedure was not included in the ease of use evaluation of the
test kit. Once the analysis was complete, the remaining solutions and well contents were
disposed of in the regular laboratory trash. Since waste disposal requirements vary from state-to-
state, the reader is encouraged to consult with state government agency for proper waste disposal
requirements.
According to the vendor, the kit has a 6-month shelf life as received and should be stored at 4 - 8
°C. Of the 96-wells on one plate, eight are needed for calibration samples. The remaining 88 are
for sample analyses that are performed in duplicate (44 total samples). Other equipment and
consumables not included in the kit are pipettes, pipette tips, DI water, a photometer capable of
reading at 405 nanometers, and the supplies needed for filtering and lysing of the sample. The
price for the MicroCystest at the time of the verification test was $450 per 96-well plate kit.
41
-------
Chapter 8
References
1. Test/Quality Assurance Plan for Verification ofMicrocystin Test KitsTest/Quality
Assurance Plan for Verification ofMicrocystin Test Kits. U.S. Environmental
Technology Verification Program, Battelle, July 2010.
2. Quality Management Plan for the ETVAdvanced Monitoring Systems Center, Version 7.
U.S. Environmental Technology Verification Program, Battelle, November 2008.
3. Cong, L.H., B.; Chen, Q.; Lu, B.; Zhang, J.; Ren, Y., Determination of trace amount of
microcystins in water samples using liquid chromatography coupled with triple
quadrupole mass spectrometry. Anal. Chim. Acta, 2006. 569 (1-2): p. 157-168.
4. Cassada, D., Standard Operating Procedure (SOP) Determination ofalgaltoxin residues
in water extracts by solid phase extraction (SPE), liquid chromatography (LC)-
atmosphericpressure electrospray ionization tandem mass spectrometry (MS/MS). July,
2010, Water Sciences Laboratory, University of Nebraska.
5. "Guidelines Establishing Test Procedures for the Analysis of Pollutants. ", USEP A,
Editor. 2000, U.S. Code of Federal Regulations.
6. SOP# SWS-2320.1A: Microcystin Analysis Using the Abraxis ELISA (Enzyme-Linked
Immuno-Sorbent Assay) Method. Nebraska Department of Environmental Quality.
7. Loftin, K.A., et al., Comparison of Two Cell Lysis Procedures for Recovery of
Microcystins in Water Samples from Silver Lake in Dover, Delaware, with Microcystin
Producing Cyanobacterial Accumulations, in USGS Open-File Report 2008 -1341. 2008.
p. 9.
42
-------
APPENDIX A
Reference Laboratory Method Detection Limit Memo
July 14, 2010
To: Anne Gregg and Ryan James, Battelle Laboratories
From: Daniel Snow and David Cassada, UNL Water Sciences Laboratory
Re: Summary of Microcystin SPE method validation - July 13-14, 2010
Microcystins LA, LR and RR were spiked into water and extracted using solid phase extraction (SPE) to
evaluate method accuracy and precision, and method detection limits. The method described in Cong et
al. 2006 was modified to allow for extraction of a larger sample by using higher capacity polymeric
(Waters Oasis, HLB) SPE cartridges. Briefly, 400-milliliter (mL) of purified reagent water was fortified
with 1500 uL of a diluted mixed stock (0.1 ng/uL) obtained from Battelle to produce 0.375 ug/L of each
analyte. Nodularin (1600 uL of a 0.10 ng/uL solution) was also added to produce a concentration of 0.40
ug/L. Eight 50 mL portions of this fortified water were weighed into 125 mL amber glass bottles and each
portion separately spiked with 100 uL of the enkephalin-Leu internal standard (IS) solution (0.1 ng/uL) to
give a concentration of 2.0 ug/L. A single method blank was prepared by spiking with IS and surrogate
only.
After capping and shaking each solution to equilibrate, samples were drawn under vacuum through pre-
conditioned 200 mg Oasis HLB SPE cartridges at a rate of approximately 10 mL/min. When the sample
had completely passed through the cartridge, it was allowed to air-dry under vacuum, removed from the
extraction apparatus and prepared for elution. Ten (10) milliliters of high purity methanol (Fisher Optima
Grade) were used to elute analyte, IS and surrogate compounds from the cartridges. The methanol was
evaporated under nitrogen to approximately 0.4 mL and the extracts transferred to low volume inserts for
analysis on the LCQ ion trap tandem LC/MS system. Calibration solutions (5, 10, 30, 60 and 75 ng/mL)
were prepared in water from the same mixed stock as the spiking solutions. A table summarizing the
results of the validation is copied below (Table A-l.).
A second 10 mL aliquot of methanol was passed through 4 of SPE cartridges and collected separately to
check for completeness of analyte elution. These second aliquots were reduced to the same 0.40 mL
volume as the MDL eluents and analyzed. The resulting absolute areas of the analyte, surrogate, and
internal standard peaks obtained were approximately 1% of the areas obtained in the first portion. This
suggests that lower elution volumes can result in decreased analyte recovery.
References
Cong, L.; Huang, B.; Chen, Q.; Lu, B.; Zhang, J.; Ren, Y. (2006) Determination of trace amount of
microcystins in water samples using liquid chromatography coupled with triple quadrupole mass
spectrometry. Anal. Chim. Acta, 569 (1-2), 157-168.
43
-------
Table A-l. Average, standard deviation, method detection limits (MDL = S x tN-1) and recoveries
of microcystins obtained from extraction and analysis of eight fortified reagent water (0.375 jig/L)
samples.
50 mL
sample
Aliquot
MDL 1
MDL 2
MDL 3
MDL 4
MDL 5
MDL 6
MDL 7
MDL 8
AVG
STD DEV
MDL
%REC
Expected
value
Amount obtained (ng)
Nodularin
23.994
24.647
22.716
23.157
26.361
19.618
20.254
19.889
22.580
2.460
7.371
112.9
20.0
MC-RR
19.679
19.661
17.660
19.715
19.731
18.214
14.533
15.247
18.055
2.113
6.333
96.3
18.75
MC-LR
18.084
21.985
20.524
21.022
20.462
18.322
20.046
17.518
19.745
1.586
4.753
105.3
18.75
MC-LA
19.913
21.752
18.404
20.304
21.182
18.393
21.490
14.614
19.507
2.360
7.072
104.0
18.75
Concentration (ug/L)
Nodularin
0.480
0.493
0.454
0.463
0.527
0.392
0.405
0.398
0.452
0.049
0.147
112.9
0.4
MC-RR
0.394
0.393
0.353
0.394
0.395
0.364
0.291
0.305
0.361
0.042
0.127
96.3
0.375
MC-LR
0.362
0.440
0.410
0.420
0.409
0.366
0.401
0.350
0.395
0.032
0.095
105.3
0.375
MC-LA
0.398
0.435
0.368
0.406
0.424
0.368
0.430
0.292
0.390
0.047
0.141
104.0
0.375
44
-------
APPENDIX B
ZEU-INMUNOTEC MicroCystest Raw Data
Table B-l. ZEU-INMUNOTEC MicroCystest Raw Data
Sample Description
Reagent Blank
Reagent Blank
Reagent Blank
Reagent Blank
Reagent Blank
Reagent Blank
Positive Control 1
Positive Control 2a
Positive Control 2b
Positive Control 3
Positive Control 4
Positive Control 5a
Positive Control 5b
Positive Control 6
StdODiffLot
StdO.lDiffLot
StdO.SDiffLot
StdO.SDiffLot
Std2.5DiffLot
0.1 LR
0.1 LR
0.1 LR
0.5 LA
0.5 LA
0.5 LA
0.5 LR
0.5 LR
0.5 LR
0.5 RR
0.5 RR
0.5 RR
I.OLA
I.OLA
I.OLA
1.0 LR
1.0 LR
Variant
RB
RB
RB
RB
RB
RB
LR
LR
LR
LR
LR
LR
LR
LR
LR
LR
LR
LR
LR
LR
LR
LR
LA
LA
LA
LR
LR
LR
RR
RR
RR
LA
LA
LA
LR
LR
Mean Cone, (ppb)
Range?
Range?
Range?
0.151
0.137
0.127
0.548
0.651
0.751
0.621
0.794
0.662
0.604
0.564
0.128
0.236
0.457
1.2
2.636
Range?
0.325
0.384
0.631
0.571
0.601
0.616
0.503
0.653
0.446
0.78
0.605
1.489
1.409
1.381
1.746
1.691
Standard Deviation (ppb)
Range?
Range?
Range?
0.046
0.01
0.034
0.165
0.048
0.009
0.024
0.021
0.005
0.027
0.069
0.031
0.022
0.005
0.099
0.039
Range?
0
0
0.006
0.016
0.017
0.028
0.032
0.026
0.024
0.045
0.039
0.025
0.107
0.043
0.065
0.012
cv%
Range?
Range?
Range?
30.1
7.1
26.7
30.2
7.3
1.2
3.8
2.6
0.8
4.5
12.2
23.9
9.2
1.1
8.3
1.5
Range?
0
0
0.9
2.8
2.8
4.6
6.3
4
5.3
5.8
6.4
1.7
7.6
3.1
3.7
0.7
45
-------
Table B-l. ZEU-INMUNOTEC MicroCystest Raw Data Continued
Sample Description
1.0 LR
1.0 RR
1.0 RR
1.0 RR
2.0 LA
2.0 LA
2.0 LA
2.0 LR
2.0 LR
2.0 LR
2.0 RR
2.0 RR
2.0 RR
4.0 RR
4.0 RR
4.0 RR
1.25 LA
1.25 LA
1.25 LA
1.25 LA
1.25 LA
1.25 LA
1.25 LA
1.25 LR
1.25 LR
1.25 LR
1.25 LR
1.25 LR
1.25 LR
1.25 LR
1.25 RR
1.25 RR
1.25 RR
1.25 RR
1.25 RR
1.25 RR
1.25 RR
2 ppb Chloro LA
2 ppb Chloro LA
2 ppb Chloro LA
2 ppb Chloro LA lOx
2 ppb Chloro LA lOx
Variant
LR
RR
RR
RR
LA
LA
LA
LR
LR
LR
RR
RR
RR
RR
RR
RR
LA
LA
LA
LA
LA
LA
LA
LR
LR
LR
LR
LR
LR
LR
RR
RR
RR
RR
RR
RR
RR
LA
LA
LA
LA
LA
Mean Cone, (ppb)
1.538
1.703
1.717
1.646
2.498
2.619
2.553
2.82
2.741
2.564
1.99
2.159
2.177
2.418
2.357
2.335
1.924
1.941
1.831
1.694
1.92
1.893
1.867
2.087
2.348
2.398
2.394
2.238
2.192
2.135
1.593
1.38
1.492
1.883
2.093
2.218
1.868
2.46
2.439
2.463
2.606
2.532
Standard Deviation (ppb)
0.028
0.04
0.013
0.047
0.108
0.028
0.013
0.091
0.081
0.117
0.269
0.031
0.018
0.001
0.118
0.022
0.158
0.058
0.035
0.283
0.014
0.059
0.022
0.054
0.041
0.027
0.029
0.173
0.094
0.033
0.314
0.035
0.075
0.339
0.003
0.091
0.216
0.113
0.008
0.186
0.065
0.03
cv%
1.8
2.3
0.7
2.9
4.3
1.1
0.5
3.2
3
4.6
13.5
1.4
0.8
0
5
0.9
8.2
o
3
1.9
16.7
0.7
3.1
1.2
2.6
1.7
1.1
1.2
7.7
4.3
1.5
19.7
2.5
5
18
0.2
4.1
11.6
4.6
0.3
7.6
2.5
1.2
46
-------
Table B-l. ZEU-INMUNOTEC MicroCystest Raw Data Continued
Sample Description
2 ppb Chloro LA lOx
2 ppb Chloro LR
2 ppb Chloro LR
2 ppb Chloro LR
2 ppb Chloro LR lOx
2 ppb Chloro LR lOx
2 ppb Chloro LR lOx
2 ppb Chloro RR
2 ppb Chloro RR
2 ppb Chloro RR
2 ppb Chloro RR lOx
2 ppb Chloro RR lOx
2 ppb Chloro RR lOx
2 ppb Matrix LA
2 ppb Matrix LA
2 ppb Matrix LA
2 ppb Matrix LA lOx
2 ppb Matrix LA lOx
2 ppb Matrix LA lOx
2 ppb Matrix LR
2 ppb Matrix LR
2 ppb Matrix LR
2 ppb Matrix LR lOx
2 ppb Matrix LR lOx
2 ppb Matrix LR lOx
2 ppb Matrix RR
2 ppb Matrix RR
2 ppb Matrix RR
2 ppb Matrix RR lOx
2 ppb Matrix RR lOx
2 ppb Matrix RR lOx
RWl(20xdil)
RWl(20xdil)
RWl(20xdil)
RW 2 (20x dil)
RW 2 (20x dil)
RW 2 (20x dil)
RW 3 (20x dil)
RW 3 (20x dil)
RW 3 (20x dil)
RW 4 (4x dil)
RW 4 (4x dil)
Variant
LA
LR
LR
LR
LR
LR
LR
RR
RR
RR
RR
RR
RR
LA
LA
LA
LA
LA
LA
LR
LR
LR
LR
LR
LR
RR
RR
RR
RR
RR
RR
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Mean Cone, (ppb)
2.39
2.567
2.642
2.759
2.606
2.506
2.556
1.909
2.014
2.498
2
1.979
2.042
2.584
2.608
2.722
2.536
2.53
g2.129
2.74
2.697
2.723
2.681
2.692
2.737
2.365
2.664
2.443
2.217
2.272
2.222
2.007
2.05
2.071
2.054
1.964
1.493
1.923
2.089
1.985
1.264
1.224
Standard Deviation (ppb)
0.001
0.078
0.017
0.249
0.019
0.064
0.075
0.001
0.019
0.574
0.209
0.146
0.073
0.026
0.017
0.034
0.073
0.011
0.07
0.049
0.104
0.019
0.121
0.016
0.028
0.037
0.306
0.042
0.094
0.054
0.019
0.049
0.064
0.097
0.076
0.056
0.496
0.046
0.11
0.008
0.096
0.03
cv%
0.1
3
0.7
9
0.7
2.6
2.9
0.1
1
23
10.5
7.4
3.6
1
0.6
1.3
2.9
0.5
3.3
1.8
3.9
0.7
4.5
0.6
1
1.6
11.5
1.7
4.3
2.4
0.8
2.4
3.1
4.7
3.7
2.9
33.2
2.4
5.3
0.4
7.6
2.4
47
-------
Table B-l. ZEU-INMUNOTEC MicroCystest Raw Data Continued
Sample Description
RW 4 (4x dil)
RW 5 (4x dil)
RW 5 (4x dil)
RW 5 (4x dil)
RW 6 (2x dil)
RW 6 (2x dil)
RW6 dissolved
RW6 dissolved
RW6 dissolved
RW6 filtrate
RW6 filtrate
RW6 filtrate
RW6 intracellular
RW6 intracellular
RW6 intracellular
RW7
RW7
RW7 dissolved
RW7 dissolved
RW7 dissolved
RW7 filtrate
RW7 filtrate
RW7 filtrate
RW7 intracellular
RW7 intracellular
RW7 intracellular
RW8
RW8
RW8 dissolved
RW8 dissolved
RW8 dissolved
RW8 filtrate
RW8 filtrate
RW8 filtrate
RW8 intracellular
RW8 intracellular
RW8 intracellular
RW9
RW9
RW9
Variant
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Mean Cone, (ppb)
1.202
1.572
1.53
1.568
1.248
1.363
0.334
0.283
Range?
0.25
0.443
0.369
2.016
2.065
2.025
0.102
Range?
Range?
Range?
0.228
Range?
Range?
Range?
Range?
Range?
0.092
0.66
0.714
Range?
0.281
Range?
Range?
Range?
Range?
0.574
0.632
0.577
0.69
0.77
0.694
Standard Deviation (ppb)
0.018
0.112
0.081
0.026
0.192
0.001
0.064
0
Range?
0
0
0.056
0.079
0.05
0.04
0
Range?
Range?
Range?
0
Range?
Range?
Range?
Range?
Range?
0
0.006
0.032
Range?
0.009
Range?
Range?
Range?
Range?
0.013
0.022
0.046
0.024
0.005
0.087
cv%
1.5
7.1
5.3
1.7
15.4
0.1
19.1
0
Range?
0
0
15.2
3.9
2.4
2
0
Range?
Range?
Range?
0
Range?
Range?
Range?
Range?
Range?
0
0.9
4.5
Range?
3.4
Range?
Range?
Range?
Range?
2.2
3.4
7.9
3.5
0.6
12.6
48
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